The Molecular Characterization and Expression of New Human SLC26 Anion Transporters Minna Kujala-Myllynen

Helsinki University Biomedical Dissertations No. 69

Department of Medical Genetics University of Helsinki Finland

Academic dissertation

To be publicly discussed with the permission of the Faculty of Medicine, University of Helsinki, in the large lecture hall of Haartman Institute, Haartmaninkatu 3, Helsinki, on November 18th 2005, at 12 noon. Supervised by: Juha Kere, Professor Department of Biosciences at Novum Karolinska Institute Stockholm, Sweden and Department of Medical Genetics University of Helsinki Helsinki, Finland

Reviewed by: Hannu Jalanko, MD, PhD, Adjunct Professor Hospital for Children and Adolescents University of Helsinki Helsinki, Finland and

Anne Räisänen-Sokolowski, MD, PhD, Adjunct Professor Department of Pathology University of Helsinki, and Helsinki University Central Hospital Helsinki, Finland

Official opponent: Per-Henrik Groop, MD, DMSc, Adjunct Professor Folkhälsan Research Center University of Helsinki Helsinki, Finland

ISSN 1457-8433 ISBN 952-10-2735-5 (paperback) ISBN 952-10-2736-3 (PDF) Yliopistopaino Helsinki 2005 To My Family

Table of Contents

List of Original Publications ...... 13 Abstract ...... 16 Introduction ...... 19 Review of the Literature ...... 23 1. Anion Exchanger Genes and Proteins ...... 23 1.1 The Classic Family of Anion Exchangers Belongs to Solute Carrier Family 4 (SLC4) Bicarbonate Transporters ...... 23 1.1.1 SLC4A1 ...... 24 1.1.2 SLC4A2 ...... 24 1.1.3 SLC4A3 ...... 25 1.2 The Solute Carrier Family 26 (SLC26) Anion Transporter Family ...... 25 1.2.1 SLC26A1 Was Originally Found in Rats ...... 26 1.2.2 Mutations of SLC26A2 Cause Several Bone and Cartilage Diseases of Varying Severity ...... 26 1.2.3 SLC26A3 is Defective in Congenital Chloride Diarrhea ...... 28 1.2.4 Certain Forms of Deafness Result from Flawed SLC26A4 ...... 29 1.2.5 The Cochlear Motor Protein SLC26A5 is Associated with Non-syndromic Deafness ...... 30 1.2.6 The New Members SLC26A6-A11 ...... 31 2. Interactions of Several Proteins Define Overall Ion Transport ...... 32 2.1 Cystic Fibrosis Transmembrane Conductance Regulator . 32 2.2 Sodium/Hydrogen Exchanger 3 ...... 33 2.3 Vacuolar H+ ATPase ...... 33 2.4 Carbonic Anhydrase II ...... 34 2.5 PDZ Domain Containing Proteins ...... 35 3. The Human Kidney ...... 36 3.1 Structure of the Human Kidney ...... 36 3.2 Kidneys Have Many Functions ...... 37 3.2.1 Kidneys Regulate pH of the Body ...... 38 3.2.2 Reabsorption of Na+ and Cl- in the Kidney Tubules ...... 39 3.2.3 Kidneys Can Autoregulate Their Filtration via Juxtaglomerular Complexes ...... 39 3.3 Polycystic and Dysplastic Kidney Diseases ...... 40 3.4 Expression of SLC26A4 in the Kidney ...... 41 4. The Epididymis ...... 42 4.1 Structure of the Human Epididymis ...... 42 4.2 Function of the Epididymis ...... 42 4.2.1 Absorption of Na+, Cl- and Water During the Flow Through the Epididymis ...... 44 4.2.2 Strict Bicarbonate Control is Needed in the Epididymal Ducts for Keeping Sperm Quiescent ...... 44 Aims of the Study ...... 47 Materials and Methods ...... 49 1. Computational Sequence Analysis (I-II) ...... 49 2. Chromosomal Mapping Using Radiation Hybrids (I) ...... 49 3. Cloning of the SLC26A6-A9 Genes (I-II) ...... 50 4. Polyacrylamide Gel Electrophoresis Analysis of Single Base Polymorphism (I) ...... 50 5. Expression Analyses Using Northern Blotting (I-II) ...... 51 6. Expression Analyses Using PCR Panels (I-II) ...... 51 7. Expression of SLC26A6 and A9 in Cell Lines (I-II) ...... 51 8. Functional Transport Measurements in Xenopus Laevis Oocytes (II) ...... 52 9. Tissue Samples (I-IV) ...... 53 10. RT-PCR (III) ...... 53 11. In Situ Hybridization (I-II) ...... 54 12. Antibodies (I-IV) ...... 54 13. Transfection, Immunofluorescence and Peptide Competition (III) ...... 55 14. Western Blotting (III-IV) ...... 56 15. Immunohistochemistry (I-IV) ...... 56 Results ...... 59 1. Detection and Chromosomal Localization of New SLC26 Genes (I) ...... 59 2. Cloning of New SLC26 Genes ...... 60 2.1 SLC26A1 (I) ...... 60 2.2 SLC26A6 (I) ...... 60 2.3 SLC26A7 (I-II) ...... 61 2.4 SLC26A8 (I-II) ...... 62 2.5 SLC26A9 (II) ...... 63 2.6 SLC26A10 and SLC26A11 ...... 64 3. Functional Characterization of the SLC26A7-A9 Transporters (II) .. 64 4. Characterization of the Antibodies (III-IV) ...... 65 4.1 The SLC26A6 Antibodies ...... 65 4.2 The SLC26A7 Antibodies (III) ...... 65 4.3 The SLC26A8 Antibodies (IV) ...... 65 5. Expression Profiles of SLC26A6-A9 (I-II) ...... 66 5.1 SLC26A6 is Expressed in Various Tissues (I) ...... 66 5.2 SLC26A7 Was Observed in the Human Kidneys (II) ...... 67 5.3 SLC26A8 Expression is Restricted to the Testes (II) ...... 68 5.4 SLC26A9 is Located in the Pulmonary Epithelium (II) ...... 68 6. Distinct Expression of SLC26A6 and SLC26A7 in the Human Kidney (III) ...... 68 6.1 SLC26A6 and SLC26A7 mRNA Expression in the Kidneys .... 69 6.2 Specific Expression of the SLC26A6 and SLC26A7 Proteins in Distinct Human Kidney Structures ...... 69 7. Expression of Selected SLC26 and Other Ion Transport Associated Proteins in the Human Epididymis (IV) ...... 72 Discussion ...... 75 1. The SLC26 Family Grew By Several New Structurally Homologous Members ...... 75 2. The New SLC26 Members May Have Important Functions in Certain Tissues ...... 80 3. SLC26A6 and A7 Have Distinct Expression Patterns and Putative Roles in the Human Kidney (III) ...... 83 4. Diverse, Partly Co-localized Expression of the SLC26 Anion Transporters and Interaction Partners in the Human Epididymis (IV) 86 Conclusion and Future Prospects ...... 93 Yhteenveto (Finnish Summary) ...... 94 Sammanfattning (Swedish Summary) ...... 96 Acknowledgements ...... 99 References ...... 102

List of Original Publications

This thesis is based on the following original publications that are referred to in the text by their Roman numerals. In addition, some unpublished data are presented.

I Lohi H., Kujala M., Kerkelä E., Saarialho-Kere U., Kestilä M. and Kere J.: Mapping of Five New Putative Anion Transporter Genes in Human And Char- acterization of SLC26A6, a Candidate Gene for Pancreatic Anion Exchanger. Genomics. 2000 Nov 15; 70(1):102-112.

II Lohi H., Kujala M., Mäkelä S., Lehtonen E., Kestilä M., Saarialho-Kere U., Markovich D. and Kere J.: Functional Characterization of Three Novel Tissue- speciﬁc Anion Exchangers SLC26A7, -A8 and -A9. J Biol Chem. 2002 Apr 19; 277(16):14246-54.

III Kujala M., Tienari J., Lohi H., Elomaa O., Sariola H., Lehtonen E. and Kere J.: The SLC26A6 and SLC26A7 Anion Exchangers Have Distinct Distribution in Human Kidney. Nephron Exp Nephrol 2005;101:50-58.

IV Kujala M., Hihnala S., Tienari J., Kaunisto K., Hästbacka J., Holmberg C., Kere J. and Höglund P.: Expression of Ion Transport Associated Proteins in Human Effer- ent and Epididymal Ducts. Submitted.

Publications I and II have also been included in the thesis of Hannes Lohi (2002).

13 Abbreviations aa amino acid ACG1B achondrogenesis type IB ADPKD autosomal dominant polycystic kidney disease AE anion exchanger AMRC apical mitochondria rich cells ATP adenosine triphosphate AOII atelosteogenesis type II, also known as neonatal osseus dysplasia AQP aquaporin bp base pair CAII carbonic anhydrase II cAMP cyclic adenosine monophosphate CD collecting duct of the kidney CD10 common acute lymphocytic leukemia antigen (CALLA) cDNA complementary DNA CF cystic ﬁbrosis CFTR cystic ﬁbrosis transmembrane conductance regulator CK7 cytokeratin 7 CLD congenital chloride diarrhea DCT distal convoluted tubule of the kidney DFNB4 a form of congenital non-syndromic deafness, also known as neurosensory non-syndromic recessive deafness 4 (NSRD4) DIDS 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid, an inhibitor of several anion channels and transporters dRTA distal renal tubular acidosis DTD diastrophic dysplasia EMC extraglomerular mesangial cells EST expressed sequence tag GADPH glyceraldehyde 3-phosphate dehydrogenase, a housekeeping gene IC intercalated cell of the kidney collecting duct kb kilo base pairs MCDK multicystic dysplastic kidney mRNA messenger RNA NHE Na+/H+ exchanger NHERF-1 Na+/H+ exchanger regulatory factor 1 NHE3 Na+/H+ exchanger 3 nt nucleotide PCR polymerase chain reaction PKC protein kinase C PNRA proximal nephrogenic renal antigen PT proximal tubule of the kidney rMED recessive multiple epiphyseal dysplasia RT-PCR reverse transcriptase polymerase chain reaction

14 SLC solute carrier SLC26A1-A11 solute carrier family 26, members 1-11 TAL thick ascending limb of the loop of Henle in the kidney TH Tamm-Horsfall glycoprotein UTR untranslated region V-ATPase vacuolar proton transporting ATPase

The names refer to the human ortholog when written in all upper case (e.g. SLC26A6), and to other mammalians when in title case (e.g. Slc26a6). The gene names are written in italics (e.g. SLC26A6), the protein names in regular font (e.g. SLC26A6).

15 Abstract

Appropriate control of intracellular ion concentrations is essential for every living cell. Different kinds of cells need distinct ion microenvironments, which are regulated by tissue and cell specific ion transporters. Anion transporters play key roles in several physiological processes, including control of the acid-base balance, regulation of cell growth, volume, metabolism, contractility, intracellular pH, and ion gradi- ents, among other things. When this work was initiated, only two human members of the anion transporter family SLC26 (solute carrier family 26) were known, both of which had distinct tissue specificities. Both genes were associated with rare recessively inherited diseases that belong to the Finnish disease heritage; diastrophic dysplasia and congenital chloride diarrhea. Since the simple nematode Caenorhabditis elegans was found to have seven genes encoding amino acid sequences homologous to the known human SLC26 proteins, we considered it extremely likely that several human SLC26 members were as of yet uncharacterized, and began the search for them. The ultimate goal was to find new SLC26 genes, to characterize their structure, expression and function, and to study their cell specific expression in selected physiologically relevant tissues.

New human SLC26 genes were identified utilizing a human genome-based sequence homology approach with several publicly available databases and computer programs. The putative new gene sequences found were further analyzed by RT-PCR and sequencing, and their chromosomal location was detected by radiation hybrid mapping. Alto- gether seven novel human SLC26 genes (SLC26A1, A6-A11) were identified, their nucleotide and amino acid sequences were inspected with computer programs, and their general tissue specific expression patterns were explored by Northern blotting and PCR of multiple tissue cDNA panels (I). We then focused especially on the molecular characterization of four new genes, SLC26A6-A9.

The SLC26A6 gene, encoding a 738 aa protein, was mapped to chromosome 3p21.3. It was highly homologous in structure with the previously known SLC26 members transporting anions, suggesting that SLC26A6 may function as an anion exchanger as well. Its highest expression levels were in the human kidney and pancreas, but lower levels were found in several other tissues. Utilizing immunohistochemistry, the SLC26A6 protein was localized in human kidney to the distal parts of the proximal tubules, some of the thin and thick ascending segments of the Henle’s loops, macula densa cells, distal convoluted tubules and a subpopulation of intercalated cells in collecting ducts, suggesting important roles in e.g. Cl- reabsorption and tubuloglomerular feedback. In addition, SLC26A6 was detected in a subgroup of cysts in ADPKD and multicystic dysplastic kidneys. In human epididymis, SLC26A6 was located on the luminal edge of the non-ciliated cells of the efferent ducts, together with Cl- channel

16 CFTR, Na+/H+ exchanger NHE3, and their common regulator NHERF-1, suggesting that they form an essential cooperative unit which reabsorbs Na+ and Cl- and controls osmotic water absorption in these structures. Moreover, SLC26A6 was found in apical forms of AMRC in human epididymal ducts, possibly ﬁne-tuning the intraluminal pH and osmolality (I,III,IV).

The SLC26A7 gene was located on chromosome 8q23 and encoded a 656 aa transmembrane protein. Its mRNA was found in the kidneys, testes, and placenta. When - 2- expressed in Xenopus laevis oocytes, SLC26A7 demonstrated Cl , SO4 , and oxalate transport activity. RT-PCR and immunoblotting showed a stronger signal from medullary areas of the human kidney rather than cortical. SLC26A7 was localized to the basolateral side of type A intercalated cells of collecting ducts and extraglomerular mesangial cells in the human kidney using immunohistochemistry, imply- - ing important roles in HCO3 reabsorption and tubuloglomerular feedback, respectively. In human epididymis, SLC26A7 was expressed in a subgroup of basal cells, possibly taking part in the regulation of the principal cells (I-IV).

SLC26A8 and A9, located on chromosomes 6p21 and 1q31-q32 respectively, both showed high tissue specific expression. Both of them were demonstrated to transport - 2- at least Cl , SO4 , and oxalate when expressed in Xenopus laevis oocytes. SLC26A8 was expressed in testes only, more specifically in the spermatocytes and spermatids, as confirmed by in situ hybridization and immunohistochemistry. Therefore, it might have an important role in the meiotic phase of sperm development, and possibly cause impaired male fertility if mutated. Using RT-PCR, Northern blotting, and immunohistochemistry, SLC26A9 was located to the alveolar and bronchial epithelium of human lungs, suggesting a part in maintaining the airway surface liquid needed for defense against bacterial infections (I,II,IV).

The new SLC26 genes are presumed to participate in several fundamental physiological processes involving anion transport in different human organs. Furthermore, they serve as good candidates for as of yet unidentiﬁed hereditary diseases.

Introduction

The ability to maintain the right concentration of different ions is vital for every living cell in all tissues. Strict regulation of ions is required e.g. for controlling the normal electrical potential across the cell membrane. The rapid control of the membrane potential is especially important for achieving contraction of muscle cells and signal transmission of neural cells. In addition, all polarized cells need several ion transporting systems in order to maintain their intracellular polarity and normal functions (Guyton 1991c).

In animal cells, the quantity of different ions in intracellular and extracellular spaces affects the cell volume through osmotic forces. If the concentration of ions inside a cell diminishes considerably compared to extracellular space, the osmolality of the cell diminishes. To balance the osmotic pressure across the cell membrane, the osmotic forces then drive water outside the cell. Consequently, the cell shrinks. If the concentration of intracellular ions compared to extracellular increases strongly, the osmotic pressure causes water to collect inside the cell. Thereupon, the cell swells (Guyton 1991a).

Even slight changes in pH can markedly alter the rates of chemical reactions in a cell. Therefore, the accurate control of pH is one of the most important aspects in maintaining cellular homeostasis. The most important mechanism for maintaining the acid-base homeostasis in the human body is the bicarbonate buffer system. While - HCO3 concentration alters when balancing the acid-base homeostasis, another anion, Cl-, is needed to adjust the shifted anion load in body fluids. Cl- is one of the main electrolytes in the human body, together with cations Na+ and K+. As the most abundant anion in extracellular fluids, Cl- plays an important role in regulating body fluids’ osmolarity as well (Guyton 1991a, Mutanen and Voutilainen 1993).

- - 3- Besides HCO3 , Cl , and PO4 other inorganic anions have speciﬁc important roles 2- in the human body as well. For example, SO4 is needed for proper cell growth and development of an organism, as it is required for cell matrix synthesis and the mainte- 2- nance of cell membranes. SO4 is involved in many detoxiﬁcation processes of endo- and exogenous compounds as well. I-, for its part, is an essential constituent of the thyroidal hormones thyroxine (T4) and triiodothyronine (T3) (Mutanen and Vouti- lainen 1993, Morris and Sagawa 2000).

The lipid bilayer of the cell membrane acts as a barrier, blocking the movement of most water molecules and water-soluble substances such as inorganic ions. The balance of intracellular ions is regulated through numerous different transporter proteins.

19 The transport of ions can occur passively through ion channels by diffusion (Figure 1a) or through active transport. Active transport can be further divided to primary and secondary forms. Primary active transport happens through diverse ion pumps, requiring energy from the breakdown of high energy phosphate compounds such as adenosine triphosphate (ATP) (Figure 1b). Secondary active transport uses the concentration gradient of a certain ion to transport another ion together with it against the gradient of the latter (Figure 1c). Secondary active transport can occur as co-transport, where both ions move in the same direction through the cell membrane, or as counter-transport where the ions move in opposite directions. Usually, each transporter protein is very speciﬁc for the ions that it transports (Guyton 1991c). a b

ADP+Pi ATP

c Figure 1. Diagrams of a) the function of an ion channel, b) primary active transport through an ion pump, and c) secondary active transport through a counter-transport type ion transporter.

This study focused on the anion transporter family SLC26 (solute carrier family 26), whose members transport negatively charged ions across the cell membrane through secondary active transport. The fact that the four ﬁrst characterized human SLC26 genes all cause severe recessively inherited diseases when mutated, conﬁrms the importance of these transporters. A failure in anion transport can manifest in very different ways, depending on the SLC26 transporter affected, as mutations of this gene family can cause at least skeletal dysplasias, congenital chloride diarrhea, Pendred syndrome with deafness and goiter, and non-syndromic deafness (Hastbacka et al. 1994, Hastbacka et al. 1996, Hoglund et al. 1996b, Superti-Furga et al. 1996, Everett et al. 1997, Superti-Furga et al. 1999, Liu et al. 2003).

20 Introduction When this work was started, only two SLC26 gene family members in man were known (SLC26A2 and SLC26A3) (Schweinfest et al. 1993, Hastbacka et al. 1994), and soon the third human member was cloned (SLC26A4) (Everett et al. 1997). How- ever, the simple nematode Caenorhabditis elegans was noted to have seven SLC26 genes (The C. Elegans Sequencing Consortium 1998, Everett and Green 1999, Kere et al. 1999), and thus it was likely that several human SLC26 anion transporters were still uncharacterized.

Introduction 21

Review of the Literature

1. Anion Exchanger Genes and Proteins

1.1 The Classic Family of Anion Exchangers Belongs to Solute Carrier Family 4 (SLC4) Bicarbonate Transporters The classic family of anion exchangers (AEs) consists of at least three distinct proteins that have important roles in the transport of CO2 by erythrocytes, the absorption or + - secretion of H or HCO3 by various epithelia, and the regulation of intracellular volume and pH. The group of AEs form a part of the superfamily of bicarbonate transporters named solute carrier family 4 (SLC4). So far 10 mammalian SLC4 genes have been identiﬁed (SLC4A1-A5, SLC4A7-A11), and the family has been subdivided into three groups according to their functions. The ﬁrst three SLC4 members (SLC4A1- A3, also known as AE1-AE3, respectively, see Table 1) transport purely anions, pre- + - - dominantly the electroneutral Na independent Cl /HCO3 exchange. The remaining + - known members are subdivided into electrogenic and electroneutral Na /HCO3 cotransporters. All SLC4s are structurally membrane proteins with 10-14 transmembrane segments (Romero et al. 2004, McKusick OMIM).

Table 1. The classic family of anion exchangers.

Symbol Also Chromosomal Anions Major Associated Known as Location Transported Expression Diseases Sites - - SLC4A1 AE1, Band 3 17q21-q22 Cl , HCO3 erythrocytes, elliptocytosis, protein kidney ovalocytosis, distal renal tubular acidosis - - SLC4A2 AE2 7q35-q36 Cl , HCO3 ubiquitous not known - - SLC4A3 AE3 2q36 Cl , HCO3 brain, retina, not known heart, smooth muscle

23 1.1.1 SLC4A1

Anion exchanger SLC4A1 (AE1) is located on chromosome 17q21-q22. It is also known as the band 3 protein for its position in SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins. It is the most common membrane glycoprotein in red blood cells. In addition to erythrocytes, it is also expressed in the kidney and at lower levels in the heart and colon. There are two distinct tissue speciﬁc splice variants of SLC4A1, one expressed mainly in erythrocytes and the other in the kidney (Kopito and Lodish 1985, Alper et al. 2002, Romero et al. 2004, McKusick OMIM).

SLC4A1 is one of the first transporters of any type that was physiologically characterized. It has several important roles in the human body. In erythrocytes, SLC4A1 - - mediated Cl /HCO3 exchange increases the CO2 carrying capacity of the blood about five-fold. In addition, it maintains biconcave disk shape of red blood cells. In the kidney, SLC4A1 affects the acidification of the urine in the distal nephron (Romero et al. 2004, Shayakul and Alper 2004).

Distinct mutations in SLC4A1 cause two main types of diseases: morphological changes of erythrocytes (elliptocytosis or ovalocytosis) leading to hemolytic anemia, and distal renal tubular acidosis (dRTA). Interestingly, in some populations originating from malaria areas, SLC4A1 mutations leading to elliptocytosis occur at high frequencies. These changes in SLC4A1 seem to protect individuals from malaria when inherited in heterozygous form. In the kidney, defective SLC4A1 presents either in a milder dominant or a more severe recessive form of inherited dRTA. The recessive form presents typically either with acute illness or with growth failure in the early years of life. dRTA is characterized by hyperchloremic metabolic acidosis of renal origin with - - high urinary pH (>5.5), low plasma HCO3 , raised plasma Cl , and normal anion gap (Shayakul and Alper 2004, Wagner et al. 2004, McKusick OMIM).

1.1.2 SLC4A2

SLC4A2 (AE2), with gene position 7q35-q36, is the most widely distributed of the SLC4 anion exchangers. It has been found virtually in all tissues studied, but its expression level is especially high in gastric parietal cells, choroid-plexus epithelial cells, apical enterocytes of the colon, and the renal collecting duct. In most epithelial cells, SLC4A2 is located on the basolateral membrane. SLC4A2 is believed to contribute to the regulation of intracellular pH by responding to alkali loads by exporting - - HCO3 . It may also have a role in the regulation of cell volume by the uptake of Cl . So far no genetic diseases have been associated with SLC4A2 (Demuth et al. 1986, Palumbo et al. 1986, Romero et al. 2004, McKusick OMIM).

24 Review of the Literature 1.1.3 SLC4A3

Anion exchanger SLC4A3 (AE3) has been mapped to 2q36. It is mainly expressed in excitable tissues such as brain, retina, heart and smooth muscle, although it can be found, e.g., in some epithelial cells of the kidney and gastrointestinal tract as well. SLC4A3 has not been linked to any diseases yet (Kopito et al. 1989, Su et al. 1994, Romero et al. 2004, McKusick OMIM).

1.2 The Solute Carrier Family 26 (SLC26) Anion Transporter Family The SLC26 anion transporters were first called sulfate transporters since the first two 2- mammalian members Slc26a1 and SLC26A2 were originally found to transport SO4 (Bissig et al. 1994, Hastbacka et al. 1994, Satoh et al. 1998). As new members of this 2- gene family were characterized, it became evident that it was not only SO4 but rather several different anions that they transport with different specificities, thus the name of the protein family was changed to solute carrier family 26 (SLC26). Members of the SLC26 family form a structurally homologous group that is clearly distinct from the SLC4 family (Everett and Green 1999, Kere et al. 1999). Therefore, the SLC26 family is also known as the second anion exchanger family.

The ﬁrst three human SLC26 anion transporters SLC26A2-A4 (Table 2) were mostly found by positional cloning of rare recessive diseases: diastrophic dysplasia, congenital chloride diarrhea and Pendred syndrome (Hastbacka et al. 1994, Hoglund et al. 1996b, Everett et al. 1997), although SLC26A3 had been previously cloned as a putative tumor suppressor gene (Schweinfest et al. 1993). While these three inherited disorders are clinically very dissimilar, the associated genes turned out to belong to the same gene family encoding structurally conserved anion transporters. An intriguing fact was that two of these diseases, diastrophic dysplasia and congenital chloride diarrhea, happened to both belong to the Finnish disease heritage, a group of rare inherited diseases that are clustered in the Finnish population (Norio 2003).

Expression of the SLC26 family members differs dramatically. A good example of this are SLC26A2 and SLC26A3: while the ﬁrst is found ubiquitously in nearly all human tissues studied, the second seems to have extremely strict tissue speciﬁc expression pattern (Hastbacka et al. 1994, Haila et al. 2000, Haila et al. 2001).

Review of the Literature 25 Table 2. The characteristics of the four human SLC26s not cloned in this work.

Symbol Also Known Chromosomal Anions Major Associated as Location Transported Expression Diseases Sites 2- - SLC26A2 DTDST 5q32-q33.1 SO4 , Cl ubiquitous achondrogenesis type IB, atelosteogenesis type II, diastrophic dysplasia, recessive multiple epiphyseal dysplasia - 2- SLC26A3 CLD, DRA 7q22-q31.1 Cl , SO4 , colon congenital chlo- - - HCO3 , OH , ride diarrhea oxalate - - - SLC26A4 PDS, Pendrin 7q31 I , Cl , HCO3 , thyroid, Pendred syn- OH-, formate kidney, drome, congenital cochlea non-syndromic deafness, enlarged vestibular aqueduct syndrome - - SLC26A5 Prestin 7q22.1 (Cl , HCO3 ) cochlea recessive non-syndromic neurosensory deafness

1.2.1 SLC26A1 Was Originally Found in Rats The first mammalian member of SLC26 anion transporters, Slc26a1, was originally cloned from rat liver mRNA in 1994 (Bissig et al. 1994). Since it was found to mediate + 2- Na independent SO4 transport, it was originally named sulfate anion transporter-1 (sat-1) (Bissig et al. 1994, Markovich et al. 1994). Northern blot analysis indicated a strong signal for rat Slc26a1 mRNA in the liver and kidney, and a weaker one in muscle and brain (Bissig et al. 1994). Slc26a1 protein was localized specifically to the basolateral membrane of the proximal tubules in rat kidney, and it was shown to at 2- least mediate SO4 and oxalate transport (Karniski et al. 1998). The human ortholog SLC26A1 was not characterized until our group published it in 2000 (I). Our results were confirmed and extended upon in 2003 by Regeer et al., who described the structure of human SLC26A1 in more detail (Regeer et al. 2003).

1.2.2 Mutations of SLC26A2 Cause Several Bone and Cartilage Diseases of Varying Severity

The SLC26A2 gene was found by Hastbacka et al., when they were seeking the gene responsible for diastrophic dysplasia by positional cloning using ﬁne-structure linkage

26 Review of the Literature disequilibrium mapping. Therefore the gene was first named diastrophic dysplasia sulfate transporter (DTDST) (Hastbacka et al. 1994). Diastrophic dysplasia (DTD, DD; OMIM #222600) is a globally rare autosomal recessive osteochondrodysplasia. However, it is prevalent in the Finnish population due to a founder effect; less than 500 patients have been described worldwide, but almost 200 of them are from Fin- land (Hastbacka et al. 1994, Norio 2003). DTD is a congenital disease with clinical features of short limbs, short stature, stiffness of the big joints, flexion limitation of the finger joints, scoliosis, kyphosis, clubfoot and hitchhiker’s thumb malformations, deformation of ear lobes, and often cleft palate. Generalized dysplasia of the joints frequently leads to motility restrictions and early arthrosis. The severity of the disease can even vary intrafamilially (Norio 2003, McKusick).

The SLC26A2 gene is located on chromosome 5q32-q33.1 (McKusick OMIM). It consists of 2833 nucleotides encoding a 739 aa transmembrane protein with a predicted molecular mass of 82 kDa and 12 transmembrane domains (Hastbacka et al. 1994). It is expressed widely in the human body, and the protein has been immunolocalized to cartilage, colon, eccrine sweat glands, bronchial glands, tracheal epithelium, placenta, and exocrine pancreas. In these tissues, many distinct cell types produce SLC26A2, but the protein is mostly located in secretory structures (Haila et al. 2001). In addition, by PCR, SLC26A2 mRNA has been detected in several tissues that did not stain with anti-SLC26A2 antibodies in immunohistochemistry, including e.g. kidney, testis, and prostate. That probably indicates that in these tissues SLC26A2 expression level is very low or it is restricted to small areas or structures (Hastbacka et al. 1994, Haila et al. 2001).

Because of the structural similarity to the previously characterized Slc26a1, SLC26A2 2- 2- was suggested to be a SO4 transporter, and a defect in SO4 transport could indeed be demonstrated in ﬁbroblasts of a DTD patient (Hastbacka et al. 1994). Further ver- iﬁcation was brought by Xenopus laevis oocyte experiments showing that SLC26A2 + 2- mediates DIDS-sensitive Na independent SO4 transport that could be inhibited by thiosulfate, oxalate and Cl-. The results indicated that SLC26A2 would function as 2- - a SO4 /Cl exchanger (Satoh et al. 1998). Even though SLC26A2 is likely to trans- 2- port other anions as well, direct transport studies of ions other than SO4 are lacking. Thus the full combination of its substrate anions is still unknown (Mount and Romero 2004).

Soon after the cloning of SLC26A2, mutations in this gene were associated with other bone and cartilage diseases as well. Achondrogenesis type IB (ACG1B, OMIM # 600972) is a perinatally lethal autosomal recessive disease characterized by extremely short limbs caused by poor skeletal development. Typical radiological findings are defi- cient ossification in the lumbar vertebrae and absent ossification in the sacral, pubic and ischial bones (Borochowitz et al. 1988, McKusick OMIM). The finding that one 2- ACG1B patient had impaired SO4 metabolism, leading to a reduction in sulfated proteoglycans (Superti-Furga 1994), motivated Superti-Furga et al. to test the newly

Review of the Literature 27 found SLC26A2 for possible mutations in ACG1B patients. Indeed, mutations were found, conﬁrming DTD and ACG1B to be allelic disorders with very different clinical severity (Superti-Furga et al. 1996).

Atelosteogenesis type II (AOII, also known as neonatal osseus dysplasia) is a neona- tally lethal chondrodysplasia that clinically and histologically resembles the less severe disease DTD. It is presumed to be an autosomal recessively inherited disease. AOII patients have strikingly short limbs, small chest, clubfeet, spinal abnormalities, cleft palate, and abducted thumbs and toes. In radiography, prevalent findings include bifid distal humerus, rounded distal femur, cervical kyphosis, scoliosis, lumbar hyperlor- dosis, and disproportion of certain metatarsals and metacarpals. Collapse of the air- ways and pulmonary hypoplasia caused by too small rib cage inflict respiratory insufficiency, and AOII patients die soon after birth (Hastbacka et al. 1996, McKusick OMIM). Interestingly, it was shown that AOII is also caused by specific mutations in 2- the SLC26A2 gene, leading to faulty uptake of inorganic SO4 and inadequate sul- fation of macromolecules (Hastbacka et al. 1996).

A fourth disease has been associated with SLC26A2. An autosomal recessive form of multiple epiphyseal dysplasia (rMED, MED4; OMIM # 226900) can clinically manifest as clubfoot only, with normal body height. With X-rays epiphyseal dysplasia can be detected. However, rMED patients may have short limbs, short stature, movement limitations or stiffness of joints, double-layered patella, and hip dysplasia as well (Superti-Furga et al. 1999, Huber et al. 2001, Makitie et al. 2003, McK- usick OMIM).

Over 30 mutations of SLC26A2 have been detected so far. Interestingly, genotype – phenotype correlations are observable, since patients with the most severe disease ACG1B tend to be homozygous, and AOII patients heterozygous for loss-of-function mutations, which cause a truncated protein or a non-conservative amino acid substitu- tion in a transmembrane domain. Respectively, individuals with milder diseases DTD or rMED are typically homozygous for alleles encoding a protein with some residual activity, having non-transmembrane amino acid substitutions and splice site mutations (Hastbacka et al. 1996, Karniski et al. 1998, Rossi and Superti-Furga 2001).

1.2.3 SLC26A3 is Defective in Congenital Chloride Diarrhea

SLC26A3 was ﬁrst identiﬁed by Schweinfest et al. as a gene that was down-regulated in colon adenomas and adenocarcinomas, hence it received the name DRA (down- regulated in adenoma) (Schweinfest et al. 1993). It is located on chromosome 7q22- q31.1 spanning over 39 kb (Taguchi et al. 1994, McKusick OMIM). The SLC26A6 mRNA includes 2881 bp corresponding to a 764 aa open reading frame of a 84,5 kDa transmembrane protein with predicted 10, 12 or 14 transmembrane domains (Schweinfest et al. 1993, Byeon et al. 1996, Haila et al. 1998).

28 Review of the Literature The SLC26A3 protein has a strict tissue speciﬁc expression pattern, and so far it has been immunolocalized only in epithelium of colon, eccrine sweat glands, and seminal vesicles in humans (Haila et al. 2000). It has been shown to function as a Na+ independent 2- - - - anion exchanger transporting at least SO4 , Cl , HCO3 , OH , and oxalate. Its function can be inhibited by the anion transporter inhibitor DIDS (Silberg et al. 1995, Byeon et al. 1998, Melvin et al. 1999, Moseley et al. 1999, Jacob et al. 2002).

Congenital chloride diarrhea (CLD, CCD; OMIM #214700) is a rare autosomal recessive disease with voluminous watery diarrhea that already begins in utero. Clin- ical findings include polyhydramnios, premature birth, excessive weight loss, byper- bilirubinemia and dehydration of the affected newborn. These are caused by exten- - - - sive loss of Cl through the stool caused by defective Cl /HCO3 exchange in the colon. The treatment of CLD by compensating for the lost Cl- is lifelong. However, if the right diagnosis is not made and treatment not started, the affected individuals die of severe electrolyte perturbation within the first weeks of life (Gamble et al. 1945, Hol- mberg et al. 1975, Norio 2003). Worldwide, CLD is an extremely rare disease. How- ever, it is part of the Finnish disease heritage, and the incidence is about 1:20,000 in Finland. It is also common in some Arabic countries, e.g. Kuwait, where as many as 1:3,200 newborns may have CLD (Badawi et al. 1998, Makela et al. 2002). Interest- ingly, it was noted recently, as the first properly treated CLD patients have reached reproductive age, that the male patients appear to be subfertile. Despite normal spermatogenesis, CLD patients were found to have oligoasthenoteratozoospermia, and high Cl- but low pH in seminal plasma as well as spermatoceles. This suggests that the gene responsible for CLD might have a role in the proximal male reproductive tract in addition to the seminal vesicles (Hoglund et al. 2005).

Using a candidate gene approach, Kere et al. found the gene responsible for CLD to locate near, but to be different from, the cystic fibrosis transmembrane regulator (CFTR) gene on chromosome 7 (Kere et al. 1993). The localization of the CLD gene was further refined by a linkage disequilibrium study in the Finnish founder population and on a physical map (Hoglund et al. 1995, Hoglund et al. 1996a), and finally mutations in SLC26A3 were shown to cause CLD (Hoglund et al. 1996b). Hence the gene is also known as CLD. The SLC26A3 mutations identified in CLD patients have been shown to cause loss-of-function in vitro assays with Xenopus laevis oocytes (Moseley et al. 1999). No diseases other than CLD have been associated with the SLC26A3 gene (Makela et al. 2002).

1.2.4 Certain Forms of Deafness Result from Flawed SLC26A4

SLC26A4 was found by positional cloning of the gene mutated in Pendred syndrome (OMIM # 274600). Thus the gene, and the protein it encodes, ﬁrst received the names PDS and Pendrin, respectively (Everett et al. 1997). Pendred syndrome is the most common form of inherited syndromic hearing loss, accounting for up to 10%

Review of the Literature 29 of hereditary deafness. It is an autosomal recessive disease, characterized by sensorineural hearing loss and euthyroid goiter. Deafness may exist at birth or develop during the early years of childhood. Some Pendred syndrome patients have vestibular dysfunction or mental retardation. Development of thyroid carcinoma has also been reported (Everett et al. 1997, McKusick OMIM).

In addition to Pendred syndrome, mutations in SLC26A4 have been associated with other forms of recessive hearing impairment. Both a form of congenital non-syndromic deafness (DFNB4, also known as neurosensory nonsyndromic recessive deafness 4 NSRD4; OMIM #600791), and enlarged vestibular aqueduct syndrome (EVA; OMIM #603545) characterized by ﬂuctuating and sometimes progressive sensorineural hearing loss and disequilibrium symptoms, are in some cases caused by mutations in this gene (Li et al. 1998, Pryor et al. 2005, McKusick OMIM).

SLC26A4 maps to chromosome 7q31, less than 50 kb from SLC26A3. These genes are also structurally very similar, even within the SLC26 family, suggesting evolutionary relationship (Everett et al. 1997, Kere et al. 1999). SLC26A4 is a 780 aa transmembrane protein with highly tissue speciﬁc expression. It has been located to the thyroid gland and the inner ear, as expected from the Pendred syndrome phenotype, but also to Sertoli cells, fetal brain and both fetal and adult kidneys (Everett et al. 1997, Lac- roix et al. 2001). Interestingly, the exact expression of SLC26A4 in distinct speciﬁc kidney tubules is debated, and differences between species possibly exist in cell types producing this anion transporter (Lacroix et al. 2001, Royaux et al. 2001, Soleimani et al. 2001, Kim et al. 2002, Wall et al. 2003). Since none of the Pendred syndrome patients have been reported to have any kidney symptoms, it is possible that some other anion transporters may replace its function in the kidney.

2- Functional experiments have revealed that SLC26A4 does not transport SO4 , as the three first mammalian SLC26 members do. Instead, it is capable of mediating I-, Cl-, - - HCO3 , OH , and formate transport (Kraiem et al. 1999, Scott et al. 1999, Bogazzi et al. 2000, Scott and Karniski 2000, Soleimani et al. 2001). The mutations associated with Pendred syndrome cause complete loss of Cl- and I- transport in the Xenopus laevis oocyte experimental model, while the alleles leading to DFNB4 only reduce the transport efficiency of these anions. It is probable that the residual transport capability is sufficient to avert the onset of goiter in DFNB4 (Scott et al. 2000).

1.2.5 The Cochlear Motor Protein SLC26A5 is Associated with Non-syndromic Deafness

Slc26a5 was ﬁrst cloned from a gerbil by Zheng et al., when seeking the motor protein of the outer hair cells of the cochlea. It was ﬁrst designated Prestin after the musical notation ‘presto’ (Zheng et al. 2000). The outer hair cells are non-neuronal epithelial cells that mechanically amplify the acoustic signals entering the inner ear. When

30 Review of the Literature their membrane potential changes, the outer hair cell stiffness and length is altered rapidly, causing variation in the cell shape of up to 5% (Kachar et al. 1986, Ashmore 1987, Zheng et al. 2000). Slc26a5 was located speciﬁcally to the outer hair cells only. Most importantly, it was able to cause cell shape changes when expressed in cultured - - human kidney cells (Zheng et al. 2000). Intracellular Cl and HCO3 anions were shown to act as extrinsic voltage sensors, binding to Slc26a5, thereby triggering con- formational changes in outer hair cells (Oliver et al. 2001). Thus, Slc26a5 was con- cluded to act as the motor protein of the outer hair cells (Zheng et al. 2000, Oliver et al. 2001). This suggestion was supported by the ﬁnding that the targeted deletion of Slc26a5 caused the loss of outer hair cell electromotility and hearing impairment in mice (Liberman et al. 2002).

Human SLC26A5 is located on chromosome 7q22.1 and contains 21 exons. It has at least four different splicing variants, encoding proteins having 7-12 predicted transmembrane domains, and varying between 335 and 746 aa in size. SLC26A5 mRNA expression was found only in the cochlea of the 13 human tissues tested. Interest- ingly, mutations in SLC26A5 have been found in some Caucasian probands with recessive non-syndromic neurosensory deafness (DFNB61; OMIM +604943) (Liu et al. 2003, NCBI Entrez).

1.2.6 The New Members SLC26A6-A11

In this thesis, the identification and molecular characterization of six new human SLC26 anion transporters is described (I-II) and the specific expression of selected proteins in human kidney and epididymis is studied in detail (III-IV). Simultaneously with our work, other groups have cloned some of these genes as well. A few months after our article tentatively describing and mapping these six genes for the first time came out in Genomics (I), Waldegger et al. published identification of human SLC26A6 in the same journal (Waldegger et al. 2001). While we were preparing our second man- uscript presenting SLC26A7-A9 in more detail (II), Touré et al published the human SLC26A8 and named it Tat1 for Testis Anion Transporter 1 (Toure et al. 2001). An article about molecular cloning of human SLC26A7 by Vincourt et al. was printed in February 2002 (Vincourt et al. 2002), the same month when our SLC26A7-A9 article was published on the Web (II). A year later Vincourt et al. reported the further molecular and functional characterization of human SLC26A11 (Vincourt et al. 2003). So far, no totally new mammalian members of the SLC26 family have been delineated since our first article (I).

Review of the Literature 31 2. Interactions of Several Proteins Define Overall Ion Transport

Net ion transport to and from a cell is a sum of the actions of several distinct transporters, channels, and their regulators. Several proteins have been shown either to interact directly with SLC26 members or to share the same interaction partners, suggesting that they form functional cooperation units. The interaction partners identi- ﬁed so far, and one functionally reasonable potential interaction partner of the SLC26 anion transporters are introduced below (Table 3).

Table 3. The presumable interaction partners of the SLC26 proteins.

Symbol Also Chromosomal Protein Type Major Associated Known as Location Expression Diseases Sites CFTR 7q31.2 Cl- channel ubiquitous cystic ﬁbrosis NHE3 SLC9A3 5p15.3 Na+/H+ kidney, intestine not known exchanger V-ATPases ATP6 multiple subunits H+ pump ubiquitous recessive dis- located in differ- tal renal tubu- ent chromosomes lar acidosis, recessive infantile malignant osteopetrosis CAII 8q22 zinc metallo- ubiquitous osteopetro- enzyme sis with renal tubular acidosis and cerebral calciﬁcation NHERF-1 SLC9A3R1, 17q25.1 apical PDZ ubiquitous not known EBP50 protein

2.1 Cystic Fibrosis Transmembrane Conductance Regulator Cystic fibrosis (CF; OMIM # 219700) is an autosomal recessive condition and the most common lethal genetic disease in Caucasian populations, with an incidence of 1:2,000 live births. However, it is much rarer in Finland, with only 1-2 new cases per year. CF is associated with a widespread dysfunction in secretory processes of several exocrine glands all over the human body (Savilahti 1997, Kinnunen et al. 2005, McKusick OMIM). It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a Cl- channel (Riordan et al. 1989). Clinical manifestation of CF varies enormously, from a life threatening lung and pancreas insufficiency at early age to mild forms not detected before middle age. Possible

32 Review of the Literature symptoms are, inter alia, meconium ileus, liver disease with biliary duct obstruction, pancreatic insufﬁciency, recurrent pulmonary infections, congenital absence of vas deferens, and infertility. There is also evidence that CFTR mutations can cause poor sperm quality without any other clinical symptoms. CFTR genotype determines the biochemical abnormality, which further deﬁnes the clinical phenotype of CF (Bien- venu et al. 1993, Sheppard et al. 1993, van der Ven et al. 1996, Savilahti 1997, Wong et al. 2002, McKusick OMIM).

CFTR is located on chromosome 7q31.2 and is expressed in numerous different epithelia. It encodes a cAMP-regulated Cl- channel that controls the regulation of other ion transport processes as well (Riordan et al. 1989, Anderson et al. 1991a, Ander- son et al. 1991b, Lee et al. 1999, Mendes et al. 2004, McKusick OMIM). Recently, it was shown that the CFTR and SLC26 transporters have a reciprocal activation capability for each other. This interaction is mediated by the binding of the STAS (sulphate transporter and anti-sigma antagonist) domain of SLC26 family members with the regulatory (R) domain of CFTR, and is facilitated by their PDZ ligands (Ko et al. 2004).

2.2 Sodium/Hydrogen Exchanger 3

Na+/H+ Exchanger 3 (NHE3, SLC9A3) is an apical Na+/H+ exchanger involved in transepithelial electroneutral Na+ absorption (Tse et al. 1992, Tse et al. 1993). It belongs to the gene family of Na+/H+ exchangers (NHE, also known as SLC9) that all catalyze the exchange of one extracellular Na+ for one intracellular H+ across the plasma membrane. Today, nine human members of the NHE family have been iden- tiﬁed (Zachos et al. 2005, McKusick OMIM).

NHE3 is located on human chromosome 5p15.3 and expressed in renal and intestinal epithelia (Brant et al. 1993, McKusick OMIM). So far, no human disease has been associated with this gene (McKusick OMIM). NHE3 activity is controlled by recycling it between the cell membrane and subapical endosomes (Janecki et al. 1998, Zachos et al. 2005). There is an indication that NHE3 may operate together with some of the SLC26 proteins, since expression of Slc26a3 mRNA was modestly upregulated in the colon of mice lacking NHE3, suggesting that these transporters normally act together to absorb Na+ and Cl- (Melvin et al. 1999).

2.3 Vacuolar H+ ATPase

The vacuolar-type proton ATPases (V-ATPases, ATP6) are ubiquitous multi-subunit proteins. They are expressed practically in every eukaryotic cell, located on the membranes of intracellular organelles, such as endosomes and lysosomes. Their main function is the pH regulation of these structures by acidiﬁcation. In addition, V-ATPases

Review of the Literature 33 are also involved in hydrogen ion transport across the plasma membrane into the extracellular space in some specific cell types, e.g., in the epididymis where seminal fluid is acidified, or in the kidney where V-ATPases are involved in acid-base transport, thus contributing to overall body homeostasis (Breton et al. 1996, Sun-Wada et al. 2004, Wagner et al. 2004, McKusick OMIM).

V-ATPases consist of two main domains, a peripheral catalytic V1 domain (640 kDa;

ATP6V1) and a membrane-bound V0 domain (240 kDa; ATP6V0), together forming a protein complex of approximately 900 kDa. The cytosolic V1 domain is com- posed of eight subunits, named A-H (ATP6V1A-H), containing three catalytic sites for ATP hydrolysis. The V0 sector, containing up to ﬁve subunits (a, c, cV, cU and d; ATP6V0A-D), translocates protons across the membranes. Some of the subunits further have several distinct isoforms. The genes encoding for the different subunits and isoforms are spread all over the human genome and show different expression patterns (Sun-Wada et al. 2004, Wagner et al. 2004, McKusick OMIM).

At least three different inherited diseases are associated with distinct isoforms of V- ATPase subunits. V-ATPase subunit B1 (ATP6V1B1) is located on chromosome 2p13, and expressed in the inner ear, intercalated cells of the kidney, epididymis, and cil- iar body. When mutated, it causes a syndrome of recessive distal renal tubular acidosis with sensorineural hearing loss (dRTA with SNHL). This disorder inﬂicts severe metabolic acidosis, together with perturbations of K+ balance, urinary Ca2+ solubility, bone physiology, and growth. The hearing loss is progressive in nature, with the age at the diagnosis of hearing loss quite variable (Karet et al. 1999, Alper 2002, Wagner et al. 2004). Mutations in the V-ATPase subunit a4 encoding gene (ATP6V0A4) cause recessive distal renal tubular acidosis dRTA. This subunit is located almost solely on the apical surface of type A intercalated cells in the human kidney (Smith et al. 2000, Alper 2002, Wagner et al. 2004).

The a3 subunit of V-ATPase (ATP6V0A3), expressed chieﬂy in osteoclasts, has been shown to be essential for the maintenance of normal bone turnover. Mutations in the a3 subunit cause a subset of autosomal recessive infantile malignant osteopetrosis (OMIM # 259700). This disease is characterized by macrocephaly, progressive deafness and blindness, hepatosplenomegaly, and severe anemia beginning in early infancy or in utero (Frattini et al. 2000, McKusick OMIM).

2.4 Carbonic Anhydrase II

Carbonic anhydrases (CAs) form a large family of zinc-containing metalloenzymes + - that catalyze the reversible hydration of carbon dioxide and water to H and HCO3 , thus playing roles in acid-base regulation. The tissue distribution of the different CAs shows a high degree of species variation. CAs are encoded by members of 3 independent CA gene families; alpha-CA, beta-CA, and gamma-CA.

34 Review of the Literature Carbonic anhydrase II (CAII) is a highly active cytoplasmic member of the alpha- CA family. It is ubiquitously expressed in human tissues (Alper 2002, McKusick OMIM). CAII interacts with the carboxyl terminus of anion exchanger SLC4A1, - forming a membrane protein complex involved in the regulation of HCO3 metabolism and transport. This interaction enhances anion transport activity and allows maximal transport (Vince et al. 2000, Sterling et al. 2001). In addition, CAII may - be needed for full-efﬁcacy HCO3 transport by anion transporter SLC26A3 (Sterling - et al. 2002). Interestingly, CAII was very recently shown to form a HCO3 transport metabolon complex with SLC26A6 (Alvarez et al. 2005).

Mutations in CAII lead to the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calciﬁcation. In this syndrome, renal tubular acidosis appears in a mixed form, showing both proximal and distal components. Bone marrow stem cell transplantation can correct the osteopetrosis and halt progression of cerebral calciﬁcation, but not the renal symptoms (Sly et al. 1985, McMahon et al. 2001, Alper 2002, McKusick OMIM).

2.5 PDZ Domain Containing Proteins

For any epithelium to gain directional secretory and absorptive functions, correct localization of distinct transporters to speciﬁc membranes and their strict regulation is crucial. Recent studies have shown that PDZ (PSD-95/Disc-large/ZO-1, named after the ﬁrst three proteins found to contain PDZ domains) domain containing proteins (PDZ proteins) have important roles in both of these tasks. They retain proteins on the correct membrane and bring them close to each other, enabling their interactions and the formation of transducisomes, which are spatially restricted units of functionally connected proteins such as transporters, receptors or kinases. Several different PDZ proteins targeted either for the apical or basolateral membrane have been characterized so far. Human genome analysis suggested the presence of 540 distinct PDZ domains in 306 different proteins, making the PDZ domain one of the most abundant modular domains found in human proteins. Multiple PDZ domains, together with other protein-interaction domains, within a single protein further expand the possible functional variety of protein networks. PDZ ligands, for their own part, are sequences a few amino acids long usually located at the target proteins’ carboxyl terminus which are needed for interactions with PDZ domain containing proteins (She- nolikar et al. 2004, Brone and Eggermont 2005).

One of the most studied apical PDZ domain containing proteins is the Na+/H+ exchanger regulatory factor 1 (NHERF-1). It was originally found to be a regulator of NHE3 (SLC9A3) (Weinman et al. 1995), and thus it is also called SLC9A3 regulatory factor 1 (SLC9A3R1). A third name for NHERF-1 is 50kDa ezrin-radixin- moesin binding phosphoprotein (EBP50) (Reczek et al. 1997, McKusick OMIM).

Review of the Literature 35 NHERF-1 is expressed widely in human tissues, however most abundantly in polarized epithelia tissues such as kidney, small intestine, placenta and liver. NHERF-1 is needed at least for the cAMP mediated inhibition of NHE3, but NHE3 is able to function as a transporter even without NHERF-1. In addition, NHERF-1 has been shown to interact e.g. with SLC26A6, CFTR, and H+V-ATPase subunit B1, that all include carboxyl terminal consensus PDZ binding sequences (Weinman et al. 1995, Yun et al. 1997, Wang et al. 1998, Breton et al. 2000, Weinman et al. 2000, Lohi et al. 2003, Shenolikar et al. 2004, Brone and Eggermont 2005).

Thus far, no human diseases have been directly associated with NHERF-1. However, psoriasis, a chronic inflammatory dermatosis of multifactorial ethiology, has been associated in or near the NHERF-1 gene in one study (Helms et al. 2003), but that association could not be replicated by another group (Stuart et al. 2005). Interestingly, some mutations in human disease genes that encode NHERF-1 target proteins have been associated with impaired NHERF-1 binding. For example, some mutations in CFTR cause decreased affinity for NHERF-1, possibly inflicting reduced targeting of the mutant CFTR proteins to the apical surface of epithelial cells and affecting the regulation of Cl- transport (Moyer et al. 2000, Shenolikar et al. 2004).

3. The Human Kidney

3.1 Structure of the Human Kidney A human kidney contains over one million functional units called nephrons. A single nephron consists of a glomerulus, which ﬁltrates primary urine, and a uriniferous tubule, which modulates the primary urine into the ﬁnally excreted urine. The uriniferous tubule is further divided into distinct segments according to the tubule shape and epithelium: proximal tubule (PT) consisting of proximal convoluted tubule and proximal straight tubule, thin segment including descending and ascending thin limbs, thick ascending tubule, macula densa, distal convoluted tubule, connecting tubule, and collecting duct (Figure 2). The descending proximal straight tubule, the thin segment and the distal thick ascending tubule together form the loop of Henle. The collecting duct can be further subdivided into cortical, outer medullary, and inner medullary segments (Ross and Romrell 1989).

While the proximal segments consist mainly of one cell type each, there are at least three distinct cell types in the connecting tubules and collecting ducts: principal cells, and at least two subtypes of intercalated cells (IC), namely types A, and B (or alpha, and beta, respectively). Intercalated cells make up approximately 40% of the overall cell population in the cortical collecting duct and in the outer medulla, while in the inner medulla they are only found in the initial segment (Wagner and Geibel 2002).

36 Review of the Literature Figure 2. A schematic diagram of the nephron segments.

1 = afferent arteriole; 2 = efferent arteriole; 3 = glomerulus; 4 = urinary space; 5 = Bowman’s capsule; 6 = proximal convoluted tubule; 7 = thick descending limb (proximal straight tubule); 8 = thin descending limb; 9 = thin ascending limb; 10 = thick ascending limb (distal straight tubule); 11 = macula densa; 12 = extraglomerular mesangial cells; 13 = distal convoluted tubule; 14 = connecting tubule; 15 = collecting duct. Units consisting of several segments: 3 + 4 + 5 = renal corpuscle; 6 + 7 = proximal tubule; 7 + 8 + 9 + 10 = the loop of Henle; 10 + 11 + 13 = distal tubule.

3.2 Kidneys Have Many Functions

Kidneys have several important physiological functions, including the regulation of water and electrolyte balance, controlling of acid-base homeostasis, excretion of metab- olites and foreign chemicals, as well as participation in the regulation of the blood pressure. Different nephron segments have specific roles in these tasks. The glomeruli together filtrate about 180 liters of primary urine within 24 hours. Over 99% of the water filtered through the glomeruli is reabsorbed in the nephron tubules. Since water transport in the kidney tubules occurs by osmotic diffusion, successful reabsorption of

Review of the Literature 37 osmotic particles such as ions is of great signiﬁcance. Active secretion of ions into the produced urine occurs during its ﬂow through the tubules as well (Guyton 1991a).

3.2.1 Kidneys Regulate pH of the Body

The normal pH of arterial blood is 7.4, whereas in venous blood and interstitial ﬂuids it is about 7.35. If the pH is lower, a person is considered to have acidosis, and with higher pH values alkalotic. The normal intracellular pH ranges between 6.0 and 7.4 in various cell types. The human body is normally able to maintain body liquid pH values with the help of three compensatory mechanisms. The quickest way, acting within a fraction of a second, is the acid-base buffer systems present in all body liquids, - of which HCO3 is the most common. According to the physiochemical equilibrium, - HCO3 is able to rapidly balance the pH by fusing with hydrogen to form water and carbon dioxide, or vice versa, in the reaction catalyzed by carbonic anhydrases:

+ - H + HCO3 ⇌ H2CO3 ⇌ H2O + CO2

If the amount of hydrogen is changed substantially, it stimulates the respiratory center, leading to an adjustment in the breathing rate within minutes. As a consequence, excretion of carbon dioxide from the body is changed (Guyton 1991a).

The third, and most powerful, compensatory mechanism is the excretion of either acidic or alkaline urine by the kidneys, requiring from several hours to many days for restoring the overall body pH balance. This is achieved by three mechanisms: reabsorp- - tion of most of the ﬁltered HCO3 , titration of non-bicarbonate buffers, mainly phosphate, and generation and excretion of ammonium cations (Guyton 1991a, Capasso et al. 2002).

Distinct metabolic processes of the body yield acidic end products, in other words, surplus H+. Therefore under normal conditions kidneys actively excrete H+. The final urine pH is a consequence of the original pH in the glomerular filtrate and a series of several complex transport processes occurring along the diverse nephron segments. - Approximately 70-90% of HCO3 is reabsorbed in the proximal tubule, predominantly in the initial parts of them, coupled to H+ secretion. Large amounts of H+ are secreted as a result of countertransport with significant Na+ reabsorption, chiefly through the apical NHE3. Smaller quantities of H+ are transported Na+ independently through the proton pump V-ATPase (Laghmani et al. 2002). In the loop of Henle (mainly in the thick ascending limb) and distal convoluted tubule, an additional 5-15% and 5- - 9% of the filtrated HCO3 is reabsorbed, respectively, with a proposed major role of H+/Na+ exchange again.

The collecting duct serves as the ﬁnal regulator of the urine pH and H+ excretion, affected by several factors like hormones and diet. The two types of intercalated cells

38 Review of the Literature + - are the major sites of H and HCO3 secretion or reabsorption. Type A intercalated - - cells have the Cl / HCO3 -exchanger SLC4A1 on the basolateral membrane and the proton pump, i.e., vacuolar H+-ATPase on the apical side, thus secreting H+ into the - + urine and thereupon reabsorbing HCO3 . In contrast, type B cells express H -ATPase - - at the basolateral edge and a Cl / HCO3 -exchanger at the luminal pole resulting in - HCO3 secretion into the urine (Capasso et al. 2002, de Mello-Aires and Malnic 2002, Wagner and Geibel 2002).

3.2.2 Reabsorption of Na+ and Cl- in the Kidney Tubules

The amount of the Na+ and Cl- ﬁltered into the primary urine corresponds to 1,7 kg of NaCl, and the majority of it is reabsorbed in the proximal tubules. Similarly, about 65% of the water of the primary urine is reabsorbed there. The Na+ absorption in all nephron segments is mainly driven by basolateral Na+,K+-ATPase, but the major apical Na+ transporter differs in distinct segments. In the proximal tubules, the quanti- tatively most important Na+ transporter is the Na+/H+ exchanger NHE3. The rate of Cl- reabsorption is higher in the distal portion of proximal tubules when compared with the initial segment. Although there is passive paracellular transport of Cl-, an important fraction of the Cl- reabsorption in proximal tubules is transcellular and occurs in part via Cl-/oxalate and Cl-/formate exchange. Oxalate/carbonate and oxa- 2- - late/SO4 exchangers are suggested to have important roles in the Cl reabsorption process of proximal tubules by recycling oxalate (Aronson and Giebisch 1997, Greger 2000). In addition, there is evidence for Na+-independent Cl-/OH- exchange on the apical membrane of the proximal tubule cells (Shiuan and Weinstein 1984, Kurtz et al. 1994, Aronson and Giebisch 1997).

In the thick ascending limb of the loop of Henle, about 20-30% of the ﬁltered Na+ and Cl- are reabsorbed via the apical furosemide and bumetanide sensitive Na+-2Cl--K+ cotransporter SLC12A1 (NKCC2). Active Na+ and Cl- transport takes place in the distal convoluted tubules as well. The majority of the Na+ and Cl- uptake in this segment is mediated by thiazide-sensitive Na+-Cl- cotransporter (NCC, SLC12A3), but there is also evidence for apical thiazide-insensitive Cl-/formate, Cl-/oxalate and - - Cl /HCO3 exchange in distal convoluted tubules (Wang et al. 1993, Greger 2000, Reilly and Ellison 2000). In the collecting ducts, principal cells participate in the ﬁne tuning of the major electrolytes by Na+ reabsorption and Cl- secretion (Wagner and Geibel 2002).

3.2.3 Kidneys Can Autoregulate Their Filtration via Juxtaglomerular Complexes

The kidneys autoregulate their incoming blood ﬂow and thus glomerular ﬁltration rate via the juxtaglomerular complexes (also known as the juxtaglomerular apparatus),

Review of the Literature 39 which consist of the macula densa, mesangial cells and juxtaglomerular cells of the afferent and efferent arterioles of an individual nephron. This autoregulation happens through two negative feedback mechanisms, where the macula densa functions as the sensor of the tubular fluid’s Na+ and Cl- concentrations. If the glomerular filtration gets too low, both Na+ and Cl- are overreabsorbed in the proximal tubules and the loop of Henle, causing their concentrations to decrease in the macula densa. Diminished concentrations trigger signals from the macula densa via extraglomerular mesangial cells to the granular and smooth muscle cells of the afferent arteriole, causing arteriole dilation. This increases glomerular blood flow, amplifying the glomerular pressure. In addition, decreased intratubular Na+ and Cl- concentrations at the macula densa lead juxtaglomerular cells to release renin. It further induces the production of angiotensin II, which constricts the efferent arteriole, thus increasing glomerular pressure. The accretion of glomerular pressure induces an increase in the glomerular filtration rate back towards the normal level (Guyton 1991a, Goligorsky et al. 1997, Peti-Peterdi 2005).

3.3 Polycystic and Dysplastic Kidney Diseases

Polycystic renal diseases are one of the leading causes for endstage renal failure. They are characterized by multiple cysts arising from different segments of the nephron tubules, progressive expansion of these cysts and therefore a loss of normal renal structure and function. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of these diseases, affecting up to 1:800 individuals. Half of the ADPKD patients develop endstage renal failure by the age of 40, requiring dialysis and a kidney transplantation. Typical early symptoms include hypertension, poly- uria, recurrent urinary tract infections, urinary stones, and back pain. In addition, ADPKD patients may develop cysts in the liver or pancreas, diverticular disease of colon, intracerebral or aortic aneurysms, and heart valve defects. (Wilson 2004a, Wil- son 2004b, Zhang et al. 2004)

ADPKD is caused by mutations in one of the two polycystin genes, whose protein products are co-distributed in the primary cilia of the kidney epithelial cells. Polycys- tin-1 (PKD1), located on chromosome 16p13.3, encodes a membrane receptor capable of binding and interacting with several proteins, carbohydrates, and lipids. It is believed to act together with its interaction partners as a sensor of the extracellular environment and to elicit intracellular responses through phosphorylation pathways to regulate gene transcription in the nucleus. Polycystin-2 (PKD2) on chromosome 4q21–22, encodes a Ca2+ channel interacting with PKD1 (Wilson 2004a, Wilson 2004b, Zhang et al. 2004). Interestingly, anion transport is also altered in polycystic kidney diseases. Active Cl- transport drives ﬂuid secretion in cysts in ADPKD. There is evidence of CFTR activity being involved in the rate of ﬂuid and Cl- secretion in ADPKD tissue (Sullivan et al. 1998, Sutters and Germino 2003).

40 Review of the Literature Renal dysplasia is the major cause of chronic renal failure in children. However, little is known about its pathophysiology. The majority of renal dysplasias are associated with early embryonic urinary tract obstruction. They can be further divided into two distinct phenotypes depending on the anatomical level of the obstruction; multicystic dysplastic kidney (MCDK) affects especially kidneys, while obstructive dysplasia is characterized by lower urinary tract obstruction, thus having a postrenal reason. MCDK is caused by failed coordination of the metanephros and the branch- ing ureteric bud during development, leading to a low number of branches of collecting tubules, in addition to cystic dilatation of the ampullary portion of the ureteric buds. Obstruction usually occurs at an early embryonic stage, leading to a non-func- tioning kidney. Hereditary components may be involved, since 9% of relatives of patients with bilateral renal agenesis or dysplasia have renal malformations (Nagata et al. 2002, Vanderheyden et al. 2003).

3.4 Expression of SLC26A4 in the Kidney

Two of the SLC26 family members were reported to be expressed in the mammalian kidney before our work (III). Slc26a1 is located in the basolateral membranes of proximal tubules of the rat kidney (Karniski et al. 1998). SLC26A4 is expressed in mammalian kidneys, however the exact segment and cell speciﬁc expression is debated. SLC26A4 could not be detected in the proximal tubules of mice, rats or humans (Roy- aux et al. 2001) or in mice or rats (Kim et al. 2002). However, in addition to Solei- mani et al., who found Slc26a4 in proximal tubules (and cortical collecting ducts) in rats (Soleimani et al. 2001), Wall et al. found Slc26a4 mRNA in mouse glomeruli, proximal tubules and cortical thick ascending limbs, but could not detect the corresponding protein in these structures by immunohistochemistry (Wall et al. 2003). In addition, Wall et al. detected at least ﬁve-fold higher expression of Slc26a4 mRNA in connecting tubules and cortical collecting ducts, compared to the more proximal segments (Wall et al. 2003). Thus, it seems that Slc26a4 is most abundant in the type B and non-A-non-B intercalated cells of collecting ducts, but minor expression in other segments can not be excluded.

In humans, SLC26A4 is reported in the thick ascending limb of the Henle’s loop and distal convoluted tubules by real-time PCR (Lacroix et al. 2001) and in cortical collecting ducts (Royaux et al. 2001). However, Royaux et al. mentioned that in their work, only one piece of human kidney cortex was used, thus their results are based mostly on data derived from rodents. All things considered, orthologous SLC26A4 proteins may have distinct expression in speciﬁc nephron segments between different species.

Even though SLC26A4 is abundantly expressed in the kidney, Pendred syndrome patients have not been reported to suffer from renal defects. Therefore, it is possible that other anion transporter/transporters replace the function of SLC26A4 in the kidney. Other members of the SLC26 protein family are good candidates for this task.

Review of the Literature 41 4. The Epididymis

4.1 Structure of the Human Epididymis The epididymis can be divided into the caput, corpus, and cauda regions by macroscopic inspection (Figure 3). The caput of the human epididymis consists mainly of efferent ducts that can be further divided into ﬁve different tubule types based on their morphology. Type I epithelium has an irregular proﬁle as the result of the variable height of the epithelial cells. Efferent duct type II epithelium is regular low cuboidal in histology, and only has very few vacuoles. Type III epithelium is distinguishable by its vacuolated epithelial cells which form tubules with a regular outline. Cuboidal type IV epithelium only lines, with mosaic pattern, parts of short communal cavities. Type V efferent duct epithelium consists of ciliated columnar cells. Only two of these tubule types form longer segments (types I and III), while type II is mainly found in short blind end tubules, type V in brief distal segments close to epididymal ducts, and type IV in short transition segments only. Each of the efferent duct tubule types has both ciliated and non-ciliated cells. In addition, basal cells can be found in tubule types I-III (Yeung et al. 1991).

The corpus and cauda epididymis consist of epididymal ducts (epididymis proper). In human epididymal ducts, at least three morphologically and histochemically different cell types have been detected: principal cells, apical mitochondria rich cells (AMRC), and basal cells, but in other mammals clear and halo cells are also present (Regadera et al. 1993). AMRC are divided further into two histologically separable cell types: narrow cells, having a slender cytoplasm extending all the way from the basement membrane to the luminal edge, and apical cells, with a short cone-shaped morphology on the luminal side of the epithelium, with possibly no continuation to the basement membrane. It has been debated whether they are two distinct cell types or different forms of the same cell type (Palacios et al. 1991, Regadera et al. 1993, Martinez-Gar- cia et al. 1995, Adamali and Hermo 1996, Hermo and Robaire 2002).

4.2 Function of the Epididymis

The two major functions of the epididymis are to serve as favorable surroundings for the further maturation of sperm cells and to store them. The right microenvironment in each segment of the reproductive tract is essential for the successful maturation and correctly-timed capacitation (transforming of the sperm to be fertile) of sperm cells, and thereby for male fertility. Sertoli cells excrete testicular fluid that car- ries immotile, immature spermatozoa to efferent ducts (Setchell 1967, Setchell 1969, Wong et al. 2002). The major role of the efferent ducts is to absorb most of the fluid entering them, thus concentrating sperm (Tuck et al. 1970, Clulow et al. 1998, Wong et al. 2002). During the transport through the epididymal ducts, the luminal fluid composition is further changed by gradual absorption and secretion (Turner 2002, Wong et al. 2002).

42 Review of the Literature 2 3 1 6 4 5

Figure 3. A diagram of the distinct macroscopic segments of the human testis and epididymis indicated with different graphical patterns.

1 = tunica albuginea; 2 = septum; 3 = seminiferous tubules; 4 = rete testis; 5 = efferent ducts; 6 = caput of the epididymis; 7 = corpus of the epididymis; 8 = cauda of the epididymis; 9 = ductus deferens.

The direct influence of different intraluminal ions on sperm maturation has not been studied much, and would be difficult to study directly (Turner 2002). There is evidence that alterations in intraluminal ion concentrations of rat cauda epididymis have effects on the secretion of organic compounds, e.g. proteins, by epithelial cells (Wong et al. 1980). If the same were true for all segments of epididymis, intraluminal ion concentrations could influence sperm maturation indirectly by affecting the secretion of organic compounds by epithelial cells (Wong et al. 1980, Turner 2002).

Review of the Literature 43 4.2.1 Absorption of Na+, Cl- and Water During the Flow Through the Epididymis

Both Cl- and Na+ concentrations decrease from over 100 mmol/l in the seminiferous tubules and rete testis to about 25 mmol/l or less in the distal epididymis (Jenkins et al. 1980, Turner 1984, Turner 2002). For a long time, Cl- has been thought to be the driving force for the remarkable water reabsorption in the efferent ducts (Turner 2002). However, a recent study demonstrated that both both Cl- and Na+ transport are needed for ﬂuid reabsortion in rat efferent ducts, and that these ions are transported interdependently. The results suggested the presence of an apical anion anti- - - + + porter, probably exchanging Cl for HCO3 , acting in parallel with a Na /H antiporter (Hansen et al. 2004). In addition, it has been proposed that transepithelial Cl- transport would be important in maintaining the electrolyte composition in the epididymal ﬂuid (Leung and Wong 1992).

In rat epididymis, the Na+/H+ exchanger NHE3 is found on the apical side of the non-ciliated cells of the efferent ducts and the principal cells (Pushkin et al. 2000, Bagnis et al. 2001, Leung et al. 2001b, Kaunisto and Rajaniemi 2002). In addition, CFTR has been found on the apical membrane of non-ciliated cells in rat efferent ducts. In rat epididymal ducts, CFTR has a cell and region speciﬁc expression pattern, with the strongest immunostaining in the principal cells, and weak-to-moderate reaction in the clear cells of the corpus and cauda regions (Leung et al. 2001a, Ruz et al. 2004). In the human male reproductive tract, CFTR mRNA has been found in the epithelium of the epididymis and vas deferens (Patrizio and Salameh 1998). Before the work described in this thesis (IV), none of the SLC26 anion transporters had been localized to the epididymis.

4.2.2 Strict Bicarbonate Control is Needed in the Epididymal Ducts for Keeping Sperm Quiescent

- There is evidence that at least in rats, the luminal pH and HCO3 concentration increase in the proximal segments of the efferent ducts, decrease in the distal segments of efferent ducts, and diminish further along the epididymis proper (Levine and Kelly 1978, - Newcombe et al. 2000). The epididymal fluid is low in HCO3 (< 4 mM) compared - to seminal and prostatic fluids (25 mM) and the oviduct (> 20 mM). HCO3 has an essential role in inducing sperm hypermotility and initiating capacitation. It evokes cascades, increasing the membrane fluidity and membrane lipid scrambling in mature sperm cells; reactions that are needed for the sperm cell to be able to fuse with the - oocyte in fertilization (Litvin et al. 2003, Gadella and Van Gestel 2004). HCO3 stimulates soluble adenylyl cyclase that increases the intracellular cAMP amount, which in turn activates a protein kinase A –dependent protein phosphorylation cascade (Oka- mura et al. 1985, Litvin et al. 2003, Gadella and Van Gestel 2004). Changes in the external pH values cause the intracellular pH of the spermatozoa to move in the same

44 Review of the Literature - direction as the external pH (Gatti et al. 1993), and HCO3 is the major pH buffer. - Even small intracellular changes in pH and /or HCO3 concentration may cause significant changes in cAMP concentration in spermatozoa (Litvin et al. 2003), inducing sperm hypermotility and cell membrane instability. Therefore, an accurate con- - trol of HCO3 concentration in the epididymal fluid by the epididymal epithelium is needed to avoid premature capacitation that might lead to impaired fertility (Litvin et al. 2003, Gadella and Van Gestel 2004). The proton pump H+V-ATPase is expressed widely in the different parts of the male reproductive tract of the rat, including the non-ciliated cells of the efferent ducts and the narrow, apical and clear cells in specific parts of the epididymis (Herak-Kramberger et al. 2001). It has been found in a subgroup of epididymal cells in humans as well (Herak-Kramberger et al. 2001), but the exact localization of H+V-ATPase has not been studied in human efferent ducts before. Several different carbonic anhydrases have been detected in mammalian epididymis, including the CA isoenzyme II (CAII) (Kaunisto et al. 1995, Ekstedt et al. 2004, Hermo et al. 2005). It has also been located to some cells of the human epididymis, but the exact cell type is not known so far (Kaunisto et al. 1990). In mouse and rat epididymis, CAII has been partly co-localized with H+V-ATPase within the narrow cells (Hermo et al. 2000).

Review of the Literature 45

Aims of the Study

The principal object of this study was to determine whether there are more than three human SLC26 anion transporter genes, and if there were, to characterize them and their expression in humans.

The speciﬁc aims were:

- to identify and clone new human SLC26 genes

- to study the expression and function of the new SLC26 genes and proteins in human tissues

- to further characterize the detailed expression of selected SLC26 proteins in human tissues with strictly regulated ion transport, speciﬁcally the kidneys and the epididymal ducts

Materials and Methods

1. Computational Sequence Analysis (I-II)

New members of the SLC26 family were found by searching for homologs to the human SLC26A2, SLC26A3, and SLC26A4 nucleotide and protein sequences in different databases with the BLASTP, BLASTX, TBLASTN, TBLASTX, and FastA algorithms (Pearson 1994, Altschul et al. 1997, Zhang et al. 1997). SLC26A9 was ﬁrst found by searching for the sulfate transport motif (PS01130) of the human SLC26A3 protein in NCBI’s high throughput genomic sequence (htgs) database with the TBLASTN algorithm (Pearson 1994, Altschul et al. 1997, Zhang et al. 1997). GENSCAN, FGENES, and GRAIL2 were used to predict the coding exons from genomic clones (Xu et al. 1994, Solovyev et al. 1995, Burge and Karlin 1997). Mul- tiple sequence alignment was performed using ClustalW (Thompson et al. 1994), GENEDOC (http://www.cris.com/~Ketchup/genedoc.shtml), and ClustalX programs, and BoxShade Server 3.21 (www.ch.embnet.org/software/BOX_form.html). Transmembrane topology of the SLC26A6 protein was predicted by the MEMSAT program (Jones et al. 1994) and transmembrane topologies of the SLC26A7–A9 proteins were predicted by the TMHMM (Krogh et al. 2001) and PSIpred programs (Jones et al. 1994). Putative N-glycosylation sites were analyzed by the PROSITE program (Bairoch et al. 1997).

2. Chromosomal Mapping Using Radiation Hybrids (I) Chromosomal locations were assigned by PCR with speciﬁc primers for each EST using the GeneBridge 4 Radiation Hybrid and Stanford G3 panels (Research Genet- ics, Inc, Huntsville, AL). PCRs were performed in 25 µl volumes using 25 ng radiation hybrid clone DNA as a template, using the following conditions: 94°C for 3 min, 30 cycles of 94°C for 30 s, 55–70°C for 30 s, and 72°C for 40 s to 2 min followed by 72°C for 10 min. PCR products were run on a 2% agarose/EtBr gel, and results were analyzed by publicly available RH-mapper servers at www-genome.wi.mit.edu/cgi- bin/contig/rhmapper.pl and www-shgc.stanford.edu/RH/rhserverformnew.html.

49 3. Cloning of the SLC26A6-A9 Genes (I-II) The coding region of each gene was assembled from several overlapping PCR fragments, which were amplified by gene-specific primers designed for GENSCAN-predicted exons. The overlapping PCR fragments were amplified by PCR either from a pooled cDNA mix (Clontech, Palo Alto, CA) or the first strand cDNAs of human kidney, testis, and lung for SLC26A6, A7, A8 and A9, respectively. The first strand cDNAs were synthesized from 1 µg of poly(A)+ RNA (CLONTECH, Palo Alto, CA) using the SMART RACE cDNA Amplification Kit (CLONTECH) according to the manufacturer’s instructions. The SMART RACE method was used to amplify the 5’ and 3’ ends of the cDNAs. In addition, the cloning of the 3’ ends of the genes was also verified by PCR with primers designed for the regions that matched the expressed sequence tags (EST) annotated in GenBank. PCRs were performed in 25 µl volumes using 2.5-5 µl of cDNA mix as a template and the following conditions: 94°C for 3 min, 35 cycles of 94°C for 30 s and 68°C for 1-2 min, followed by 72°C for 10 min. PCR products were run on agarose/EtBr gels and either purified with the Qiagen Gel Extraction Kit for sequencing or subcloned with the TOPO-TA cloning system (Invitrogen) or the pCR-2.1 plasmid (Invitrogen) before sequencing. The PCR products were sequenced using dye-terminator chemistry (Sanger et al. 1977) and an auto- mated sequencer (ABI 373A or 377, Applied Biosystems).

4. Polyacrylamide Gel Electrophoresis Analysis of Single Base Polymorphism (I) During the cloning of the SLC26A6 gene, several alternative sequences were discov- ered, suggesting alternative splicing of the gene. To find different splice forms in the beginning of exon 17, this region was amplified by PCR using exon 16 and 17 specific primers. First-strand cDNA, used as a template for PCR, was synthesized from 1 µg of human kidney total poly(A) RNA (Clontech) using the SMART RACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions. The PCR products were separated on a 6% polyacrylamide gel and visualized by sil- ver staining. The different splicing forms of SLC26A6 were further verified by cloning and sequencing the PCR products. Genetic polymorphism was excluded by analyzing genomic DNA samples of 100 people in the same way.

50 Materials and Methods 5. Expression Analyses Using Northern Blotting (I-II)

To screen for tissues expressing the mRNA of the new SLC26 genes, Northern analyses were done using the CLONTECH (Palo Alto, CA) Multiple Tissue Northern Blots (MTN 7780-1 and MTN 7760-1). The tissues surveyed included heart, brain, placenta, lung, liver, pancreas, skeletal muscle, kidney, small intestine, colon, thymus, spleen, peripheral blood leukocyte, uterus, prostate and testis. A 928 bp PCR amplified probe corresponding to the 3’ sequence nt 1769-2697 of SLC26A6, a 1984 bp probe corresponding to nt 198–2181 of SLC26A7, a 551 bp probe corresponding to the 5’ sequence nt 156–707 of SLC26A8, and a 2362 bp probe corresponding to the open reading frame sequence nt 115–2476 of SLC26A9 were radiolabeled with [32P]dCTP with the Rediprime Kit (Amersham Biosciences) according to the manufacturer’s instructions. The gene specific probes were hybridized to Northern blot filters in Express Hyb solution (Clontech) from about 2 h to overnight, followed by several washes. Autoradiography was performed on X-ray films at -20°C.

6. Expression Analyses Using PCR Panels (I-II) To further verify tissues producing the mRNA of the new SLC26 genes, PCR analyses were performed using the Clontech PCR ready human Multiple Tissue cDNA (MTC) panels I and II (K1420–1 and K1421–1) with gene speciﬁc primers. The tissues examined were heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and peripheral blood leukocytes. The primers for the housekeeping control gene GADPH (glyceraldehyde 3-phosphate dehydrogenase) were included in the kit. All PCR products were veriﬁed by sequencing.

7. Expression of SLC26A6 and A9 in Cell Lines (I-II) The expression of SLC26A6 was analyzed in HEK 293 cells and two human pancreatic duct cell lines, Capan-1 and Capan-2 (obtained from ATCC, Manassas, VA), and the expression of SLC26A9 was analyzed in two human lung speciﬁc epithelial cell lines, NCI-H358 and A549 (ATCC, Manassas, VA) by RT-PCR. The HEK 293, NCI-H358 and A549 cells were grown in Dulbecco’s modiﬁed Eagle’s medium (DMEM, Gibco BRL, Gaithersburg, MD) with 50 U/ml penicillin, 2 mM sodium pyruvate, 2 mM glutamine, and 5% fetal bovine serum. Capan-1 cells were grown

Materials and Methods 51 in Iscove’s modified Dulbecco’s medium with 4 mM L-glutamine, 50 U/ml penicillin, and 20% fetal bovine serum, and Capan-2 cells in McCoy’s 5a medium with 1.5 mM L-glutamine, 50 U/ml penicillin, and 10% fetal bovine serum. The cells were grown at 37°C under a 5% CO2 atmosphere to achieve full confluence. Total RNA was isolated from cells with the Qiagen RNA Isolation Kit according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1-2 µg of total RNA using the SMARTRACE cDNA Amplification Kit (Clontech) according to the manufacturer’s instructions. The presence of the SLC26A6 and A9 genes was established by PCR with gene specific primers and by analyzing the products on agarose gels. The identity of the PCR products was verified by sequencing.

8. Functional Transport Measurements in Xenopus Laevis Oocytes (II) Mature Xenopus laevis females were purchased from the African Xenopus Facility C.C., Noordhoek, South Africa. Stage V and VI oocytes from Xenopus laevis were maintained at 17°C in modified Barth’s solution. Oocytes were injected with either 50–100 nl of water (control) or 7–12 ng of the SLC26A7–A9 cRNA using a Nano- jet automatic injector (Drummond Scientific Co., Broomall, PA). For cRNA synthesis, pcDNA3.1:SLC26A7 or pcRII:SLC26A7, pcDNA3.1:A8 or pNKS2:SLC26A8, and pcRII:SLC26A9 plasmids were linearized by EcoRV, NotI, or XhoI digestions; the cDNAs were in vitro transcribed using T7 or SP6 RNA polymerases (Promega), and the resulting capped cRNA was dissolved in MilliQ water before use. Transport of [35S]sulfate, [36Cl]chloride, and [14C]oxalate uptake was performed 3 days after injection. Briefly, 10 oocytes (per data point) were washed at room temperature for 1–2 min in solution A (115 mM sodium gluconate, 2.5 mM potassium gluconate, 4 mM calcium gluconate, 10 mM HEPES/Tris, pH 7.4) or solution B (100 mM choline chloride, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 20 mM Hepes/Tris, pH 7.5) and then placed into 100 µl of solution A containing 2.5 mM NaCl with 36Cl− or 0.1 mM oxalate with 2–5 µCi/ml [14C]oxalate, or into 100 µl of solution B containing 35 2− 0.1 mM K2SO4 with 10 µCi/ml SO4 (PerkinElmer Life Sciences) for 30–60 min at room temperature. The oocytes were washed 4 times with ice-cold solution B, lysed with 4% SDS, dissolved in scintillant (BCS, Amersham Biosciences), and counted 2- by liquid scintillation spectrometry. Inhibition of the SO4 uptake of SLC26A9 was performed by adding either 1 mM DIDS, 5 mM thiosulfate, 5 mM oxalate, or 5 mM glucose to the uptake solution B. All isotopes were purchased from PerkinElmer Life Sciences. Statistical analyses of the transport results were performed using the prism statistic package, version 3.0 (GraphPad software Inc., San Diego, CA). The degree of statistical significance between two groups was calculated using the unpaired t test, with p < 0.05 considered significant.

52 Materials and Methods 9. Tissue Samples (I-IV) Formalin-ﬁxed, parafﬁn-embedded human specimens were obtained from the Depart- ment of Pathology, HUCH Laboratory Diagnostics, Helsinki University Central Hospital. Tissues examined included microscopically normal pieces of adult human kidneys, testes, epididymises, pancreases, and lungs. In addition, kidney samples diag- nosed with ADPKD and MCDK were studied.

More speciﬁcally, in study III altogether 10 normal adult kidneys, 5 ADPKD adult kidneys and 11 multicystic dysplastic kidneys from individuals from the age of 17 embryonic weeks to 8 years were examined. Five of the patients with MCDK had bilateral disease and two of them had atresia of the urethra in addition, while six of the patients were reported to have a unilateral cystic kidney. Altogether, in study IV we examined 18 histologically normal adult epididymises removed because of testicular tumors, which included: 10 seminomas, 2 teratocarcinomas, 1 mature teratoma, 1 embryonal carcinoma and seminoma, 1 cysta epidermalis, 1 cystadenoma of rete testis, 1 cicatrix, and 1 normal ﬁnding. These patients were 22-71 years old (mean 37) at the time of the operation. Normal spermatogenesis was found in all samples except for two seminomas.

Fresh human kidney samples were obtained from nephrectomies. The samples were taken from macroscopically normal areas, cortical and medullary samples separately. The fresh samples were snap frozen in liquid nitrogen and stored at –70oC.

10. RT-PCR (III) Fresh cortical and medullary kidney samples from two patients were used for mRNA extraction. mRNA was separated using the RNeasy Mini kit (Qiagen Sciences, MD, USA) according to manufacturer’s instructions. 1 µg of each RNA sample was transcribed to cDNA with random hexamer primers using M-MLV Reverse Transcriptase (Promega, WI, USA) according to manufacturer’s instructions. To verify the quality of the transcribed cDNA, a control PCR with the housekeeping gene GADPH was performed (primers from Applied Biosystems, Foster City, CA). cDNAs were used as templates for the SLC26A6 and A7 PCR, 1 µl for each 25 µl reaction. Primers 5’-GCCTATGCCCTTCTGCTCCAACA-3’ and 5’-TGGGCAG- GTCATGGAAGAGTTCC-3’ were used for SLC26A6 and primers 5’-AATGGA- CAGTGAAACCCTGCAGCAG-3’ and 5’-GGAAGCTGTACAATGGGCTAAC-3’ for SLC26A7, for the expected product sizes of 1126 bp and 358 bp, respectively. To avoid errors from genomic contamination, primers were designed to bridge an intron and give a clearly larger band (SLC26A6) or no band at all (SLC26A7) from possible genomic DNA. The PCR protocol for SLC26A6 was 94oC for 3 min, 35 cycles of 94 oC for 40 s, 68 oC for 40 s, 72 oC for 2 min followed by 72 oC for 10 min.

Materials and Methods 53 For SLC26A7 the PCR was 94 oC for 3 min, 31 cycles of 94 oC for 30 s and 68 oC for 2 min, followed by 72 oC for 10 min. The PCR products were analyzed by electrophoresis on 2% agarose gels stained with ethidium bromide. The products were puriﬁed by ExoSAP-IT (USB, Ohio, USA) and sequenced in both directions by an ABI PRISM 377 sequencer.

11. In Situ Hybridization (I-II) To illustrate the expression of the mRNA of the new SLC26 genes in vivo, in situ hybridization was performed. Formalin-fixed, paraffin-embedded specimens of normal adult human kidney and testes were used. A 471 bp fragment corresponding to nt 621–1091 of the SLC26A6 cDNA, and a 538 bp fragment corresponding to nt 1629– 2166 of the SLC26A8 cDNA were amplified by PCR and used to transcribe sense and antisense RNA probes (Saarialho-Kere et al. 1994). Deparaffinized 5 µm tissue sections were digested with 1 µg/ml proteinase K for 30 min at 37°C and washed in 0.1 M triethanolamine containing 0.25% acetic acid. Sections were hybridized with 35S-labeled probes (4 x 104 cpm/µl and 4 x 105 cpm/µl of hybridization buffer, for SLC26A6 and A8 respectively) at 55°C (for SLC26A6) or 52°C (for SLC26A8) for at least 18 h in a humidified chamber. The slides were then washed under stringent conditions, including a treatment with RNaseA to remove any unhybridized probe (Prosser et al. 1989), and dipped in LM-1 emulsion (Amersham Biosciences). After 35–54 days of autoradiography for SLC26A6 and 10–30 days for SLC26A8 at 4°C, the photographic emulsion was developed, and the slides were stained with hematoxylin and eosin. The sense cRNA probe was used as a negative control.

12. Antibodies (I-IV) Antisera were raised in rabbits against the following synthetic SLC26 peptides: TVRD- SLTNGEYCKKEEEN for SLC26A2 (anti-A2), MDLRRRDYHMERPLLNQEHL for aminoterminal (anti-A6N) and TFALQHPRPVPDSPVSVTRL for carboxytermi- nal (anti-A6C) sequence of SLC26A6, and SHIHSNKNLSKLSDHSEV for SLC26A7 (anti-A7), VEEVWLPNNSSRNSSPGLPD for SLC26A8 (anti-A8), and ELSLYD- SEEDIRSYWDLEQE for SLC26A9 (anti-A9) corresponding to amino acids 689-706 (anti-A2), 1-20 (anti-A6N), 718-737 (anti-A6C), 639-656 (anti-A7), 680–699 (anti- A8), and 758–777 (anti-A9), respectively. Two rabbits were immunized with each peptide. Peptide synthesis and antibody production were purchased from Research Genetics (anti-A2) and Sigma Genosys Ltd (anti-A6, -A7, -A8, -A9). In the two first studies (I-II) the antisera were used as such, but in the two last studies (III-IV) the anti-A2, -A6N, -A6C and -A7 antibodies were purified from whole serum by affinity chromatography with the corresponding peptide coupled to N-hydroxysuccin- imide-Sepharose 4B according to the manufacturer’s instructions (Amersham Phar- macia Biotech).

54 Materials and Methods The following monoclonal mouse antibodies were used: anti-human proximal nephrogenic renal antigen (PNRA; Zymed Laboratories Inc., CA, USA), anti-human CD10 (Novocastra laboratories Ltd, UK), anti-human Tamm-Horsfall protein (TH; Cedar- lane laboratories, Canada), anti-human high molecular weight cytokeratin (34βE12; DAKO, CA, USA), anti-human cytokeratin 7 (CK7; DAKO, Denmark), and anti- human cystic ﬁbrosis transmembrane regulator (CFTR, Ab-2 MM13-4; Neo Markers, CA, USA). The polyclonal rabbit antibodies used were anti-rat aquaporin-2 (AQP2, AQP21-A; Alpha Diagnostics, TX, USA), anti-human carbonic anhydrase II (CAII, 200-401-136; Rockland Immunochemicals, PA, USA), anti-rat Na+/H+ exchanger 3 (NHE3, NHE31-A; Alpha Diagnostics, TX, USA), anti-human vacuolar type H+- ATPase B1/2 (H+V-ATPaseB, sc-20943; Santa Cruz Biotechnology, Germany), anti- human vacuolar type H+-ATPase E (H+V-ATPaseE, sc-20946; Santa Cruz Biotech- nology, Germany), and anti-human Na+/H+ exchanger regulating factor (NHERF, EBP50, PA1-090; Afﬁnity BioReagents, CO, USA).

13. Transfection, Immunofluorescence and Peptide Competition (III) The specificity of the N-terminal affinity-purified anti-SLC26A6 antibodies was analyzed by immunofluorescence with the SLC26A6a-isoform (Lohi et al. 2003) transfected or untransfected COS-1 cells. Specificity was also shown by competition with the antigen peptides. The cDNA of SLC26A6a was amplified by PCR and subcloned to eukaryotic pcDNA3.1/V5/His-TOPO plasmid (Invitrogen). For immunofluorescence, COS-1 cells plated on glass coverslips were transiently transfected using Fugene6 (Roche Molecular Biochemicals) with either the clones or with water as a control, following the manufacturer´s instructions. The cells were grown on 6 cm plates in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO-BRL, MD, USA) with 50 U/ml penicillin, 2 mM sodium pyruvate, 2 mM glutamine, and 5% fetal bovine serum at 37°C in 5% CO2 atmosphere. After 48 h, cells were fixed with 3% paraformaldehyde in phosphate-buffered saline (PBS), washed twice with PBS, permeabilized with 0.1% Triton in PBS for 30 min, and blocked in 3% goat serum in PBS. Affinity-purified antibodies were then added (1-5 µg/ml) to 1% goat serum in PBS and incubated for 1 h at room temperature. After three washes with 3% goat serum in PBS, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgGs (Sigma) were added and incubated for 1 h. The coverslips were then washed 5 times with PBS and mounted on glass slides using Immu-mount medium (Shandon, Pittsburgh, PA). For peptide competition analysis, affinity purified N-terminal antibodies were incubated with ~100-fold weight excess of N-terminal A6 peptides at 37oC for two hours before immunostaining.

Materials and Methods 55 14. Western Blotting (III-IV) Fresh cortical and medullary human kidney or rat testis samples were snap frozen in liquid nitrogen, and stored at -18oC. The frozen samples were homogenized, centri- fuged at 15,800 g at +4oC for 15 min, the supernatants were collected and protein concentrations determined using Bio-Rad Protein Assay (Bio-Rad, CA, USA). Laemmli buffer and β-mercaptoethanol were added. The testis samples were boiled at 100oC for 5 min. The samples were run on 7.5-8% SDS-PAGE gels, 30 µg of protein in each lane. The separated proteins were transferred electrophoretically to Hybond-C extra membranes (Amersham Life Sciences). After blocking in 5% non-fat dry milk in TBST for 30 min (for A7) or 5% non-fat dry milk and 1% bovine serum albu- min (BSA) in TBST for 60 min (for A8), the filters were incubated with either A7 antibody (10 µg/ml) in 2.5% non-fat dry milk in TBST or with A8 antiserum (1:50) in 2.5% non-fat dry milk and 0.5% BSA in TBST for 1 h. Horseradish peroxidase (HRP) conjugated swine anti-rabbit IgG (Dako, Denmark) was used as a secondary antibody. The corresponding rabbit preimmune sera (10 µg/ml or 1:50, respectively) were used as negative controls. In addition, for A7 a peptide competition control was done. The protein bands were visualized by chemiluminesence (ECL Western Blot- ting Detection Reagents, Amersham Biosciences, UK) detected on X-ray films. For peptide competition analysis, affinity purified A7 antibodies were incubated with 20- fold molar excess of corresponding A7 peptides at room temperature for 1 h before immunostaining.

15. Immunohistochemistry (I-IV) Deparaffinized 5 µm human tissue sections were used in the experiments. Peroxidase- antiperoxidase labeling kits (Vectastain Elite ABC Kit, Vector Laboratories) and two- step peroxidase kits (EnVision+ System HRP, DAKO) were used. The best suitable pretreatment for each antibody was carefully studied using several separate experiments for each antibody. The best adequate pretreatment for each of the antibodies was: boiling in a microwave oven in 10 mM citrate buffer (pH 6.0) for anti-A2, anti-A6N, anti-CAII, anti-CD10, anti-CK7, anti-34βE12, anti-CFTR, anti-NHE3 and anti-NHERF, no specific pretreatment for anti-A6C, anti-A6N, anti-A7, anti- A8, anti-A9, anti-PNRA, anti-TH, anti-VATPaseB and anti-VATPaseE. For some of the antibodies, pretreatment with 1% SDS or heating at 95oC in 0.01 M EDTA buffer (pH 8.0) were also tested, but none of the antibodies benefited from these treatments. Different pretreatment times were tried out also.

The pretreatment was followed by blocking of endogenic peroxidase activity with

H2O2, according to the kit manufacturer’s directions. The antisera were used in dilutions of 1:50-1:200 for anti-A6N, 1:1000–1:2000 for anti-A7, 1:750-1:2000 for anti- A8, and 1:1000–1:2000 for anti-A9. The afﬁnity puriﬁed antibodies were used at concentrations of 0.1-0.5 µg/ml for anti-A2, 35 µg/ml for anti-A6C, 1-5 µg/ml for

56 Materials and Methods A6 V-ATPaseB A7

Figure 4.

An example of a comparison of three consecutive sections from a human kidney, stained with anti-A6N, the tubular marker V-ATPaseB, and A7 (left column from above downwards, respectively), focusing on a selected collecting tubule. This method enables the dissection of individual cells with several distinct antibodies. All of the antibodies used here give off a positive signal in a speciﬁc intercalated cell. The original pictures of V-ATPaseB and A7 have been published in study III, and are reprinted here with the permission of S. Karger AG, Basel.

Materials and Methods 57 anti-A6N, and 20 µg/ml for anti-A7. The concentrations used for the commercial antibodies were: 1:100 for anti-CD10, 1:100 for anti-CK7, 1:500 for anti-TH, 1:100 for anti-PNRA, 1:50 for anti-34βE12, 2 µg/ml for anti-CFTR, 1 µg/ml for anti- NHE3, 0.1-0.5 µg/ml for anti-NHERF, 0.5-1 µg/ml for anti-CAII and 0.2 µg/ml for both anti-H+V-ATPaseB and anti-H+V-ATPaseE. Diaminobenzidine (DAB) was used as the chromogenic substrate with Mayer’s hematoxylin counterstaining. Pre- immune sera, normal rabbit IgG or normal mouse IgG were used as the corresponding negative controls, with similar dilutions of the sera as for the antisera, or similar concentrations of IgG as for the puriﬁed antibodies.

In study III, to identify the specific segments positive for SLC26A6 and A7, consecutive sections were stained for defined tubule markers. The stainings were performed so that each of the marker series included three slides in the following order: SLC26A6, the marker, SLC26A7, enabling their comparison (Figure 4). Analyzing of the different types of efferent ducts in study IV, was done by following the definitions by Yeung et al (Yeung et al. 1991).

58 Materials and Methods Results

1. Detection and Chromosomal Localization of New SLC26 Genes (I)

The discovery of new human SLC26 members was based on a protein and nucleotide homology approach. The search was done predominantly in 1997-1999, using the publicly available databases, mainly NCBI BLAST server. The programs mainly utilized were BLAST and FastA. Both nucleotide and protein sequences of human genes SLC26A2-A4 (U14528, L02785, and AF030880, respectively), the rat gene Slc26a1, and selected segments of them were used as templates in the homology searches. Sev- eral signiﬁcant matches against expressed sequence tags (ESTs) and genomic sequences (BLASTX E-value > 10-20) were found, suggesting candidate genes for new members of the SLC26 family. Chromosomal locations for each promising putative new gene were deﬁned by the GeneBridge4 and Stanford G3 radiation hybrid panels and matches to mapped and sequenced genomic clones deposited in GenBank. In addition, preliminary tissue expression of each candidate gene was explored using Clon- tech’s multiple tissue cDNA panels.

Altogether, seven new putative SLC26 genes were found in the human genome (Table 4), including the human ortholog for rat Slc26a1. Thereafter, ﬁve of them were cloned and characterized in more detail.

Table 4. The characteristics of the new human members of the SLC26 anion transporter family.

Symbol Also Chromosomal Anions Transported Major Expression known as Location Sites 2- - SLC26A1 SAT1 4p16.3 SO4 , Cl , oxalate liver, kidney, pancreas, brain - 2- - - SLC26A6 PAT1, 3p21.3 Cl , SO4 , HCO3 , OH , oxa- ubiquitous CFEX late - 2- - SLC26A7 8q23 Cl , SO4 , HCO3 , oxalate kidney, testis, placenta - 2- SLC26A8 TAT1 6p21 Cl , SO4 , oxalate testis - 2- SLC26A9 1q32 Cl , SO4 , oxalate lung SLC26A10 12q13 pseudogene? brain, other? 2- SLC26A11 17q25 SO4 ubiquitous

59 2. Cloning of New SLC26 Genes

2.1 SLC26A1 (I) The 412 bp long EST AW001016 was mapped to chromosome 4p16.3. Its translated amino acid sequence showed the highest homology (84%) to the rat sulfate anion transporter-1 (sat-1, Slc26a1), suggesting it to be the probable human ortholog SLC26A1. The matching of the identiﬁed EST to a genomic sequence in GenBank accelerated cloning of the whole, approximately 3.6 kb long, human SLC26A1 sequence (Gen- Bank AF297659), containing three exons. The corresponding protein consists of 701 aa, with 75% sequence similarity to rat Slc26a1. PCR analysis from multiple tissue cDNA samples showed abundant SLC26A1 expression in human liver, kidney, pancreas, and brain, in accordance with the known expression of the rat Slc26a1 protein (Bissig et al. 1994, Markovich et al. 1994, Karniski et al. 1998).

2.2 SLC26A6 (I)

The carboxy-terminal regions of the human SLC26A3 and SLC26A4 sequences indicated high homology to the translated sequence of the ovarian tumor EST AA411587 and identical embryonal EST AA779900. The putative new SLC26 gene was located to chromosome 3p21.3 by radiation hybrid mapping. The full length sequence, gained by sequencing the corresponding 1.2 kb IMAGE clone 755184, matched the 88 kb human genomic 3p21 PAC RPCI5-751E10 (Roswell Park Cancer Institute Human PAC Library) sequence AC005923 in homology searches. The genomic sequence found was analyzed by the GENSCAN, FGENES, and GRAIL2 exon prediction programs, and these results were veriﬁed by sequencing RT-PCR products. We initially named the gene for putative anion transporter-1 (PAT1), and subsequently it was assigned the symbol SLC26A6 by the HUGO Gene Nomenclature Committee (HGNC).

The SLC26A6 cDNA (Accession No. AF279265) sequence analysis demonstrated an open reading frame of 2217 bp encoding a 738 aa protein. The putative ATG translation start site is located within the Kozak consensus sequence (Kozak 1987), and four in-frame stop codons precede that methionine. The SLC26A6 gene contains 20 exons ranging in size from 55 to 177 bp. The general AG-GT rule is followed in all exon– intron boundaries except for exon 15, which ends with a GC. A 280-bp 3’ untranslated region precedes the consensus polyadenylation signal AATAAA. It is interesting that 12 of the 20 SLC26A6 exons are exactly the same size as corresponding ones of the SLC26A3 and SLC26A4 genes, which share as many as 15 exons of similar size.

The amino acid homology of SLC26A6 compared to the SLC26A3 and SLC26A4 proteins is only about 50%, while it shows the best sequence similarity (56%) to the cochlear motor protein SLC26A5. Comparison of SLC26A6 and SLC26A5 sulfate transporter motifs (PROSITE PS01130) revealed that two of three amino acid differences

60 Results (F, Y, and M), suggested to give distinct properties for SLC26A5 (Zheng et al. 2000), are identical in the SLC26A6 protein sequence (F and Y), and only the third nonpolar residue M is replaced by another nonpolar residue, I. Interestingly, several conserved residues were found by multiple alignment of SLC26A3, SLC26A5, and SLC26A6 protein sequences. SLC26A6 has two putative N-glycosylation sites in a similar region compared to SLC26A3, and one of them is fully conserved. The MEMSAT program suggested SLC26A6 to have a 12-transmembrane structure with intracellular N- and C-terminal domains, resembling the topology predicted for the rat Slc26a1 and human SLC26A2 and SLC26A3 proteins (Bissig et al. 1994, Hastbacka et al. 1994, Byeon et al. 1996, Kere et al. 1999). Instead, SLC26A4 has been suggested to have a diverging topology with only 11 transmembrane domains (Everett and Green 1999). However, it should be remembered that topology predictions are only spec- ulative and should be used with caution, since results vary when different programs are used. Thus these predictions need to be veriﬁed experimentally.

While cloning the full length cDNA sequence of SLC26A6, several transcripts turned up repeatedly in the RT-PCR and Northern blots, indicating the possibility of alternative splicing of the gene. Sequencing of the different fragments revealed a 3 bp polymorphism in the exon 17. Further investigation demonstrated two consensus splice acceptor sites that were separated by three nucleotides, leading to the presence or absence of a glutamine residue at position 611, thus suggesting alternative splicing. To rule out allelic variation, the boundary of the exon 17 was sequenced from genomic DNA samples of 100 non-related individuals. No sequence variation was detected in these genomic samples, further supporting alternative splicing to be the mechanism for the single-residue polymorphism.

2.3 SLC26A7 (I-II)

The EST AA992584 was found to have over 50% similarity to the previously known human SLC26 members. It mapped to chromosome 8q23. The EST was first expanded by sequencing cDNA IMAGE clone AI275738 and the human genomic draft sequence RP11-353D5, which were found to match it. The gene structure found, predicted with GENSCAN program, encoded a 376 aa protein showing 48% similarity to the first 230 amino acids of SLC26A2. The predicted exons were verified by sequencing overlapping PCR fragments. RT-PCR was used to uncover the 5’ and 3’ regions of the putative new gene. The complete gene was found to contain a 1971 bp open reading frame encoding a 656 aa protein. The full cDNA sequence was submitted to GenBank (accession no AF331521) and named SLC26A7.

The SLC26A7 gene spans about 100 kb of genomic sequence. The exon-intron boundaries were determined by aligning the cDNA sequence with genomic clones from the Human Genome Project and Celera Genomics (PAC clone RP11–353D5 Gen- Bank accession no AC017061, and Celera’s clone GA_x2HTBL2W902) (Venter et

Results 61 al. 2001), and all of them were detected to obey the general AG-GT rule. The open reading frame is divided into 19 exons ranging from 55 to 306 bp in size. Ten of the 19 SLC26A7 exons are of exactly the same size as those of the SLC26A3 and SLC26A4 genes, which share 15 exons of similar size. The putative ATG translation start site of SLC26A7 (GAAAAATGACA) is ﬂanked by the -3 purine but not +4 guanine residues of the Kozak consensus sequence (Kozak 1987), and four in-frame stop codons precede that methionine. There is a 3024 bp 3’-untranslated region before the consensus polyadenylation signal AATAAA. Actually, several alternative poly(A) signals were identiﬁed at the 3’ end of the SLC26A7 fusion sequence.

The SLC26A7 protein has 50% similarity compared to the SLC26A2 and A3 amino acid sequences (BLASTP E value ~10-80), and several conserved residues displayed by their multiple alignment. Two putative N-glycosylation sites at Asn125 and Asn131 were suggested for SLC26A7 by the PROSITE program. The TMHMM and PSIpred programs predicted a 10- or 12-transmembrane structure with intracellular N- and C- terminal domains, similar to the suggested topology of SLC26A3 (Kere et al. 1999). Analysis of the SLC26A7 protein sequence with ProﬁleScan pinpointed two domains that are commonly shared by the other SLC26 family members as well: sulfate transporter family domain (ST family, predicted between aa ~200 and 500, PF00916), and sulfate transporters and anti-sigma antagonists (STAS, between aa ~500 and 700, PS50801). The putative NTP-binding STAS domain suggests that anion transport of SLC26A7 might be regulated by intracellular nucleotides (Aravind and Koo- nin 2000). The carboxyl terminal end of SLC26A7 protein contains the consensus PDZ ligand sequence (SEV), opening the possibility that SLC26A7 may be linked to PDZ interaction networks.

2.4 SLC26A8 (I-II)

Homology searches indicated the genomic PAC clone 179N16, located on chromosome 6p21.1–p21.33, to have similarities with known SLC26 sequences. However, this clone included only about 300 aa of the amino terminal head of SLC26A8. A little later, another genomic sequence (PAC clone RP11–482O9, accession AL133507) appeared in GenBank, enabling the prediction of the gene’s carboxyl terminal end with GENSCAN as well. The predicted exons were veriﬁed by sequencing overlapping PCR fragments, and the 5’ and 3’ ends of the mRNA were revealed by RT-PCR. Sequence analysis showed a 1971 bp long open reading frame encoding a 970 aa protein. The complete cDNA sequence was submitted to GenBank (accession no AF331522), and the gene was named SLC26A8. While our work was in progress, Touré et al. published the characterization of TAT1 (testis anion transporter 1) gene and protein (Toure et al. 2001), which are identical to our SLC26A8 sequences.

The SLC26A8 gene spans about 80 kb of genomic sequence. The sequence surrounding the putative ATG translation start site (AGGAATGGCA) contains the Kozak

62 Results consensus sequence -3 purine and +4 guanine residues (Kozak 1987), and a 361 bp long UTR precedes the consensus polyadenylation signal AATAAA at the gene’s 3’ end. SLC26A8 includes 20 exons ranging between 49 and 369 bp in size. The general AG-GT rule is obeyed by all exon-intron boundaries. A few exons show conser- vation of size, although there are more alterations in SLC26A8 exon structure compared to the other human SLC26 family members.

The amino acid similarity of SLC26A8 to the other known SLC26 proteins is over 50%, with the highest values to SLC26A3 and SLC26A6 sequences (BLASTP E value 2 x 10-78 and 1 x 10-73, respectively). SLC26A8 protein has 200–300 amino acids more than the other family members, located between amino acids 600–652 and in the carboxyl terminal region. As expected, multiple alignment with the other SLC26 proteins indicated SLC26A8 to include several conserved sequence blocks in addition to several unconserved residues. Eight proposed N-glycosylation sites were found at Asn52, Asn192, Asn277, Asn384, Asn595, Asn651, Asn687, and Asn688. Interestingly, the TMHMM program predicted SLC26A8 to have 11 transmembrane domains with intracellular amino terminal and extracellular carboxyl terminal ends, differing from the majority of the predictions for other SLC26 proteins, whereas the PSIpred program failed to predict any kind of structures for SLC26A8. ProﬁleScan analysis of the amino acid sequence found ST (aa 212–521) and STAS (aa 544–791) domains.

2.5 SLC26A9 (II)

When the sulfate transport signature motif of the human SLC26A3 protein was run with the TBLASTN algorithm against the NCBI’s htgs database, a hit for the genomic PAC clone RP11–370I5 (GenBank accession no AL360009) on chromosome 1q32 was found (E value = 0.001). Again, GENSCAN was used to predict the exons, which were further ascertained by sequencing overlapping PCR products. The SMART RACE method was used to reach the 5’ and 3’ ends of the gene. The complete cDNA sequence, containing an open reading frame of 2373 bp encoding a 791 aa protein, was submitted to GenBank (accession no AF331525) and named SLC26A9.

The SLC26A9 gene spans about 25 kb of the genomic sequence. The -3 purine but not +4 guanine residues of the Kozak consensus sequence (Kozak 1987) were found around the putative ATG translation start site (CAGATATGAGC). A 115 bp long UTR, including the first exon, precedes that methionine. In addition, a TATA box was found 25 bp upstream from the first exon. A 2308 bp 3’ UTR is followed by the consensus polyadenylation signal AATAAA. The open reading frame of SLC26A9 contains 21 exons ranging from 55 to 282 bp in size, and all exon-intron boundaries obey the general AG-GT rule. Altogether 14 of the 20 SLC26A9 exons are of exactly the same size as the corresponding ones in the SLC26A3 and SLC26A4 genes. Sequenc- ing verification revealed that the second or fourth exon of SLC26A9 is spliced out,

Results 63 resulting in truncation of the ﬁrst 89 amino terminal aa or deletion of 37 aa (aa 90– 126) of the protein, respectively.

The SLC26A9 protein is highly conserved among the known family members, having the highest similarity (56%) with the SLC26A6 sequence. Two putative N-glycosylation sites were found at Asn153 and Asn156. ProﬁleScan analysis revealed ST (aa 187–497) and STAS (aa 520– 733) domains in SLC26A9. A PDZ interacting motif is located at the carboxyl terminus. Both the PSIpred and TMHMM programs predicted SLC26A9 to have 9 transmembrane segments with intracellular amino terminal and extracellular carboxyl terminal domains.

2.6 SLC26A10 and SLC26A11

In addition to the ﬁve new human SLC26 genes described above, we mapped two additional SLC26 gene candidates. Homology searches identiﬁed a 3008 bp unspliced cDNA (Accession No. AL050358) derived from human fetal brain. It was mapped to chromosome 12q13. Its predicted 427 aa protein, named SLC26A10, showed about 60% similarity to the SLC26A3 and SLC26A4 amino acid sequences. PCR experiments with multiple tissue cDNA panels suggested wide tissue distribution, with the strongest signals from brain.

The sequence homology approach pointed out a 214 bp infant adrenal gland EST (Accession no. AA348883) as well. This putative gene was mapped to chromosome 17q25. Its nucleotide sequence was expanded by sequencing the corresponding 1.7 kb IMAGE clone 59444. Interestingly, the predicted ﬁrst 300 amino acids of this gene, named SLC26A11, were found to have the strongest homology to the Drosoph- ila melanogaster sulfate transporter (Accession No. AF180728) and several plant sulfate transporters. SLC26A11 expression was found in all 16 tissues included in the multiple tissue cDNA panel.

3. Functional Characterization of the SLC26A7-A9 Transporters (II) The high structural homology of the new SLC26 proteins to the previously known family members capable of transporting anions suggested that the new ones might have similar functions. To characterize the possible anion transport mediated by the new SLC26A7-A9 proteins, in vitro transcribed cRNAs of SLC26A7–A9 were injected into Xenopus laevis oocytes, and [35S]sulfate, [36Cl]chloride, and [14C]oxalate uptakes - 2- were measured. All three proteins studied were able to induce Cl , SO4 , and oxalate 2- transport above water-injected control oocytes when expressed separately. The SO4 transport mediated by SLC26A9 could be inhibited by the anion exchanger inhibitor

64 Results DIDS and thiosulfate but not by oxalate or glucose. These results demonstrated that SLC26A7–A9 proteins indeed function as anion transporters, and are able to medi- - 2- ate at least Cl , SO4 , and oxalate transport.

4. Characterization of the Antibodies (III-IV)

4.1 The SLC26A6 Antibodies The specificity of the carboxyl terminal (anti-A6C) and amino terminal (anti-A6N) SLC26A6 antibodies was first shown by immunoblotting in our study not included in this thesis (Lohi et al. 2003). Both antibodies detected specific bands of the expected size in Western blots made of SLC26A6 transfected Cos-1 cell lysates. In addition, anti-A6C showed a single clear band of the appropriate size in an immunoblot made of in vitro translated SLC26A6 protein (Lohi et al. 2003). The specificity of anti- A6N was further confirmed by immunofluorescence labeling of transfected COS-1 cells in study III. Anti-A6N stained the SLC26A6a isoform transfected cells, showing a strong signal both following the cell membrane and in the cytoplasm. The results were ensured by a peptide competition assay, which completely blocked the anti-A6N staining of the protein in the transfected cells. In immunohistochemistry, both anti- A6C and anti-A6N gave similar results.

4.2 The SLC26A7 Antibodies (III)

Specificity of SLC26A7 antibodies (anti-A7) was confirmed by immunoblots made of human kidney cortex and medulla lysates. Anti-A7 bound to an approximately 100 kDa band in both lysates. The competition assay with corresponding SLC26A7 peptide inhibited the staining completely, further confirming the specificity of anti-A7. The single 100 kDa band detected with anti-A7 was about 30 kDa larger than predicted for SLC26A7 on the grounds of its structure (656 aa). However, it was of the same size as the specific band of 90-94 kDa seen in the anti-mouse Slc26a7 immunoblots (Petrovic et al. 2004). Glycosylation or other post-translational modifica- tion of the protein may explain the larger than expected size of the SLC26A7 seen in the samples.

4.3 The SLC26A8 Antibodies (IV)

The speciﬁcity of the SLC26A8 antiserum (anti-A8) was shown by a western blot made of adult rat testis known to express this protein (Toure et al. 2001). A strong specific band of approximately 100 kDa was seen when stained with anti-A8,

Results 65 corresponding well to the known 970 aa size of the SLC26A8 protein. In addition, a less intense 60 kDa band could be observed, probably originating from a degrada- tion product of the SLC26A8 protein.

5. Expression Profiles of SLC26A6-A9 (I-II) The general expression profiles of the new SLC26 genes were first characterized by Northern hybridization and by PCR with CLONTECH’s multiple tissue cDNA panels, including 16 different tissues: brain, colon, heart, kidney, leukocytes, liver, lung, ovary, pancreas, placenta, prostate, skeletal muscle, small intestine, spleen, testis, and thymus. In situ hybridization was used to detect the mRNA more specifically at tissue level. When antibodies against the cloned new members SLC26A6-A9 had been done, we performed preliminary immunohistochemical studies on the tissues predicted to be their major expression sites by the aforementioned techniques. Later, the expression of selected SLC26 proteins was studied in more detail in two human tissues where strict anion control was known to be critical: the kidney and the epididymis.

5.1 SLC26A6 is Expressed in Various Tissues (I)

Northern analysis with a SLC26A6 probe showed a distinct 3 kb band, corresponding well with the size of 2748 bp of the full-length SLC26A6 cDNA. This band was strongest in the kidney, but was evident in the pancreas, placenta, and skeletal muscle as well. In addition, a few bands of different sizes could be observed for several tissues, eliciting the possibility of alternative splicing. PCR analysis indicated even wider tissue distribution, suggesting the most abundant expression in the kidneys and pancreas, but a fainter positive reaction could be detected in the colon, liver, lungs, prostate, small intestine, thymus, and testes as well (Figure 5). PCR experiments supported the possibility of alternative splicing of SLC26A6, since several bands of different sizes could be observed with speciﬁc primer pairs (Figure 5). To conﬁrm the expression results, RT-PCR was performed on two human pancreatic duct cell lines, Capan-1 and Capan-2, and the human embryonal kidney cell line HEK293. All of the three cell lines studied expressed SLC26A6 mRNA.

In addition, preliminary histological studies of SLC26A6 expression were done by in situ hybridization and immunohistochemistry on human kidney and pancreas specimens. The SLC26A6 speciﬁc antisense cRNA probe displayed focal expression of the SLC26A6 mRNA in a subgroup of cells in the kidney tubules. Immunostaining with anti-A6N antibodies revealed positive staining on apical and basolateral sides of kidney tubule cells and on the brush border of the pancreatic duct.

66 Results Figure 5. Results from PCR experiments with multiple tissue cDNA panel, showing the distinct expression patterns of SLC26A6-A8.

5.2 SLC26A7 Was Observed in the Human Kidneys (II)

In Northern blot analysis, the SLC26A7 probe detected an approximately 5 kb band, which corresponds well to the 5250 bp length of SLC26A7 cDNA. The gene clearly had the most abundant expression in the kidney. A weaker transcript of about 3.0 kb could also be seen, possibly derived from alternative polyadenylation of the gene. The long untranslated region in the 3’ head of SLC26A7 sequence contains several consensus AAUAAA or AUUAAA sites that may serve as alternative polyadenylation signals in a reasonable concordance with the size of the less abundant approximately 3.0 kb transcript. Repeated SLC26A7 PCR analysis revealed the most abundant expression in the kidneys, placenta, and testes (Figure 5), supporting the Northern blot results well. In immunohistochemical staining of human kidney, SLC26A7 protein was located in the distal nephron segments, while the proximal tubules remained non-reactive.

Results 67 5.3 SLC26A8 Expression is Restricted to the Testes (II)

The SLC26A8 gene was found to have a strictly testis speciﬁc distribution both by Northern and PCR analyses (Figure 5). The 3371 bp size of the full-length SLC26A8 cDNA matched the observed 3.5 kb size in Northern hybridization. Microscopic localization of SLC26A8 in human testes was analyzed by both in situ hybridization and immunohistochemistry. In situ hybridization with an SLC26A8 antisense cRNA probe revealed abundant mRNA expression in the seminiferous tubules. The SLC26A8 mRNA signal was concentrated on the luminal side of the tubuli where the spermatocytes and the spermatids are located, whereas the basal side of the tubuli, containing the spermatogonia, seemed negative. Similarly, immunohistochemistry revealed the SLC26A8 protein in developing spermatocytes and spermatids. Our data suggests that the expression of SLC26A8 is limited to the meiotic phase of the germ cells in the human testis.

5.4 SLC26A9 is Located in the Pulmonary Epithelium (II) Northern blotting suggested lung specific expression for SLC26A9, with a specific band of 4.8 kb corresponding to the gene’s 4815 bp full-length cDNA. The SLC26A9 PCR product was overwhelmingly strongest from the lungs, albeit weaker bands were detected from the pancreas and prostate. To refine the expression results, RT-PCR of two human lung specific epithelial cell lines, NCI-H358 and A549 (from bronchoal- veolus and alveolus, respectively) were studied. Both cell lines were found to express SLC26A9 mRNA. In human lung specimens, immunohistochemistry with anti-A9 antisera revealed strong cytoplasmic immunoreaction in the bronchiolar epithelium. Apparent membrane-associated concentration of the signal was observed in the alveolar epithelium as well.

6. Distinct Expression of SLC26A6 and SLC26A7 in the Human Kidney (III) The preliminary expression studies had revealed SLC26A6 and A7 in specific tubules of the human kidney (I-II), each of which are known to have distinct tasks in important physiological anion transport processes. As the first step towards understanding the possible physiological role of SLC26A6 and A7 in human kidneys, we studied their expression in different tubule segments in detail. In order to specify the distinct kidney tubule segments, we utilized several known marker proteins whose exact localization in different tubule cell types has been previously verified. In addition to normal human kidneys, we examined SLC26A6 and A7 expression in ADPKD and MCDK.

68 Results 6.1 SLC26A6 and SLC26A7 mRNA Expression in the Kidneys Both SLC26A6 and A7 mRNA were found in human kidney medulla and cortex samples by RT-PCR. The results were veriﬁed by sequencing the RT-PCR products. The expression level of the housekeeping gene GADPH was equal in all samples studied. No genomic contamination was observed.

Interestingly, when approximated from the bands detected on the agarose gels, the SLC26A6 RT-PCR products seemed more abundant in samples from the cortex than from the medulla, but the intensity of the products was completely opposite for SLC26A7, whose bands derived from the medullary cDNA samples were stronger than the ones from the cortex. The results were consistent in repeated experiments using separate samples from kidneys of two individuals. In immunoblots with SLC26A7 antibody, repeatedly the bands observed were modestly stronger in the medullary than in the cortical samples, further supporting the RT-PCR results and indicating that SLC26A7 is more abundantly translated in the medulla than in the cortex.

6.2 Specific Expression of the SLC26A6 and SLC26A7 Proteins in Distinct Human Kidney Structures Next we used the SLC26A6 and A7 antibodies to further characterize the cell type speciﬁc expression of these proteins in the segments of the human kidney.

Glomeruli

The glomeruli did not stain for SLC26A6 antibodies. On the contrary, a clear SLC26A7 signal was seen in extraglomerular mesangial cells.

Proximal Tubules

In normal human kidneys, CD10 is expressed in the epithelium of all proximal tubule (PT) segments (Holm-Nielsen and Pallesen 1988), while PNRA is found in the luminal surface of Bowman’s capsule adjoining the outgoing PT and the brush border of PT epithelium (Yoshida and Imam 1989). All CD10 and PNRA positive PT were negative for SLC26A7. Apically emphasized staining of SLC26A6 was observed in a subgroup of PT. The initial regions of PT, recognized by beginning directly at the urinary pole, were negative for SLC26A6, while the more distal segments of PT, including the descending thick limbs of Henle’s loops, expressed SLC26A6 protein.

Results 69 Henle’s Loops and Distal Tubules

The TH antigen is absent from macula densa but present in the tubule segments around it: the thick ascending limbs of the loops of Henle (TAL) and in the distal convoluted tubules (DCT) (Sikri et al. 1981), while PNRA, CD10, 34βE12 and CK7 are not expressed in any of these structures (Gown and Vogel 1984, Ramaekers et al. 1987, Holm-Nielsen and Pallesen 1988, Yoshida and Imam 1989). SLC26A6 was expressed in the TAL and DCT, since it co-located with TH. In addition, macula densa cells as well as some of the thin segments of the loops of Henle were positive for SLC26A6 antibodies. In contrast, SLC26A7 was negative in the TH-expressing tubules, macula densa, and the thin loops of Henle.

Collecting Ducts

CK7 and 34βE12 are located in the collecting ducts (CD) of human kidneys (Gown and Vogel 1984, Ramaekers et al. 1987). Both SLC26A6 and A7 are present in CD, since they were found in speciﬁc cells in CK7 and 34βE12 positive segments. In CD, Aquaporin 2 (AQP2) is expressed exclusively by the principal cells (Nielsen et al. 2002), while H+V-ATPase is located in the type A and type B intercalated cells (IC) (Wagner et al. 2004). SLC26A6 and AQP2 did not co-localize, demonstrat- ing that principal cells of CD do not express SLC26A6. A subgroup of IC expressing H+V-ATPase showed apically emphasized SLC26A6 positive staining. The SLC26A7 expression in CD was mostly detected in H+V-ATPase positive, AQP2 negative, IC. Particularly, a subgroup of the IC that had mainly apical staining for H+V-ATPase, thus most probably type A IC (Wagner et al. 2004), showed a predominantly basal expression pattern for SLC26A7. Antibodies against different B and E subunits of H+V-ATPase gave identical results. Interestingly, SLC26A6 and A7 showed partial co-expression in a subgroup of IC along the CD.

ADPKD

Both SLC26A6 and A7 expression were variable in ADPKD. The proteins could be detected in the epithelium of a subgroup of cysts while others remained negative (Fig- ure 6, a and b). Some of the positive cysts expressed only either SLC26A6 or A7, some both of them. The proportion of positive cysts varied between different patients for both anion transporters studied.

70 Results MCDK

SLC26A6 was explicitly expressed in the epithelium of small cysts (less than 5-10x normal tubule diameter) in MCDK (Figure 6d). When the cyst diameter was larger, the epithelium appeared as if it gradually lost SLC26A6 expression (Figure 6e), and the largest cysts were negative for this anion transporter (Figure 6f). All of the MCDK cysts were negative for SLC26A7 (Figure 6c).

Figure 6 (unpublished data). Expression of SLC26A6 and SLC26A7 in polycystic and dysplastic kidneys. In ADPKD both a) SLC26A6 and b) SLC26A7 were expressed in the epithelium of some of the cysts (arrows), while others remained negative (arrowheads). In MCDK c) SLC26A7 was not express in any dysplastic cysts. d) SLC26A6 was detected in the epithelium of small dysplastic cysts. e) In medium-sized dysplastic cysts a subgroup of the epithelial cells expressed SLC26A6. f) Large cysts were negative for SLC26A6 in MCDK. Scale bars 50 µm.

Results 71 7. Expression of Selected SLC26 and Other Ion Transport Associated Proteins in the Human Epididymis (IV)

Since the strict control of intraluminal ion composition in the epididymis is known to be essential for male fertility, SLC26 proteins might have physiological tasks there. As a primary step towards understanding their possible roles in maintaining the epididymal and efferent duct microenvironment, we studied the cell speciﬁc expression of selected SLC26 members in these structures in man. While seeking marker proteins for different epithelial cell types of efferent and epididymal ducts, we found out that expression of many important proteins in these structures has been studied mainly in rodents, and only very limitedly in humans. Therefore, we picked out several proteins that might have relevant physiological interactions with SLC26 members in efferent or epididymal ducts, and included them into our studies.

SLC26A2

The anti-A2 antibody detected SLC26A2 speciﬁcally in the cilia of the ciliated cells of the efferent ducts of testis. In the epididymal ducts, anti-A2 did not have a signal in any cell types.

SLC26A6

SLC26A6 was expressed on the apical side of the non-ciliated cells of the type I and II efferent ducts, while the type III efferent ducts did not give off any signal. In the epididymis proper, SLC26A6 was detected in a subgroup of AMRC. Most of the narrow type AMRC and the early phase ﬂat apical cells were negative with anti-A6, while the later phase apical cells extruding to the lumen were stained apically. Interestingly, the positive signal in the efferent ducts required 10 min microwaving in citrate buffer to become visible, but the staining in the epididymis proper was seen clearest with no pretreatment of the slides.

SLC26A7

SLC26A7 was not observed in any type of efferent ducts. However, it was present in a subgroup of basal cells of the epididymal ducts. The distinct positive staining with anti-A7 followed the cell membrane of these basal cells. The rest of the epididymal cell types were negative for the SLC26A7 antibody.

72 Results SLC26A8

Anti-A8 did not give off any speciﬁc signal in the immunohistochemical stainings of human efferent ducts or epididymis proper, regardless of the numerous different pre- treatments and concentrations of the antiserum used.

CFTR

In the efferent ducts, CFTR was explicitly expressed in type I and II epithelium, but only a portion of type III tubules gave off a weak signal for it, and type V epithelium remained totally negative. The positive staining was concentrated on the apical edge of the non-ciliated cells, while the ciliated cells remained negative. In the epididymis proper, a distinct positive signal was seen on the apical border of the apical form of AMRC. The other cell types of the epididymal ducts remained unstained.

NHE3

A strong positive signal on the luminal edge of the non-ciliated cells was detected with anti-NHE3 in type I and II efferent ducts. The majority of type III efferent ducts remained negative, but in some non-ciliated cells a weak staining could be seen. No speciﬁc signal was observed with the NHE3 antibody in the epididymal ducts with any of the antigen retrieval methods used.

NHERF-1 (EBP50)

In all of the different efferent duct epithelium types detected, NHERF-1 was found on the apical side of virtually all of the cells, both ciliated and non-ciliated. The positive staining was generally slightly darker in the non-ciliated than the ciliated cells. In addition, NHERF-1 was found in the cytoplasm of both the narrow and apical forms of AMRC, and in a few basal cells of epididymal ducts.

V-ATPase

V-ATPase was not observed in any type of efferent duct epithelium. Instead, it was strongly expressed both in the narrow and apical forms of AMRC in the epididymis proper. Both antibodies, targeted against the B and E subunits of V-ATPase, gave similar results.

Results 73 CAII

CAII was not found in any human efferent duct type. Instead, in the epididymal ducts, the apical and narrow AMRC forms stained pancytoplasmically for the CAII antibody, while the other epididymal cell types remained unstained.

74 Results Discussion

1. The SLC26 Family Grew By Several New Structurally Homologous Members

The systematic characterization of gene families utilizing the homology of sequences between the family members opens up a rich source for expanding the knowledge of human physiology. In this study, we have focused on the newly delineated second family of anion exchanges, SLC26, which has earned attention particularly among physiologists. In addition, specific interest towards this gene family has been created by the fact that four SLC26 members are associated with distinct human diseases; SLC26A2, SLC26A3, SLC26A4, and SLC26A5 have been identified to be mutated in several bone and cartilage dysplasias, congenital chloride diarrhea, Pendred syndrome, and hereditary deafness, respectively. Identification of new tissue specific SLC26 members, which might also be associated with human diseases, has further increased the motivation to study this gene family (Everett and Green 1999, Kere et al. 1999, Kere 2005). The growing attention towards this subject can be illustrated by PubMed searches: with the keyword ”slc26*” altogether 266 citations were found in July 2005, of which 70 were published before our first article (I) in November 2000. As a comparison, with the keyword ”slc4*” for the classical anion exchanger family, 176 citations came up of which 43 appeared before ours. In this thesis, we have identified seven novel human SLC26 genes, further characterized five of them, and studied the expression of four SLC26 proteins in detail.

All SLC26 members are highly homologous (Figure 7), and comparison of the amino acid sequences revealed at least two common conserved domains within the family: the sulfate transporter family (ST) and the sulfate transporters and anti-sigma antagonist (STAS) domains. The functional signiﬁcance of the ST domain is still unclear.

Instead, understanding of the highly conserved STAS domains has improved lately. They are found both in SLC26 proteins and in a variety of bacteria, where they have been predicted to form a α-helical handle-like structure. At least in bacteria, STAS domains bind NTPs and are believed to be regulated by them. Because of the high homology, SLC26 proteins have been suggested to be regulated by intracellular GTP and/or ATP concentrations as well (Aravind and Koonin 2000). It was recently demonstrated that CFTR has reciprocal regulatory interaction at least with SLC26A3 and SLC26A6, and that the STAS domain of the SLC26 proteins is essential for binding to the R-domain of CFTR and thus for enabling the whole functional interaction (Ko et al. 2004). Moreover, deletion of SLC26A3 STAS domain prevents the

75 Figure 7. A multiple alignment of the human SLC26A1-A11 amino acid sequences. The identical residues are shown in black and conserved ones in gray.

SLC26A1 1 ...... MDESPEPLQQGRGPVPVRRQRPAPRGLREMLKARLWCSCSCSVLCVRALVQDLLPATRWLRQYRPREYLAG SLC26A2 1 MSSESKEQHNVSPRDSAEGNDSYPSGIHLELQRESSTDFKQFETNDQCRPYHRILIERQEKSDTNFKEFVIKKLQKNCQCSPAKAKNMILGFLPVLQWLPKYDLKKNILG SLC26A3 1 ...... MIEP.FGNQYIVARPVYSTNAFEENHKKTGRHHKTFLDHLKVC.....CSCSPQKAKRIVLSLFPIASWLPAYRLKEWLLS SLC26A4 1 ...... MAAPGGRSEPPQLPE.YSCSYMVSRPVYSELAFQQQHERRLQERKTLRESLAKC.....CSCSRKRAFGVLKTLVPILEWLPKYRVKEWLLS SLC26A5 1 ...... MDHAEENEILA.ATQRYYVERPIFSHPVLQERLHTKDKVPDSIADKLKQA.....FTCTPKKIRNIIYMFLPITKWLPAYKFKEYVLG SLC26A6 1 ...... MDL.RRRDYHMERPLLNQEHLEEL..GRWGSAP.RTHQWRTW.....LQCSRARAYALLLQHLPVLVWLPRYPVRDWLLG SLC26A9 1 ...... MSQ.PRPRYVVDRAAYSLTLFDDEFEKKDRTYP.VGEKLRNA.....FRCSSAKIKAVVFGLLPVLSWLPKYKIKDYIIP SLC26A8 1 ...... MAQLERSAISGFSSKSRR.NSFAYDVKREVYNEETFQQEHKRKASSSGNMNINITTFRHHVQCRCSWHRFLRCVLTIFPFLEWMCMYRLKDWLLG SLC26A7 1 ...... MTGAKRKKKSMLWSKMHTPQCEDIIQWCRRRLPILDWAPHYNLKENLLP SLC26A10 1 ...... MRLDLASLMSAPKSLGSAFKSWRLD SLC26A11 1 ...... MAPSACCCSPAALQRRLPILAWLPSYSLQ.WLKM Consensus 1 ...... C...... LP.L.WLP.Y.LK.WLL.

SLC26A1 72 DVMSGLVIGIILVPQAIAYSLLAG..LQPIYSLYTSFFANLIYFLMGTSRHVSVGIFSLLCLMVGQVVDRELQLAGFDPSQDGLQPG..ANSSTLNGSAAMLDCGRDCYA SLC26A2 111 DVMSGLIVGILLVPQSIAYSLLAG..QEPVYGLYTSFFASIIYFLLGTSRHISVGIFGVLCLMIGETVDRELQKAGYDNAHSAPSLGMVSNGSTLLNHTSDRICDKSCYA SLC26A3 76 DIVSGISTGIVAVLQGLAFALLVD..IPPVYGLYASFFPAIIYLFFGTSRHISVGPFPILSMMVGLAVSGA...... VSKAVPDRNATTLGLPNNSNNSSLLDDERVR SLC26A4 87 DVISGVSTGLVATLQGMAYALLAA..VPVGYGLYSAFFPILTYFIFGTSRHISVGPFPVVSLMVGSVVL.S...... MAPDEHFLVSSSNGTVLNTTMIDTAARDTAR SLC26A5 83 DLVSGISTGVLQLPQGLAFAMLAA..VPPIFGLYSSFYPVIMYCFLGTSRHISIGPFAVISLMIGGVAVRL...... VPDDI.....VIPGGVNATNGTE..ARDALR SLC26A6 72 DLLSGLSVAIMQLPQGLAYALLAG..LPPVFGLYSSFYPVFIYFLFGTSRHISVGTFAVMSVMVGSVTESL...... APQ...... ALNDSMINETARDAAR SLC26A9 74 DLLGGLSGGSIQVPQGMAFALLAN..LPAVNGLYSSFFPLLTYFFLGGVHQMVPGTFAVISILVGNICLQL...... APESK....FQVFNNATNESYVDTAAMEAER SLC26A8 95 DLLAGISVGLVQVPQGLTLSLLARQLIPPLNIAYAAFCSSVIYVIFGSCHQMSIGSFFLVSALLINVLKVS...... PFNNGQ..LVMGSFVKNEFSAPSYLMGYNKS SLC26A7 50 DTVSGIMLAVQQVTQGLAFAVLSS..VHPVFGLYGSLFPAIIYAIFGMGHHVATGTFALTSLISANAVERI...... VPQNM.....QNLTTQSNTSVLGLSDFEMQR SLC26A10 26 KAPSPQHTFPSTSIPGMAFALLAS..VPPVFGLYTSFFPVLIYSLLGTGRHLSTGTFAILSLMTGSAVERL...... VPEPLVGNLSGIEKEQLDAQR SLC26A11 34 DFVAGLSVGLTAIPQALAYAEVAG..LPPQYGLYSAFMGCFVYFFLGTSRDVTLGPTAIMSLLVSFYT...... FHE Consensus 14 D..SG.S.G....PQGLA.ALLA....PPV.GLY.SFFP..IYF.LGTSRH.S.G.FA..SLM.G..V.R...... N.S...... R

SLC26A1 178 IRVATALTLMTGLYQVLMGVLRLGFVSAYLSQPLLDGFAMGASVTILTSQLKHLLGVRIPRHQGPGMVVLTWLSLLRGAGQANVCDVVTSTVCLAVLLAAKELSDRYRHR SLC26A2 219 IMVGSTVTFIAGVYQVAMGFFQVGFVSVYLSDALLSGFVTGASFTILTSQAKYLLGLNLPRTNGVGSLITTWIHVFRNIHKTNLCDLITSLLCLLVLLPTKELNEHFKSK SLC26A3 176 VAAAASVTVLSGIIQLAFGILRIGFVVIYLSESLISGFTTAAAVHVLVSQLKFIFQLTVPSHTDPVSIFKVLYSVFSQIEKTNIADLVTALIVLLVVSIVKEINQRFKDK SLC26A4 186 VLIASALTLLVGIIQLIFGGLQIGFIVRYLADPLVGGFTTAAAFQVLVSQLKIVLNVSTKNYNGVLSIIYTLVEIFQNIGDTNLADFTAGLLTIVVCMAVKELNDRFRHK SLC26A5 176 VKVAMSVTLLSGIIQFCLGVCRFGFVAIYLTEPLVRGFTTAAAVHVFTSMLKYLFGVKTKRYSGIFSVVYSTVAVLQNVKNLNVCSLGVGLMVFGLLLGGKEFNERFKEK SLC26A6 160 VQVASTLSVLVGLFQVGLGLIHFGFVVTYLSEPLVRGYTTAAAVQVFVSQLKYVFGLHLSSHSGPLSLIYTVLEVCWKLPQSKVGTVVTAAVAGVVLVVVKLLNDKLQQQ SLC26A9 170 LHVSATLACLTAIIQMGLGFMQFGFVAIYLSESFIRGFMTAAGLQILISVLKYIFGLTIPSYTGPGSIVFTFIDICKNLPHTNIASLIFALISGAFLVLVKELNARYMHK SLC26A8 195 LSVVATTTFLTGIIQLIMGVLGLGFIATYLPESAMSAYLAAVALHIMLSQLTFIFGIMISFHAGPISFFYDIINYCVALPKANSTSILVFLTVVVALRINKCIRISF.NQ SLC26A7 145 IHVAAAVSFLGGVIQVAMFVLQLGSATFVVTEPVISAMTTGAATHVVTSQVKYLLGMKMPYISGPLGFFYIYAYVFENIKSVRLEALLLSLLSIVVLVLVKELNEQFKRK SLC26A10 116 VGVAAAVAFGSGALMLGMFVLQLGVLSTFLSEPVVKALTSGAALHVLLSQLPSLLGLSLPRQIGCFSLFKTLASLLTALPRSSPAELTISALSLALLVPVKELNVRFRDR SLC26A11 103 PAYAVLLAFLSGCIQLAMGVLRLGFLLDFISYPVIKGFTSAAAVTIGFGQIKNLLGLQNIPRPFFLQVYHTFLRIAETRVGDAVLGLVCMLLLLVLKLMRDHVPPVHPEM Consensus 60 ..VAA...FL.G.IQL.$GVL.LGF...YLSEP...GFT.AAA..!..SQLK.LLGL..P...G..S...T...... L...LL.LV.L..VKELN..F...

SLC26A1 288 LR...... VPLPTELLVIVVATLVSHFGQLHKRFGSSVAGDIPTGFMPPQVPEPRLMQRVALDAVA...... LALVAAAFSISLAEMFARSHGYSVRAN SLC26A2 329 LK...... APIPIELVVVVAATLASHFGKLHENYNSSIAGHIPTGFMPPKVPEWNLIPSVAVDAIA...... ISIIGFAITVSLSEMFAKKHGYTVKAN SLC26A3 286 LP...... VPIPIEFIMTVIAAGVSYGCDFKNRFKVAVVGDMNPGFQPPITPDVETFQNTVGDCFG...... IAMVAFAVAFSVASVYSLKYDYPLDGN SLC26A4 296 IP...... VPIPIEVIVTIIATAISYGANLEKNYNAGIVKSIPRGFLPPELPPVSLFSEMLAASFS...... IAVVAYAIAVSVGKVYATKYDYTIDGN SLC26A5 286 LP...... APIPLEFFAVVMGTGISAGFNLKESYNVDVVGTLPLGLLPPANPDTSLFHLVYVDAIA...... IAIVGFSVTISMAKTLANKHGYQVDGN SLC26A6 270 LP...... MPIPGELLTLIGATGISYGMGLKHRFEVDVVGNIPAGLVPPVAPNTQLFSKLVGSAFT...... IAVVGFAIAISLGKIFALRHGYRVDSN SLC26A9 280 IR...... FPIPTEMIVVVVATAISGGCKMPKKYHMQIVGEIQRGFPTPVSPVVSQWKDMIGTAFS...... LAIVSYVINLAMGRTLANKHGYDVDSN SLC26A8 304 YP...... IEFPMELFLIIGFTVIANKISMATETSQTLIDMIPYSFLLPVTPDFSLLPKIILQAFS...... LSLVSSFLLIFLGKKIASLHNYSVNSN SLC26A7 255 IK...... VVLPVDLVLIIAASFACYCTNMENTYGLEVVGHIPQGIPSPRAPPMNILSAVITEAFG...... VALVGYVASLALAQGSAKKFKYSIDDN SLC26A10 226 LP...... TPIPGEVVLVLLASVLCFTSSVDTRYQVQIVGLLPGGFPQPLLPNLAELPRILADSLP...... IALVSFAVSASLASIHADKYSYTIDSN SLC26A11 213 PPGVRLSRGLVWAATTARNALVVSFAALVAYSFEVTGYQPFILTGETAEGLPPVRIPPFSVTTANGTISFTEMVQDMGAGLAVVPLMGLLESIAVAKAFASQNNYRIDAN Consensus 110 .P...... PIP.#...V..A...... Y....VG.IP.GFPPP..P...... AF...... ALVG...S.SLA...A.K..Y.!D.N

SLC26A1 375 QELLAVGCCNVLPAFLHCFATSAALAKSLVKTATGCRTQLSSVVSATVVLLVLLALAPLFHDLQRSVLACVIVVSLRGALRKVWDLPRLWRMSPADALVWA.GTAATCML SLC26A2 416 QEMYAIGFCNIIPSFFHCFTTSAALAKTLVKESTGCHTQLSGVVTALVLLLVLLVIAPLFYSLQKSVLGVITIVNLRGALRKFRDLPKMWSISRMDTVIWF.VTMLSSAL SLC26A3 373 QELIALGLGNIVCGVFRGFAGSTALSRSAVQESTGGKTQIAGLIGAIIVLIVVLAIGFLLAPLQKSVLAALALGNLKGMLMQFAEIGRLWRKDKYDCLIWI.MTFIFTIV SLC26A4 383 QEFIAFGISNIFSGFFSCFVATTALSRTAVQESTGGKTQVAGIISAAIVMIAILALGKLLEPLQKSVLAAVVIANLKGMFMQLCDIPRLWRQNKIDAVIWV.FTCIVSII SLC26A5 373 QELIALGLCNSIGSLFQTFSISCSLSRSLVQEGTGGKTQLAGCLASLMILLVILATGFLFESLPQAVLSAIVIVNLKGMFMQFSDLPFFWRTSKIELTIWL.TTFVSSLF SLC26A6 357 QELVALGLSNLIGGIFQCFPVSCSMSRSLVQESTGGNSQVAGAISSLFILLIIVKLGELFHDLPKAVLAAIIIVNLKGMLRQLSDMRSLWKANRADLLIWL.VTFTATIL SLC26A9 367 QEMIALGCSNFFGSFFKIHVICCALSVTLAVDGAGGKSQVASLCVSLVVMITMLVLGIYLYPLPKSVLGALIAVNLKNSLKQLTDPYYLWRKSKLDCCIWV.VSFLSSFF SLC26A8 391 QDLIAIGLCNVVSSFFRSCVFTGAIARTIIQDKSGGRQQFASLVGAGVMLLLMVKMGHFFYTLPNAVLAGIILSNVIPYLETISNLPSLWRQDQYDCALWM.MTFSSSIF SLC26A7 342 QEFLAHGLSNIVSSFFFCIPSAAAMGRTAGLYSTGAKTQVACLISCIFVLIVIYAIGPLLYWLPMCVLASIIVVGLKGMLIQFRDLKKYWNVDKIDWGIWV.STYVFTIC SLC26A10 313 QEFLAHGASNLISSLFSCFPNSATLATTNLLVDAGGKTQLAGLFSCTVVLSVLLWLGPFFYYLPKAVLACINISSMRQVFCQMQELPQLWHISRVDFLLQVPGLCILSYP SLC26A11 323 QELLAIGLTNMLGSLVSSYPVTGSFGRTAVNAQSGVCTPAGGLVTGVLVLLSLDYLTSLFYYIPKSALAAVIIMAVAPLF.DTKIFRTLWRVKRLD.LLPLCVTFLLCF. Consensus 142 Q#LLA.GL.N...S.F.CFP...AL.RT.V....GG.TQ.AGL.....V$LV.L.LG.LFY.LPK.VLA.III..L...L.Q..DLP.LWR....D.L.W...TF..S..

SLC26A1 484 VSTEAGLLAGVILSLLSLAGRTQRPRTALLARIGDTAFYEDATEFEGLVPEPGVRVFRFGGPLYYANKDFFLQSLYSLTGLDAGCMAARRKEGGSETGVGE...... SLC26A2 525 LSTEIGLLVGVCFSIFCVILRTQKPKSSLLGLVEESEVFESVSAYKNLQTKPGIKIFRFVAPLYYINKECFKSALYKQTVNPILIKVAWKK..AAKRKIKE...... SLC26A3 482 LGLGLGLAASVAFQLLTIVFRTQFPKCSTLANIGRTNIYKNKKDYYDMYEPEGVKIFRCPSPIYFANIGFFRRKLIDAVGFSPLRILRKRNKALRKIRKLQKQG...... SLC26A4 492 LGLDLGLLAGLIFGLLTVVLRVQFPSWNGLGSIPSTDIYKSTKNYKNIEEPQGVKILRFSSPIFYGNVDGFKKCIKSTVGFDAIRVYNKRLKALRKIQKLIKSG...... SLC26A5 482 LGLDYGLITAVIIALLTVIYRTQSPSYKVLGKLPETDVYIDIDAYEEVKEIPGIKIFQINAPIYYANSDLYSNALKRKTGVNPAVIMGARRKAMRKYAKEVGNA...... SLC26A6 466 LNLDLGLVVAVIFSLLLVVVRTQMPHYSVLGQVPDTDIYRDVAEYSEAKEVRGVKVFRSSATVYFANAEFYSDALKQRCGVDVDFLISQKKKLLKKQEQLKLKQ...... SLC26A9 476 LSLPYGVAVGVAFSVLVVVFQTQFRNGYALAQVMDTDIYVNPKTYNRAQDIQGIKIITYCSPLYFANSEIFRQKVIAKTGMDPQKVLLAKQKYLKKQEKRRMRP...... SLC26A8 500 LGLDIGLIISVVSAFFITTVRSHRAKILLLGQIPNTNIYRSINDYREIITIPGVKIFQCCSSITFVNVYYLKHKLLKEVDMVKVPLKEEEIFSLFNSSDTNLQGGKICRC SLC26A7 451 FAANVGLLFGVVCTIAIVIGRFPRAMTVSIKNMKEMEF..KVKTEMDSETLQQVKIISINNPLVFLNAKKFYTDLMNMIQKENACNQPLDDISKCEQNTLL...... SLC26A10 423 TPLYFGTRGQFRCNLEWHLGLGEGEKETSKPDGPMVAVAEPVRVVVLDFSGVTFADAAGAREVVQVRERLASRCRDARIRLLLAQCNALVQGTLTRVGLLDRVTPDQLFV SLC26A11 430 WEVQYGILAGALVSLLMLLHSAARPETK.VSEGPVLVLQPASGLSFPAMEALREEILSRALEVSPPRCLVLECTHVCSIDYTVVLGLGELLQDFQKQGVALAFVGLQVPV Consensus 199 ..L..GL..GV...LL....R...P....L...P...... G.KI...... N...... L...... K......

SLC26A1 585 ...... GGPAQGEDLGPVSTRAALVPAA...... A SLC26A2 624 ...... KVVTLGGIQDEMSVQLSHDPLE...... SLC26A3 586 ...... LLQVTPKGFIC.TVDTIKDSDEELDNNQ...... IEVLDQPINTTDLPFHIDWNDDLP...... LNIEVPK...... I SLC26A4 596 ...... QLRATKNGIISDAVSTNNAFEPDEDIED...... LEELDIP..TKEIEIQVDWNSELP...... VKVNVPK...... V SLC26A5 586 ...... NMANA...... TVVKADAEVDGEDATK...... PEEEDGEVKYPPIVIKSTFPEEMQ...... RFMPPG...... D SLC26A6 570 ...... LQKEEKLRKQAASPKGASVSINVNTSLEDMRSNNVEDCKMMQVS....SGDKMEDATANGQEDSKAP.DGSTL...... KALGLPQ...... P SLC26A9 580 ...... TQQRRSLFMKTKTVSLQELQQDFENAPPTDPNNNQTPANGTSVSYITFSPDSSSPAQSEPPASAEAPGEPSDM...... LASVPPF...... V SLC26A8 610 FCNCDDLEPLPRILYTERFENKLDPEASSINLIHCSHFESMNTSQTASEDQVPYTVSSVSQKNQGQQYEEVEEVWLPNNSSRNSSPGLPDVAESQGRRSLIPYSDASLLP SLC26A7 550 ...... NSLSNGNCNEEASQSCPNEKCY...... SLC26A10 533 SVQDAAAYALGSLLRGSSTRSGSQEALGCGK...... SLC26A11 539 LRVLLSADLKGFQYFSTLEEAEKHLRQKPGTQPYNIREDSILDQKVALLKA...... Consensus 216 ......

SLC26A1 608 GFHTVVIDCAPLLFLDAAGVSTLQDLRRDYGALGISLLLACCSPPVRDILSRGGFLGEGPGDTAEEEQLFLSVHDAVQTARARHRELEATDAHL...... SLC26A2 646 .LHTIVIDCSAIQFLDTAGIHTLKEVRRDYEAIGIQVLLAQCNPTVRDSLTNGEYCKK.....EEENLLFYSVYEAMAFAEVSKNQKGVCVPNGLSLSSD...... SLC26A3 645 SLHSLILDFSAVSFLDVSSVRGLKSILQEFIRIKVDVYIVGTDDDFIEKLNRYEFF.DGE...VKSSIFFLTIHDAVL.HILMKKDYSTSKFNPSQEKDGKIDFTINTNG SLC26A4 654 PIHSLVLDCGAISFLDVVGVRSLRVIVKEFQRIDVNVYFASLQDYVIEKLEQCGFF.DDN...IRKDTFFLTVHDAIL.Y.LQNQVKSQEGQGSILETITLIQDCKDTLE SLC26A5 638 NVHTVILDFTQVNFIDSVGVKTLAGIVKEYGDVGIYVYLAGCSAQVVNDLTRNRFF.ENP...ALWELLFHSIHDAVLGSQLREALAEQEASAPPSQEDLEPNATPATPE SLC26A6 646 DFHSLILDLGALSFVDTVCLKSLKNIFHDFREIEVEVYMAACHSPVVSQLEAGHFF.DAS...ITKKHLFASVHDAVTFALQHPRPVPDSPVSVTRL...... SLC26A9 661 TFHTLILDMSGVSFVDLMGIKALAKLSSTYGKIGVKVFLVNIHAQVYNDISHGGVFEDGS...LECKHVFPSIHDAVLFAQANARDVTPGHNFQGAPGDAELSLYDSEED SLC26A8 720 SVHTIILDFSMVHYVDSRGLVVLRQICNAFQNANILILIAGCHSSIVRAFERNDFF.DAG...ITKTQLFLSVHDAVLFALSRKVIGSSELSIDESETVIRETYSETDKN SLC26A7 572 ....LILDCSGFTFFDYSGVSMLVEVYMDCKGRSVDVLLAHCTASLIKAMTYYGNLDS...... EKPIFFESVSAAISHIHSNKNLSKLSDHSEV...... SLC26A10 ...... SLC26A11 ...... Consensus 216 ..H...LD.....F.D..G...L...... A.C...... F.S...A......

SLC26A1 ...... SLC26A2 ...... SLC26A3 750 GLRNRVYEVPVETKF...... SLC26A4 758 LIETELTEEELDVQDEAMRTLAS...... SLC26A5 744 A...... SLC26A6 ...... SLC26A9 768 IRSYWDLEQEMFGSMFHAETLTAL...... SLC26A8 826 DNSRYKMSSSFLGSQKNVSPGFIKIQQPVEEESELDLELESEQEAGLGLDLDLDRELEPEMEPKAETETKTQTEMEPQPETEPEMEPNPKSRPRAHTFPQQRYWPMYHPS SLC26A7 ...... SLC26A10 ...... SLC26A11 ...... Consensus ......

SLC26A1 ...... SLC26A2 ...... SLC26A3 ...... SLC26A4 ...... SLC26A5 ...... SLC26A6 ...... SLC26A9 ...... SLC26A8 936 MASTQSQTQTRTWSVERRRHPMDSYSPEGNSNEDV SLC26A7 ...... SLC26A10 ...... SLC26A11 ...... Consensus ......

76 Discussion anion transport function of the protein (Chernova et al. 2003). In addition, the STAS 2- domain was shown to be essential for the SO4 transport function in SULTR1.2, an Arabidopsis thaliana SLC26 sulfate transporter, strengthening the hypothesis that the 2- STAS domain is needed for protein-protein interactions controlling SO4 (or anion) transport (Rouached et al. 2005). The important role of the STAS domains is further supported by several disease causing mutations in the STAS domain sequence of SLC26A2 and SLC26A4 (Aravind and Koonin 2000). Thus, the STAS domain appears to be necessary for selected protein-protein interactions as well as for the anion transport function of the SLC26 proteins.

In addition, the sequence analyses revealed that at least SLC26A2, A3, A6, A7, and A9 have consensus PDZ binding sequences at their carboxyl termini (II). This offers the possibility for these SLC26 proteins to connect with PDZ interaction networks, which play important roles in polarized epithelial cells by targeting and retaining membrane proteins asymmetrically on distinct membrane domains. Furthermore, PDZ proteins bring different proteins close to each other, enabling their interactions and formation of transducisomes, which are spatially restricted units of functionally connected proteins such as transporters, receptors, or kinases (Brone and Egg- ermont 2005). A German group has proved that SLC26A3 is able to interact with a PDZ protein Na+/H+ exchanger regulatory factor 2 (NHERF-2, also known as SLC9A3R2) (Lamprecht et al. 2002). Together with them, we ﬁrst showed in a study not included in this thesis that SLC26A6 also binds to two PDZ proteins, NHERF-1 and NHERF-2, in vitro (Lohi et al. 2003), and then in this thesis we demonstrated that SLC26A6 and NHERF-1 are co-localized in human efferent and epididymal ducts in vivo (IV). Further studies on the possible PDZ interactions of other SLC26 members are still needed.

Phylogenetic analyses of amino acid sequences of the human, Drosophila melanogaster and Caenorhabditis elegans SLC26 proteins illustrates their relationships (Figure 8). Some of the relationships in Figure 8 made in July 2005, diverge slightly from the illustration in study II from year 2002, possibly due to conceivable differences in the facultative and version speciﬁc computational options. In addition, Figure 8 contains all 11 human SLC26 proteins, while in study II only the members SLC26A1-A9 were included. However, the results are essentially similar. The SLC26 family members are divided mainly into three separate groups according to species (Figure 8). Interestingly, SLC26A11 is located in a cluster where other members originate from D. melanogaster. SLC26A8, which has a notably longer sequence that of the other human members, is in its own branch, being somewhat closer to the D. melanogaster sequences than other human members. SLC26A3 and A4, constituting a small cluster of their own inside the human branch, are located within 40 kb on chromosome 7. Their high homology and close spatial location have prompted the idea that they might result from gene duplication during evolution (Everett et al. 1997).

Discussion 77 In addition, the genomic structures of human SLC26A1 and SLC26A2 with only three to four exons differ markedly from the other human members that consist of about 20 exons each. The diverging exon structure of SLC26A1 and -A2 suggests that their common origin may have separated from the other SLC26 members early during evolution, leaving them with only a few introns when others have gained several new introns after this separation. Moreover, the similarity of the exon-intron structure suggests that SLC26A1 and -A2 might have originated from a gene duplication, although they have unconnected chromosomal locations at 5q32 and 4p16, respectively. Supporting the genomic disparity, the corresponding proteins make up their own branch in the phylogenetic tree.

Figure 8. A phylogenetic tree showing the homology between the human, Drosophila melanogaster, and Caenorhabditis elegans SLC26 proteins’ amino acid sequences. The length of the branches between distinct proteins reﬂects their evolutionary distance. The analysis was made at http://www.genebee.msu.su/clustal/basic.html

78 Discussion We cloned SLC26A1, the human ortholog for rat Slc26a1, which was found ﬁrst of the SLC26 genes (Bissig et al. 1994). Later, an Australian group characterized both human and mouse genes in more detail, and showed the corresponding proteins 2- - to transport SO4 , Cl and oxalate (Lee et al. 2003, Regeer et al. 2003). Human SLC26A1 mRNA was found most abundantly in the kidneys and liver, but also in the pancreas, brain, testis, small intestine, colon and lungs (Regeer et al. 2003), supporting our previous results (I).

In this study, we reported the chromosomal loci and initial tissue distribution of SLC26A10 and A11. SLC26A10 has remained poorly characterized so far, and it has actually been suggested to be a pseudogene (Mount and Romero 2004). Vincourt et al. + 2- showed later that SLC26A11 indeed encodes a Na independent DIDS sensitive SO4 transporter, with mRNA expression in the placenta, kidneys, brain, and high endothelial venules. They proposed that SLC26A11 might be linked to hereditary hearing loss, since three distinct hereditary hearing impairment diseases had been mapped to the same region on chromosome 17q25 where SLC26A11 is located (Vincourt et al. 2003). However, soon afterwards it was shown that patients with the diseases in question have mutations in other genes located in the same region (Weil et al. 2003, Zhu et al. 2003), thus the possible diseases caused by SLC26A11 remain unclear.

Discussion 79 2. The New SLC26 Members May Have Important Functions in Certain Tissues

Despite the structural similarities, a characteristic quality of the SLC26 family is the unique tissue specific expression of each of the members. Interestingly, the diseases caused by mutations in SLC26 genes are very diverse, reflecting the major expression sites of the respective genes. Flawed SLC26A2 causes growth defects, being found quite ubiquitously, but particularly in cartilage (Haila et al. 2001). Mutations in SLC26A3 produce congenital chloride diarrhea, coinciding with the colon being the gene’s major expression site (Haila et al. 2000). SLC26A4 mutations inflict hearing loss and goiter, while the protein is located in the inner ear and thyroid (Everett et al. 1997, Everett et al. 1999). A few cases of hereditary deafness have been linked to SLC26A5, expressed strictly in the outer hair cells of cochlea (Liu et al. 2003).

A general question in studying human tissues is the normality of the samples. For example, the kidneys and the epididymises are routinely extirpated from a living patient only in the case of a tumor located in or near the tissue in question. Thus, there is always a possibility that some alterations in the surrounding, normal appearing, tissue have occurred due to the tumor, and that they might alter the expression of proteins in otherwise normal looking cells. In addition, the ischemic time with human samples is always longer compared to animal samples, which can be ﬁxed at the time blood perfusion stops. Despite these difﬁculties, it is important to get information about human tissues as well, since the expression and function of several orthologous proteins vary between species.

SLC26A6 was found in several human tissues studied, but the mRNA signal appeared to be strongest in the kidneys and pancreas. The high structural homology to the known SLC26 members strongly supported the idea that SLC26A6 would function as an anion transporter (I). Later, we and others have demonstrated that this hypothesis is true: SLC26A6 is indeed an anion exchanger. Interestingly, it has turned out that the human and mouse SLC26A6 orthologs have remarkable differences in their anion selectivity, transport mechanism and regulation (Chernova et al. 2005), - 2- - - although both orthologs are able to transport at least Cl , SO4 , HCO3 , OH and oxalate anions (Jiang et al. 2002, Ko et al. 2002, Wang et al. 2002, Xie et al. 2002, Lohi et al. 2003, Chernova et al. 2005). In addition, mouse Slc26a6 mediates Cl- / formate exchange (Knauf et al. 2001, Xie et al. 2002).

The primary function of the apical epithelium of the pancreatic duct is to secrete large amounts of NaHCO3 into the duodenum in order to neutralize the acidic gastric contents emptied into it (Guyton 1991b, Steward et al. 2005). It seems most likely that - - - HCO3 secretion is mediated via a Cl /HCO3 exchanger operating in parallel with a Cl- channel. This apical Cl- channel in the pancreatic ducts was established to be CFTR (Gray et al. 1993, Steward et al. 2005). Interestingly, some pathogenic CFTR

80 Discussion mutants are able to reach the cell membrane and function normally as Cl- channels but yet are associated with pancreatic insufﬁciency, while others are defective as Cl- channels but still support relatively normal pancreatic function. Expression studies in HEK293 cells demonstrated that CFTR mutants associated with normal pancre- - - atic function are still able to activate Cl /HCO3 exchange, whereas those associated with pancreatic failure are not (Choi et al. 2001, Steward et al. 2005). Our results of SLC26A6 expression in the pancreas, together with further functional results from - - other laboratories, suggest that the major Cl /HCO3 exchanger in the pancreatic ducts is very likely to be SLC26A6, probably acting together with SLC26A3. The in vitro evidence of reciprocal activation of CFTR and these two SLC26 transporters supports the physiological observations in microperfused pancreatic ducts, where stimulation - - of CFTR upregulated the activity of the apical Cl /HCO3 exchanger and the in vivo observations in CF patients (Lee et al. 1999, Greeley et al. 2001, Ko et al. 2002, Ko et al. 2004, Steward et al. 2005).

- A recent study demonstrated that SLC26A6 binds CAII, thus forming a HCO3 - - transport metabolon, where CAII activates the Cl /HCO3 exchange mediated by SLC26A6. The SLC26A6-CAII binding is disrupted by phosphorylation of SLC26A6 by protein kinase C (PKC), leading to the displacement of CAII from the vicinity of - SLC26A6. CAII dislocation causes a possible fall in local HCO3 concentration near SLC26A6, leading to decreased anion transport by SLC26A6 (Alvarez et al. 2005). - Interestingly, in the pancreas, PKC is known to inhibit HCO3 secretion. Thus the disruption of the SLC26A6-CAII transport metabolon offers a model of regulation - of pancreatic HCO3 secretion (Alvarez et al. 2005, Hegyi et al. 2005). Taking all - data into account, SLC26A6 mutations might affect the HCO3 secretion of the pancreas, causing pancreatic dysfunction.

The preliminary expression analyses showed that SLC26A7 mRNA was located in human kidneys, placenta, and testes. We further localized the SLC26A7 protein to the basolateral border of the type A intercalated cells of the collecting ducts and extraglomerular mesangial cells in human kidneys, a subgroup of basal cells in the epididymal ducts, and macrophages (III, IV, Kujala et al., unpublished results). Functional - 2- characterization revealed that human SLC26A7 is capable of transporting Cl , SO4 , and oxalate (II). Later studies with our collaborators showed that it mediates DIDS- - - sensitive Cl /HCO3 exchange as well, suggesting that SLC26A7 plays an important - role in HCO3 reabsorption in the collecting ducts, especially during dehydration (Petrovic et al. 2003, Barone et al. 2004, Petrovic et al. 2004).

Interestingly, the testis speciﬁc SLC26A8 gene mapped to human chromosome 6p21, which is homologous to the distal inversion of the mouse t-complex containing a cluster of genes expressed speciﬁcally in the testis (Tirosvoutis et al. 1995). With all methods used, SLC26A8 was detected strictly in seminiferous tubules. More precisely, it seems to be restricted to the meiotic phase of the spermatogenic cells, since immunostaining was detected in the spermatocytes and during the round and early elongating

Discussion 81 phases of spermatids only. We demonstrated that SLC26A8 is capable of transport- - 2- ing Cl , SO4 , and oxalate anions at least, suggesting that it functions as a male germ cell speciﬁc anion exchanger. These results, together with the extensive morphological changes observed during the meiotic phase of spermatogenesis, when SLC26A8 is expressed, suggest that adequate regulation of intracellular ions might be a critical component in this differentiation process.

Furthermore, SLC26A8, which proved to be the same gene as TAT1 charactherized by a French group, was shown to interact with MgcRacGAP (male germ cell GATPase activating protein for RhoGATPases) (Toure et al. 2001). Interestingly, MgcRacGAP seems to play an essential role in cell division processes (Hirose et al. 2001). In addition, MgcRacGAP shows strong structural similarity with the D. melanogaster protein RotundRacGAP, known to have an essential role in male fertility in the fruit fly (Agnel et al. 1992, Bergeret et al. 2001). All this suggested that MgcRacGAP and its interaction partner SLC26A8 might have important tasks in human male fertility, and prompted a mutational analysis of the coding region of the SLC26A8 gene in infertile male patients. Among the 83 patients studied, six novel coding sequence variations were identified, five of which resulted in amino acid substitutions. How- ever, all of these genomic variants were observed in control individuals with similar frequencies as in patients, suggesting no causality for infertility. Thus, it was con- cluded that SLC26A8 mutations are probably not a common cause of human male infertility at least (Makela et al. 2005).

SLC26A9 has a very tissue speciﬁc expression, with the most abundant production in human bronchial and alveolar epithelium. Its mRNA was detected in lower levels in the pancreas and prostate as well. We showed in study II that SLC26A9 is able - 2- to transport Cl , SO4 , and oxalate at least. Later, the rodent ortholog Slc26a9 was + located to mouse and rat stomach and trachea, and shown to mediate NH4 inhibited Cl-/HCO- exchange (Xu et al. 2005). Accurate epithelial secretion of water and ions, especially Cl- and Na+, is important for the formation of airway surface liquid, which is needed for normal lung function and defense against bacterial infections. An example of this is again CF, where the impaired Cl- secretion diminishes airway surface liquid volume, causing thickened mucus plaques that serve as a suitable environment for bacterial growth. Chronic bacterial infections lead to bronchiectasis and ﬁnally respiratory failure (Boucher 2002, Boucher 2004). Interestingly, SLC26A9 has a putative PDZ ligand at its carboxyl terminal end, offering the possibility of common regulatory mechanism with CFTR through PDZ interaction networks. Further studies on the possible interaction of SLC26A9 and CFTR are highly warranted.

82 Discussion 3. SLC26A6 and A7 Have Distinct Expression Patterns and Putative Roles in the Human Kidney (III)

The human kidney has numerous essential physiological roles directly associated with ion transport. However, several specific transporters and physiological processes are still poorly characterized on a cellular level. As the first step toward understanding their functional role, we studied in detail the expression of two novel anion transporters, SLC26A6 and A7 in normal human kidneys, in ADPKD and renal dysplasia. Our results indicate that both transporters have distinct and specific expression patterns, both in the different segments of the normal kidney tubules and in pathological cysts, suggesting non-complementary roles for these two transporters in human kidneys. Regardless, it must be recalled that expression studies do not directly evidence any function for the proteins. Our results differ partly from the expression data reported in earlier studies of rodents, indicating that results from rodents cannot be directly converted to humans.

SLC26A6 is widely expressed in different segments of human kidney tubules. While glomeruli, proximal segments of the PT, and principal cells of CD seemed to be negative, the protein was found in distal parts of the PT, some of the thin and thick ascending segments of the Henle’s loops, macula densa cells, DCT and a subpopulation of IC in CD. Yet, the mouse ortholog Slc26a6 has only been located to the apical membrane of a portion of PT (Knauf et al. 2001). The expression pattern of orthologous proteins may vary between species. Moreover, it was recently demonstrated that the anion selectivity, transport mechanism and regulation of human and mouse SLC26A6 orthologs differ remarkably (Chernova et al. 2005), although both have been shown - 2- - - to at least transport Cl , SO4 , HCO3 , OH and oxalate anions (Jiang et al. 2002, Ko et al. 2002, Wang et al. 2002, Xie et al. 2002, Lohi et al. 2003, Chernova et al. 2005). In addition, mouse Slc26a6 is able to perform Cl-/formate exchange (Knauf et al. 2001, Xie et al. 2002). Functional data are usually derived from experiments on rodents, but as these expression data and previous functional results on SLC26A6 implicate, such conclusions should be extended to humans with caution.

The majority of the Cl- in glomerular ﬁltrate is already reabsorbed in the PT. The rate of Cl- reabsorption is higher in the distal part of the PT than in the initial segment. An important portion of the Cl- reabsorption in the PT has been shown to be active transcellular and occur at least in part by Cl-/oxalate and Cl-/formate exchange, - - although there is some passive paracellular transport of Cl as well. Oxalate/HCO3 2- - and oxalate/SO4 exchangers are proposed to have key roles in the Cl reabsorption process of the PT by recycling oxalate (Aronson and Giebisch 1997). Moreover, there is evidence for Na+-independent Cl-/OH- exchange on the apical membrane of PT epithelial cells (Shiuan and Weinstein 1984, Kurtz et al. 1994, Aronson and Giebisch

Discussion 83 1997). Our ﬁnding, that SLC26A6 is located on the apical membrane of the distal portion of the PT in human kidneys, is harmonious with these physiological obser- - - 2- - vations, since SLC26A6 mediates transport of Cl , OH , SO4 , HCO3 , and oxalate anions at least (Wang et al. 2002, Lohi et al. 2003, Chernova et al. 2005), suggesting a signiﬁcant role for SLC26A6 in Cl- reabsorption in the distal parts of the PT in human kidneys.

Active Na+ and Cl- transport occurs in the DCT also. The greater part of Na+ and Cl- uptake in this segment goes through the thiazide-sensitive Na+-Cl- cotransporter (NCC, SLC12A3), but there is evidence for apical thiazide-insensitive Cl-/formate, - - - Cl /oxalate and Cl /HCO3 exchange in the DCT as well (Wang et al. 1993, Reilly and Ellison 2000). Again, our results are congruent with these earlier functional data, proposing a role for SLC26A6 in Cl- transport of the DCT.

Our results indicate that in human kidneys, SLC26A7 expression is more restricted than SLC26A6 expression and in part non-overlapping. SLC26A7 seems to be absent from virtually all of the PT, loop of Henle and distal tubule. Instead, it is found in some of the extraglomerular mesangial cells and the CD, in which its expression is generally stronger in medullary than cortical tubules. Cells with predominantly basal staining for SLC26A7 showed strong immunoreactivity for H+V-ATPase near their apical membrane in the CD, proposing these cells to be type A IC. However, peroxidase based immunohistochemistry can only suggest the subcellular localization of a protein. We noticed that SLC26A6 and A7 were expressed partly in the same IC along the CD (Figure 4). This is especially interesting when paying attention to the observation that many of the transporters co-occur in tissues in pairs (Ko et al. 2002).

The rodent ortholog Slc26a7 is found on the basolateral membrane of type A IC in the outer medullary CD (OMCD) in rat kidneys (Barone et al. 2004, Petrovic et al. 2004), in unison with our results in humans. However, we observed human SLC26A7 in extraglomerular mesangial cells as well, thus having a wider expression than the rat ortholog. We showed that human SLC26A7 is capable of transporting - 2- Cl , SO4 and oxalate anions when expressed in Xenopus laevis oocytes (II). Type A IC + - - - secretes acid via apical H V-ATPase and absorbs HCO3 via a basolateral Cl /HCO3 exchanger. In addition to the well studied anion exchanger SLC4A1, other basolat- - - - eral Cl /HCO3 exchanger mechanisms can contribute to the net HCO3 absorption of IC type A, as has been shown for the rodent orthlog Slc26a7 (Barone et al. 2004, Petrovic et al. 2004). The recent observation that mouse Slc26a7 may function as a Cl- channel (Kim et al. 2004) is especially remarkable in this context, since both type A and B IC have basolateral Cl- channels (Wagner and Geibel 2002). Further functional experiments with human SLC26A7 are needed.

Our experiments located both SLC26A6 and A7 in the juxtaglomerular apparatus, an important part of the tubuloglomerular feedback attending the autoregulation of renal blood ﬂow and glomerular ﬁltration. To our knowledge, expression of these anion

84 Discussion transporters had never before been reported in the juxtaglomerular apparatus of any species. We observed SLC26A6 in the macula densa (MD) cells, while SLC26A7 was localized to the extraglomerular mesangial cells (EMC) in human kidneys. The MD functions as a tubular sensor, where alterations in the intratubular fluid Na+ and Cl- concentrations trigger signals from the MD via the EMC to the granular and smooth muscle cells of the afferent arteriole. Cl- is essential especially in the first steps of this cascade; it is thought that initiation of the signal takes place via MD Cl- transport. The Cl- concentration of the juxtaglomerular interstitium changes linearly with the Cl- concentration of intraluminal fluid at MD level. The EMC are especially sensitive to changes in ambient Cl- concentration, which further modulates EMC functions (Goligorsky et al. 1997).

An increase in the intratubular Na+ concentration in turn causes signiﬁcant intracellular alkalinization of the MD, mediated by the apical Na+/H+ exchanger NHE2 (Peti-Peterdi 2005). High cytosolic pH of the MD seems to be an important regulator of tubuloglomerular feedback via the neural nitric oxide synthase (Wang et al. 2003). Recent data from rabbit kidney MD suggests the presence of a basolateral Na+- - - independent Cl /HCO3 exchanger that can contribute to MD intracellular pH regulation and participate in MD signaling. Anion exchanger SLC4A2 (AE2) has been located to MD cells, but the members of the SLC26 family could be involved as well (Komlosi et al. 2005). Thus, MD expression of SLC26A6, capable of transporting, - - e.g., Cl and HCO3 , and EMC expression of SLC26A7, capable of transporting, e.g., Cl-, support previous functional knowledge and suggest roles for SLC26A6 and A7 in tubuloglomerular feedback. Functional studies of these anion transporters in tubuloglomerular feedback are required.

Both SLC26A6 and A7 are good candidates for important anion exchange operations - in the human kidney. The HCO3 transport capability makes SLC26A6 and A7 good candidates for genetically uncharacterized forms of tubular acidosis. Failure of the anion transporters to function may cause excessive amounts of anions, such as oxalate, to concentrate into the urine. Together with pH alterations caused by impaired - HCO3 transport, this may predispose to the formation of urinary stones.

In ADPKD, the cysts may arise at any point between Bowman’s capsule and the terminal collecting ducts (Sutters and Germino 2003). We found that only a subset of cysts expressed either SLC26A6 or A7 in ADPKD. This is likely to reﬂect the origin of an individual cyst. Active Cl- secretion into the cyst lumen is needed for cyst enlargement in ADPKD. The Cl- channel CFTR mediates Cl- secretion in this disease (Sutters and Germino 2003). Since CFTR and SLC26 anion transporters interact (Ko et al. 2004), both SLC26A6 and A7 might have a role in cyst expansion in ADPKD through their Cl- transport capability and reciprocal regulation of CFTR. Further studies on this subject are warranted.

Discussion 85 Renal dysplasia is a common cause of chronic renal failure in children. However, little is known about ion trafﬁcking in its pathophysiology. Here we show that SLC26A6 is expressed in small dysplastic cysts, and the expression is gradually lost in larger cysts. This phenomenon might be due to dedifferentiation of the cyst epithelium when the cysts expand. Interestingly, in contrast to the distinct expression of SLC26A7 in ADPKD, this anion transporter was not expressed in any of the dysplastic cysts. This may suggest that in renal dysplasia, the cysts develop exclusively from SLC26A7-negative segments. Alternatively, it is possible that the production of SLC26A7 starts at a later stage of kidney development than that of SLC26A6. Since formation of dysplastic cysts already begins during kidney development, this could result in a situa- tion where the cysts form and the epithelial cells degenerate before the expression of SLC26A7 begins.

Our results do not support that SLC26A6 or A7 could be used as diagnostic markers for specifying the nature of cystic renal disorders. The possible functional role of these anion transporters in different kinds of cystic lesions of the human kidney requires further studies.

4. Diverse, Partly Co-localized Expression of the SLC26 Anion Transporters and Interaction Partners in the Human Epididymis (IV) Although epididymal and efferent ducts are fundamental for male fertility, only little is known about the detailed roles of the several specific cell types identified in these ducts, especially in man. As a primary move toward defining their role in modifying the ion composition of the luminal fluid, we examined the expression of SLC26A2, A6, A7, and A8 in human efferent and epididymal ducts. In addition, we immunolocalized the Cl- channel CFTR, the proton secretion involved NHE3 and V-ATPase, their common regulator the PDZ domain containing NHERF-1, and the acid-base balance regulating CAII. The expression of the SLC26 anion exchangers and NHERF- 1 has not been described in these structures of any species before, and the specific localization of CFTR, NHE3, and V-ATPase was novel in humans.

We found that the SLC26A6 anion transporter was co-localized with CFTR, NHE3 and NHERF-1 on the apical border of non-ciliated cells in human efferent ducts (Fig- ure 9). All of them were expressed most abundantly in type I epithelium, that is, in the initial segments of the ducts. SLC26A2 was located speciﬁcally in the cilias of the ciliated cells in the efferent ducts. SLC26A7, A8, CAII, or B and E subunits of V-ATPase were not observed in any of these ducts.

86 Discussion Lumen CO2 + H2O

- - + Cl HCO3 H AQP A6 NHE3 CFTR

NHERF-1

- + Cl Na H2O

Figure 9. A diagram of the possible interactions of SLC26A6 (A6), CFTR, NHE3, and NHERF-1 in a non-ciliated cell of the efferent ducts of the human testis. Aquapo- rins (AQP) are needed in order for water to move across the cell membrane.

Discussion 87 The main function of the efferent ducts is to absorb most of the luminal fluid released from the testis. It presumably involves both paracellular and transcellular pathways (Clulow et al. 1998, Hess 2002). Fluid reabsorption in rat efferent ducts is a function of interdependent transport of both Cl- and Na+. It has been proposed that there would be an apical Na+/H+ antiporter acting in parallel with an anion antiporter, - - which could exchange Cl for HCO3 (Hansen et al. 2004). Our results suggest that - - SLC26A6, capable of Cl /HCO3 exchange (Wang et al. 2002, Chernova et al. 2005), and the Na+/H+ antiporter NHE3 might be these apical transporters needed for fluid reabsorption in the efferent ducts. This idea is supported by their co-expression on the apical surface of the non-ciliated cells, together with their common regulator NHERF-1, which could enable their close spatial location and interaction (Fig- ure 9). It is believed that the non-ciliated cells take up more fluid in the initial zone of the efferent ducts, thus mainly representing type I epithelium, while in more distal segments these cells absorb more particulate matter (Hess 2002). Even this coin- cides with our suggestion, since both SLC26A6 and NHE3 were found especially in the initial type I efferent duct epithelium.

- - There is evidence of extensive HCO3 and Cl reabsorption by the efferent ducts, but the epithelium is also capable of anion secretion (Clulow et al. 1994, Clulow et al. 1998). Presence of both the anion exchangers SLC26A2 and A6, and the Cl- channel CFTR in the efferent ducts conforms to these previous functional results. SLC26A6 - - - - can mediate multiple anion exchange operations, including Cl /HCO3 , Cl /OH , and bidirectional oxalate ﬂux (Wang et al. 2002, Chernova et al. 2005). SLC26A6 and CFTR have reciprocal activation capability for each other (Ko et al. 2004). Since they are coexpressed on the apical border of the non-ciliated cells, this interaction might have an important role in controlling the net anion and water transport from and to the intraluminal ﬂuid in human efferent ducts.

Different pretreatment methods are known to have a great effect on the sensitivity to detect distinct forms of some proteins (Haila et al. 2001). An example of this are the SLC26A6 antibodies, which showed speciﬁc staining of apical cells without pretreatment, but required microwaving for the signal in non-ciliated cells to become visible. This could result from dissimilar interactions of SLC26A6 in various cell types.

The speciﬁc expression of SLC26A2 in the cilias of the ciliated cells in the efferent ducts differs from all other transporters studied in this work. Human SLC26A2 is - 2- capable of Cl /SO4 exchange (Satoh et al. 1998), but studies on its ability to transport other anions are lacking, although it is likely to function in transport processes of other anions as well (Mount and Romero 2004). Thus, the major role of SLC26A2 in human efferent ducts might involve transporting other anions in addition to Cl- 2- and SO4 . Further studies on the function of SLC26A2 are needed.

88 Discussion We found SLC26A2 positive staining in the narrow but not in the apical forms of AMRC, while SLC26A6 and CFTR were co-localized to the apical pole of only the apical forms of the AMRC. All forms of AMRC expressed NHERF-1, CAII and both B and E subunits of V-ATPase. The observation that SLC26A2, A6 and CFTR were located only in subgroups of AMRC could be explained by different stages of evolution of the AMRC (Palacios et al. 1991). We did not detect either NHE3 or SLC26A8 in the epididymal ducts. None of the proteins analyzed in this study were found in the principal cells of human epididymal ducts, in contrast to some previous reports from rodents (Pushkin et al. 2000, Bagnis et al. 2001, Leung et al. 2001a, Ruz et al. 2004).

SLC26A7 was found in a subgroup of basal cells of the human epididymal ducts. The function of the basal cells is still partly unclear. It has been proposed, e.g., that they would be a form of tissue fixed macrophages and have a role in local immune defense, since they are reactive for macrophage specific antibodies (Yeung et al. 1994). Our results coincide with these previous findings because SLC26A7 is also expressed in macrophages (unpublished results). A recent study demonstrated that at least in vitro the basal cells are able to regulate the electrogenic anion secretion of principal cells in cell cocultures derived from rats (Cheung et al. 2005). This is especially interesting since SLC26A7 is expressed in EMC of human kidney glomeruli, as well (III). EMC are especially sensitive for changes in ambient Cl- concentration, which further modulates EMC functions and triggers signals to the granular and smooth muscle cells of the afferent arteriole (Goligorsky et al. 1997). Thus, we propose that SLC26A7, capa- - 2- ble of transporting Cl , SO4 and oxalate anions at least, might have similar roles in mediating the information about ambient anion concentrations in EMC and basal cells of the epididymis proper. This suggestion needs further experimental verification in the future.

AMRC have been suggested to have a critical role in regulating the intraluminal pH - (Palacios et al. 1991), and thus HCO3 concentration in human epididymal ducts. - Our results support this idea, since these cells express both the HCO3 transporting SLC26A6 and the proton transporting V-ATPase, together with CAII catalyzing the formation of these ions (Figure 10). SLC26A6 may help V-ATPase in fine tuning the acidic intraluminal pH. The recent finding that CAII and SLC26A6 form a - transport metabolon together that regulates the HCO3 transport by SLC26A6 further strengthens this suggestion (Alvarez et al. 2005). In addition to substantial reab- - - sorption, epididymal epithelium can actively secrete Cl and HCO3 when stimulated by neurohumoral factors. Because both absorptive and secretory processes influence water movement across the epithelium, anion secretion may act in the fine controlling of the osmolality of the intraluminal fluid where sperm is bathed (Wong et al. 2002). Our results suggest that SLC26A6 and CFTR, both expressed on the apical side of the apical forms of AMRC (Figure 10), might have roles in this fine adjusting process.

Discussion 89 - HCO 3 H + - Cl

CFTR A6 CO 2 H2O Lumen VATPase CAII ADP - H+ Cl - ATP HCO3

NHERF-1

Figure 10. A diagram of SLC26A6 (A6), CFTR, CAII, NHERF-1, and V-ATPase (VATPase) in an apical form of AMRC of human epididymal ducts and their possible interactions.

The influence of different intraluminal ions on sperm maturation have not been studied much, and would be difficult to study directly (Turner 2002). There is evidence that alterations in intraluminal ion concentrations of rat cauda epididymis have effects on the secretion of organic compounds, e.g. proteins, by epithelial cells (Wong et al. 1980). If the same is true for all segments of the epididymis, intraluminal ion concentrations could indirectly influence sperm maturation by affecting the secretion of organic compounds by epithelial cells (Wong et al. 1980, Turner 2002). This further emphasizes the importance of the different ion transporters studied here for the fertility of men.

90 Discussion Cystic fibrosis is a good example of the importance of the right anion microenvironment for male fertility. Different mutations in CFTR cause distinct phenotypes varying from CF with inter alia severe pulmonary and pancreatic insufficiency and infertility to a congenital absence of the vas deferens (McKusick). There is also evidence that CFTR mutations can cause poor sperm quality without any other clinical symptoms (van der Ven et al. 1996, Wong et al. 2002). In the rat, Cftr has been found on the apical membrane of principal cells in the efferent ducts, and to have cell and region specific expression in the epididymis (Leung et al. 2001a, Ruz et al. 2004). In the human male reproductive tract, CFTR mRNA has previously been found in the epithelium of the epididymis and vas deferens (Patrizio and Salameh 1998). We showed that the CFTR protein is localized on the apical surface of the non-ciliated alias principal cells in human efferent ducts, similarly to previous results derived from rats (Leung et al. 2001a, Ruz et al. 2004). However, in human epididymal ducts, we found CFTR located specifically to the apical form of AMRC. This differs remarkably from the earlier findings in rats, where Cftr was immunolocalized to the principal cells of the epididymis, and the staining was strongest in the corpus and cauda regions. Narrow and apical cells were negative for Cftr in rats, while clear cells showed a weak-to-moderate reaction in the corpus and cauda (Ruz et al. 2004). Functional studies are usually performed in rodents, and the results are often easily thought to be universal in different mammals. The discrepancy in the CFTR expression in our results in humans compared to previous findings in rats is a reminder that even functional results derived from rodents should be converted to man with caution.

The asymmetric distribution of distinct membrane proteins, including transporters, is essential for any epithelial cell to create and maintain polarity. PDZ domain containing proteins retain other proteins on the correct membrane and bring them close to each other, enabling their interactions and the formation of transducisomes, spatially restricted units of functionally connected proteins, like transporters (Brone and Eggermont 2005). NHERF-1 is an apical PDZ domain containing protein, shown to interact, e.g., with SLC26A6, CFTR, NHE3, and the V-ATPase subunit B1, that all include carboxyl terminal consensus PDZ binding sequences (Yun et al. 1997, Wang et al. 1998, Breton et al. 2000, Lohi et al. 2003, Brone and Eggermont 2005). We demonstrated that NHERF-1 co-localizes with SLC26A6, CFTR and NHE3 on the apical side of the non-ciliated cells in human efferent ducts, and with SLC26A2, A6, CFTR and V-ATPase in the AMRCs in the epididymal ducts. Our in vivo localization data corresponded well with the previous functional in vitro results and further supports them. Although only SLC26A6 of the SLC26 family members has been studied for NHERF-1 interactions so far (Lohi et al. 2003), we found SLC26A2, A3, A7 and A9 to have consensus PDZ binding sequences at their carboxyl terminus too, offering them a possibility to interact with PDZ networks (II). Further studies on these possible interactions are warranted.

Discussion 91

Conclusion and Future Prospects

This study is an example of the possibilities that the genome projects have enabled. The anion transporter family SLC26 was expanded with seven novel human members utilizing a genome-driven sequence homology approach. All of the new genes found have distinct tissue specific expression. Characterization of the structure and function of the new proteins encoded by these genes suggested that they function as anion exchangers. Moreover, cell specific polarized expression of SLC26A6-A9 pro- - - teins suggested that they may have specific roles in, e.g., Cl and HCO3 reabsorption in the kidney, concentration of the intraluminal fluid in the epididymis, meiosis phases of the developing sperm cell, and regulation of the airway surface liquid volume.

The preliminary characterization of the new SLC26 members performed in this work offers a rich source for further physiological studies. It is possible that the new SLC26 genes found cause distinct but rare hereditary diseases when mutated. Speculatively, common functional polymorphisms might exist in some of the new SLC26 family genes that would with a low risk predispose their carriers to common, complex disorders such as hypertension, duodenal ulcers or urinary stones. Such a possibility could be explored in the future using targeted genetic association studies. If and when disease associated mutations or polymorphisms in the new SLC26 genes turn up, the preliminary work done in this thesis will help the understanding of the patophysiol- ogy of these diseases.

93 Yhteenveto (Finnish Summary)

Solunsisäisten ioneiden pitoisuuksien kontrollointi on elintärkeää jokaiselle solulle. Eri- tyyppiset solut tarvitsevat ionipitoisuuksien suhteen erilaiset pienoisympäristöt, joita säädellään kudos- ja soluspesiﬁsillä ioninkuljetinproteiineilla eli ionitransporttereilla. Anionitransportterit ovat avainasemassa monissa elimistön normaaliin toimintaan liit- tyvissä tapahtumissa, kuten koko elimistön happo-emästasapainon kontrolloimisessa ja solujen kasvun, tilavuuden, aineenvaihdunnan, supistuvuuden, solunsisäisen pH:n ja ionigradienttien säätelyssä. Kun tämä tutkimus aloitettiin, anionitransportteriperhe SLC26:sta (engl. solute carrier family 26) tunnettiin vain kaksi ihmisessä ilmenty- vää jäsentä. Kumpikin näistä geeneistä oli liitetty yhteen suomalaiseen tautiperimään kuuluvaan harvinaiseen peittyvästi periytyvään tautiin: diastroﬁseen dysplasiaan ja synnynnäiseen kloridiripuliin. Koska yksinkertaiselta sukkulamadolta (Caenorhabdi- tis elegans) oli löydetty seitsemän geeniä, jotka koodittavat hyvin samankaltaisia ami- nohapposekvenssejä kuin tuolloin tunnetut ihmisen SLC26-geenit, pidimme erittäin todennäköisenä, että ihmisellä olisi useita toistaiseksi tunnistamattomia SLC26-per- heenjäseniä perimässään ja aloitimme niiden etsinnän. Pohjimmainen tavoite oli löy- tää uusia SLC26-geenejä, selvittää niiden rakenteet, ilmentyminen ja toimintaa, sekä tarkemmin tutkia niiden ilmentymistä eri solutyypeissä tietyissä fysiologisesti mer- kityksellisissä kudoksissa.

Uusia SLC26-geenejä tunnistettiin etsimällä ihmisen genomista tunnettujen geenien kanssa samankaltaisia sekvenssejä. Tässä käytettiin hyväksi useita erilaisia julkisesti käytössä olevia tietokantoja ja tietokoneohjelmia. Näin löydetyt mahdolliset uusien geenien sekvenssit analysoitiin tarkemmin RT-PCR-tekniikalla ja sekvensoimalla ja niiden sijainti kromosomistossa selvitettiin radiation hybrid mapping –menetelmällä. Yhteensä seitsemän uutta ihmisen SLC26-geeniä (SLC26A1, A6-A11) löydettiin, niiden nukleotidi- ja aminohapposekvenssit tutkitiin tietokoneohjelmilla ja niiden yleistasoi- nen kudoskohtainen ilmentyminen selvitettiin Northern blotting -tekniikalla ja usei- den kudosten cDNA näytteitä PCR-reaktioissa käyttämällä (I). Sen jälkeen keskityimme erityisesti neljän uuden geenin, SLC26A6-A9, molekyylibiologian selvittämiseen.

SLC26A6-geeni, joka koodaa 736 aminohapon proteiinia, paikallistettiin kromosomiin 3p21.3. Sen rakenne oli erittäin samankaltainen kuin aiemmin tunnettujen anionikuljettimina toimivien SLC26-perheenjäsenten, viitaten SLC26A6:nkin olevan mahdollisesti anionitransportteri. Se ilmentyi voimakkaimmin ihmisen munuaisissa ja haimassa, mutta pienempiä määriä löytyi useista muistakin kudoksista. Immunohis- tokemian avulla SLC26A6-proteiini paikallistettiin ihmisen munuaisessa proksimaa- listen kiemuratiehyiden distaaliosiin, osaan Henlen lingon ohut- ja paksuseinäisistä osista, makula densan soluihin, distaalisiin kiemuratiehyisiin ja osaan kokoojatiehyiden interkalaarisista soluista, viitaten tärkeisiin rooleihin mm. Cl-:n takaisinimeytymisessä

94 ja tubuloglomerulaarisessa takaisinkytkennässä. SLC26A6:a havaittiin myös osassa ADPKD- ja monirakkulaisten dysplastisten munuaisten rakkuloista. Ihmisen lisäki- veksessä SLC26A6 paikallistettiin kiveksen efferenttien duktusten värekarvattomien solujen apikaaliselle reunalle yhdessä Cl- kanava CFTR:n, Na+/H+ vaihtaja NHE3:n ja niiden yhteisen säätelijä NHERF-1:n kanssa. Tämä tulos viittasi näiden proteiinien mahdollisesti muodostavan tärkeän yhteistyöyksikön, joka imee takaisin Na+- ja Cl-- ioneita ja säätelee veden takaisinimeytymistä näissä rakenteissa. Lisäksi SLC26A6 löy- dettiin ihmisen lisäkiveksen AMRC-solujen apikaalimuodoista, joissa se mahdollisesti osallistuu lisäkiveksen ontelonsisäisen pH:n ja osmolaliteetin hienosäätöön (I, III, IV).

SLC26A7-geeni paikallistettiin kromosomiin 8q23 ja sen havaittiin koodaavan 656 aminohapon mittaista solukalvoproteiinia. Sen mRNA:ta löytyi munuaisista, kivek- sistä ja istukasta. Kun SLC26A7:ä tuotettiin Xenopus laevis –sammakon munasoluissa, - 2- sen todettiin kuljettavan Cl :a, SO4 :a ja oksalaattia. RT-PCR:llä ja immunoblotta- uksella havaittiin ihmisen munuaisen ytimessä kuorikerrosta voimakkaampi signaali. Immunohistokemialla SLC26A7 paikallistettiin ihmisen munuaisissa kokoojatiehyiden A-tyypin interkalaaristen solujen basolateraaliosiin ja ekstraglomeulaarisiin mesan- - giaalisoluihin, viitaten vastaavasti tärkeisiin rooleihin HCO3 :n takaisinimeytymi- sessä ja tubuloglomerulaarisessa takaisinkytkennässä. Ihmisen lisäkiveksissä SLC26A7 ilmentyi osassa basaalisoluja, joissa se saattaa osallistua viereisten prinsipaalisolujen säätelyyn (I-IV).

SLC26A8 ja SLC26A9, jotka paikantuivat vastaavasti kromosomeihin 6p21 ja 1q31- q32, olivat erittäin kudosspesiﬁsesti ilmentyviä. Kummankin osoitettiin kuljettavan - 2- ainakin Cl :a, SO4 :a ja oksalaattia, kun niitä tuotettiin Xenopus laevis –sammakon munasoluissa. SLC26A8 ilmentyi ainoastaan kiveksissä, ja in situ hybridisaatiolla ja immunohistokemialla se paikannettiin tarkemmin spermatosyytteihin ja spermati- deihin. Tästä syystä sillä voi olla tärkeä rooli siittiösolujen meioottisessa jakautumis- vaiheessa ja sen mutaatiot saattavat aiheuttaa alentunutta hedelmällisyyttä miehellä. RT-PCR-, Northern blotting- ja immunohistokemia-menetelmillä SLC26A9 paikannettiin keuhkorakkuloiden ja keuhkoputkien epiteelisoluihin. Tämä viittaa kyseisen proteiinin mahdollisesti osallistuvan hengitysteiden pintanesteen ylläpitoon, joka on tärkeää bakteeritulehduksilta suojautumiseksi (I,II,IV).

Uusien SLC26-geenien oletetaan osallistuvan useisiin olennaisiin anionivaihtoon liit- tyviin fysiologisiin tapahtumiin ihmisen eri elimissä. Lisäksi ne ovat hyviä tautigee- nikandidaatteja joillekin perinnöllisille taudeille, joiden geenivirheitä ei toistaiseksi ole kyetty selvittämään.

95 Sammanfattning (Swedish Summary)

Noggrann reglering av de intracellulära jonkoncentrationerna är väsentlig för varje levande cell. Celler behöver specifika jonmikroomgivningar, som regleras av vävnads- och cellspecifika jontransportörer. Anjontransportörer spelar betydande roller vid flera fysiologiska processer, såsom reglering av syra-basbalansen, celltillväxt, volym, metabolism, kontraktilitet, upprätthållande av intracellulärt pH och jongradienter. Då detta arbete påbörjades var enbart två medlemmar av SLC26 familjens (eng. solute carrier family 26) anjontransportörer kända hos människa, och båda uppvisade dis- tinkt vävnadsspecificitet. Generna var associerade med ovanliga recessivt nedärvda sjukdomar som tillhör det finska sjukdomsarvet; diastrofisk dysplasi och medfödd kloriddiarré. Eftersom den primitiva nematoden Caenorhabditis elegans hade visats ha sju gener som kodade för aminosyrasekvenser homologa med de humana SLC26 proteinerna, ansåg vi det vara sannolikt att det fanns flera humana SLC26 medlemmar som ännu inte upptäckts. Vi började därför leta efter dessa. Målet var att finna nya SLC26 gener, karakterisera deras struktur, expression och funktion och att stu- dera deras cellspecifika expression i utvalda, fysiologiskt relevanta vävnader.

För att identifiera nya humana SLC26 gener utfördes en sökning av homologa sek- venser i det mänskliga genomet med hjälp av flera offentligt tillgängliga databaser och datorprogram. De nyfunna sekvenserna analyserades ytterligare med hjälp av RT-PCR och sekvensering, och deras kromosomala läge detekterades genom radiation hybrid mapping -tekniken. Sammanlagt identifierades sju nya humana SLC26 gener (SLC26A1, A6-A11), vars nukleotid- och aminosyrasekvenser undersöktes med datorprogram. De sju nya genernas vävnadsspecifika expressionsmönster analyserades med Northern blottning och PCR med en mångfald vävnadsspecifika cDNA pane- ler (I). Vi fokuserade sedan på den molekylära karakteriseringen av fyra av de nya generna, SLC26A6-A9.

SLC26A6 genen, som kodar för ett 738 aminosyror-långt protein, kartlades till kromosom 3p21.3. Dess struktur var i hög grad homologt med tidigare kända SLC26 medlemmar som transporterar anjoner, vilket tyder på att SLC26A6 även fungerar som en anjontransportör. Expressionen av SLC26A6 hos mänska var högst i njurarna och buk- spottkörteln, men lägre expressionsnivåer konstaterades också i ﬂera andra vävnader. Med hjälp av immunohistokemi fastställdes SLC26A6 proteinets läge i njurarna hos människa till de distala delarna av proximala tubuli, delar av tunna och tjocka upp- åtstigande segmenten av Henles slynga, macula densa celler, distala vindlande tubuli och en subpopulation av interkalerande celler i samlingsrören. Detta tyder på viktig

96 funktion vid Cl- upptaget och tubuloglomerulära återkopplingen. Vidare, observerades SLC26A6 i en undergrupp av cystor i ADPKD (autosomal dominant polycys- tisk njursjukdom) och i multicystisk dysplastisk njure. I bitestikel hos människa, var SLC26A6 beläget i den luminala delen av icke-cilierade celler i utförsgångarna, tillsammans med Cl- kanalen CFTR, Na+/H+ pumpen NHE3, samt deras gemensamma reglerare NHERF-1. Detta indikerar att dessa tillsammans bildar en nödvändig funk- tionell enhet som återupptar Na+ ach Cl- samt reglerar det osmotiska vattenupptaget. SLC26A6 fanns även i apikala former av AMRC i bitestikel gångarna, där det sannolikt ﬁnjusterar det intraluminala pH och osmolaliteten (I,III,IV).

SLC26A7 genen är belägen på kromosom 8q23 och kodar för ett 656 aminosyror långt transmembrant protein. Dess mRNA observerades in njurarna, testiklarna och i moderkakan. Då SLC26A7 uttrycktes i äggceller av Xenopus laevis uppvisade det Cl-, 2- SO4 och oxalat transportaktivitet. RT-PCR och immunoblottning gav hos män- niska starkare signal i njurmärgen än i njurbarken. Genom immunohistokemisk ana- lys konstaterades SLC26A7 vara lokaliserat till den basolaterala sidan av typ A interkalerande celler i samlingsrören och extraglomerulära mesangialceller, vilket antyder - att det spelar en avgörande roll i HCO3 upptaget respektive tubuloglomerulära åter- kopplingen. I bitestiklarna uttrycktes SLC26A7 i en undergrupp av basalceller, där det eventuellt deltar i regleringen av principalceller (I-IV).

SLC26A8 och A9, belägna på kromosomerna 6p21 respektive 1q31-q32, visade båda - 2- hög grad av vävnadsspeciﬁk expression. Bägge konstaterades transportera Cl , SO4 och oxalat då de uttrycktes i äggceller av Xenopus laevis. SLC26A8 uttrycktes endast i spermatocyter och spermatider i testiklarna, vilket bekräftades med in situ hybridi- sering och immunohistokemi. Därför antas det ha en viktig funktion vid den meio- tiska fasen av spermieutveckligen och kan i muterad form eventuellt orsaka nedsatt fertilitet hos män. Genom RT-PCR, Northern blottning och immunohistokemi loka- liserades SLC26A9 till det alveolära och bronkiala epitelet i lungorna hos människa. Dess läge tyder på en roll vid bibehållande av ytvätska i luftvägarna som behövs mot angrepp av bakteriella infektioner (I, II, IV).

De nya SLC26 generna antas delta i ﬂera grundläggande fysiologiska processer som involverar anjontransport i olika organ i människokroppen. Vidare är de goda kandida- ter till gener som i muterad form kan tänkas orsaka hittills okända ärftliga sjukdomar.

Acknowledgements

This study was carried out at the Department of Medical Genetics, Haartman Insti- tute and Biomedicum Helsinki, University of Helsinki, Finland. I wish to express my deepest gratitude to all my coworkers and friends who have helped and supported me during these years.

I want to thank my supervisor, Professor Juha Kere, for providing the fundamental idea for this project and good laboratory facilities for performing the studies. His unfailing optimism, enthusiasm, and admirable capability of creating new ideas have inspired me. He has taught me a lot about science and the scientiﬁc world during this whole thesis process.

I am grateful to the former and present heads of the department, Professors Juha Kere, Leena Palotie, Pertti Aula, Anna-Elina Lehesjoki, Kristiina Aittomäki, and Päivi Pel- tomäki, for providing excellent research facilities.

The reviewers of this thesis, Adjunct Professors Hannu Jalanko and Anne Räisänen- Sokolowski, are sincerely acknowledged for valuable discussions and important com- ments. In addition, I wish to warmly thank Anne for being a member of my thesis committee and for always being there for me when I needed good advice during the rough times. I am grateful to Prof. Hannu Sariola for being a member of my thesis committee, for spurring me on, and for tutoring me whenever I needed help with kidney histology.

I wish to thank all my coauthors for their contributions during these years. I express my special thanks to Adjunct Professor Eero Lehtonen for his irreplaceable support for me by teaching me histology and immunohistochemistry, and most importantly by continuously encouraging me. I thank Prof. Hannu Sariola, Prof. Christer Holm- berg, Adj. Prof. Ulpu Saarialho-Kere, Outi Elomaa PhD, Kari Kaunisto MD PhD, Johanna Hästbacka MD PhD, and Erja Kerkelä PhD for their collaboration, important advice, and educational discussions. Dr. Daniel Markovich is acknowledged for his collaboration in functional studies. Adj. Prof. Marjo Kestilä and Pia Höglund MD PhD are thanked for their invaluable help and guidance in the beginning phases of this project. Collaboration with Pia became even more fruitful in the late phases of this project, and I am particularly grateful for her advice on scientiﬁc writing. Jukka Tienari MD PhD is acknowledged for his expertise in histopathology, valuable criti- cism and discussions, and continuous interest in this work. Doctors Siru Mäkelä and Satu Hihnala are thanked for their pleasant collaboration and for sharing the joy and frustration of immunohistochemistry. My special thanks to Hannes Lohi PhD for

99 his productive collaboration, priceless discussions, and tireless efforts in anion transporter research.

I am thankful to my friends Sanna Lehtonen PhD and Annina Lyly MSc for their helpful advice on protein chemistry. Matti Vierula PhD is acknowledged for his valuable discussions about the histology of the epididymis. Pathologist Markus Nyberg is thanked for his assistance with the preparation of some tissue samples. I wish to thank the current and former laboratory technicians Ranja Eklund, Riitta Känkänen, Riitta Lehtinen, and Johanna Lahtinen from Juha’s lab and Ulla Kiiski, Tuulikki Leinonen, and Alli Tallqvist from the collaborators’ labs for all the help and advice on the laboratory techniques during these years. Hannu Turunen is thanked for his assistance with computers.

I am grateful to Morag Dixon for revising the English language of my thesis and Annette Lindroos for the Swedish translation. I wish to thank Lauri Ylimaa for his help with the graphics processing and for the make-up of this thesis.

The staff at the Department of Medical Genetics, Helsinki Biomedical Graduate School, and HUSLAB, especially Pirjo Koljonen, Minna Maunula, Aija Kaitera, and Terhi Kulmala, are thanked for their assistance with practical matters.

During this process I have had the opportunity to get to know many young, as well as more experienced, scientists, and I want to thank all of them for the pleasant moments together. I especially want to acknowledge the current and former members of Juha’s group: Anna, Elina, Elisabeth, Hannele, Hannes, Hannu, Harriet, Inkeri, Jaana, Johanna, Katariina, Kati, Lotta, Marja, Marjo, Markus, Morag, Outi, Paula, Pia, Päivi A., Päivi H., Ranja, Riitta K., Riitta L., Sari, Satu H., Satu K., Sini, Siru, Tarja, Ulla, and Ville, for all the help and relaxing breaks. My special thanks and hugs go to Nina Kaminen for her friendship and priceless support, which have meant a lot to me. All the people at Sariola lab, Ulpu’s lab, and the third ﬂoor of Biomedi- cum who have assisted me in various matters, are thanked for their kind efforts and our enjoyable times together.

Special thanks and warm hugs go to my colleagues Hanni Alho and Johanna Savikko for their friendship and encouragement during both joyful and depressing times in the scientiﬁc and clinical world, and in life in general. It has been important to have friends who you can always count on and who understand exactly what you mean.

During these years I had the chance every now and then to take a bracing break from the laboratory and work as a physician at the Department of Urology at Maria Hospi- tal. I am grateful to all my colleagues and friends there for their interest in my study, encouragement, and for heartily welcoming me to the clinic.

100 Acknowledgements I extend my sincere thanks to all my friends for their support and the innumerable hilarious moments during these years. Spending time together, having long talks about life, and just their existence have been a relaxing balance to work.

I wish to express my deepest gratitude to my whole family. My dearest mother Tarja and father Anssi are thanked for being the best parents in the world and for all the love and support throughout my life. I want to thank my dear brother Markus and his girlfriend Annina for being true friends. I also want to thank my parents-in-law Liisa and the late Pertti for inspiring me in the ﬁelds of medicine and science, and the whole Myllynen family for their support.

Finally, I want to direct my dearest thanks to my husband Miika for always stand- ing by me. His love, encouragement, and understanding have aided me through the ups and downs of this work. In addition, I thank Miika for being my personal computer support.

This study was ﬁnancially supported by Biomedicum Helsinki Foundation, Chancel- lor of the University of Helsinki, Clinical Research Fund of Helsinki University Cen- tral Hospital, Finnish Medical Foundation, Finska Läkaresällskapet, Helsinki Bio- medical Graduate School, Kidney Foundation, Research and Science Foundation of Farmos, and The Finnish Cultural Foundation.

Helsinki, October 2005

Minna Kujala-Myllynen

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