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Podocyte-Specific Deletion of Integrin-Linked Kinase Results in Severe Glomerular Basement Membrane Alterations and Progressive Glomerulosclerosis

Chiraz El-Aouni,* Nadja Herbach,†† Simone M. Blattner,* Anna Henger,* ʈ Maria P. Rastaldi,† George Jarad,‡ Jeffrey H. Miner,‡ Marcus J. Moeller,§ Rene St-Arnaud, Shoukat Dedhar,¶ Lawrence B. Holzman,** Ruediger Wanke,†† and Matthias Kretzler* *Klinikum der Universita¨t Mu¨nchen, Medizinische Poliklinik-Innenstadt, Nephrologisches Zentrum, Mu¨nchen, Germany; †Renal Immunopathology Laboratory, Associazione Nuova Nefrologia and Fondazione D’Amico per la Ricerca sulle Malattie Renali, c/o San Carlo Borromeo Hospital, Milan, Italy; ‡Department of Internal Medicine and Cell Biology, Washington University School of Medicine, St. Louis, Missouri; §Department of Internal Medicine, Rheinisch- ʈ Westfa¨lische Technische Hochschule, Aachen, Germany; Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada; ¶British Columbia Cancer Research Center, Vancouver, British Columbia, Canada; **Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; and ††Institut fu¨r Veterina¨rpathologie, Ludwig- Maximilians Universita¨t, Mu¨nchen, Germany

Alterations in glomerular podocyte cell–cell and cell–matrix contacts are key events in progressive glomerular failure. Integrin-linked kinase (ILK) has been implicated in podocyte cell–matrix interaction and is induced in proteinuria. For evaluation of ILK function in vivo, mice with a Cre-mediated podocyte-specific ILK inactivation were generated. These mice seemed normal at birth but developed progressive focal segmental glomerulosclerosis and died in terminal renal failure. The first ultrastructural lesions that are seen at onset of albuminuria are glomerular basement membrane (GBM) alterations with a significant increase in true harmonic mean GBM thickness. Podocyte foot process effacement and loss of slit diaphragm followed with progression to unselective proteinuria. No significant reduction of slit membrane molecules (podocin and nephrin), key GBM components (fibronectin, laminins, and collagen IV isoforms), or podocyte integrins could be observed at onset of proteinuria. However, ␣3-integrins were relocalized into a granular pattern along the GBM, consistent with altered integrin-mediated matrix assembly in ILK-deficient podocytes. As the increased GBM thickness precedes structural podocyte lesions and key components of the GBM were expressed at comparable levels to controls, these data suggest an essential role of ILK for the close interconnection of GBM structure and podocyte function. J Am Soc Nephrol 17: 1334–1344, 2006. doi: 10.1681/ASN.2005090921

idney function depends on an intact glomerular filtra- laminin, and proteoglycans (1). Endothelial cells and glomeru- tion barrier retaining macromolecules in the blood lar epithelial cells (podocytes) are attached via specific recep- K and allowing the excretion of potentially hazardous tors, integrins, to the GBM. Integrins are heterodimeric trans- small molecules into the urine. The glomerulus is composed of membrane molecules that are assembled of ␣- and ␤-chains. three different cell types (mesangial cells, endothelial cells, and Integrins mediate cell adhesion and link the ECM to the cell’s podocytes) that are anchored in the specialized extracellular cytoskeleton. Each glomerular cell type expresses a specific matrix (ECM) of the glomerular basement membrane (GBM) combination of ␣- and ␤-subunits that determine the specificity and the mesangial matrix. The GBM is a gel-like meshwork of to ECM ligands and define cellular phenotypes, including pro- cell adhesive glycoproteins, including collagen IV, fibronectin, liferation, differentiation, survival, and ECM assembly (2,3). In the glomerular capillaries, the arteriolar intravascular pressure forces the plasma through a sequential sieve. The sieve Received September 8, 2005. Accepted February 18, 2006. consists of the endothelial fenestrae, the meshwork of the GBM and the slit diaphragm that connects interdigitating podocyte Published online ahead of print. Publication date available at www.jasn.org. foot processes. A tightly controlled interaction of the podocyte C.E.-A. and N.H. contributed equally to this work. foot processes with each other and with the matrix components The current affiliation for S.M.B., A.H., and M.K. is University of Michigan, Ann of the GBM is crucial to maintain the filtration barrier against Arbor, Michigan. this high transcapillary pressure gradient. The cytoskeleton in Address correspondence to: Dr. Matthias Kretzler, Division of Nephrology, the foot processes of podocytes is highly dynamic and serves as Department of Internal Medicine, University of Michigan, 1570 MSRB II, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0676. Phone: 734-764-3157; Fax: a central scaffold for the integrity of the filtration barrier (4). 734-763-0982; E-mail: [email protected] Molecules that regulate the interaction of the cytoskeleton with

Copyright © 2006 by the American Society of Nephrology ISSN: 1046-6673/1705-1334 J Am Soc Nephrol 17: 1334–1344, 2006 Podocyte-Specific ILK Deletion 1335 the extracellular matrix via podocyte matrix receptors therefore Urine and Plasma Analysis are considered to be of critical importance for an intact glomer- Urine protein excretion was detected by SDS-PAGE followed by ular filtration barrier (5). Coomassie blue staining and quantified using the Bradford colorimetric Integrin-linked kinase (ILK) is a good candidate for regulat- assay (BioRad, Munich, Germany), expressed as ratios to urine creati- ing integrin function in podocytes. Initial evidence of ILK acti- nine. Serum values were obtained from EDTA blood samples at time of killing for blood urea nitrogen, total protein, albumin, and cholesterol vation in glomerular filtration barrier failure was obtained by a using a Hitachi autoanalyzer (Hitachi, Tokyo, Japan). mRNA expression screen of glomeruli from children with con- genital nephrotic syndrome of the Finnish type (6). Further- more, Wu et al. (7) found an activation of ILK in human diabetic Histology and Morphometry glomeruli and in isolated glomerular mesangial cells that were Mice were killed by ether inhalation, and tissue was fixed for histol- cultured with high glucose medium. We previously reported ogy via orthograde vascular perfusion with 3% glutaraldehyde and an induction of ILK in murine models of glomerular disease in processed for plastic embedded light microscopy (16) and scanning and glomeruli and microdissected podocytes (6). In vitro studies transmission electron microscopy as described previously (17). demonstrated a role of ILK for focal contact assembly, cell The GBM thickness was determined by the orthogonal intercept attachment, cytoskeletal organization, proliferation, and sur- method (18) as described by Ramage et al. (19). Using a transparent vival of podocytes (6,8–10). grid, the shortest distance between the endothelial cytoplasmic membrane and the outer lining of the lamina rara externa underneath the These data suggest an important role of ILK for the function cytoplasmic membrane of the epithelial foot processes was measured of the filtration barrier, serving multiple roles at the cell contact with a logarithmic ruler (19) where gridlines transected the GBM. The sites of podocyte foot processes. With the plethora of ILK apparent harmonic mean thickness (lh) was calculated, from which the function shown in vitro, the role of ILK in podocytes had to be true harmonic mean thickness (Th) was estimated by the following evaluated in the glomerular context using genetic models. As equation ILK deletion results in early embryonic lethality, the Cre-Lox system was used for a podocyte-specific excision of ILK. The 8 106 Th ϭ ϫ ϫ lh resulting phenotype was evaluated on a functional, structural, 3␲ M and molecular level to define the sequence of events that lead to the progressive filtration barrier failure seen with ILK deletion where M represents the final print magnification. On average, 800 in podocytes. intercepts determined in six glomeruli per animal (range: 660 to 980) were measured. The filtration slit frequency (FSF) was determined by counting the Materials and Methods number of epithelial filtration slits divided by the length of the periph- Targeted Inactivation of ILK in Podocytes eral capillary wall at the epithelial interface. On average, 324 filtration For selective deletion of ILK in glomerular podocytes, transgenic slits (range 265 to 387) were counted per mouse. The mean glomerular mice that express Cre-recombinase specifically in podocytes were volume was determined from the mean glomerular profile area accord- crossed with “floxed” mice, which contain loxP sites upstream of exon ing to the method developed by Weibel and Gomez using a Videoplan 5 and downstream of exon 12 of the ILK gene (11). 2.5P-Cre mice image analysis system (Zeiss-Kontron, Munich, Germany) (20–22). (podocinCre/Cre) with the Cre-recombinase cassette under the regulation of a fragment of the human NPHS2 promoter of the podocin gene, leading to podocyte-specific expression of the Cre-recombinase, were Immunohistology obtained from L.B. Holzman (University of Michigan, Ann Arbor, MI) Unfixed renal tissue was embedded in OCT compound (Miles Sci- flox/flox (12). ILK mice were provided by S. Dedhar (University of British entific, Naperville, CA), snap-frozen in a mixture of isopentane and dry Cre/Cre Columbia, Vancouver, BC, Canada) (11). The podocin mice were ice, and stored at Ϫ80°C. Five- to 7-␮m sections were placed on gelatin- Cre/Ϫ crossed with homozygous floxed ILK mice. The F1 podocin / coated slides and stored at Ϫ20°C until immunostaining. For fixations flox/Ϫ flox/flox ILK bitransgenic mice were crossed to homozygous ILK / and primary antibodies used, see Supplementary Table 2 (references Ϫ/Ϫ Creϩ/Ϫ flox/flox podocin mice, generating homozygous podocin -ILK [41–48]). Confocal imaging was performed on a Zeiss LSM510 confocal Ϫ Ϫ mice (podoILK / ). microscope at ϫ600 magnification. Rabbit anti-chicken integrin ␣3 se- Genotyping was performed by PCR as described (11–13), using the rum was a gift from Dr. C. Michael Dipersio (Albany Medical College, following oligonucleotide primers for the floxed ILK locus: Fr-Lox Albany, NY), and rat anti-mouse entactin clone ELM1 was purchased Ј Ј Ј 5 -CAAGGCTGACATCAATGC-3 and Rv-Lox 5 -GTGCCACCTG- from Chemicon (Temecula, CA). Alexa-Fluor 488– and Cy3-conjugated Ј CAAATTAC-3 . All animal experiments were conducted according to goat anti-rabbit antibodies were used as secondary antibodies (Molec- institutional and national guidelines. ular Probes, Eugene, OR; and Invitrogen, Carlsbad, CA). Negative controls were performed concurrently by substituting buffer or isotype Isolation of RNA and Real-Time Reverse Transcription–PCR control immunoglobulins (rabbit primary antibody isotype control; Analysis Invitrogen) for the primary antibody. Steady-state mRNA expression levels were quantified with real-time ⌬⌬ reverse transcription–PCR (RT-PCR) as described (8,14) using the Ct technique (15). Controls that consisted of ddH2O were negative in all Statistical Analyses runs. All real-time RT-PCR reagents were supplied by Applied Biosys- Data are given as mean Ϯ SD. A minimum of four mice were used for tems (Foster City, CA). Except for 18S rRNA, the primers were cDNA each analysis, unless stated otherwise. Statistical analysis was per- specific and did not amplify genomic DNA. PCR oligonucleotide formed by using Mann-Whitney U test (SPSS-PC Version 12; SPSS, Inc., primer and probes are given in Supplementary Table 1. Chicago, IL); significance was determined at P Ͻ 0.05. 1336 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1334–1344, 2006

Results Podocyte-Specific Deletion of ILK Because ILK null embryos die at time of implantation (23), mice with flanking loxP sites (floxed) of the ILK exons 5 to 12 (ILKflox/flox) were crossed with mice that express the Cre- recombinase under the control of a podocyte-specific promoter ϩ Ϫ (2.5P-Cre / ) for targeted ILK inactivation in podocytes (Fig- ure 1A). Resulting bitransgenic mice were crossed with homozygous ILKflox/flox mice to obtain mice with the genotype ϩ Ϫ ILKflox/flox/2.5P-Cre / (called podoILKϪ/Ϫ). To evaluate the ability of the podocin-Cre recombinase to excise the floxed ILK alleles, we examined kidney cortex DNA by PCR, and the excised floxed ILK allele was detected only in ϩ Ϫ kidney but not tail DNA of ILKflox/flox/2.5P-Cre / mice, confirming tissue-specific recombination of the loxP sites within the ILK locus (Figure 1B). In microdissected glomeruli from podoILKϪ/Ϫ mice, steady-state ILK mRNA was reduced by 74% compared with Cre-negative litter mates (podoILKϩ/ϩ 2.29 Ϯ 1.00 ϫ 10 to 3 (n ϭ 7) versus podoILKϪ/Ϫ 0.59 Ϯ 0.53 ϫ 10 to 3 (n ϭ 4); ratios to 18S rRNA; Figure 1C). Because ILK has been shown to be expressed in glomerular mesangial cells and glomerular podocytes (6,7), intraglomeru- lar ILK protein expression was localized by immunohistochemistry. In podoILKϩ/ϩ mice, an ILK signal is seen in the mesangium and along the outer aspects of the GBM that are derived from the podocyte foot processes that are attached to the GBM. In podoILKϪ/Ϫ mice, the mesangial staining remained unchanged, but the GBM-associated signal was lost, confirming loss of podocyte ILK expression (Figure 1D). Figure 1. Targeted inactivation of integrin-linked kinase (ILK) in podocytes. (A) Strategy and map for podocyte-specific inacti- PodoILKϪ/Ϫ Mice Show Rapid Progression of Proteinuria vation of ILK. 2.5P-Cre mice that express the Cre-recombinase to Terminal Renal Failure under the control of the NPHS2 promoter in a podocyte-spe- Mice of all genotypes were born at the expected Mendelian fre- cific manner were bred to mice that carry a floxed ILK locus. Ϫ quency. Heterozygous mice for the floxed ILK allele (ILKflox/ /2.5P- Podocyte-specific Cre activation resulted in deletion of ILK ϩ Ϫ Cre / ) had no overt phenotype, and urine analysis showed no exons 5 through 12. (B) Genotypic analysis of Cre-mediated excision of the floxed ILK allele in podocytes. DNA was pre- albuminuria up to 15 mo of age. pared from kidney cortex and analyzed by PCR, demonstrating PodoILKϪ/Ϫ mice seemed normal at birth but showed a Cre-mediated excision of the ILK locus in ILKflox/flox/2.5P- ϩ Ϫ Ϫ Ϫ drastically reduced life span with a median age of death at 19 Cre / (podoILKϪ/Ϫ) but not ILKflox/flox/2.5P-Cre / wk (Figure 2A). Sequential urine analysis for proteinuria by (podoILKϩ/ϩ) mice. (C) ILK mRNA levels in microdissected Bradford assay and SDS-PAGE revealed the appearance of glomeruli. Microdissected glomeruli of podoILKϪ/Ϫ mice selective albuminuria at 2 to 4 wk of age, progressing to un- showed a reduction of ILK mRNA by 74% in real-time reverse selective proteinuria at 4 to 12 wk (Figure 2, B and C). Biochem- transcription–PCR, expressed as ratio to 18S rRNA as the ref- ical analysis at 12 wk was consistent with massive proteinuria erence gene. (D) Glomerular ILK protein expression. In 2.5P- Ϫ/Ϫ and impaired renal function (blood urea nitrogen 79 Ϯ 62 versus Cre –negative mice (podoILKϩ/ϩ), ILK was detected by 27 Ϯ 5 mg/dl; albumin 2.5 Ϯ 0.4 versus 3.4 Ϯ 0.3 g/dl; total immunohistochemistry in the mesangium (arrowhead) and cholesterol 400 Ϯ 190 versus 78 Ϯ 8 mg/dl; podoILKϪ/Ϫ versus along the filtration barrier in podocyte foot processes (perime- sangial and pericapillary glomerular basement membrane podoILKϩ/ϩ, n ϭ 4 for each group, respectively). Macroscop- [GBM]; arrow). In homozygous floxed ILK/heterozygous ically, 16-wk-old podoILKϪ/Ϫ mice showed nephrotic, end- podocin-Cre littermates (podoILKϪ/Ϫ), the ILK signal was lost stage kidneys with rough surface and yellow appearance (Fig- at the filtration barrier, whereas the ILK staining in the mesan- ure 3A). gium remained unaltered. For orientation, the corresponding glomerular cross-sections are given in the inserts. Magnifica- PodoILKϪ/Ϫ Mice Develop Progressive Focal Segmental tions: ϫ400 in D; ϫ100 in D inserts. Glomerulosclerosis Histologic examination of kidneys from 1- to 16-wk-old mice revealed the development of progressive glomerulosclerosis in of postnatal renal development and were indistinguishable podoILKϪ/Ϫ mice (Figures 3, A and B, and 4). Kidneys from 1- from those of podoILKϩ/ϩ mice, consistent with normal and 12-d-old podoILKϪ/Ϫ mice showed the typical gradient nephrogenesis in podoILKϪ/Ϫ mice. J Am Soc Nephrol 17: 1334–1344, 2006 Podocyte-Specific ILK Deletion 1337

Figure 3. Structural alterations in podoILKϪ/Ϫ mice. (A) Kid- neys from podoILKϩ/ϩ and ILKϪ/Ϫ mice at 16 wk of age. PodoILKϪ/Ϫ mice showed end-stage fibrotic kidneys with rough surface and yellow appearance but comparable size to podoILKϩ/ϩ kidneys (scale bar in millimeters). PodoILKϪ/Ϫ mice showed sclerosed glomeruli, severe tubulointerstitial lesions with cystic dilation of tubules, proteinaceous casts, tubu- Figure 2. Podocyte-specific inactivation of ILK results in pro- lar atrophy, and interstitial fibrosis. (B) Progressive glomerulo- gressive loss of the glomerular filtration barrier. (A) Kaplan- sclerosis and tubulointerstitial fibrosis in podoILKϪ/Ϫ mice. Meier survival curve for podoILKϩ/ϩ and podoILKϪ/Ϫ In 3-wk-old mice with selective albuminuria, no differences in mice. PodoILKϪ/Ϫ mice had a median survival of 19 wk and histology between podoILKϪ/Ϫ and podoILKϩ/ϩ mice could a 100% mortality at 32 wk. (B and C) Development of protein- be detected. In 4-wk-old mice with unselective proteinuria, uria in podoILKϪ/Ϫmice. (B) Protein-to-creatinine ratio juxtamedullary glomeruli of podoILKϪ/Ϫ mice frequently showed progressive proteinuria in podoILKϪ/Ϫ mice. (C) In demonstrated prominent podocytes and segmental mesangial SDS-PAGE, selective albuminuria was first seen at 2 wk of age expansion with increased matrix deposition and distorted cap- and progressed rapidly to unselective proteinuria (4- and 16- illaries. At 12 wk of age, glomerulosclerosis with severe podo- wk-old mice). A, albumin standard. cyte lesions, tuft adhesions to Bowman’s capsule, crescent formation, mesangial expansion with increased matrix deposition, distorted capillaries, and glomerular obsolescence were found. Tubulointerstitial lesions included tubular atrophy, interstitial fi- At the onset of selective albuminuria at 2 to 3 wk of age, brosis, and mononuclear infiltration. Magnifications: ϫ10 in A. podoILKϪ/Ϫ mice showed occasionally prominent single podocytes, predominantly in juxtamedullary glomeruli. Mean glomerular volume, as a parameter for overall glomerular ar- prominent podocytes and occasional segmental mesangial ex- chitecture, was not different between podoILKϪ/Ϫ and pansion with increased matrix deposition and distorted capil- podoILKϩ/ϩ littermates at this stage (podoILKϩ/ϩ 91,300 Ϯ laries. 15,600 versus podoILKϪ/Ϫ 78,500 Ϯ 8600 ␮m3; NS). At 4 wk, In podoILKϪ/Ϫ mice that ranged from 8 to 16 wk of age, juxtamedullary glomeruli of podoILKϪ/Ϫ mice demonstrated more advanced glomerular damage with characteristic focal 1338 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1334–1344, 2006

manner, and showed electron-lucent areas in the lamina densa (Figure 5). At 8 to 12 wk of age, the podocyte lesions had progressed further in podoILKϪ/Ϫ mice with vacuolization, microvillous transformation, widespread foot process effacement, and focal detachment from the basement membrane. The GBM exhibited diffuse and irregular thickening and showed electron-lucent areas (Figure 5). Because the GBM alterations were the first ultrastructural lesions seen, morphometric analysis of the GBM width was performed at 3 wk of age in mice with selective albuminuria. Using orthogonal intercepts, the true harmonic mean GBM thickness was found to be significantly increased from 225.4 Ϯ 4.7 nm in podoILKϩ/ϩ mice to 291.4 Ϯ 22.9 nm in podoILKϪ/Ϫ (n ϭ 4 mice in each group; P Ͻ 0.05; Figure 6A). The frequency of filtration slits that were encountered along a given GBM distance was determined to evaluate podocyte foot process spacing as a parameter for podocyte effacement. No decreased FSF was encountered in the glomeruli of podoILKϪ/Ϫ mice with selective albuminuria compared with Figure 4. Ϫ Ϫ Structural alterations in podoILK / mice. Glomer- podoILKϩ/ϩ littermates (1655.47 Ϯ 107.21 versus 1714.18 Ϯ ular architecture in semithin sections of podoILKϪ/Ϫ.At3wk 81.57 filtration slits/mm GBM; n ϭ 4 mice in each group; NS). of age, juxtamedullary glomeruli of podoILKϪ/Ϫ mice With progressive proteinuria, podocyte foot process architec- showed single prominent podocytes compared with podoILKϩ/ϩ mice. At 4 wk, juxtamedullary glomeruli of ture deteriorated. Analysis of the mice with unselective pro- podoILKϪ/Ϫ mice demonstrated prominent podocytes with teinuria by scanning electron microscopic analysis revealed protrusions and microvillous transformation of the epithelial progressive podocyte changes with age, including multiple surface. At 12 wk, podocytes of podoILKϪ/Ϫ mice were en- luminal microvilli and widespread flattening of the cell bodies larged and showed protrusions and microvillous transforma- and major processes, thereby covering major parts of the filtration. Foot processes were effaced and covered a broadened tion area with rarely interdigitating foot processes (Figure 6B). GBM. Magnification, ϫ100. Slit Diaphragm and Associated Molecules Are Altered with Progressive Proteinuria in podoILKϪ/Ϫ Mice and segmental sclerotic lesions evolved and was associated Because the podocyte slit diaphragm is considered to be a with tubulointerstitial changes. End-stage kidney lesions were key element of the filtration barrier, electron microscopic anal- characterized by diffuse glomerulosclerosis and tubulointersti- ysis focused on slit diaphragm alterations at onset of albumin- Ϫ ϩ Ϫ tial inflammation and fibrosis. ILKflox/ /2.5P-Cre / and uria (3 wk) and could not detect ultrastructural alteration at this 2.5P-Cre–negative littermates of the different age groups stage (Figures 5 and 6A). With progression to unselective pro- showed no renal pathology. teinuria, loss of slit diaphragm and foot process effacement could be encountered. To further evaluate the slit diaphragm, PodoILKϪ/Ϫ Mice Show GBM Alteration at Onset of we evaluated the expression level and localization of two key Albuminuria SD components, nephrin and podocin, in glomeruli from Because albuminuria preceded the development of the focal podoILKϪ/Ϫ. Steady-state mRNA levels for both molecules segmental glomerulosclerosis (FSGS) lesions, the ultrastructure did not show a significant difference using microdissected glo- of the filtration barrier was evaluated starting at the first sign of meruli from podoILKϪ/Ϫ mice versus podoILKϩ/ϩ litter- altered glomerular function in podoILKϪ/Ϫ and thereafter. At mates at disease onset (Table 1). Immunofluorescence studies 1 and 12 d of age, no differences between podoILKϪ/Ϫ and also demonstrated comparable signal intensities and distribu- podoILKϩ/ϩ mice were observed. tion for both molecules (Figure 7). At later stages (12 wk), a At 3 wk of age and concomitant with the onset of albumin- significant reduction in mRNA steady-state levels of nephrin, uria, prominent podocytes in the juxtamedullary glomeruli of synaptopodin, ␣-actinin-4, and WT-1 could be found (Table 1), podoILKϪ/Ϫ mice demonstrated occasional vacuoles and fo- consistent with loss of podocytes from the glomeruli or repres- cal microvillous transformation. Foot processes showed a nor- sion of these podocyte-specific molecules. mal architecture, and slit diaphragm remained morphologically intact. The GBM was homogeneously thickened (Figure 5). Podocyte Matrix Receptors in podoILKϪ/Ϫ Mice At 4 wk of age, prominent podocytes of podoILKϪ/Ϫ mice Because ILK has been reported to be involved in matrix showed pseudocysts and vacuolization, focal microvillous assembly via transmembrane matrix receptors (24), concentra- transformation, and multifocal foot process effacement. The tion and distribution of integrin-␣3 and -␤1 were evaluated. By GBM was thickened, both homogeneously and in a nodular immunofluorescence studies, no difference in the distribution J Am Soc Nephrol 17: 1334–1344, 2006 Podocyte-Specific ILK Deletion 1339

Figure 5. Ultrastructural analysis of the glomerular filtration barrier in podoILKϪ/Ϫ mice. Transmission electron micrographs of glomeruli from 3-, 4-, and 12-wk-old mice. At 3 wk, no obvious differences in ultrastructure between podoILKϪ/Ϫ and podoILKϩ/ϩ podocytes could be seen, but the GBM appeared homogenously thickened (see inserts). At 4 wk of age, podoILKϪ/Ϫ podocytes showed focal effacement of foot processes, and the GBM was clearly thickened, both homogeneously and in a nodular manner and showed electron-lucent areas in the lamina densa. At 12 wk of age, glomeruli of podoILKϪ/Ϫ mice displayed prominent podocytes, exhibiting microvillous transformation and protrusions of the epithelial surface. Widespread foot process effacement and elongated, flattened primary processes were seen. The GBM exhibited diffuse and irregular thickening and showed electron-lucent areas.

of integrin-␤1 in mesangial or endothelial cells and podocytes difference in GBM staining for murine IgA, IgG, or IgM was could be observed (data not shown). Confocal microscopy anal- found (data not shown). However, in mice with progressive ysis found in wild-type mice a partial overlap of the podocyte- filtration barrier failure and glomerular scarring (12 wk of age), derived integrin-␣3 signal with entactin staining marking the increased levels of fibronectin and collagen type I ␣1 could be GBM. In 3-wk-old podoILKϪ/Ϫ mice, overall signal intensity found, consistent with induction of these molecules during for integrin-␣3 seemed unchanged, but co-localization with mesangial expansion and glomerulosclerosis (Table 1). The entactin was lost, consistent with an ILK-dependant redistribu- mRNA for the GBM molecules collagen IV ␣3 through 5, in tion of this integrin at the onset of albuminuria (Figure 8). contrast, were reduced in these advanced stages of glomerular damage. GBM Composition in podoILKϪ/Ϫ Mice To define the alterations in the molecular composition of the GBM, we evaluated the expression levels and distribution of Discussion the major GBM components by real-time RT-PCR and immu- For maintaining an intact filtration barrier against the high nofluorescence. Analysis of laminin-␣ 1, 2, 4, and 5; laminin-␤ transcapillary pressure gradient of the glomerulus, an intimate 1 and 2; collagen type IV ␣ 1 through 6; agrin; perlecan; nido- molecular cross-talk between podocyte foot processes and the gen-1; and fibronectin did not reveal an increase on the mRNA GBM is crucial (25). ␣3/␤1-Integrins are the main transmem- and/or protein level for any of these molecules in 3-wk-old brane matrix receptors of podocytes, and collagen IV and lami- mice with selective albuminuria (Table 1, Figure 9). Also, no nins are their GBM ligands. Integrins regulate cell function and 1340 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1334–1344, 2006

high degree of context dependence of ILK function. ILK deletion in Caenorhabditis elegans resulted in a phenotype that re- sembled some aspects of ␤1-integrin deletion, underscoring ILK as a key adaptor between the cytoskeleton and integrins (27). Complete knockout of ILK results in early embryonic lethality (23). Tissue-specific deletion of ILK in chondrocytes (11,28) and endothelial cells in mice and zebra fish (29) demonstrated key roles of ILK for integrin-mediated cell adhesion and spreading, actin stress fiber formation, cell survival, and proliferation (11,28,29). A podocyte-specific Cre-lox system was used to evaluate ILK function in the glomerular context in vivo (13). ILK excision was detectable at birth as mature glomeruli are found in the juxta- glomerular region of newborn mouse kidneys. At 3 wk of age, a loss of the ILK signal from podocyte foot processes at the GBM could be shown with unchanged ILK expression in the mesangium (Figure 1). ILK mRNA levels were reduced by 74%, consistent with detectability of the mesangial ILK mRNA in the glomerular preparation after Cre excision. PodoILKϪ/Ϫ mice developed the first functional alteration (selective albuminuria) 2 to 3 wk after birth (Figure 2). Albuminuria quickly progressed to nonselective proteinuria and progressive FSGS with terminal renal failure in all mice examined to date. This well-defined and easy-to-monitor onset of podocyte damage allowed the dissec- Figure 6. Ultrastructural analysis of the glomerular filtration bar- tion of early events after ILK loss and to segregate them from rier in podoILKϪ/Ϫ mice. (A) True harmonic mean GBM thick- late, unspecific lesions that were associated with FSGS. The ness but not filtration slit frequency (FSF) is altered in delay between ILK genomic deletion and initially detectable podoILKϪ/Ϫ mice at onset of albuminuria. In orthogonal inter- functional alterations could be a consequence of a long ILK cepts, the true harmonic mean GBM thickness increased by 22.6% protein half-life in podocytes. Alternatively, ILK-negative Ϫ Ϫ in 3-wk-old podoILK / mice with selective albuminuria com- podocytes could display a stable phenotype postpartum but pared with podoILKϩ/ϩ (n ϭ 4 mice in each group; P Ͻ 0.05, decompensate with increasing demands on the filtration barrier top). In contrast, in the same glomeruli, no significant difference of the FSF, determined as number of slit diaphragms encountered in adolescence. per millimeter of GBM, could be detected (n ϭ 4 mice in each Because glomerular filtration barrier loss has been linked to group; NS, bottom). (B) Scanning electron micrographs of glomer- alterations in the slit diaphragm and podocyte foot process uli from 12-wk-old mice. View of a capillary loop from Bowman’s effacement, this crucial unit of the filter was studied at disease space. Bar ϭ 1 ␮m. (Left) PodoILKϩ/ϩ. (Middle and Right) onset. In 3-wk-old mice with selective albuminuria, ultrastruc- PodoILKϪ/Ϫ. Podocytes demonstrated a spectrum of changes tural and molecular analysis could not detect visible alterations from irregular foot process interdigitation (middle) to complete of the slit diaphragms (Figure 5). Elements of the podocyte foot process effacement, cell body attenuation, and microvillous cytoskeleton also were found to be normally expressed without transformation (right). Magnification, ϫ10,000 in B. ultrastructural evidence of significant podocyte foot process effacement as determined by FSF at the GBM (Figure 6A). matrix assembly via a protein complex that is associated with These results, however, do not exclude a simultaneous role of their cytoplasmic tail in focal adhesion plaques (26). ILK has ILK at the slit diaphragm, as interference with slit diaphragm emerged as a multifunctional protein in this complex. In cul- function resulting in proteinuria has been demonstrated with- tured podocytes, ILK has been shown to orchestrate a wide out visible changes in ultrastructure (30). After progression to array of functions, including focal adhesion plaque assembly nonselective proteinuria, slit diaphragm loss and foot process (6); F-actin cytoskeletal organization (6,9); membrane proximal effacement became evident (Figure 5). No detachments of initiation of signal transduction via Akt, GSK-3␤, and ␤-catenin podocytes from the GBM were seen, and transferase-mediated (6,8); regulating cell phenotype and survival (6,8,9); and inte- dUTP nick-end labeling staining did not reveal an increased grin binding affinity and avidity that are responsible for podo- staining of podocytes in podoILKϪ/Ϫ at3and4wkofage cyte adhesion and extracellular matrix assembly (6,9). Because (data not shown), consistent with ILK’s not being essential for ILK levels have been found to be induced in progressive glo- podocyte survival in vivo. merular damage (6,7), the physiologic role of ILK for podocyte The first lesions that consistently were found at onset of albumin- function had to be addressed in the glomerular environment in uria were GBM alterations with an increase in thickness, followed by vivo to understand the consequences of ILK induction in dis- splitting and massive extension at later stages (Figure 5). Possible ease. Recent studies using genetic models have demonstrated a mechanisms for this surprising GBM phenotype could be an in- J Am Soc Nephrol 17: 1334–1344, 2006 Podocyte-Specific ILK Deletion 1341

Table 1. Glomerular gene expression of GBM, podocyte slit membrane molecules, and cell–matrix receptorsa

4Wk 12Wk

PodoILKϩ/ϩ PodoILKϪ/Ϫ PodoILKϩ/ϩ PodoILKϪ/Ϫ Target (n ϭ 6) (n ϭ 5) (n ϭ 4) (n ϭ 6) P P Mean SD Mean SD Mean SD Mean SD

Procollagen I, ␣1 3.0610-6 2.7210-6 2.6910-6 1.6610-6 NS 0.3310-6 0.1810-6 3.1710-6 4.2210-6 Ͻ0.05 Procollagen IV, ␣1 2.1810-3 2.1010-3 1.4710-3 1.2110-3 NS 5.1810-4 2.2910-4 7.2210-4 5.0910-4 NS Procollagen IV, ␣2 4.7310-5 3.1410-5 4.1310-5 3.3010-5 NS 0.6210-5 0.3010-5 1.3510-5 0.8810-5 NS Procollagen IV, ␣3 7.8410-5 5.9110-5 12.2010-5 11.7010-5 NS 9.3610-5 3.3610-5 1.6910-5 1.3410-5 Ͻ0.05 Procollagen IV, ␣4 1.8310-3 1.1610-3 1.7510-3 1.5210-3 NS 7.6510-4 3.1610-4 2.6310-4 1.8910-4 Ͻ0.05 Procollagen IV, ␣5 1.1210-4 6.1210-4 1.0310-4 0.7810-4 NS 3.3410-5 0.9410-5 1.3410-5 0.7810-5 Ͻ0.05 Fibronectin 1 7.6010-5 6.9910-5 4.6310-5 3.8110-5 NS 0.1510-4 0.0510-4 1.4610-4 1.4010-4 Ͻ0.05 Laminin, ␤2 3.9710-5 2.7810-5 3.0810-5 2.2810-5 NS 1.7910-5 0.5610-5 1.1710-5 0.2610-5 NS Agrin 5.2810-1 2.9910-1 3.5410-1 1.7010-1 NS 2.5210-1 1.4910-1 3.0210-1 1.7110-1 NS Perlecan 2.7210-4 1.9910-4 1.3110-4 0.8910-4 NS 8.3710-5 3.3610-5 9.6810-5 5.7310-5 NS Nidogen 1 4.8910-4 7.2210-4 2.5010-4 1.7710-4 NS 9.7210-5 4.3710-5 7.2810-5 4.5310-5 NS Integrin ␣3 2.7110-4 2.0810-4 3.3010-4 2.4210-4 NS 1.9310-4 0.3010-4 1.4710-4 1.0010-4 NS Integrin ␤1 6.6010-4 5.6010-4 5.2010-4 3.0010-4 NS 4.1910-4 2.1010-4 4.1610-4 2.5010-4 NS Dystroglycan 9.9810-4 9.3110-4 9.8610-4 5.6610-4 NS 3.7710-4 1.5810-4 3.4110-4 2.3510-4 NS Nephrin 5.3810-4 4.9010-4 21.7010-4 14.3010-4 NS 1.0110-3 0.6610-3 0.1810-3 0.1010-3 Ͻ0.05 Podocin 1.8210-4 1.0910-4 1.3610-4 0.7810-4 NS 1.3310-4 0.7910-4 0.6010-4 0.2910-4 NS P-Cadherin 4.1310-6 2.4310-6 3.1910-6 2.7010-6 NS 2.9710-6 1.7710-6 9.1810-6 8.6410-6 NS ZO-1/Tjp1 7.2610-4 5.9210-4 7.2210-4 5.3910-4 NS 3.5310-4 1.1310-4 2.0110-4 0.7610-5 NS WT-1 2.1610-4 1.3810-4 1.8610-4 1.5710-4 NS 1.3610-4 0.4710-5 0.7610-5 0.2710-5 Ͻ0.05 ␣-Actinin 4 5.1510-4 4.0310-4 7.6410-4 3.3610-4 NS 3.8510-4 2.2710-4 1.4210-4 0.6910-5 Ͻ0.05 Synaptopodin 2.7010-4 3.5410-4 1.2610-4 0.7610-5 NS 6.3310-5 1.9910-5 1.6910-5 0.6410-6 Ͻ0.05

amRNA levels were determined by real-time mRNA quantification from 4- and 12-wk-old mice. Significant expression induction is displayed in bold; significant expression repression is displayed in italics. At 4 wk in proteinuric mice, no significant differences are seen. At 12 wk, in mice that showed progressive glomerulosclerosis, repression of podocyte-specific molecules and induction of the matrix molecules Col I ␣1 and fibronectin are detected. GBM, glomerular basement membrane; Ϫ Ϫ ϩ Ϫ podoILKϩ/ϩ, ILKflox/flox/2.5P-Cre / ; podoILKϪ/Ϫ, podocinCre / -ILKflox/flox mice.

An analysis of key components of the GBM at early disease stage did not demonstrate an increased concentration of GBM component transcripts or—accepting the inherent limitations of immunofluorescence studies for drawing quantitative conclu- sions—protein level. Also, no persistence of a developmental pattern of collagen IV or laminin isoforms could be found (Figure 8, Table 1). These findings do not indicate an increased synthesis of matrix molecules or decreased production of matrix-modifying enzymes, despite that ILK has been reported to be involved in these processes in in vitro models (10). The significant induction of fibronectin during the later stages of progression in the podoILKϪ/Ϫ has been observed in several animal models and human diseases and might be part of an Ϫ Ϫ Figure 7. Slit membrane–associated proteins in podoILK / ILK-independent scarring process (31). mice. In 4-wk-old mice, two key elements of the glomerular A second alternative would be passive deposition of circu- filtration barrier, podocin and nephrin, were evaluated by immunofluorescence studies and showed comparable staining lating molecules into the GBM. This is a frequent event in intensities and signal distribution along the GBM. For corre- autoimmune renal disease, resulting in subendothelial or sub- sponding mRNA analysis, see Table 1. Magnification, ϫ40. epithelial immune complexes at the GBM (32). However, immune complex deposits do show a typical ultrastructure that is not observed in the podoILKϪ/Ϫ mice. In addition, direct creased matrix synthesis or decreased degradation, deposition of staining for Ig was not able to reveal a difference between circulating proteins in the GBM, or alterations in GBM assembly. control and podoILKϪ/Ϫ GBM (data not shown). 1342 Journal of the American Society of Nephrology J Am Soc Nephrol 17: 1334–1344, 2006

Figure 8. GBM components and matrix receptors in podoILKϪ/Ϫ mice. Immunofluorescence of ␣3-integrin. High-resolution confocal fluorescence microscopic images of integrin-␣3 (red) and entactin as a GBM marker (green). In wild-type mice (podoILKϩ/ϩ, left) a partial co-localization of integrin-␣3 with the GBM staining is seen, resulting in the yellow signals in the merge. In podoILKϪ/Ϫ mice (right), a separation of the two signals is observed, consistent with re-localization of integrin-␣3at the earliest stages of disease onset in podoILKϪ/Ϫ mice.

An alternative explanation for the GBM phenotype could a small molecular ILK kinase inhibitor, preliminary data be an altered matrix assembly with impaired integrin func- indicate impaired integrin function in vitro with reduced tion (33). Matrix molecules could be less densely packed, migratory ability of differentiated cultured podocytes in causing a broadened and split GBM parallel to the conse- transfilter migration assays after ILK inhibition (M.K. et al., quences of some of the collagen IV mutations in Alport 2005, unpublished observations). syndrome (34). Podocytes are crucial for GBM dynamics and maintenance in health and disease (35,36). In in vitro systems, ILK has been implicated in matrix assembly via modulation Conclusion of integrin function by altering focal contact structure, inte- The gradual onset of the glomerular phenotype in Ϫ Ϫ grin activity state, and cell migration (7,24,29,37–39). ILK podoILK / mice allowed the dissection of the sequence of depletion in endothelial cells via RNA interference impaired structural lesions that leads to filtration barrier failure. The the ability to recruit ␣5/␤1-integrins to fibrillar adhesions (as GBM phenotype preceded subsequent podocyte damage, pro- defined by Geiger et al. [26]) and the maturation of the gressive glomerulosclerosis, and death. Because the key com- adhesions to competent matrix-forming structures (39). Fi- ponents of the GBM are expressed at comparable levels to bronectin matrix production remained unchanged in ILK controls, an alteration in matrix assembly subsequent to ILK knockdown, but the integration of fibronectin into a com- deletion via modified integrin function seems to be an attractive plex, focal adhesion–associated matrix was impaired. A po- hypothesis for this phenotype. tential mechanism of integrin- mediated matrix adhesions is the unmasking of self-assembly sites in the fibronectin mol- Acknowledgments ecule by activated integrins, allowing spontaneous polymer- This study was supported by DFGKr1492/6-2ϩ3 and EUFPV:QLG1- ization into densely packed fibers (26,40). The hypothesis of CT-2002-01215 to M.K. Ϫ Ϫ a GBM matrix assembly defect in ILK / podocytes is We thank A. Siebert, A. Ciolovan, I. Edenhofer, and I. Bayer for excellent difficult to test. As initial evidence of altered integrin GBM technical assistance; D. Schlo¨ndorff for critical review of the manuscript; E. interaction in ILK-deficient glomeruli, dissociation of inte- Freitag for the design of the logarithmic ruler; and W. Amselgruber for the grin-␣ 3 signal from the GBM was detected (Figure 8). Using opportunity to perform scanning electron microscopy. J Am Soc Nephrol 17: 1334–1344, 2006 Podocyte-Specific ILK Deletion 1343

Figure 9. GBM components and matrix receptors in podoILKϪ/Ϫ mice. Immunofluorescence of GBM molecules. The distribution and signal intensity of the major components of the murine GBM was evaluated and did not show any consistent difference between podoILKϩ/ϩ and proteinuric podoILKϪ/Ϫ mice at 4 wk of age. Stainings for developmental and mature laminins and collagen type IV minor chains are shown. For corresponding mRNA expression analysis see Table 1. Magnification, ϫ40.

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Supplementary Table 1:

Real-time RT-PCR oligonucleotide primer and probe sequences Gene Primer Accession No. Alpha-Actinin 4 FP 5'-AGTGCATGGTCCCTCTTTGG-3' AJ289242 RP 5'-CGCTGAGAGCAATCACATCAA-3' Probe FAM 5'-ACCAGCTGCTGCACCTTCTCCCA-3' Agrin AoD, Context Seq 5´-GACTGAGAGTGAGAAAGCGCTGCAG-3´ NM_021604 Coll1alpha1 FP 5'-TGCTTTCTGCCCGGAAG A-3' X54876 RP 5'-GGGATGCCATCTCGTCCA-3' Probe FAM 5'-CCAGGGTCTCCCTTGGGTCCTACATCT-3' Coll4 alpha 1 AoD, Context Seq 5´-GGCTATTCCTTCGTGATGCACACCA-3´ NM_009931 Coll4 alpha 2 AoD, Context Seq 5´-TTGGCCAGGAAGGGGAGCCAGGCCG-3´ NM_009932 Coll4 alpha 3 AoD, Context Seq 5´-TCACCCAGGAAAACCAGGTCCTGCT-3´ NM_007734 Coll4 alpha 4 AoD, Context Seq 5´-ATCAAGATCTTGGTTTGGCAGGCTC-3´ NM_007735 Coll4 alpha 5 AoD, Context Seq 5´-TGTCAGACATGTTCAACAAACCTCA-3´ NM_007736 Coll4 alpha 6 AoD, Context Seq 5´-CCAGGATCTGGGATTTGCTGGCTCC-3´ NM_053185 Dystroglycan AoD, Context Seq 5´-GGGAGATCATCAAGGTGTCTGCAGC-3´ NM_010017 Fibronectin 1 AoD, Context Seq 5´-GTTTCGGAGGCCAGCGGGGCTGGCG-3´ NM_010233 ILK FP 5'-ATGAGAATCATTCTGGAGAGCTTTG-3' U94479 RP 5'-TGTACTCCAGTCTCGAACCTTCAG-3' Integrin alpha3 AoD, Context Seq 5´-GCCTCGCTCAGCTTAATGAATCATC-3´ NM_013565 Integrin beta 1 AoD, Context Seq 5´- GTGGAGCCTGCAGGTGCAATGAGGG-3´ NM_010578 Laminin beta2 AoD, Context Seq 5´-GAGGCTGAGAAACAACTACGGGAAC-3´ NM_008483 Nephrin AbD, FP 5'-ACCCTCCAGTTAACTTGTCTTTGG-3' AF168466 AbD, RP 5'-ATGCAGCGGAGCCTTTGAA-3' AbD, Probe FAM 5'-TCCAGCCTCTCTCC-3' Nidogen-1 AoD, Context Seq 5´-TGGGTGGATGCAGGCACCCATAGGG-3´ NM_010917 P-Cadherin FP 5'-GCTCTACCACGACGGCAGAG-3' X06340 RP 5'-GCCTCATACTTCTGCGGCTC-3' Probe FAM 5'-CCTTGATGCCAACGATAACGCTCCG-3' Perlecan AoD, Context Seq 5´-GTACGGCCAAGAGCAAATCCCCAGC-3´ M77174 Podocin AbD, FP 5'-GGGACATCTGCTTCCTGGAA-3' AY050309 AbD, RP 5'-TGATAGGTGTCCAGACAGGGTAAAA-3' AbD, Probe FAM 5'-AACAGGCCAGGACCT-3' 18S rRNA PDAR Synaptopodin AbD, FP 5'-TTCCTTGCCCTCACTGTTCTG-3' AF077003 AbD, RP 5'-TCCTAGCAGCAATCCACATCTG-3' AbD, Probe FAM 5'-CCTAGCTTTCTAAAGGAC-3' WT-1 AbD, FP 5'-CAGCTCAAAAGACACCAAAGGA-3' M55512 AbD, RP 5'-CGCTGACAAGTTTTACACTGGAAT-3' AbD, Probe FAM 5'-ACACAGGTGTGAAACC-3' ZO-1 AoD, Context Seq 5´-TCTGAGGGGAAGGCGGATGGTGCTA-3´ NM_009366

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Supplementary Table 2:

Antibodies and fixations used for IHC Species Source Fixation Nephrin Rabitt polyclonal L. B. Holzman (41) Cryosection Podocin Rabitt polyclonal C. Antignac (42) Laminin alpha1 Rat mAb 8B3 D. Abrahamson (43) 2% Laminin alpha2 Rat mAb 4H8-2 Alexis Biochem.(44) Paraformaldehyde Laminin alpha5 Rabbit polyclonal 8948 J. Miner (45) Laminin beta1+2 Rabitt polyclonal T.Sasaki (46) Coll IV alpha 3,4,5 Rabitt polyclonal J.Miner (47) Urea (47) Coll IV alpha 1,2 Rat mAbs H11 and H12 Y.Sado,Y.Ninomiya (48)

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