Acknowledgements and Dedication

Firstly, I would like to thank Prof. Karl Kunzelmann for accepting me as a student, thus allowing me to develop my scientific skills in a successful and organized environment. Also, for the continuous and close supervision over this project.

In the second place, I would like to show my appreciation to Prof. Margarida Amaral for the co- supervision of this work and communication over the year. Additionally, I would like to thank Prof. Rainer Schreiber for the constant supervision, help and all his suggestions.

I also want to specially thanks Ji for always being available to help with anything for her constant suggestions and ultimately to turn me into a better student.

A big thanks to all my fellow colleagues. To Inês, for being supportive even before my arrival to the last day of work. Also for the major contribution she had on this work and for always keeping a positive attitude and a good mood even when things seemed to go off track. To Roberta for never being too busy to help. For being officially a good friend and for never leaving me on the side with her formal invitations. Also for being the best gaming partner: “Here is my thank you Roberta, take it”. To Raquel, for all the supportive attitude and for helping me every time I need, even if for a short time. To Madalena, for her contribution to this work, but also for her kindness and sympathy. A big thanks for all the help with the thesis!

To all the technicians (Silvia, Tini, Patricia, and Brigitte) for all the effort and major support to develop this work.

I would like to show my appreciation to the Department of Physiology of the University of Regensburg where this work was developed since October 2017 until September 2018.

À Rita, agradeço toda a sua paciência durante este ano. Por todo o apoio incondicional que tiveste. Por todas as tuas palavras e pela tua presença durante este ano. Por toda a ajuda e pela tua contribuição para esta tese o meu obrigado.

A toda a minha família agradeço o apoio que me proporcionaram desde o início e pelo incentivo durante todo este ano.

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Summary

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of bilateral renal cysts that continuously expand and lead to an impaired renal function. ADPKD is originated by mutations in PKD1 or PKD2 genes, whose protein products are collectively called polycystins.

Polycystin-1 (PC1) encodes a membrane protein with large extracellular domains, which probably works as a receptor for an unidentified ligand. Polycystin-2 (PC2) is also a membrane-associated protein that works as a non-selective ion channel with high affinity to calcium ions (Ca 2+ ). Together, these proteins are thought to form a functional complex, which can regulate intracellular Ca 2+ signaling.

One of the hallmarks of ADPKD is the transepithelial Cl - secretion towards the cyst lumen. This secretion is thought to occur, mainly, through cAMP-gated channel Cystic Fibrosis Transmembrane Regulator (CFTR). CFTR is expressed in the apical membrane of epithelial cells in several tissues, namely in the kidney, and its function is suggested to be upregulated in cystic epithelial cells. However, heterogeneity in CFTR expression suggests that this channel may not be the only one responsible for transepithelial chloride (Cl -) secretion in ADPKD.

Additionally, Calcium-activated Cl - channels (CaCC) activated through purinergic receptors seem to play a fundamental role in this process. TMEM16A, a CaCC expressed in kidney epithelial cells, can be, together with CFTR, responsible for Cl - secretion towards the cyst lumen, and, therefore, be involved in cyst development.

Having a better understanding of TMEM16A and CFTR role in cyst growth can help in the development of new therapies in order to surpass ADPKD impaired Cl - secretion, hence controlling cyst formation and progression.

Here, we show that in the M1 cell line and in primary cultures of kidney cells derived from the renal cortex and medulla, the knockdown of either PKD1 or PKD2 is sufficient to generate a cystic phenotype on cellular level. ADPKD cells have an upregulation of constitutive and stimulated Cl - secretion, mainly through CFTR and TMEM16A. Increase Cl - secretion by CFTR and TMEM16A is due to an enhanced Ca 2+ signaling. ADPKD representative cells are characterized by an increased Ca 2+ concentration in the cytoplasm as well in the ER Ca 2+ stores

Remarkably, the downregulation of TMEM16A expression rescued the cells from the cystic phenotype, showing a major impact of TMEM16A in this disease, and shedding light towards new therapeutic approaches.

Key words : ADPKD, TMEM16A, CFTR, Cl - secretion, Ca 2+ signaling

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Resumo

A doença renal policística autossómica dominante (ADPKD) é caracterizada pelo desenvolvimento de cistos renais bilaterais que se expandem continuamente e levam a um comprometimento da função renal, sendo responsável por 10% dos casos que requerem transplante renal . A ADPKD é originada por mutações nos genes PKD1 ou PKD2, cujos produtos proteicos são coletivamente denominados policistinas. É a doença monogénica mais comum a nível mundial (incidência de 1:400-1:1000 indivíduos).

A Policistina-1 (PC1) é uma proteína de membrana cuja função não é completamente conhecida. Pensa-se que pode atuar como receptor para um ligando não identificado. Está presente na membrana plasmática, no cílio primário e nos complexos de adesão de células epiteliais polarizadas. A Policistina-2 (PC2 ou TRPP2) é uma proteína associada à membrana, que pertence à família de receptores de potencial transientes (TRP). É caracterizada como um canal catiónico não seletivo com alta afinidade para iões de cálcio (Ca 2+ ). Ainda que PC2 seja encontrada em localizações comuns a PC1, como a membrana plasmática e o cílio primário, encontra-se maioritariamente em organelos intracelulares como o Complexo de Golgi (GC) e o Retículo Endoplasmático (ER). Diversos estudos sugerem que PC1 e PC2 interagem formando o complexo policístico. No cílio primário pensa-se que estas duas proteínas regulam o fluxo de urina primária que passa ao longo do nefrónio. PC1 funciona então como um sensor graças ao seu amplo domínio extracelular, ativando assim PC2 o que leva a uma entrada de Ca 2+ na célula.

Uma das principais características da ADPKD é secreção de fluído transepitelial em direção ao lúmen do cisto. Ainda que este não seja um fenómeno comum dos túbulos renais, conhecidos por reabsorverem mais de 99% do filtrado glomerular, vários estudos mostram uma aptidão do epitélio de células com ADPKD para secretarem fluido. É possível que vários transportadores e canais iónicos possam estar alterados em células com fenótipo cístico, como os canais apicais de cloreto (Cl -), que assumem particular relevância nesta situação. O aumento dos níveis de monofosfato cíclico de adenosina (cAMP) nestas células induz a secreção de Cl - através de CFTR (do inglês: Cystic Fibrosis Transmembrane conductance Regulator ). Este processo encontra-se descrito como sendo uma das principais alterações nas células epiteliais de doentes com ADPKD. Contudo, existe alguma heterogeneidade na expressão deste canal iónico no tecido renal, o que levou à hipótese de que outros tipos de canais de Cl - poderiam estar envolvidos nesta doença. A existência de sinalização autócrina e parácrina no rim levou à hipótese da ativação dos canais de Cl - ativados por Ca 2+ (CaCC).

Mutações no CFTR dão origem à Fibrose Quística (CF), a doença autossómica recessiva com maior mortalidade a nível mundial. Este canal é expresso na membrana apical de vários órgãos, incluindo o rim, nomeadamente nos túbulos proximais, distais e ducto coletor. A sua ativação requer um processo de fosforilação dependente de cAMP. CFTR apresenta uma interação funcional com outros canais iónicos, nomeadamente os CaCC.

Os CaCC são canais iónicos membranares específicos para o transporte de aniões. São expressos numa grande variedade de tecidos e desempenham diversos papeis na célula, incluindo secreção epitelial. Podem ser ativados através de um fluxo de Ca 2+ transportado através da membrana plasmática ou por libertação de Ca 2+ de organelos intracelulares.

As proteínas transmembranares 16 (TMEM16) constituem uma família de dez proteínas homólogas (TMEM16A-K) caracterizadas pela elevada conservação das suas sequências. O membro mais bem estudado desta família é a proteína transmembranar 16 A (TMEM16A). Vários estudos

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demonstram que TMEM16A conduz correntes de CaCC em vários tipos de tecido, incluindo o rim. Neste órgão, TMEM16A desempenha um papel fundamental na reabsorção de proteínas pelos túbulos proximais e na secreção de protões.

Uma interação funcional foi descrita entre CFTR e TMEM16A ao nível do intestino e vias aéreas. Estudos demonstraram que TMEM16A aumenta a libertação de Ca 2+ do retículo endoplasmático (ER) ativando assim a sinalização por cAMP. É possível que esta dependência funcional ocorra também a nível renal, particularmente nos pacientes com ADPKD que tem secreção apical de Cl - aumentada e sinalização de Ca 2+ anormal.

Foi previamente demostrado que tanto PKD1 como PKD2 desempenham um papel fundamental na regulação da homeostase intracelular de Ca 2+ . No ER, PKD2 interage com o receptor de inositol trifosfato (IP3R), aumentando assim a libertação de Ca 2+ deste organelo, ao contrário do PKD1 que regula negativamente esta interação. PKD2 também regula a concentração de Ca 2+ intracelular 2+ ([Ca ]i) ao formar heteodímeros com canais TRP presentes tanto na membrana plasmática como no 2+ cílio primário. Assim foi proposto que mutações em PKD1 ou PKD2 , via desregulação da [Ca ]i, podem levar à ativação de vias regulatórias anormais presentes na ADPKD. Contudo os mecanismos moleculares por detrás destes processos não se encontram claros.

Deste modo, o objetivo deste trabalho é estudar o processo de desenvolvimento dos cistos, nomeadamente a secreção de Cl - desregulada pela qual a ADPKD é caracterizada. Assim, diferentes técnicas de detecção de transporte iónico foram aplicadas, tendo sido utilizadas para o efeito diferentes modelos de estudo.

Em primeiro lugar, foi utilizada uma linha celular M1, originária do ducto coletor de rato com expressão diminuída de Pkd1 e Pkd2, de modo a induzir nas células um fenótipo cístico. As propriedades ao nível de transporte iónico foram medidas utilizando diferentes abordagens. De modo a simular a fisiologia do epitélio renal, as monocamadas celulares foram cultivadas em filtros que permitiram que as células polarizassem e que o transporte de eletrólitos fosse avaliado na Câmara de Ussing. Foram também utilizados ensaios com células isoladas, nomeadamente patch clamp e o ensaio de quenching de YFP. No geral, foi possível que observar que menores níveis de Pkd1 e Pkd2 levam à existência de maiores correntes elétricas, geradas por uma maior atividade de canais iónicos, tanto a nível basal como depois de estimulada a secreção de Cl - via CFTR ou CaCC. Visto que a sinalização 2+ 2+ de Ca aparenta estar alterada nas células com ADPKD, a [Ca ]i foi medida nestas células. Menor 2+ expressão de Pkd1 levou a maior [Ca ]i tanto a nível basal como depois de estimulação por agonistas 2+ 2+ purinérgicos. Estas células também aparentam ter uma maior concentração de Ca no ER ([Ca ] ER ).

Os resultados obtidos foram então confirmados utilizando culturas primárias de tecido renal de diferentes modelos animais: ratinhos wild-type (wt ) utilizados como controlo; ratinhos com Pkd1 Knockout (KO) específico para o rim, de modo a obter um modelo da doença; e ratinhos com Pkd1 e Tmem16a KO específicos para o rim, que permite perceber a relevância neste CaCC na ADPKD. Estas células foram isoladas a partir do córtex e também da medula renal, o que permitiu perceber se haveria alguma diferença entre as células provenientes de diferentes regiões do rim no que diz respeito ao desenvolvimento de um fenótipo cístico. Utilizando as mesmas técnicas acima mencionadas foi possível verificar que a secreção de Cl - se encontrava aumentada, tanto a nível basal como após estimulação dos CaCC e de CFTR, nas células dos animais com KO no Pkd1. Este efeito é revertido quando as células apresentam o duplo KO para Pkd1 e Tmem16a, mostrando assim a importância deste CaCC para a ADPKD. Os níveis intracelulares de Ca 2+ foram também analisados de maneira

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semelhante ao que foi descrito para a linha celular M1. As células primárias que apresentavam KO de 2+ 2+ Pkd1 apresentaram uma maior [Ca ]i e de [Ca ]ER . Este aumento foi abolido nas células primárias provenientes do animal com Pkd1/Tmem16a KO.

Em suma, este estudo permitiu demonstrar que o CFTR e TMEM16A colaboram no aumento da secreção de Cl -, um fenótipo característico da ADPKD. Um aumento dos níveis de Ca 2+ tanto a nível citoplasmático como do ER em células com KO de Pkd1 sugere que nestas existe uma maior ativação de TMEM16A que, através da sua interação funcional com CFTR, aumenta também a sua atividade. 2+ Especula-se que o aumento de [Ca ]i se deve à falta de regulação de PKD2 pela PKD1 que se encontra mutada, levando a uma deslocalização de PKD2 do cílio primário e uma maior presença 2+ desta proteína quer a nível da membrana plasmática, quer no ER, o que leva a uma maior [Ca ]i nestas células. A compreensão da função conjunta que o CFTR e TMEM16A aparentam desempenhar no epitélio de células com fenótipo cístico pode impulsionar o conhecimento acerca desta doença e permitir o desenvolvimento de novas terapias.

Palavras-chave: ADPKD, TMEM16A, CFTR, Secreção de Cl -, Sinalização de Ca 2+

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Table of Contents

Acknowledgements and Dedication i Summary ii Resumo (Summary – Portuguese version) iii Table of Contents vii List of Acronyms and Abbreviations xi Index of Figures and Tables xv 1. Introduction 1 1.1. Autossomal dominant polycystic kidney disease 1 1.2 What is the function of Polycystins? 1 1.2.1 Polycystins role in flow sensing 2 1.3 Cyst formation in ADPKD 3 1.4 Impaired transport in ADPKD 3 1.5 CFTR and TMEM16A 5 1.5.1 Crosstalk between CFTR and TMEM16A 7 1.6 Regulation of Ca 2+ signaling by polycystins 7

2. Objectives 9 3. Materials and Methods 11 3.1 Cell Culture 11 3.1.1 Mammalian Cell Lines and Culture Conditions 11 3.1.2 Cell Culture on filter support 12 3.1.3 Transient Transfections 12 3.1.4 Production of lentivirus and stable cell lines generation 12 3.2 Molecular Biology 13 3.2.1 cDNA and siRNA 13 3.2.2 RT-PCR 13 3.3 Animal models 13 3.3.1 Isolation of Primary Kidney cells from cortical preparation 13 3.3.2 Isolation of Primary Kidney cells from medullary preparation 14 3.4 Analysis of protein expression and histology 14 3.4.1 Immunocytochemistry on primary kidney cells 14 3.4.2 Histology of renal cryosections 14 3.4.3 Western Blot 15 3.5 Measurements of Ion channel activity and Ca 2+ signaling 15

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3.5.1 Assesment of Cl - permeability through YFP Quenching assay 15 3.5.2 Whole-cell current measurements 16 3.5.3 Measurements of transepithelial ion transport 16 3.5.4 Measurements of free intracellular Ca 2+ Signaling using FURA-2 AM 17 3.6 Statistical Analysis 17

4. Results 19 4.1 Downregulation of Pkd1 induced a cystic phenotype which involves activation of Tmem16a 19 4.2 Mouse Cortical Collecting Duct cell line as a model of ADPKD 19 4.2.1 Polarized M1 cells produce cAMP-stimulated and CaCC short-circuit currents 20 4.2.2 Downregulation of Pkd1/2 enhances basal and CaCC currents. 21 4.2.3 Knockdown of Pkd1 increases Tmem16a-dependent I - uptake 23 4.2.4 Downregulation of endogenous Pkd1 or Pkd2 upregulates intracellular Ca 2+ signaling in M1 cells 27 4.3 Isolation of primary kidney cells from the renal medulla and renal cortex 28 4.4 Characterization of primary cells isolated from the renal cortex 29 4.4.1 Polarized primary cortical cells did not show increased secretion after induction of a cystic phenotype 29 4.4.2 Primary Pkd1 KO cortical cells had a higher basal and purinergic stimulated conductance, for which Tmem16a is essential 31 4.4.3 Pkd1 KO in cortical primary cells enhances I - uptake through CaCC 32 4.4.4 In primary cortical cells Pkd1 KO upregulates Ca 2+ signaling, in a Tmem16a-dependent manner 35 4.6 Primary Kidney Cells isolated from medullary preparations 37 4.6.1 Pkd1 KO enhances CaCC activity in primary medullary cells 37 4.6.2 Pkd1 KO in medullary primary cells induces an enhanced secretory phenotype that is rescued in the absence of Tmem16a 39 4.6.3 Medullary cells with Pkd1 KO have an enhanced I - permeability after cAMP and purinergic stimulation for which TMEM6A and CFTR are essential. 39 4.6.4 In primary medullary cells, Ca 2+ signaling is upregulated by Pkd1 KO in a Tmem16a- dependent manner 42

5. Discussion 45 5.1 Phenotypical switch of ADPKD cyst lining cells 45 5.2 M1 cell line and primary cultures of isolated kidney cells as models to study fluid secretion in cyst development 45 5.3 Proposed model for impaired Cl - secretion in ADPKD 46

6. Future Perspetives 50

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7. References 52 8. Appendices 62 Appendix I –siRNA 62 Appendix II – Primers and RT-PCR 62 Appendix III – Analysis of protein expression 63 Appendix IV – mRNA expression analysis by RT-PCR 65 Appendix V – YFP fluorescence measurements 66 Appendix VI – General compendium of the results obtained during this work 67

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List of Acronyms and Abbreviations

• Ohm

A Microampere (10 -6 A)

g Microgram (10 -6 g)

M Micromolar (10 -6 M) m Micrometer (10 -6 m)

2+ ([Ca ]i) Intracellular calcium concentration ABC ATP Binding Cassete ADPKD Autosomal dominant polycystic kidney disease ANOVA Analysis of variance AQP Aquaporin ATP Adenosine triphosphate BSA bovine serum albumin °C Degree celsius Ca 2+ Calcium ion CaCC Ca 2+ -activated Cl- channels cAMP Cyclic adenosine monophosphate CD8 Cluster of Differentiation 8 cDNA Complementary Deoxyribonucleic acid CF Cystic Fibrosis CFTR Cystic fibrosis transmembrane conductance regulator

CFTR inh CFTR inhibitor Cl - Chloride ion cm 2 Square Centimeter

CO 2 Carbon Dioxide CPA Cyclopiazonic acid C-terminal Carboxyl- terminal DAG Diacylglycerol ddH 2O Double distilled water DF Dilution Factor dH 2O Distilled water DMEM Dulbecco's Modified Eagle Medium DNA Deoxyribonucleic acid EGF Epidermal Growth Factor

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EGTA Ethylene Glycol Tetraacetic Acid ENaC Epithelium sodium channel ER Endoplasmic reticulum ERS Electrical Resistance System ESRD End-stage renal disease EtOH Ethanol eYFP Enhanced Yellow Fluorescent Protein FBS Fetal Bovine Serum Fsk Forskolin FURA-2 AM Fura-2-acetoxymethyl ester GAPDH Glyceraldehyde 3-phosphate dehydrogenase GPCR G protein-coupled receptor h Hour(s) H+ Hydrogen ion HBSS Hank’s Balanced Salt Solution HEK 293T Human Embryonic Kidney 293T (cell line) I- Iodide ion IBMX 3-isobutyl-1-methylxanthine Ig Immunoglobulin IP3 Inositol-1,4,5 triphosphate IP3R Inositol 1,4,5-triphosphate receptor

Isc Short-circuit currents ITS Insulin-Transferrin-Selenium K+ Potassium ion

K2HPO 4.3H 2O Dipotassium hydrogen phosphate trihydrate KCa3.1 Calcium-activated potassium channel 3.1 KCl Potassium chloride

KH 2PO 4 Potassium dihydrogen phosphate KO Knockout Ksp kidney specific cadherin promoter Min Minute(s) mg Milligram (10 -3 g) Mg 2+ Magnesium ion

MgCl 2.6H 2O Magnesium chloride hexahydrate mL Millilitre (10 -3 L) mm Millimetre (10 -3 m) mM Millimolar (10 -3 M)

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mmol Millimol (10 -3 mol) mRNA Messenger RNA mTMEM16 Mouse Transmembrane Protein 16 mV Millivolt (10 -3 V) Na + Sodium ion Na +/K +- ATPase Sodium-potassium pump NaCl Sodium chloride NaH2PO4.H2O Monosodium dihydrogen orthophosphate NaI Sodium iodide NBD Nucleotide binding domain NKCC Sodium –potassium –chloride cotransporter nM Nanomolar (10 -9 A) nm Nanometre (10 -9 m) N- terminal Amino- terminal Orai1 Calcium Release-Activated Calcium Modulator 1 ORCC Outwardly rectifying Cl - channels P2Y Purinergic G Protein-coupled Receptor 2Y P2Y2 Purinergic G Protein-coupled Receptor 2Y2 P2X Purinergic G Protein-coupled Receptor 2X PBS Phosphate Buffered Saline PC Polycystin PCR Polymerase Chain Reaction Pen Strep Penicillin-Streptomycin PFA Paraformaldehyde PIP2 Phosphatidyl inositol-diphospate PKA Protein Kinase A PKD Polycystic kidney disease PLC Phospholipase C PT Proximal tubules R-domain Regulatory Domain RNA Ribonucleic acid rpm Rotations per minute RT Room temperature RT-PCR Reverse Transcriptase Polymerase Chain Reaction

Rte Resistance RyR Ryanodine receptor s Second(s)

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scr Scrambled SEM Standard error of the mean sh Short hairpin RNA siRNA Small interfering RNA SK&F96365 1-(beta-[3-(4-methoxy-phenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride SOCE Store Operated Calcium Entry STIM1 Stromal interaction molecule-1 T3 Triiodothyronine T16A Transmembrane member16 A TER Transepithelial Electrical Resistance TMD Transmembrane domain TMEM16 Transmembrane member 16 TRP Transient receptor potential TRPC1 Transient receptor potential channel 1 TRPP2 Transient receptor potential polycystic 2 TRPV4 Transient receptor potential cation channel subfamily V member 4 UTP Uridine Triphosphate

Vte Voltage WB Western Blot wt Wildtype YFP Yellow Fluorescent Protein YM58483 4-methyl- 4• -[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3- thiadiazole-5-carboxanilide

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Index of Figures and Tables

Figure 1.1. Schematic representation of polycystin 1-polycystin 2 complex and their respective interaction...... 2 Figure 1.2. Representation of the possible physiological role of the polycystin complex ...... 3 Figure 1.3. Model of the transporters and channels involved in fluid accumulation in ADPKD ...... 5 Figure 1.4. Scheme of CFTR structure ...... 6 Figure 1.5. Representation of mTMEM16A architecture...... 7 Figure 3.1. Scheme of Iodide-mediated YFP fluorescence quenching ...... 16 Figure 4.1. The absence of Tmem16a rescues cystic phenotype induced by KO of Pkd1 ...... 19 Figure 4.2. M1 cells are a representative in vitro model for ADPKD...... 20 Figure 4.3. M1 cells show cAMP and CaCC dependent electrolyte transport ...... 21 Figure 4.4 . Knockdown of Pkd1/Pkd2 leads to a higher whole-cell current at basal state ...... 22 Figure 4.5. Knockdown of Pkd1 or Pkd2 leads to enhanced CaCC and diminished CFTR currents ...... 23 Figure 4.6. Knockdown of Pkd1 leads to enhanced Ca2+ and cAMP induced I- uptake ...... 24 Figure 4.7. I - uptake is due to CaCC and CFTR activation ...... 25 Figure 4.8. Downregulation of Pkd1 led to increased I - permeability dependent on Tmem16a ...... 26 Figure 4.9. Ca 2+ signaling is upregulated with the knockdown of Pkd1 or Pkd2 and Tmem16a plays a fundamental role in this process ...... 28 Figure 4.10. mRNA expression from Primary kidney cells from renal cortex or medulla ...... 29 Figure 4.11. Pkd1 KO did not induce a secretory phenotype in polarized primary cortical kidney cells...... 31 Figure 4.12. Pkd1 KO leads to a higher basal current and CaCC activation, for which Tmem16a is essential...... 31 Figure 4.13. Pkd1 KO leads to enhanced Ca 2+ and cAMP induced I - uptake...... 32 Figure 4.14. I - uptake occurs due to CaCC and CFTR activation ...... 35 Figure 4.15. KO of Pkd1 leads to Ca 2+ signaling upregulation ...... 36 Figure 4.16. Pkd1 KO lead to enhanced Tmem16a activation, and reduced epithelium resistance .... 38 Figure 4.17. Tmem16a is essential for both basal and purinergic stimulated currents...... 39 Figure 4.18. Pkd1 KO leads to enhanced Ca 2+ and cAMP induced I - uptake...... 40 Figure 4.19. I - uptake occurs due to CaCC and CFTR activation ...... 41 Figure 4.20. Pkd1 KO leads to intracellular Ca 2+ signaling deregulation ...... 43 Figure 5.1. Proposed model of Cl- secretion in ADPKD cystic epithelium...... 47

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Table I.1. Accession number and base pairs (bp) of the plasmids used ...... 62 Table II.1. Pair list of Primers used for RT-PCR ...... 62 Table II.2. Standard RT-PCR program...... 63 Figure III.1. Western blot analysis of PKD1/ PKD2 downregulation ...... 63 Figure III.2. Western blot analysis of PKD1, PKD2, and TMEM16A downregulation ...... 64 Figure III.3. Immunostaining analysis of TMEM16A and Acetylated tubulin in primary cells ...... 64 Figure IV.1. RT-PCR analysis of Pkd1/Pkd2 downregulation in M1 cell line...... 65 Figure IV.2. RT-PCR analysis of Pkd1 and Tmem16a expression in medullary primary cells...... 65 Figure IV.3. RT-PCR analysis of Scnn1 expression in medullary primary cells...... 66 Figure V.1. Assessment of YFP fluorescence on M1 cells...... 66 Figure V.2. Assessment of YFP fluorescence on primary cells...... 67 Table VI.1. Summary of the results obtained during this work...... 67

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1. Introduction

1.1. Autosomal dominant polycystic kidney disease

Polycystic kidney disease (PKD) constitutes a series of genetic disorders in which renal tubules become structurally abnormal, resulting in kidney cyst development and growth 1. Between several types, autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent (incidence of 1:400 to 1:1000) monogenic renal disease 2. ADPKD is characterized by an acute and bilateral development of multiple renal cysts over time that leads to the displacement and destruction of the adjacent renal parenchyma, progressive organ enlargement, and, ultimately, loss of renal function, being the cause of 10% of end-stage renal disease (ESRD) cases worldwide, which demand renal replacement therapy strategies 2-4 .

ADPKD is a systemic disorder that can have an in utero to adult onset 5,6 . Extrarenal manifestations also constitute cysts in other epithelial organs including liver and pancreas 7. Furthermore, it is a genetically heterogeneous disease originated by a germline mutation in either of two genes, PKD1 or PKD2 (Polycystic kidney disease 1 and 2) 8,9 . Mutations in PKD1 (located on chromosome 16p13.3) lead to the development of ADPKD in 85% of the patients and are linked with a more serious clinical progression while the remaining 15% of the cases are due to PKD2 mutations (located on chromosome 4q 21-23) 10 and present a milder phenotype with later onset, longer renal survival and fewer complications 11 . The protein products encoded by PKD1 and PKD2 genes are collectively named polycystins.

1.2 What is the function of Polycystins?

Polycystin-1 (PC1) is classified as an integral membrane protein and is constituted by eleven transmembrane domains, an extensive extracellular N-terminal domain, and a shorter C-terminal cytosolic domain 10 . PC1 contains a series of juxtaposed structural motifs, which suggests its involvement in protein-protein or protein-carbohydrate interactions, its functions as a receptor for an unknown ligand and its critical role in cell-cell or cell-matrix interactions 12,13 . It is mainly expressed in epithelial cells of developing and mature renal tubules. PC1 expression is regulated over time showing higher levels in fetal tissues and decreased presence in adult tissues 14 . PC1 is localized on the plasma membrane, adhesion complexes in polarized epithelial cells and on the primary cilium, a single hair- like organelle that protrudes from the surface of most mammalian cells, implicated in many cystic diseases 15-17 .

Polycystin-2 (PC2) or TRPP2 is a membrane-associated protein that belongs to the transient receptor potential family (TRP) 18 . It has 6 transmembrane domains, two EF-hands, (characteristic of Ca 2+ binding domains that allow the protein to sense changes in Ca 2+ ) an endoplasmatic reticulum (ER) retention signal and cytoplasmatic N- and C-terminal domains 9,19 . It works as nonselective cation channel with high affinity to Ca 2+ , being also permeable to Na + and K + ions. Although PC2 is found on the primary cilium and plasma membrane, where it mediates Ca 2+ entry into the cytoplasm, most of its pool is located in intracellular compartments, such as the ER and the Golgi complex, acting as Ca 2+ release channel 20,21 . PC2 interacts with other members of TRP superfamily, namely TRPC1 and TRPV4, to form heteromeric channel 22 .

The most significant partner of PKD1 is PKD2 23 , interacting through their C-terminal coiled-coil domains ( Fig. 1.1 )2,13 . Several studies suggest that PKD1 and PKD2 reciprocally affect the cellular location of each other, and defects in one of the proteins lead to a mislocalization of the other one. The

1

precise mechanism of this dependence is still not elucidated 24 . However, it is thought that this interaction is critical to create a functional ion channel. Yet, it is still unknown if this is due to the activation of PKD2 inherent channel properties or through emergent channel properties arising from the interaction between the two proteins 25 .

Figure 1.1. Schematic representation of polycystin 1-polycystin 2 complex and their respective interaction. PC1 has a large extracellular domain, 11 transmembrane domains and a shorter C-terminal tail. PC2 has a N- and C- termini domains and 6 transmembrane domains, where the ionic pore is formed. Both present a Coiled-Coil domain on the C- terminal by which they interact and mediate calcium entry into the cells. Retrieved and adapted from Patel et. al 26 .

1.2.1 Polycystins role in flow sensing

As previously mentioned, the cellular localization of these two proteins overlap, namely on the primary cilium. The primary cilium of renal tubular epithelial cells bends in response to fluid flow (primary urine) 27,28 . However, the subsequent cascade of events is still not clear and additional studies are still required.

Some authors state that the polycystin complex is implicated in fluid flow regulation, through 2+ the regulation of the concentration of free intracellular calcium ([Ca ]i), thus interfering with its 29,30 2+ homeostasis and signaling . [Ca ]i is important for the regulation of several cellular functions, including cell proliferation, cell volume regulation, ion and fluid transport, and differentiation 31 . PKD1 is essential for fluid regulation process, sensing the flow with its large extracellular domains and activating the associated PKD2 Ca 2+ channels ( Fig. 1.2 ), which may lead to a Ca 2+ -induced Ca 2+ release from intracellular stores 1,29 .

Other studies claim that the primary cilium has its own compartmentalized calcium signaling. Even with the polycystins complex promoting Ca 2+ entry into to the cells, the amount of Ca 2+ that enters through the primary cilium in response to fluid flow would be too small to trigger a visible response in cytoplasmatic Ca 2+ 32 .

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Nevertheless, in addition to this hypothesis, a study by Delling et al 33 states that there is not a ciliary specific Ca 2+ influx in response to physiological or even supraphysiological, fluid flow.

Figure 1.2. Representation of the possible physiological role of the polycystin complex. Both on the plasma membrane and on the primary cilium of renal epithelium cells PC1 and PC2 associate in a functional complex. PC1 is involved in flow sensing and PC2 is associated with Ca 2+ transport to the cytoplasm of these cells.

1.3 Cyst formation in ADPKD Continuous dilation of the tubules through enlarged cell proliferation, fluid secretion and, ultimately, separation from the parental tubule will lead to the formation of cysts 34 . It is still unclear if cysts specifically arise specifically from a determined nephron segment. Some studies report a generalized pattern, being some deep medullary and superficial cortical events also described 35 .

Even though, genetically, ADPKD is a dominant disease, it behaves as recessive at the cellular level. A human kidney is constituted of approximately a million nephrons. Nevertheless, microdissection studies indicated that cysts in ADPKD patients arise from about 1000 nephrons 36 . All cells from the patient organism possess a germline mutation in one of the polycystins genes alleles, which proves that the germline mutation alone cannot cause cystogenesis, otherwise all cells should develop cysts. A second-hit model was proposed to explain this phenomenon. This model state that every cyst emerge as a product of an individual somatic mutation event in addition to the germline mutation in either PKD1 or PKD2 alleles, leading to total loss of function of the polycystins 37-39 . This hypothesis can explain the slow advance of the pathology over several decades.

Recent studies show that late inactivation of the Pkd1 gene in adult mice kidneys resulted in a slow onset of cystogenesis 10,40-41 . The authors reported that the slow onset of the disease could not be explained by the previous hypothesis and suggested a third hit theory. This hypothesis states that a cell is not necessarily cystogenic just by having a germline mutation (first hit) and a somatic inactivation of the second allele (second hit) 42,43 , but by having an additional event that leads to cell proliferation and cyst growth (such as renal injury) 44 .

1.4 Impaired transport in ADPKD

Cyst expansion is characterized by tubular cell proliferation, defects in the extracellular matrix and transepithelial fluid secretion 45 . Since the developed cysts are physically separated from the renal tubule, this means that the intracystic fluid does not come from the glomerular filtrate, but instead

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from a net transepithelial fluid secretion towards the cyst lumen 45 . This hallmark is not common on the nephron segments, known to reabsorb approximately 99% of glomerular filtrate. Yet, several studies show an aptitude of the cystic epithelium for fluid secretion 46 .

Water channels were suggested to play a role in ADPKD impaired fluid transport 47 . Aquaporins (AQP) constitute the molecular pore for water transport in the cell membrane 48 . Most of the cysts express either (and exclusively) Aquaporin-1 (AQP1) or Aquaporin-2 (AQP2) 49 . These two proteins are located on the proximal tubules and principal cells of the collecting ducts, respectively 50 . Thus, the type of AQP expressed in a given cyst may shed a light towards its origin. In response to the osmotic gradient (driven by the upregulated Cl - secretion as explained below) water moves across the apical side of the epithelium towards the cystic lumen, where it accumulates, leading to cyst enlargment 51 .

Basolateral Cl - uptake was shown to be an important event for cyst lining cells 52 . Bumetanide is a loop diuretic that specifically inhibits NKCC1 and NKCC2 family of cotransporters (responsible for electroneutral transport of Na +, K +, and Cl -)53 . NKCC1 is expressed on the basolateral membrane of secreting epithelia 54 and several studies detected its expression on cystic cultured epithelial cells and kidneys 54,55 . Using bumetanide a reduction of luminal electronegativity and a decrease of fluid secretion was observed 52 , meaning that this transporter may be an important pathway for basolateral Cl - entrance into the cell, a crucial event for its posterior apical secretion 56 . The Na +/K +-ATPase and KCa3.1work as functional partners of NKCC1 at the basolateral membrane, recycling Na + and K + respectively 57,58 . The Na +/K +-ATPase also plays a role in the formation of intracellular negative membrane potential, providing the electrochemical driving force for apical Cl - secretion 57 .

One of the major causes of epithelial fluid secretion in ADPKD is the apical Cl - transport stimulated by cyclic adenosine monophosphate (cAMP) 59-61 . Diverse studies detected an increment of fluid secretion towards the cyst lumen and an increase in luminal electronegativity in response to cAMP enhancers 62 . Comparisons between cystic epithelium and properties of other secretory epithelia, in addition with studies where specific inhibitors were used to propose that the cystic fibrosis transmembrane conductance regulator (CFTR) channel could be the responsible for the cAMP- stimulated Cl - secretion 61,63-64 .

Several studies have reported CFTR expression in all tubular segments of the human kidney 65-67 . Moreover, cultures of ADPKD primary cells and ADPKD kidneys have also shown expression of this protein with apical membrane location 68 . Yet, some heterogeneity was observed, suggesting that other Cl - channels might be involved in fluid secretion 69-70 .

Diverse studies show that several nephron segments can release ATP into the lumen under the influence of certain stimuli , which leads to autocrine and paracrine signaling via the purinergic receptors expressed in the apical membrane of these cells 71 . Equivalent purinergic signaling has been documented in ADPKD cysts, where it could stimulate Cl - secretion towards the cysts, through CaCC activity 72,73 .

The anoctamin ion channel family (TMEM16) is constituted by 10 proteins which are all probably capable of mediating Ca 2+ activated Cl - currents 74,75 . Together with CFTR, these two proteins may be responsible for the secretion of fluid towards the cyst lumen.

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Figure 1.3. Model of the transporters and channels involved in fluid accumulation in ADPKD. Several transporters and channels are implicated in fluid accumulation in ADPKD cysts. Transepithelial Cl - secretion is supported by the basolateral cotransporter NKCC1, which requires basolateral K + (via KCa3.1) and Na + (via Na +-K +-ATPase) recycling, and occurs on the apical side through cAMP-stimulated (CFTR) and Calcium-activated Cl - channels, stimulated by purinergic signaling through P2Y and P2X receptors. Net secretion of Cl - drives water movement into the cyst facilitated by Aquaporins. Polycystin complex is involved in Ca 2+ signaling both in the primary cilium and intracellular stores. Retrieved and adapted from Terryn et al 76 .

1.5 CFTR and TMEM16A

Mutations in CFTR gene are the cause of Cystic Fibrosis (CF), the most typical and lethal autosomal recessive disease in Caucasians, characterized by an imbalance of ion and fluid homeostasis, which ultimately leads to impaired mucus production and airways infections 77 . The gene product of CFTR is a membrane glycoprotein functioning as a cAMP-regulated Cl- channel 78 .CFTR sequence is composed of 1480 amino acids residues and is symmetrically organized in two transmembrane domains (TMD1 and TMD2) and two nucleotide-binding domains (NBD1 and NBD2) separated by an R domain characteristic of the ABC family, from which the protein has evolved ( Fig. 1.4)79 . The protein is regulated by cAMP-dependent phosphorylation of serine residues in the R domain by protein kinase A (PKA) followed by ATP binding to the cytoplasmatic NBD, thus resulting in transepithelial Cl - transport 80,81 . It is expressed in the apical membrane of epithelial cells in several organs, including the kidney, namely on the proximal tubules (PT), thin limbs of the loop of Henle, distal tubules and collecting ducts 66 . CFTR has a functional interaction with other channels such as the epithelium sodium channel (ENaC or SCNN1) 82,83 , outwardly rectifying Cl - channels (ORCC) 84 and Calcium-activated Chloride Channels 85 .

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Figure 1.4. Scheme of CFTR structure. CFTR is located within the plasma membrane and is composed of two transmembrane domains (TMD1, TMD2), two NBD domains (NBD1, NBD2) and one R domain with multiple phosphorylation sites. Retrieved and adapted from UK Cystic Fibrosis Gene Therapy Consortium 86 .

CaCCs are anion-selective membrane ion channels, whose activation is dependent on a cytosolic increase of Ca 2+ levels 87 . These channels are present on a variety of cell types, namely epithelial cells88 and play diverse roles in the cell, including epithelial secretion 89 . CaCCs can be activated either through Ca 2+ influx through the plasma membrane or Ca 2+ release from the intracellular stores 88 .

TMEM16 constitutes a family of ten homologous proteins (TMEM16A-K) characterized by their highly conserved sequence, mostly around the putative pore-forming region 74 . Their function is not completely defined: some members are characterized as CaCCs, while others are described as Ca 2+ - activated scramblases of the lipid bilayer 90 . These proteins have a broad expression pattern being present in almost every cell type 91 . Their physiological role is related to epithelial Cl - secretion, volume regulation, neuronal excitability, among others 92 . Defects in TMEM16 are connected to several disorders such as the malformation of epithelial organs and bone or muscular dystrophy 74 . On the other hand, its overexpression is correlated with different kinds of cancer 74 .

The most well-studied member of the TMEM16 family is TMEM16A (T16A). This protein is located on the plasma membrane and is activated by an intracellular Ca 2+ increment 93,94 . Diverse studies showed that TMEM16A contributes to CaCC secretion in a variety of tissues 94 , including the kidney, where it plays a fundamental role in proximal tubules protein reabsorption and H + secretion 95 . TMEM16A is also described to conduct Ca 2+ -activated Cl - secretion and to stimulate proliferation on cystic epithelial cells 96 .

The most accurate structure of TMEM16A was recently determined in mouse (mTMEM16). mTMEM16A showed a homodimeric structure with both N- and C- termini located on the cytoplasmatic side of the plasma membrane. Each subunit contains ten transmembrane domains (TMD), an extracellular component, two Ca 2+ binding sites and an ion conduction pore ( Fig. 1.5 )97 .

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Figure 1.5. Representation of mTMEM16A architecture. One of the monomers is shown in grey. The cytosolic and extracellular domains are represented in yellow and red, respectively. The permeation pathway is displayed in green and the Ca 2+ ions are shown in pink. The rest of the protein is colored in blue. Retrieved and adapted from Falzone et al .97 .

1.5.1 Crosstalk between CFTR and TMEM16A

Studies have shown that P2Y purinergic receptors can couple to secondary messengers, namely 2+ 98 2+ cAMP and free intracellular Ca . Furthermore, [Ca ]i controls enzymes responsible for cAMP level regulation (adenylate cyclase and phosphodiesterases) 99 . Thus, a functional crosstalk between cAMP and Ca 2+ -activated Cl - secretion, and hence CFTR and TMEM16, has been suggested 100.

Benedetto et al .101 recently proved this functional overlap by showing an absence of CFTR- mediated Cl - secretion on airways and intestine of a Tmem16a knockout (KO) mice. TMEM16A was shown to enhance Ca 2+ release from ER store thus activating Store-Operated cAMP signaling, and Ca 2+ regulated adenyl cyclases. This functional dependence may also occur in the kidney, particularly on ADPKD patients where there is an upregulated apical Cl - secretion and impaired Ca 2+ signaling.

1.6 Regulation of Ca 2+ signaling by polycystins

It had been shown that both PKD1 and PKD2 play a key role in intracellular Ca 2+ homeostasis regulation, not only on the primary cilium (with the flow regulated Ca 2+ -sensing- described in section 1.2.1), but also on the plasma membrane and intracellular compartments 25 .

In the ER, polycystins are known to interact with the inositol 1,4,5-trisphosphate receptor (IP3R) 102,103 . IP3R is responsible for Ca 2+ release of ER internal store and it is activated by inositol- 1,4,5 trisphosphate (IP3). Intracellular IP3 accumulation is triggered through the binding of an agonist like Adenosine triphosphate (ATP) or Uridine Triphosphate (UTP) to a G protein-coupled receptor (GPCR), such as P2Y2 receptors. G protein will then bind to phospholipase C (PLC) triggering the cleavage of phosphatidylinositol-bisphosphate (PIP2) into IP3 and diacylglycerol (DAG). PKD2 can enhance Ca 2+ release from the ER by stimulating the activity of this receptor 104, whilst PKD1 suppresses this mechanism by a decrease on PKD2-IP3R interaction through a mechanism involving the stromal interaction molecule-1 (STIM1), responsible for sensing Ca 2+ levels inside the ER 105.

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PKD2 is also known to regulate the Ryanodine receptor, (RyR) responsible for ER Ca 2+ -induced Ca 2+ release 106. Furthermore, PKD2 is also able to regulate intracellular calcium on the plasma membrane and primary cilium by interacting with TRP channels, such as TRPC1 and TRPV4 107, 108 .

Consequently, it has been suggested that both PKD1 and PKD2 might regulate Ca 2+ signaling in different intracellular compartments, and its dysregulation is responsible for the activation of aberrant pathways present on ADPKD, such as the abnormal fluid secretion events. However, the precise nature of these regulatory events is not fully elucidated.

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2. Objectives

The main goal of the present work was to uncover the process of cyst development, namely the impaired Cl- secretion (by which ADPKD is characterized), mediated by CFTR and TMEM16A. For this, two different systems were adopted: a cell system (M1) and primary kidney cells isolated from renal cortex and medulla from mice with specific kidney knockout of Pkd1 or Pkd1 together with Tmem16a.

The first aim of this study was to assess if the induction of a cystic phenotype, through the knockdown of Pkd1 or Pkd2 in the M1 cell line, would alter epithelial ion transport. For this, several methodologies were employed. Transport properties were analyzed using differentiated cell monolayers on filter supports in Ussing chamber experiments and on the single-cell level using Patch Clamp and YFP-Quenching techniques.

Furthermore, results obtained with M1 cells were verified with freshly isolated renal cells. For this, we established primary cultures of kidney cells derived from renal cortex and medulla of mice with tissue-specific knockout of Pkd1 or Pkd1 and Tmem16a. Transport properties were measured as described for the M1 cell line.

As changes in intracellular Ca 2+ concentration regulate Ca 2+ activated Cl - channels, intracellular Ca 2+ levels were assessed by measurements with FURA-2 AM dye.

Better knowledge on polycystins physiological function may lead to a better understanding of impaired fluid secretion, a hallmark of ADPKD and could ultimately lead to the development of new therapeutic approaches.

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3. Materials and Methods

3.1 Cell Culture

Cell Culture was performed according to standard procedures using sterile equipment and solutions under a laminar flow cabinet. Cells were grown in 75 cm 2 cell culture flasks (Sarstedt AG &

Co. KG, Nümbrecht, Germany), and maintained in an incubator at 37° C in a 5% CO 2-95% air humidified water-saturated atmosphere. Cells were split every 3 to 5 days at 70-90% confluency and, if required, seeded in plastic plates (Sarstedt AG & Co. KG, Nümbrecht, Germany) with the proper density to execute experiments.

3.1.1 Mammalian Cell Lines and Culture Conditions

M1 cells 109 were cultured in Dulbecco’s Modified Eagle’s Medium/Ham’s Nutrient Mixture F12 (DMEM F12; Capricorn Scientific GmbH, Ebsdorfergrund, Germany), supplemented with 5% Fetal Bovine Serum (FBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 1% L-Glutamine 200mM (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), 1% Penicillin-Streptomycin (Pen Strep; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) and 1% Insulin-Transferrin-Selenium 100x (ITS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany).

M1 cells stably transfected with eYFP (Enhanced Yellow Fluorescent Protein) with Iodide increased sensitivity were cultured in DMEM F12 supplemented with 5% FBS, 1% Pen Strep,1% L- Glutamine 200mM, 1% ITS and 2,5 !g/mL Puromycin ( Thermo Fisher Scientific, Darmstadt, Germany).

Primary kidney cells were cultured in DMEM F12 supplemented with 1% FBS, 1% Pen Strep,1% L-Glutamine 200mM, 1% ITS 100x, 50 nM hydrocortisone(Sigma, Taufkirchen, Germany), 5 nM triiodothyronine (T3; Sigma, Taufkirchen, Germany) 5 nM Epidermal Growth Factor (EGF; Sigma Taufkirchen, Germany).

Primary kidney cells stably transfected with eYFP with Iodide increased sensitivity were cultured in DMEM F12 supplemented with 1% FBS, 1% Pen Strep,1% L-Glutamine 200mM, 1% ITS 100x, 50 nM hydrocortisone,5 nM T3,5 nM EGF and 2,5 !g/mL Puromycin. 110,111 Human Embryonic Kidney 293T cells (HEK 293T) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Capricorn Scientific GmbH, Ebsdorfergrund, Germany) supplemented with 10% FBS.

All cells were tested for mycoplasma infection, being mycoplasma free.

Cells were split using Trypsin. To trypsinize cells from flasks, cultured medium was first removed by aspiration and cells were washed with Ca 2+ and Mg 2+ free Dulbecco’s Phosphate Buffered Saline (PBS; Capricorn Scientific GmbH, Ebsdorfergrund, Germany). Trypsin (Life Technologies - gibco®, Karlsruhe, Germany) was added to culture flasks and incubated at 37°C, 5% CO2 for 10 min. To stop the trypsinization process, medium containing 1% FBS (Primary kidney cells and Primary kidney stable cells) or 5% FBS (M1 and M1-YFP cells) or 10% FBS (HEK 293T) was added to the cell suspension, which was then centrifuged at 20 000 rpm for 4 min. After centrifugation, the supernatant was discarded and cells were resuspended in fresh culture medium.

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3.1.2 Cell Culture on filter support

For assays which required polarized cells, specifically micro-Ussing Chamber, M1 and primary kidney cells were seeded into 12mm filter inserts with 0.4 !m Pore Polyester Membrane and 0,6 cm 2 surface area (Merck KGaA, Darmstadt, Germany). This type of inserts allow cells to polarize and, to acquire an apical and basolateral side mimicking physiological conditions. To achieve this purpose, cells were seeded on the apical side of the filter at a density of 3 x 105 cells per filter, using appropriate culture media (described in section 3.1.1). The filters were maintained in liquid-liquid interface. Cells took approximately 4-6 days to polarize.

The transepithelial electrical resistance (TER) was routinely measured using a Millicell-ERS volt- ohmmeter (Merck KGaA, Darmstadt, Germany). Experiments were performed after obtaining a stable TEER with levels above 600 "/cm2 .

3.1.3 Transient Transfections

All transient transfections were carried out using LipofectamineTM3000 transfection reagent (Invitrogen GmbH, Karlsruhe, Germany), according to manufacturer´s guidelines. Lipofection is a transfection methodology that takes advantage of the ability of cationic lipids to form unilamellar liposomes that adsorb molecules of nucleic acid (negatively charged). Use of this method allows downregulation of target genes, by small interfering RNA (siRNA), or protein expression by using plasmids.

PKD1 and PKD2 (M1 cells) and TMEM16A (M1-YFP cells) endogenous expression were downregulated using siRNAs (Ambion®, Darmstadt, Germany) allowing the downregulation of the respective encoded proteins PC1 and PC2. The decrease of expression was then confirmed by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Western Blot (WB). A negative control (scrambled; Ambion®, Darmstadt, Germany) with no sequence similarity for human, mouse or rat gene sequences was used to control the effects of the siRNA delivery. For patch clamp and single-cell YFP quenching measurements, all transfections were done using a siRNA containing PKD1, PKD2 or TMEM16A sequence linked to a CD8 receptor. Experiments and collection of RNA for RT-PCR and protein for WB were done between 24h and 72h after transfection.

3.1.4 Production of lentivirus and stable cell lines generation

Lentivirus with pLVX-puro were produced in HEK293T cells and both M1 and primary kidney cells were transduced with these viruses.

Firstly, HEK 293T cells were seeded on T75 flasks (diluted 1:6) and incubated overnight. Then, cells were transfected with 35 µg of DNA (16.66 µg of pLVX-puro-hsYFP-shScrambled or shPKD1 or shPKD2), 16.66 µg of plasmid pCMV-dR8.74psPAX2, 1.68 µg of envelop plasmid VSV- G/pMD2.G) with the methodology described in section 3.1.3. On the next day, the medium was changed to remove the transfection reagent and cells were incubated for another 30h. After the packaging, cells were discarded and the medium containing the lentiviruses was harvested, filtered with a 0,45µm filter and immediately used to transduce M1 and primary cells. Cells were infected with 7-8 mL of pLVX-puro-HS-YFP-shRNA virus with Hexamethrine bromide (Polybrene; Sigma- Aldrich, Taufkirchen, Germany) infection enhancer (8 µg/mL final concentration), the plates were incubated for 1h at room temperature (RT) and then incubated for 24h. The medium was changed to normal medium containing half of the concentration needed of antibiotic to kill all the non-infected cells (1.25 µg/mL of puromycin) and cells were incubated for 24h. After, the medium was changed

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and supplemented with 2.5 µg/mL of puromycin. Later, the medium was changed every 48h and cells were kept in culture.

3.2 Molecular Biology

3.2.1 cDNA and siRNA

All the cDNA and siRNA used and mentioned in this study are described in Appendix I .

3.2.2 RT-PCR

Expression of mRNAs encoding Cftr, Tmem16a Pkd1, Pkd2 and Scnn1 were examined in M1 and primary kidney cells by RT-PCR. Total RNA (1µg/ 50µL reaction) was reverse transcribed using a random primer (Promega, Manheim, Germany) and M-MLV Reverse Transcriptase RNase H Minus (Promega, Mannheim, Germany). Each RT-PCR reaction contained sense and antisense primer (0,5 µM) for Glyceraldehyde 3-phosphate dehydrogenase (Gapdh), Tmem16a, Pkd1, Pkd2, Scnn1 or for Cftr (see Appendix II, Table II.1 ), 0,5 µL cDNA and GoTaq Polymerase (Promega, Mannheim, Germany). RT-PCR program can be found in Appendix II, Table II.2 . PCR products were visualized by loading on ethidium bromide-containing agarose gels and analyzed using Meta Morph Vers. 6.2 (Molecular Devices, USA). As a positive control, a fraction of (GAPDH) was used. RT-PCR analysis was kindly provided by Prof. Dr. rer. nat. Rainer Schreiber.

3.3 Animal models

All experimental protocols were approved by the local councils for animal care and were conducted according to the German laws for animal care and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health.

Tamoxifen-injected animals were kindly provided by PD Dr. med. Björn Buccholz (Department of Nephrology and Hypertension, University of Erlangen-Nuremberg, Erlangen, Germany). Animal genotyping was confirmed by RT-PCR.

Two tissue-specific knockout animal models were used: Ksp Cre +/ PKD1 flox/flox and Ksp Cre +/ ANO1/PKD1 flox/flox . These mice had a specific knockout on renal tubules directed by a kidney specific cadherin promoter (Ksp) associated with a Cre expression cassette. This specific knockout required the administration of tamoxifen, a selective modifier of estrogen receptor, to induce recombinase activity.

3.3.1 Isolation of Primary Kidney cells from cortical preparation

Mice were killed by cervical dislocation after being exposed to CO2 and the kidney was removed and kept in ice-cold DMEM F12 medium on a petri dish (Thermo Fisher Scientific, Darmstadt, Germany). The capsule from the kidney was removed by squeezing, under the flow, and put in another drop of medium. With a sharp blade (Heinz Herenz,Hamburg,Germany) the cortex was cut off and chopped into smaller pieces, to facilitate the digestion in a small flask with 5 mL Hank’s Balanced Salt Solution (HBSS; (Life Technologies - gibco®, Karlsruhe, Germany) buffer (previously bubbled with 95% O2 and 5% CO2), 5 mL DMEM F12 and 10 mg Collagenase type 2 (Worthington, Lakewood, USA) then incubated for 40 min at 37 ° C with a magnetic stir. The digested tissue was passed through a 100µm cell strainer (Merck KGaA, Darmstadt, Germany) to a 50 mL falcon tube, pressed with the end of a syringe and washed out with ice-cold PBS. After centrifuged for 4 min at 5100rpm at 4 ° C,

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the pellet was re-suspended and a Percoll gradient of 45% Percoll (Ge Healthcare GmbH, Munich, Germany) and 55% 2X PBS-Glucose was run at 17500rpm for 30 min, at 4 ° C. The gradient resulted in different bands, being the second one from the bottom pipetted out and collected in a 50 mL falcon tube. After washing with ice-cold PBS three times (at 2200rpm, for 4 min at 4 °C), the pellet with isolated proximal tubules (PT) was re-suspended in 10mL of adequate media (described in section 3.1.1) in 75 cm 2 cell culture flasks and maintained in an incubator. After 24 hours, primary cells started to outgrow from the cultured tubules.

3.3.2 Isolation of Primary Kidney cells from medullary preparation

The primary medullary cells were isolated in a similar manner to the cortical preparation (described in section 3.3.1). The medulla was cut off, chopped and digested. After the digested tissue being passed by a 100 µm cell strainer, the Percoll gradient step was skipped and the tissue was centrifuged once for 4 min at 5100rpm at 4 ° C.Then the preparation was washed with ice-cold PBS and centrifuged three times (at 2200rpm, for 4 min at 4 °C). Subsequently, the pellet was re-suspend in 10mL of appropriate culture media (described in section 3.1.1) ) in 75 cm 2 cell culture flasks, and maintained in an incubator.

3.4 Analysis of protein expression and histology

3.4.1 Immunocytochemistry on primary kidney cells

TMEM16A and Acetylated tubulin protein expression were detected in primary cells ( Appendix III- Fig. III.3 ). For this, cells were washed once with PBS+ (PBS with 0.1mM Ca 2+ and 0.5mM Mg 2+) at RT. Then were fixed for 10 minutes with 4% Paraformaldehyde (PFA) solution at RT. Cells were rinsed three times with RT PBS+ to remove any leftover of the fixing solution and were incubated with 0,5% (v/v PBS+) Triton X-100 for 10 minutes at RT. After a second rinse for three times with RT PBS+, 1% (w/v PBS+) bovine serum albumin (BSA; Carl Roth Gmbh, Karlsruhe, Germany) was added and left in incubation for 40 min at RT. For co-localization of TMEM16A with primary cilium, the primary antibody rabbit anti-TMEM16A (with a dilution factor (DF) 1:300; Davids Biotechnologie, Regensburg, Germany) and mouse anti-acetylated tubulin (DF 1:300; Sigma, Taufkirchen,Germany) were incubated using PBS+ supplemented with 1% BSA 1h, at 37°C. Cells were once again washed three times with RT PBS+ and incubated with the secondary antibodies Alexa Fluor 488 conjugated Donkey Anti-Rabbit IgG (DF 1:300; Molecular Probes,Oregon, USA), Donkey Anti-Mouse (DF 1:300; Molecular Probes, Oregon,USA) and 0,1 µg/mL (v/v PBS+) Hoechst 33342 (DF 1:200; Aplichem, Darmstadt, Germany), for 1 h at RT. The cells were again rinsed three times with PBS+ and the coverslips were mounted with Fluorescence Mounting Medium (DAKO Cytomation, Hamburg, Germany).

Immunofluorescence was detected in a ApoTome Zeiss Axiovert 200M microscope (Zeiss, Oberkochen, Germany) equipped with ApoTome and Axio-Vision (Zeiss, Oberkochen, Germany).

3.4.2 Histology of renal cryosections

Cryosections were air dried for several minutes to remove moisture. Then, sections were stained with filtered 0,1% Mayers Hematoxylin (Sigma, Taufkirchen, Germany) for 10 minutes in a 50 mL conical tube. Afterwards, sections were rinsed in cool running double distilled water (ddH 2O) inside of a Coplin jar. Sections were then dipped in 0,5% Eosin for 12 times and dipped in dH 2O until eosin stops streaking. Later, sections were washed in Ethanol (EtOH) gradients progressively (50%-70%- 95%-100%) for 1 minute each. Finally, cryosections were dipped in xylene several times and slides

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ere cleaned with a kimwipe and mounted on a coverslip with Cytoseal XYL mounting media (Thermo Fisher Scientific, Darmstadt, Germany). Histology studies were kindly performed by Inês Cabrita

3.4.3 Western Blot

PKD1,PKD2 and TMEM16A expression was downregulated in M1 cells (see section 3.1.3 – Transient Transfections and 3.1.4- Production of lentivirus and stable cell line generation) and confirmed by WB. In order to accomplish this, protein was collected from cells and kept in a lysis buffer (mM: 50 Tris-HCl, 150 NaCl, 50 Tris, 100 DTT, 0.5% NP-40, 1% protease inhibitor cocktail) - Roche, Germany. Then, protein content was separated in a 7% SDS-PAGE Polyacrylamide gel and separated proteins were transferred to a nitrocellulose membrane (GE Healthcare Europe GmbH, Munich, Germany). To detect the protein, the membrane was incubated with primary antibodes (DF 1:400 – 1:1000) O/N at 4º C. Proteins were visualized using a suitable (HRP) conjugated secondary antibody (Dilution Factor of 1:10000) and ECL Detection Kit (GE, Healthcare, Munich, Germany). Protein bands were detected using a FujiFilm LAS-3000 (FujiFilm, Tokyo, Japan). Experiments were kindly performed by Silvia Materna-Reichelt.

3.5 Measurements of Ion channel activity and Ca 2+ signaling

3.5.1 Assesment of Cl - permeability through YFP Quenching assay

This methodology relies on the aptitude of a Yellow fluorescent protein to sense halide concentration on its environment. This protein can bind halide ions, namely Iodide (I -) and Chloride (Cl -) through its cavity with a specific binding site. When this ion species bind this cavity a quenching phenomenon occurs, leading to a decline of YFP fluorescence intensity. The use of this assay can provide insights about ion channel activity ( Fig. 3.2 ).

M1 cells and primary kidney cells were seeded in collagen/fibronectin 18 mm coated glass coverslips and then mounted in a chamber on a stage of an Axiovert S100 inverted microscope (Zeiss, Oberkochen, Germany). YFP fluorescence was detected using a Flua 20x objective (Zeiss, Oberkochen, Germany) and a high-speed polychromator system (VisiChrome, Visitron Systems, Puccheim, Germany), at an excitation wavelength of 485 nm, and emission 520-545 nm using a CCD- camera (CoolSnap HQ, Visitron Systems,Puccheim, Germany).

The bath was perfused at 37º C initially with Ringer 5mM Cl - solution (mmol: 140 NaCl; 0.4

KH 2PO 4; 1.6 K 2HPO 4.3H2O; 5 Glucose; 1 MgCl 2.6H 2O; 1.3 Ca-Gluconate.1H 2O; 5 Na-Gluconate) - - and posteriorly with Ringer 5mM Cl 5mM I (mmol: 140 NaCl; 0.4 KH 2PO 4; 1.6 K 2HPO 4.3H2O; 5

Glucose; 1 MgCl 2.6H2O; 1.3 Ca-Gluconate.1H 2O; 5 NaI).

The software package Meta-Fluor (Universal imaging, New York, USA) allowed imaging acquisition and data analysis.

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Figure 3.1. Scheme of Iodide-mediated YFP fluorescence quenching. When the cell is at a resting state, ion channels remain closed and I - cannot be transported across the plasma membrane. When these transporters are opened after some stimuli I- enters the cell binds to YFP and decreases (quenches) its fluorescence.

3.5.2 Whole-cell current measurements

Patch clamp allows the study of ionic currents on single cells. In order to achieve this, M1 and primary kidney cells were grown in collagen/fibronectin-coated 18 mm glass coverslips and M1 cells were transfected as described in section 3.1.3, in the presence of the CD8 receptor and downregulation was confirmed by Western Blot (Appendix III – Fig. III.1 ) and RT-PCR ( Appendix IV – Fig. IV.1 ). M1 cells were incubated 1-2 min with Dynabeads CD8 (Invitrogen, Karlsruhe, Germany), a step that allowed the identification of the transfected cells.

Afterwards, coverslips were mounted in a chamber on the stage of an inverted microscope (IM35, Zeiss, Germany) where the whole-cell current was measured. The bath was perfused continuously at 37°C initially with Ringer solution (mM: 145 NaCl; 0,4 KH2PO4; 1,6 K2HPO4 .3H2O; 5 Glucose; 1 MgCl2 .6H2O; 1,3 Ca-Gluconate .1H2O; pH 7.4). The patch pipette was filled with a solution containing 95 mM K-Gluconate, 30 mM KCl, 1.2 mM NaH2PO4 .H2O, 4.8 mM Na2HPO4 .2H2O, 5 mM Glucose, 2.38 mM MgCl2 .6H2O, 1 mM EGTA (Ethylene Glycol Tetraacetic Acid) and 0.209 mM Ca-Gluconate .1H2O, with freshly added 3 mM ATP and pH adjusted to 7.2.

Membrane voltage was clamped in steps of 20 mV from -100 to +100 mV from a holding voltage of -100 mV. Currents and voltages were recorded using a patch clamp amplifier EPC 10 and PULSE software (HEKA, Lambrecht, Germany), as well as a Chart software (AD-Instruments, Spechbach, Germany).

3.5.3 Measurements of transepithelial ion transport

Ussing chamber is an apparatus used to measure epithelial membrane properties, namely ion transport. This technique can be applied in a native tissue or in a monolayer of cells grown onto permeable support 112.

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In order to perform these experiments, M1 or primary kidney cells were seeded as described in section in 3.1.2 – Cells grown on filter support. Monolayers were mounted in modified perfused micro-Ussing chambers and analyzed under open-circuit conditions. CFTR and Anoctamins were activated with ATP/UTP 100 !M and 3-isobutyl-1-methylxanthine (IBMX) mixed with Forskolin (Fsk) 100 !M/2 !M respectively, under the presence of Amiloride (5 !M) a specific blocker of SCNN1.

The apical and basolateral surfaces of M1 and primary cells were continuously perfused with Ringer solution at a rate of 3-5 mL/min. pH was adjusted to 7.4 and the experiments were carried out at 37ºC under short circuit conditions.

Following a 15 min stabilization period, 5 !M Amiloride were added through t he entire experiment. Next, UTP, IBMX/Fsk and ATP were added at the apical side sequentially with a 5 minute washout period with Ringer solution between each one of them.

Transepithelial resistance (Rte) was determined by applying short (1 s) current puls es (I = 0.5 !A) and the corresponding changes in transepithelial voltage (Vte) were recorded continuously. The short- circuit currents (I SC ) were calculated by Ohm’s law from Vte and Rte (I SC = Vte/Rte).

3.5.4 Measurements of free intracellular Ca 2+ Signaling using FURA-2 AM

M1 cells and primary kidney cells were seeded in collagen/fibronectin 18 mm coated glass coverslips. Cells were then loaded with 2 !M Fura -2 AM (Molecular Probes, Oregon, USA) diluted in Ringer solution, in the presence of 0.02% Pluronic, (Molecular Probes, Oregon, USA) for 1h at RT. After loading, the coverslips were mounted in a cell chamber and perfused continuously with ringer solution.

Fura-2 AM is a membrane-permeable calcium indicator constituted by an ester group (AM- acetoxymethyl). When loaded into cells, the AM group is cleaved by esterases, allowing retention of the sensor in the cytosol.

In order to measure the intracellular concentration of Ca 2+ , Fura-2AM fluorescence was detected using an inverted microscope Axiovert S100 (Zeiss,Oberkochen, Germany) Flua 20x objective (Zeiss,Oberkochen, Germany) and a high speed polychromator system (VisiChrome, Visitron Systems,Puccheim, Germany), at an excitation wavelength of 340/380 nm, and emission 470-550 nm using a CCD-camera (CoolSnap HQ, Visitron Systems, Puccheim Germany).

The software package Meta-Fluor (Universal imaging, New York, USA) allowed imaging acquisition and data analysis. Intracellular Ca 2+ was calculated according to the 340/380 nm fluorescence ratio, after background subtraction and calibration. Experiments were kindly performed by Inês Cabrita

3.6 Statistical Analysis

For Statistical Analysis, student’s t-test (paired and unpaired samples) and analysis of variance (ANOVA) were used as suitable. Values were accepted as significant if p-value < 0.05.

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4. Results

4.1 Downregulation of Pkd1 induced a cystic phenotype which involves activation of Tmem16a

The first goal of this study was to assess the effect of Pkd1 and Tmem16a knockout (KO) on mouse kidney tissue. To achieve this goal, histology studies were performed and kidney cryosections from wildtype ( wt ), Pkd1 KO (PKD -/- ) and a Pkd1/Tmem16a KO (PKD -/- /T16A -/- ) mice were stained with Haematoxylin and Eosin.

As expected, the KO of Pkd1 was sufficient to induce a cystic phenotype on the kidney. Pkd1 KO cryosections showed clear tubular dilation, characteristic of ADPKD patients. Pkd1 downregulation also led to an increased kidney size. Notably, additional Tmem16a KO led to an attenuation of the previous parameters ( Fig. 4.1 ).

Figure 4.1. The absence of Tmem16a rescues cystic phenotype induced by KO of Pkd1. Haematoxylin and Eosin staining of kidney cryosections of wildtype ( wt ), Pkd1 KO and Pkd1/Tmem16a KO kidney-specific mouse models. Mice had the same gender (females), age (18 weeks) and were injected with tamoxifen at the same time (3 weeks old) to induce kidney-specific KO. Scale bar: 500µm.

4.2 Mouse Cortical Collecting Duct cell line as a model of ADPKD

The M1 cell line was chosen and characterized to further examine the impact of Pkd1, Pkd2, and Tmem16a in cyst development.

M1 cells derive from mouse cortical collecting duct, a region in which cyst development may start 113 . To understand if these cells are suitable as an in vitro model for ADPKD, expression of ADPKD related genes was investigated by RT-PCR. As shown in Fig. 4.2 , M1 cells expressed mRNA for Cftr, Tmem16a (T16a), Pkd1, and Pkd2. These cells also expressed Scnn1a subunit, a channel involved in Na + reabsorption in the collecting duct. Low mRNA expression of Scnn1b and Scnn1g subunits could be explained by the absence of corticosteroids in the culture media, necessary for upregulation of Scnn1 mRNAs ( Fig. 4.2 A, B ).

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A

B

Figure 4.2. M1 cells are a representative in vitro model for ADPKD. A) Expression of kidney epithelial ion channels (Cftr, Tmem16a, and Scnn subunits) as well as polycystins (Pkd1 and Pkd2) were assessed by RT-PCR. B) mRNA quantification relatively to Gapdh. (Values are mean ± SEM, number of replicates is shown between brackets).

4.2.1 Polarized M1 cells produce cAMP-stimulated and CaCC short-circuit currents

To investigate electrolyte transport properties in conditions that would mimic the physiological environment of the kidney epithelium, Ussing chamber experiments were performed using M1 cells grown on filter supports. Addition of amiloride (20µM), a specific SCNN1 blocker, to the apical side of the cell monolayer did not lead to a significant transepithelial voltage (V te ) deflection, which indicates an absence of SCNN1 activity ( Fig. 4.3A, B ). Activation of purinergic receptors (100µM

UTP on the apical side) induced firstly a positive deflection of V te , which could indicate an activation + of K channels. Afterwards, a transient decrease of V te followed by a plateau, characteristic of CaCC, was observed. The short-circuit current (I SC ) of the transient part was calculated ( Fig. 4.3A, B ).

Activation of adenylyl cyclase by Forskolin (2µM) and inhibition of phosphodiesterases by IBMX

(100µM) increased cAMP levels and led to a two-phase response which resulted in a V te deflection and generated an steady-state I sc , equivalent to the one generated by purinergic stimulation, through the activation of CFTR ( Fig. 4.3A, B ).

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A B

Figure 4.3. M1 cells show cAMP and CaCC dependent electrolyte transport. M1 cells were grown on filter support and mounted on micro Ussing chamber under open circuit conditions. A) Original recordings are shown after apical stimulation with 20µM amiloride,100 µM UTP and IBMX/ Forskolin (IF: 100µM/2 µM). B) Calculated short-circuit currents from experiments shown in A). (Values are mean ± SEM; the number of filter inserts is shown between brackets).

4.2.2 Downregulation of Pkd1/2 enhances basal and CaCC currents.

To determine if the induction of a cystic phenotype leads to enhanced secretion, M1 cells were transfected with a ”scrambled” non -targeting siRNA, or siRNAs targeting Pkd1 or Pkd2, and whole- cell currents were measured using patch clamp.

First, we aimed to understand if the knockdown of endogenous Pkd1 or Pkd2 leads to higher basal currents (without any stimulation or inhibition). Whole-cell current measurements showed that cells treated with siPkd1 or siPkd2 had an enhanced basal current when compared to cells treated with scrambled siRNA ( Fig .4.4 A-C ).

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Secondly, we asked if this enhanced baseline was due to an increase in Cl - conductance, mimicking one of the hallmarks of ADPKD: impaired fluid secretion. For this purpose, Ringer Cl - concentration was reduced from 145 mM to 5 mM (5 Cl -). As seen in Fig. 4.4 , the reduction in Cl - concentration abolished the current increase observed in cells treated with siRNAs targeting Pkd1 or Pkd2 .

To determine if this enhanced Cl - conductance could be explained by a higher activity of CaCC, cells were perfused with CaCC inhibitor AO1 (10µM). Cells treated with siRNAs targeting Pkd1 or Pkd2 showed a significantly higher inhibition of the baseline current by AO1 compared to control (Fig. 4.4 A,B, and E ). A B C

D

E

Figure 4.4. Knockdown of Pkd1/Pkd2 leads to a higher whole-cell current at basal state. A) Current overlays from basal current and respective inhibition with 5Cl - and AO1 in M1 cells (10µM). Knockdown of endogenous Pkd1 or Pkd2 leads to an enhanced baseline and a higher inhibition by 5Cl - and AO1. B) Current-voltage (I/V) curves from experiments displayed in A). C) Basal current densities D) Correspondent delta current densities (stimulated current density-basal current density) after cells were perfused with a 5Cl - solution. E) Correspondent delta current densities after cells were treated with 10 µM AO1. (Values are mean ± SEM; # paired t-test to basal current densities § unpaired t-test to scr cells; p-value < 0.05; the number of cells is shown between brackets).

Next, we investigated the activation of CFTR and TMEM16A by an increase of intracellular cAMP or intracellular Ca 2+ respectively, through whole-cell patch clamp experiments. Cells were stimulated using 100µM UTP or ATP. Interestingly, cells with knockdown of endogenous Pkd1 or Pkd2 showed higher Cl - secretion. These results were confirmed using Ionomycin, an ionophore which raises the intracellular Ca 2+ concentration. After addition of 2µM Ionomycin, whole-cell currents of siPkd1 or siPkd2 treated cells were significantly higher ( Fig. 4.5 ).

During cyst formation, apical Cl - secretion is mediated mainly through CFTR. Therefore, we investigated if the knockdown of Pkd1 or Pkd2 leads to enhanced currents after cAMP increase by stimulating the cells with IBMX and Forskolin (IF; 100 µM/ 2 µM). Unexpectedly, cells with lower

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expression of Pkd1 and Pkd2 showed a reduction of cAMP-stimulated current when compared to cells treated with scrambled siRNA.

A B C

Figure 4.5. Knockdown of Pkd1 or Pkd2 leads to enhanced CaCC and diminished CFTR currents. A) Current overlays obtained after stimulation with UTP, ATP, Ionomycin and IF in M1 cells. Knockdown of endogenous Pkd1 and Pkd2 leads to enhanced CaCC secretion and reduced CFTR Cl - transport. B) Current-voltage (I/V) curves from experiments displayed in A). C) Correspondent current densities from experiments shown in A). (Values are mean ± SEM; # paired t- test to basal conditions § unpaired t-test to scrambled cells; p-value < 0.05; the number of cells is shown between brackets).

4.2.3 Knockdown of Pkd1 increases Tmem16a-dependent I - uptake

Using the YFP-Quenching assay, it was possible to compare iodide (I -) permeability between scrambled M1 cells and cells with downregulation of endogenous Pkd1 or Pkd2 expression. M1 cells were stably transfected with halide sensitive YFP and with non-targeting scrambled shRNA or targeting endogenous Pkd1 or Pkd2 (see section 3.1.4). Downregulation was confirmed by WB (Appendix III- Fig. III.2 ), and the YFP fluorescence assessed using fluorescence microscopy (Appendix V- Fig. V.1 ). Cells were perfused with Ringer (145mM Cl -), followed by a solution in which 5mM Cl - was replaced by gluconate to keep the Cl- concentration constant during the experiment. To detect a basal I - conductance, gluconate was replaced by 5mM I -. The I - uptake,

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measured by the initial slope of YFP fluorescence quenching, was significantly higher after downregulation of endogenous Pkd1 or Pkd2 when compared to cells transfected with scrambled shRNA ( Fig. 4.6A, B ).

In order to activate CaCC, cells were stimulated with purinergic receptor agonists (ATP/UTP) applied together with the 5mM I - solution. Notably, cells with lower Pkd1 expression showed an enhanced I - permeability through activated CaCC either with ATP (100 µM) or UTP (100 µM) stimulation ( Fig. 4.6 A, B ).

Additionally, CFTR was stimulated using IBMX/Forskolin (100µM/2 µM). Cells with lower Pkd1 expression showed an enhanced I - uptake. However, the I - uptake was less when compared to purinergic stimulation ( Fig. 4.6 A, B ).

A

B

Figure 4.6. Knockdown of Pkd1 leads to enhanced Ca2+ and cAMP induced I- uptake. A) Original tracings showing YFP fluorescence quenching using 5 mM I -, 5 mM I - + 100 µM ATP, 5 mM I - + 100 µM UTP or 5 mM I - + 100/2 µM IF. B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -). (Values are mean ± SEM;# unpaired t-test to basal quenching, § unpaired t-test to scrambled-YFP cells, & unpaired t-test to shPkd1-YFP cells; p-value < 0.05; the number of cells is shown between brackets).

Inhibition of TMEM16A and CFTR by CaCC inhibitor AO1 (10 µM) and CFTR inhibitor 172 - (CFTR inh 172;20 µM), respectively, strongly reduced the I uptake. These data confirmed that TMEM16A and CFTR were the main players in the observed I - permeability ( Fig. 4.7 A, B and Fig. 4.7 A, C , respectively).

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A

B C

Figure 4.7. I - uptake is due to CaCC and CFTR activation. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5 mM I - + 100 µM UTP + 10 µM AO1 and 5mM I - + 100/2 µM IF + 20 µM CFTRinh172 in M1 cells. B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) using CaCC inhibitor. C) or CFTR inhibitor 172. (Values are mean ± SEM; # unpaired t-test to basal quenching, § unpaired t-test to

25

scrambled-YFP cells, & unpaired t-test to shPkd1-YFP cells, A unpaired t-test to non-inhibited cells; p-value < 0.05; the number of cells is shown between brackets).

Subsequently, Tmem16a expression was downregulated by siRNA transfection (see section 3.1.3- Transient transfections). A reduction of I - permeability was observed in cells with downregulated Pkd1 under basal conditions and also after stimulation with UTP, ATP or IBMX/ Forskolin. These results suggest that Tmem16a is the main responsible channel for the increase of I - permeability in these cells (Fig. 4.8A, B ). Tmem16a is also responsible for CaCC activation in scrambled and shPkd2 cells ( Fig. 4.8A, B).

A

B

Figure 4.8. Downregulation of Pkd1 led to increased I - permeability dependent on Tmem16a. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5mM I - , 5 mM I - + 100 µM ATP, 5 mM I - + 100 µM UTP and 5 mM I - + 100/2 µM IF. B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application I -) Initial slope (10 secs.) quantification of YFP fluorescence of experiences shown in A). (Values are mean ± SEM;# unpaired t-test to basal quenching, § unpaired t-test to scrambled-YFP cells, & unpaired t-test to shPkd1-YFP cells, A unpaired t-test to non- treated cells (without siTmem16a); p-value < 0.05; the number of cells is shown between brackets).

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4.2.4 Downregulation of endogenous Pkd1 or Pkd2 upregulates intracellular Ca 2+ signaling in M1 cells

To investigate the impact of Pkd1 or Pkd2 expression on the regulation of intracellular Ca 2+ 2+ concentration ([Ca ]i), M1 cells were stably transfected with scrambled shRNA, or with shRNA targeting Pkd1 or Pkd2.

2+ The basal [Ca ]i was markedly increased in downregulated Pkd1 cells and also, at a lower extent, in downregulated Pkd2 cells. Simultaneous downregulation of Tmem16a with Pkd1 or Pkd2 decreased 2+ levels of [Ca ]i and became similar between the different genotypes ( Fig. 4.9A, C ).

2+ Secondly, [Ca ]i was measured after stimulation with 100 µM ATP. As previously described, ATP induces Ca 2+ release from the ER stores (peak) followed by Ca 2+ influx (plateau). Downregulation of Pkd1 or Pkd2 led to a higher Ca 2+ peak after addition of a purinergic agonist. When 2+ cells were treated with siTmem16a, ATP-induced [Ca ]i was substantially decreased but remained higher in the cells with less expression of Pkd1 and Pkd2 compared to control. Downregulation of endogenous Pkd1 or Pkd2 led to an enhanced Ca 2+ influx represented by the plateau phase ( Fig. 4.9A,B ).

Ca 2+ free conditions led to a movement of this ion from the cytoplasm towards the extracellular 2+ 2+ media. [Ca ]i decrease was stronger in downregulated Pkd1 and Pkd2 cells. The Ca concentration of 2+ the ER stores ([Ca ]ER ) could be indirectly assessed by inhibition of the Sarco/Endoplasmatic reticulum Ca 2+ -ATPase (SERCA pump) by cyclopiazonic acid (CPA), which reveals ER Ca2+ leakage in the absence of extracellular Ca 2+ . CPA-induced Ca 2+ leakage from the ER stores was enhanced in Pkd1 or Pkd2 knockdown cells suggesting higher ER Ca 2+ concentration in these cells ( Fig. 4.9 D, E ).

2+ 2+ Stromal interaction molecule (STIM1) is a Ca sensor localized in the ER. If [Ca ]ER is diminished, STIM1 interacts with Ca 2+ release-activated Ca 2+ channel protein 1 (Orai1) in the plasma membrane, leading to a Ca 2+ uptake and refilling of the ER. This process is called Store-Operated Calcium Entry (SOCE) 114. When Orai1 and STIM1 were inhibited (by YM58483 and SK&F96365, respectively) a reduction of the CPA induced Ca 2+ influx was observed, indicating a reduction of SOCE ( Fig. 4.9 F-I ).

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A B C

D E

F G

H I

Figure 4.9. Ca 2+ signaling is upregulated with the knockdown of Pkd1 or Pkd2 and Tmem16a plays a 2+ fundamental role in this process. A) Original tracings showing upregulated basal and purinergic stimulated [Ca ]i in shPkd1 and shPkd2 cells as well as a general decrease when Tmem16A is downregulated in M1 cells. B) Respective 2+ quantification of ATP induced peak and plateau shown in A). C) Quantification of basal [Ca ]i represented in A). D) Original tracings displaying a higher ER and cytosolic [Ca 2+ ] on cells with Pkd1 and Pkd2 knockdown, being these parameters decreased when Tmem16A is downregulated. E) Quantification of data displayed on image D). F) Original tracings showing a reduction on ER Ca 2+ content as well as influx when cells are treated with an inhibitor of Orai1. G) Summary of data displayed in F). H) Original tracings showing a reduction on ER Ca 2+ content as well as influx when cells are treated with an inhibitor of STIM1. I) Summary of data displayed in H). (Values are mean ± SEM; # unpaired t-test to scrambled, § unpaired t-test to shPkd1, unpaired t-test to control conditions, p-value < 0.05; the number of cells is shown between brackets).

4.3 Isolation of primary kidney cells from the renal medulla and renal cortex

To confirm the results obtained from the M1 cell line, we cultured primary cells from the renal cortex and medulla. Primary cells were isolated from kidneys of wt mice to be used as a control, from tissue-specific Pkd1 knockout mice, and finally, from tissue-specific Pkd1/Tmem16a double knockout mice, which allows the study of the role of CaCC in the disease.

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Expression of ADPKD related genes and KO validation was assessed by RT-PCR ( Fig. 4.10 and Appendix IV – Fig. IV.2 ). It was possible to observe that both cortical and medullary preparations expressed epithelial ion channels involved in Cl - secretion (Cftr and Tmem16a) as well as Scnn1. Both cell types also expressed Pkd1 and Pkd2. Interestingly, cortical cells showed a higher expression of Tmem16a mRNA. Scnn1 expression was more pronounced on the medullary preparation (Appendix IV – Fig. IV.3 ) which may be explained by the high number of principal cells 115. ´ B A

cortical

D C

medullary

Figure 4.10. mRNA expression from Primary kidney cells from renal cortex or medulla. A) Expression of mRNAs from cortical primary kidney cells encoding for Tmem16a, Scnn1 subunits, Cftr, Pkd1, and Pkd2. Gapdh was used as a positive control. B) Quantification of mRNA levels relative to Gapdh. C) Expression of mRNA from medullary primary kidney cells encoding for Tmem16A, Scnn1 subunits, Cftr, Pkd1 and Pkd2. D) Quantification on mRNA levels relative to Gapdh . (Values are mean ± SEM; # unpaired t-test to cortical cells, p-value < 0.05; the number of replicates is shown between brackets).

4.4 Characterization of primary cells isolated from the renal cortex

4.4.1 Polarized primary cortical cells did not show increased secretion after induction of a cystic phenotype

In order to study electrolyte transport, primary kidney cells were grown on filter supports and mounted in a micro-perfused Ussing Chamber. 5µM Amiloride was added to inhibit Na+ uptake throughout the experiment.

+ A clear SCNN1-mediated Na transport occurred, as shown by the transient and steady-state V te increase after blocking this channel with amiloride. A higher I SC was generated on Pkd1 KO (PKD1-/-) compared to wt which was further augmented on Pkd1/Tmem16a KO (PKD1-/-/T16A-/-) ( Fig. 4.11 A,´B ).

Next, activation of CaCC was assessed by adding 100µM UTP/ATP. Surprisingly, cells isolated from Pkd1 KO mice showed an attenuated voltage deflection upon purinergic receptors stimulation. This decrease was unexpectedly corrected on the cells isolated from the double KO mice ( Fig. 4.11 A, B).

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Lastly, cAMP-induced ion transport was measured by stimulating the cells with IBMX/Forskolin

(100/2µM). Pkd1 KO showed a smaller I SC relatively to the other cells ( Fig. 4.11 A, B ). Interestingly, the knockout of Pkd1 led to a stronger transepithelial resistance, which indicates that Pkd1 may affect the composition of the tight junctions ( Fig. 4.11 C ).

A

B C

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Figure 4.11. Pkd1 KO did not induce a secretory phenotype in polarized primary cortical kidney cells. A) Original Ussing chamber recordings showing deflections on V te and R te after application of Amiloride (5 µM) UTP (100 µM) ATP (100 µM) and IBMX/Forskolin (100/2 µM) in cortical primary kidney cells. B) Summary of the calculated equivalent short circuit currents after application of each compound. C) Representation of transepithelial resistance of each animal model. (Values are mean ± SEM; # unpaired t-test to wt , $ unpaired t-test to Pkd1 KO, p-value < 0.05; the number of animals/ number of filter inserts is shown between brackets).

4.4.2 Primary Pkd1 KO cortical cells had a higher basal and purinergic stimulated conductance, for which Tmem16a is essential

In order to study the ion current properties of primary cells isolated from the renal cortex, whole- cell currents were measured using Patch Clamp. Pkd1 KO led to an enhanced baseline current for which Tmem16a did not contribute ( Fig. 4.12 A-C ). When cells were stimulated with 100 µM UTP, Pkd1 KO showed higher currents when compared to wt . However, Pkd1 and Tmem16a KO cells displayed a strongly reduced UTP generated current, highlighting the importance of Tmem16a on Cl - transport upon purinergic receptors stimulation ( Fig. 4.12 A-C ).

A

B

C

Figure 4.12. Pkd1 KO leads to a higher basal current and CaCC activation, for which Tmem16a is essential. A) Current overlays obtained showing basal and activated current after UTP stimulation in cortical primary kidney cells. B)

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Current/ voltage (I/V) curves for the described experiment showing an upregulated response to UTP on Pkd1 KO, abolished with the additional KO of Tmem16a. C) Summary of the delta current densities (current density after stimulation – current density at basal state). (Values are mean ± SEM; # unpaired t-test to wt , § unpaired t-test to Pkd1 KO, p-value < 0.05; the number of cells is shown between brackets).

4.4.3 Pkd1 KO in cortical primary cells enhances I - uptake through CaCC

I- permeability was assessed in primary cortical cells using the YFP Quenching assay. Cells were transfected with a YFP plasmid conjugated with scrambled shRNA (see section 3.1.4). YFP fluorescence was measured using fluorescence microscopy ( Appendix V- Fig. V.2A ).

Pkd1 KO did not affect the basal I - uptake. On the other hand, Tmem16a reduced the basal I - influx in these cells ( Fig. 4.13 A, B ). When cells were stimulated with 100 µM UTP, Pkd1 KO cells presented larger I - permeability, which was largely inhibited in Pkd1 and Tmem16a KO cells ( Fig. 4.13 A, B ). Next, cells were stimulated with 100 µM IBMX and 2 µM Forskolin (IBMX/Fsk). Remarkably, Pkd1 KO cells showed a significantly increased I - permeability upon IBMX/Fsk stimulation, when compared to wt cells. Additional Tmem16a KO abolished this upregulated I - influx by Pkd1 KO ( Fig. 4.13 A, B ).

A

B

Figure 4.13. Pkd1 KO leads to enhanced Ca 2+ and cAMP induced I - uptake. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5 mM I -, 5 mM I - + 100 µM UTP or 5 mM I - + 100/2 µM IF B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) (Values are mean ± SEM; # paired t-test to 5mM I -, § unpaired t-test to wt scr-YFP, & unpaired t-test to Pkd1 KO scr-YFP; number of animals/ number of cells is shown between brackets).

Afterwards, the same experiment was performed using TMEM16A or CFTR inhibitors. When AO1 was applied together with UTP, quenching was drastically reduced, showing that this I - is

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dependent on Tmem16a. I - uptake was not significantly reduced on the Pkd1 and Tmem16a KO primary cells which confirms that Tmem16a is mainly responsible for the UTP-induced I - permeability - (Fig. 4.14A, B ). Also, when CFTR specific inhibitor (CFTR inh 172) was applied, CFTR-mediated I transport was reduced, leading to a similar I - uptake in all cell types ( Fig. 4.14 A, C ).

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A

B C

34

Figure 4.14. I - uptake occurs due to CaCC and CFTR activation. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5 mM I -, 5 mM I - + 100 µM UTP + 10 µM AO1 or 5 mM I - + 100/2 µM IF + 20 µM CFTRinh172 B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) after application of AO1 C) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) after application of CFTRinh172 (Values are mean ± SEM; # paired t-test to 5mM I -, § unpaired t-test to wt scr-YFP, & unpaired t- test to Pkd1 KO scr-YFP, A unpaired t-test to non-inhibited cells; the number of animals/ number of cells is shown between brackets).

4.4.4 In primary cortical cells Pkd1 KO upregulates Ca 2+ signaling, in a Tmem16a- dependent manner

2+ As performed for M1 cells, basal [Ca ]i of primary cortical cells was first assessed. Pkd1 KO led to an increase in [Ca 2+ ]i, which was inhibited in Pkd1 and Tmem16a KO cells. This indicates that the 2+ upregulated basal [Ca ]i is dependent on Tmem16a ( Fig. 4.15 A, C ).

2+ [Ca ]i was then detected after stimulation with 100 µM ATP. Pkd1 KO cells showed a 2+ considerably higher [Ca ]i peak. Notably, additional KO of Tmem16a rescued the cells from this upregulated Ca 2+ release. ATP-induced Ca 2+ plateau has higher in Pkd1 and also Pkd1/Tmem16A KO cells ( Fig. 4.15 A, B ).

2+ 2+ Afterwards, [Ca ]ER was examined. Pkd1 KO cells presented an augmented CPA-induced Ca leakage. In the double KO this effect was not observed. After removal of Ca 2+ free solution, only the Pkd1 KO presented a higher CPA-induced Ca 2+ influx compared to the wt ( Fig. 4.15 D, E . When SOCE was inhibited by Orai1 or STIM1 inhibitors, CPA-induced ER Ca 2+ leakage was reduced, as well as Ca 2+ influx ( Fig. 4.15 F-I) .

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A B C

D E

F G

H I

2+ 2+ Figure 4.15. KO of Pkd1 leads to Ca signaling upregulation. A) Original tracings showing [Ca ]I at basal level and after stimulation with 100 µM ATP. B) Summary showing higher ATP stimulated Ca 2+ release on cells isolated from the 2+ 2+ KO mouse models as well as a bigger Ca plateau. C) Summary showing higher basal [Ca ]i on the cells isolated from both KO animals. D) Original tracings show higher ER Ca 2+ content and cytosolic refill on cells from Pkd1 KO mouse model. E) Summary of data shown in D). F) Original tracings showing a reduction of the ER Ca 2+ content as well as the cytosolic refill when cells are treated with Orai1 inhibitor. G) Summary of data displayed in F). H) Original tracings showing reduction of the ER Ca 2+ content when cells are treated with STIM1 inhibitor I) Summary of data displayed in H. (Values are mean ± SEM; # unpaired t-test to wt , § unpaired t-test to Pkd1 KO, unpaired t-test to control conditions, p-value < 0.05; number of animals/ number of cells is shown between brackets).

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4.6 Primary Kidney Cells isolated from medullary preparations

4.6.1 Pkd1 KO enhances CaCC activity in primary medullary cells

Electrolyte transport properties were investigated in polarized primary cells isolated from kidney medulla. Apical addition of 5 µM Amiloride led to a decrease in V te , showing that these cells have a clear Na + absorption via SCNN1 (Fig. 4.16 A, B ). Next, cells were stimulated with 100 µM UTP or ATP on the apical side. Pkd1 KO cells showed an increased Cl - secretion when stimulated with UTP, which was cancelled in Pkd1/Tmem16a KO cells ( Fig. 4.16 A, B ). Lastly, cAMP-stimulated Cl - secretion was activated by IBMX/Forskolin (100/2 µM). Similar I sc were obtained for primary cells isolated from the 3 animal models ( Fig. 4.16 A, B ). The absence of Pkd1 had a contrary effect on tight junctions resistance when compared to cortical primary cells. Here, Pkd1 seemed essential to generate stronger tight junctions ( Fig. 4.16 C ).

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A

B C

Figure 4.16. Pkd1 KO lead to enhanced Tmem16a activation, and reduced epithelium resistance. A) Original recordings of transepithelial voltage showing the effect of Amiloride (5µM), UTP (100 µM), ATP (100 µM) and IBMX/ Forskolin (100/2 µM) in primary medullary cells B) Summary of Amiloride, UTP, IBMX/Forskolin and ATP induced I sc . (Values are mean ± SEM; # unpaired t-test to wt , § unpaired t-test to Pkd1 KO, p-value < 0.05; the number of animals/ number of filter inserts is shown between brackets).

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4.6.2 Pkd1 KO in medullary primary cells induces an enhanced secretory phenotype that is rescued in the absence of Tmem16a

Whole-cell currents were measured by patch clamp. The basal currents seemed to be Tmem16a-dependent since cells isolated from the double KO mouse model presented a significantly smaller basal current ( Fig. 4.17 A, B ).

After stimulation with 100 µM UTP, Pkd1 KO cells produced higher currents than the wt cells. This UTP-induced response was mainly due to Tmem16a, as shown by the almost inexistent activation in Pkd1/Tmem16a KO cells ( Fig. 4.17 A-C ).

A

B

C

Figure 4.17. Tmem16a is essential for both basal and purinergic stimulated currents. A) Current overlays showing basal and UTP stimulated currents in medullary primary cells. B) IV curves showing an enhanced response to UTP on Pkd1 KO cells ablated when Tmem16a is also KO. C) Summary of delta current densities (current density after stimulation – basal current densities). (Values are mean ± SEM; # unpaired t-test to wt , $ unpaired t-test to Pkd1 KO, p-value < 0.05; the number of cells is shown between brackets).

4.6.3 Medullary cells with Pkd1 KO have an enhanced I - permeability after cAMP and purinergic stimulation for which TMEM6A and CFTR are essential.

To assess I - permeability, medullary primary cells were transfected with a YFP plasmid conjugated with a scrambled shRNA (see section 3.1.4), as previously described for cortical cells. YFP fluorescence was measured using fluorescence microscopy ( Appendix V- Fig. V.2B ).

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At basal conditions, I - uptake was similar in wt and Pkd1 KO cells. However, I - influx was reduced in Pkd1/Tmem16a cells, suggesting that Tmem16a had an important role in basal secretion ( Fig. 4.18 A, B ). CaCC activity was stimulated by 100 µM UTP. It was possible to observe that I - uptake was enhanced in Pkd1 KO cells, being this effect drastically diminished in Pkd1/Tmem16a KO cells ( Fig. 4.18 A, B ). Lastly, cAMP-stimulated quenching was investigated using IBMX/Fsk (100/2µM). Pkd1 KO cells showed a higher I - permeability when compared to wt cells, that was not dependent on Tmem16a ( Fig. 4.18 A, B ).

A

B

Figure 4.18. Pkd1 KO leads to enhanced Ca 2+ and cAMP induced I - uptake. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5 mM I -, 5 mM I - + 100 µM UTP or 5 mM I - + 100/2 µM IF B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) (Values are mean ± SEM; # paired t-test to 5mM I -, § unpaired t-test to wt scr-YFP, & unpaired t-test to Pkd1 KO scr-YFP; number of animals/ number of cells is shown between brackets).

The same experiments were then performed using CaCC and CFTR specific inhibitors. UTP- stimulated quenching was abolished with CaCC-A01 addition. However, Pkd1 KO cells still presented a significant I - permeability, indicating that UTP-induced I - movement did not occur exclusively through CaCC. cAMP-stimulated quenching was inhibited in Pkd1 and Pkd1/Tmem16a KO cells in the presence of CFTRinh172 ( Fig. 4.19 A, B ).

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A

B C _

Figure 4.19. I - uptake occurs due to CaCC and CFTR activation. A) Original tracings showing I - uptake by YFP fluorescence quenching using 5 mM I -, 5 mM I - + 100 µM UTP + 10 µM AO1 or 5 mM I - + 100/2 µM IF + 20 µM CFTRinh172 B) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) after application of AO1 C) Summary of Initial I - uptake (by Slope measurement during the first 10s after application of I -) after

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application of CFTRinh172 (Values are mean ± SEM; # paired t-test to 5mM I -, § unpaired t-test to wt scr-YFP, & unpaired t- test to Pkd1 KO scr-YFP, A unpaired t-test to non-inhibited cells; the number of animals/ number of cells is shown between brackets).

4.6.4 In primary medullary cells, Ca 2+ signaling is upregulated by Pkd1 KO in a Tmem16a-dependent manner

2+ As observed in cortical cells, Pkd1 KO increased basal [Ca ]i, which was inhibited in the double KO cells ( Fig. 4.20 A, C ).

2+ Next, to induce IP 3-mediated ER store Ca release, cells were stimulated with 100 µM ATP. Pkd1 KO cells presented a higher Ca 2+ peak after ATP stimulation, which was abolished in Pkd1/Tmem16a KO cells. In comparison to wt cells, cytosolic Ca 2+ influx, represented by the plateau, was augmented in Pkd1 KO cells and decreased in Pkd1/Tmem16a KO cells ( Fig. 4.20 A, B ).

2+ Afterwards, [Ca ]ER was investigated. Primary Pkd1 KO medullary cells showed enhanced ER 2+ 2+ Ca leakage. However, Pkd1/Tmem16a KO cells presented a smaller [Ca ]ER , comparatively to both wt and Pkd1 KO cells. Primary cells from both KO models show higher Ca 2+ influx, specially Pkd1 KO cells ( Fig. 4.20 D, E ). When the previous protocol was performed in the presence of Orai1 inhibitor, both the CPA-induced ER Ca 2+ leakage and influx parameters were diminished( Fig. 4.20 F, G). Notably, STIM1 inhibition only depressed the CPA-induced Ca 2+ ER leakage on cells from the Pkd1 KO mouse. Furthermore, the CPA-induced Ca 2+ influx was affected in primary cells originated from all the animal models ( Fig. 4.20 H, I ).

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A B C v v

D E v v

F G v v

H I v v

2+ 2+ Figure 4.20. Pkd1 KO leads to intracellular Ca signaling deregulation. A) Original recordings showing [Ca ]i at basal level and after stimulation with 100 µM ATP. B) Summary showing peak and plateau of Ca 2+ levels after ATP 2+ 2+ 2+ stimulation. C) Summary showing basal [Ca ]i. D) Original recordings showing effects on [Ca ]i after application of Ca 2+ free and CPA. E) Summary of the experiment described in D) F) Original recordings showing effects on [Ca ]i after application of Ca 2+ free and CPA on cells treated with Orai1 inhibitor (YM 58483). G) Summary of the experiment shown in 2+ 2+ F) H) Original recordings showing effects on [Ca ]i after application of Ca free and CPA on cells treated with STIM1 inhibitor (SKF96365). I) Summary of the experiment shown in H). (Values are mean ± SEM; # unpaired t-test to wt § unpaired t-test to Pkd1 KO unpaired t-test to control conditions , p -value < 0.05; the number of animals/ number of cells is shown between brackets).

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5. Discussion

This work presents evidence that CFTR and TMEM16A are fundamental ion channels involved on apical Cl - secretion in the cystic epithelium in different in vitro systems (M1 cells and Primary Kidney isolated cells).

5.1 Phenotypical switch of ADPKD cyst lining cells

Data presented in this work show an upregulated Cl - secretion either through cAMP-stimulated channel CFTR or through TMEM16A in ADPKD cells. TMEM16A activity was stimulated by an increased Ca 2+ signaling in cystic epithelial cells. However, this behavior contrasts with the normal physiological fluid movement through the nephron, which is characterized by a reabsorption of over 99% of the glomerular filtrate. Evidence suggests that most of the cysts are disconnected from their tubule of origin, forming a spherical structure afterwards 116. Additionally, cysts continue growing after separation from the renal tubule, which indicates that this fluid does not come from the glomerular filtrate but instead from an upregulated epithelial Cl - secretion. In this work, we showed that both CFTR and TMEM16A contribute for electrolyte secretion and cyst formation. Furthermore, intracellular Ca 2+ was also shown here to be a fundamental intracellular second messenger in ADPKD cells. In an opposite way to what was found in this work, the majority of the studies state that ADPKD affected cells have decreased levels of cytosolic Ca 2+ 117 . However as it is shown by Chebib et al 118, this is highly variable and it is clearly depend of the model organism and the protein downregulation/knockout pathway. Therefore the results obtained during this study seem to be plausible even though they are opposed to many other works.

Being intracellular Ca 2+ signaling responsible for the regulation of several physiological processes in the cells, it is possible that the upregulation of this second messenger in cystic epithelial cells is responsible for the ion transport phenotypic switch between wt and Pkd1 KO cells. A hypothesis would be a possible shift in DNA transcription, and therefore signaling pathways, after a mutation in Pkd1/Pkd2 119.

5.2 M1 cell line and primary cultures of isolated kidney cells as models to study fluid secretion in cyst development

M1 cells derive from renal collecting ducts, - a nephron segment where ADPKD had been previously documented – and were therefore chosen as a model to study this disease. Also, M1 cells are relatively simple to maintain, have a fast proliferation rate and a high transfection efficiency.

In this work, we found that M1 cells show CFTR and TMEM16A dependent electrolyte transport (Fig. 4.3 ). In Pkd1 knockdown cells, the basal current was upregulated ( Fig. 4.4, and Fig. 4.6 ), 2+ 2+ possibly due to a higher activity of TMEM16A, supported by the higher levels of [Ca ]i and [Ca ]ER (Fig. 4.9 ). On the other hand, the lower current produced after IBMX/Forskolin stimulation in these cells may result from an inhibition of adenylate cyclase 6 (AC6) due to higher intracellular Ca 2+ levels, which will ultimately inhibit CFTR 120

Several studies using this cell line support the results obtained in this work. Barbosa et al 121 showed that an increase in intracellular cAMP leads to a higher CFTR expression and activity. When PKD1 is overexpressed no increase in this parameter was detected. Jannson et al 122 stated that a mutation in PKD1 leads to higher activity of Na +/ K + ATPase which could lead to higher apical Cl - secretion ( Fig. 1.3 ). Lastly, Hooper et al 123 had shown that when PKD1 is mutated, ATP stimulated Cl - secretion was increased.

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Overall, the M1 cell line seems to mimic the hallmarks of the cystic epithelium, showing an enhanced Cl - movement either through CFTR or TMEM16A. M1 cells also presented a de-regulated Ca 2+ signaling, which also characterizes ADPKD cells ( Appendix VI, Table VI.1 ). However, we must take into account that the use of this model possesses the inherent limitations of an immortalized in vitro system, such as the high passage effect: after a period of growth, cell characteristics can change and may become quite different from those found in native tissue. Therefore the use of primary kidney cells promises a cell system closer to the native tissue. The primary cells are still differentiated and due to mouse models, gene-modified cells could be investigated.

Indeed, in primary cells, SCNN1 activity was enhanced compared to M1 cells. Comparison of primary cell derived from renal cortex and medulla show a higher Scnn1 mRNA expression of the subunits a, b and g and higher SCNN1 activity in cells isolated from the medulla ( Fig. 4.3,4.11,4.16 and Appendix IV- Fig. IV.3 ). This could be explained by an increasing number of principal cells in medullar preparation. In Pkd1 KO cells SCNN1 activity was further enhanced. An upregulation of the basolateral membrane-located Na +/K +-ATPase in ADPKD cells may explain this occurrence 76 ( Fig. 1.3).

In this line, primary cortical cells showed in increase TMEM16A expression compared to medullar cells ( Fig. 4.10 ). This confirmed data from Faria et al 95 , showing strong expression of TMEM16A in the proximal tubule. Here, the physiological function of TMEM16A is to facilitate proton secretion and albumin uptake. In general, both types of primary cells seem to replicate the expected ADPKD secretory phenotype ( Appendix VI, Table VI.1 ).

5.3 Proposed model for impaired Cl - secretion in ADPKD

The mechanism by which TMEM16A and CFTR control transepithelial Cl - secretion contributing to cyst development and growth is not yet fully understood. This work proposes that Pkd1 or Pkd2 KO 2+ 2+ generates an increase in [Ca ]i and an increase in [Ca ]ER (Fig.4.9 ). This may occur due to a lack of interaction between Pkd1 and Pkd2 23 . This leads to a deregulation of Pkd2 activity on the plasma membrane, ultimately resulting in a higher Ca 2+ entry into the cell and into the ER 124 . Increased [Ca 2+ ]i 2+ and [Ca ]ER leads to higher basal and enhanced activation of TMEM16A by purinergic stimulation (see Figs. 4.5, 4.6, 4.12, 4.13, 4.17, and 4.18 ). This higher activity of TMEM16A cause cyst development and polycystic kidneys shown in Fig. 4.1 .

Several studies have shown that TMEM16A colocalizes and interact with the IP3R in the ER 125,126. Here and facilitates IP3-mediated Ca 2+ release from its intracellular stores. It is also reported that Pkd2 and IP3R interact and promote Ca 2+ release from the ER 104,105.Pkd1 is thought to disrupt this interaction, thus limiting ER Ca 2+ release 105. In absence of Pkd1 function in ADPKD, Pkd2-IP3R interaction is stronger 105, and together with TMEM16A a higher Ca 2+ release through IP3R can occur. On the other side, as shown in Fig. 4.9 , downregulation of Pkd2 led to an increase of intracellular Ca 2+ concentration in the cytosol and ER. This could be explained by the localization of Pkd2 in the absence of Pkd1 in the membrane of the ER 127 . In this situation, Pkd2 could contribute to ER Ca 2+ leakages and to an increase of cytosolic Ca2+ concentration (see Fig. 4.9, 4.15, and 4.20 ).

Diverse studies state that cystic cells have a higher cAMP supply 59 that leads to a higher Cl - - secretion, which is inhibited by CFTR inh 172. Therefore, CFTR has been proven to be a major Cl secreting channel in the cystic epithelium. Indeed, in this work, we found that CFTR is upregulated in PKD1 KO cells (see Fig. 4.6, 4.13 and 4.18 ). Notably, and as reported by Benedetto et al 100, CFTR and TMEM16A seem to interact, and this inaction is necessary for CFTR activation. Indeed, we found

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in primary cortical cells, that Tmem16a KO also reduced CFTR dependent Cl- secretion, which was augmented by Pkd1 KO (see Fig.4.13 ).

In summary, a model for Cl - secretion in ADPKD epithelial cells has been proposed ( Fig. 5.1 ). Under basal conditions, Pkd1 KO deregulates Pkd2 function, both on the primary cilium and on the 2+ plasma membrane. This leads to a higher [Ca ]i and a slight increase of basal chloride conductance by TMEM16A and CFTR. After purinergic stimulation, Ca 2+ release from the ER and Ca 2+ uptake into the cell is enhanced and facilitates TMEM16A and CFTR dependent chloride secretion and cyst development in ADPKD.

A v

B v

Figure 5.1. Proposed model of Cl- secretion in ADPKD cystic epithelium. A) Under basal conditions wt cells have a constitutive Cl - secretion through CFTR and TMEM16A. When PKD1 is downregulated, PKD2 transports more Ca 2+ into the cytoplasm in ER. B) Purinergic agonist binds a P2Y receptor and causes a transient Ca 2+ release from the ER store and induces SOCE. When PKD1 is downregulated Ca2+ signaling is enhanced and cause enhanced activity of TMEM16A and CFTR, which leads to cyst growth.

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6. Future Perspetives

Having a better understanding of the impaired Cl - secretion, namely through CFTR and TMEM16A may help to manage the development of ADPKD. Thus, the continuation of the work described in this study should definitely be encouraged in order to unravel the molecular mechanisms behind these processes.

Concerning CFTR involvement on Cl- secretion towards the cyst, generation of a new animal model with a tissue-specific KO for both PKD1 and CFTR would be important to obtain a deeper knowledge for the specific functioning of this channel on ADPKD. For this, use of techniques applied in this work could be interesting in order to unravel the electrophysiological role of CFTR in the cystic epithelium.

Regarding TMEM16A, the use of a tissue-specific KO allowed a better understanding of the role of this Cl - channel in the cystic epithelium. After confirmation of the major role of this CaCC to cystic development, the next step of this investigation would be to use a drug that allows specific inhibition of TMEM16A, applying it to an in vivo model and then to monitor renal clinical parameters. Here, the ultimate goal would be to apply the TMEM16A specific inhibitor drug in ADPKD human patients. TMEM16A has also been reported to affect proliferation of renal cystic epithelial cells. Thus, proliferation and migration assays can be applied to the in vitro models used for this work.

Some improvements may also be carried out on the assays performed on this work. For example, Ca 2+ signaling, as well as whole-cell current measurements, may be executed directly on the renal tubules instead of being performed in primary cells, thus avoiding some eventual artefact introduced by cell culture manipulation.

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8. Appendices

Appendix I –siRNA

Table I.1. Accession number and base pairs (bp) of the plasmids used

Target Accession No. Size (bp)

sihPKD1 NM_013630.2 14170

sihPKD2 NM_008861 5221

Appendix II – Primers and RT-PCR

Table II.1. Pair list of Primers used for RT-PCR. Primers were designed with Probe Finder Software from Roche Applied Science (www.roche-applied-science.com).

Template Sequence Size (bp)

GTG ACA AGA CCT GCA GCT AC Tmem16a 406 GCT GCA GCT GTG GAG ATT C

GAA TCC CCA GCT TAT CCA CG Cftr 544 CTT CAC CAT CAT CTT CCC TAG

GTG GAA AGC AGG TCG GAA G Pkd1 236 TCG TCT CGT TCA GCA CCA G

GTG GAT GTA CAC AAG TGA GAA GGA GC Pkd2 454 CAC GAC AAT CAC AAC ATC CAG ACA

CCT TGA CCT AGA CCT TGA CG Scnn1 a 409 CGA ATT GAG GTT GAT GTT GAG

CAA TAA CAC CAA CAC CCA CG Scnn1 b 588 GAG AAG ATG TTG GTG GCC TG

GCA CCG ACC ATT AAG GAC C Scnn1 g 464 GCC TTT CCC TTC TCG TTC TC

GTA TTG GGC GCC TGG TCA C Gapdh 200 CTC CTG GAA GAT GGT GAT GG

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Table II.2. Standard RT-PCR program.

Target gene Temperature (!C) Time Number of cycles

95!C 2 min

95!C 30 sec Tmem16a, Cftr, Pkd1, 57!C 60 sec Pkd2, Scnn1, Gapdh 30x 72!C 1 min

72!C 7 min

8!C hold

Appendix III – Analysis of protein expression

A B v v

Figure III.1. Western blot analysis of PKD1/PKD2 downregulation. In M1 cells PKD1 (A) and PKD2 (B) expression was downregulated 48 hours after siRNA transfection.

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A B C v v v

Figure III.2. Western blot analysis of PKD1, PKD2 , and TMEM16A downregulation. In M1 cells stably transfected with a YFP plasmid conjugated with a shRNA for scrambled, Pkd1, and Pkd2 and posteriorly transfected with a siTmem16a for 48 hours, PKD1 (A), PKD2 (B) and Tmem16A (C) expression were downregulated 48 hours after siRNA A B v v

transfection.

Figure III.3. Immunostaining analysis of TMEM16A and Acetylated tubulin in primary cells. A) Immunostaining against TMEM16A and Acet. Tub. in cortical isolated Primary cells B) Immunostaining against TMEM16A and Acet. Tub. in medullary isolated Primary cells. Arrows show colocalization.

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Appendix IV – mRNA expression analysis by RT-PCR

A B v v

Figure IV.1. RT-PCR analysis of Pkd1/Pkd2 downregulation in M1 cell line. A) Expression of mRNAs encoding Pkd1 and Pkd2 B) mRNA quantification relatively to Gapdh expression. (the number of samples is shown between brackets).

A v

B v

Figure IV.2. RT-PCR analysis of Pkd1 and Tmem16a expression in medullary primary cells. A) Expression of mRNAs encoding for Pkd1, Gapdh and Tmem16a B) mRNA quantification relatively to Gapdh expression. (the number of samples is shown between brackets).

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A v

B v

Figure IV.3. RT-PCR analysis of Scnn1 expression in medullary primary cells. A) Expression of mRNAs encoding for Scnn1 subunits B) mRNA quantification relatively to Gapdh expression. (the number of filters is shown between brackets).

Appendix V – YFP fluorescence measurements

Figure V.1. Assessment of YFP fluorescence on M1 cells. Using fluorescence microscopy, it was possible to detect YFP fluorescence on M1 cells.

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A B v v

Figure V.2. Assessment of YFP fluorescence on primary cells. Using fluorescence microscopy, it was possible to detect YFP fluorescence on primary cells isolated from the A) cortex and B) medulla.

Appendix VI – General compendium of the results obtained during this work

Table VI.1. Summary of the results obtained during this work. Analyzed properties are listed vertically and the in vitro models with their respective genotype are listed horizontally.

Cell line PrC PrC PrC Cortex PrC M1 Pkd1 M1 Pkd2 PrC PrC Medulla M1 wt Cortex Pkd1/T16a Medulla KO KO Cortex wt Medulla wt Pkd1/T16a Pkd1 KO KO Pkd1 KO Property KO

Low Data not Data not Significant Higher than Similar to Significant Higher than Higher SCNN1 activity available available to control wt Pkd1 KO to control Pkd1/T16a than wt

CaCC Significant Data not Data not Significant Smaller Similar to Significant Higher than Smaller Electrolyte ISC available available to control than wt wt to control wt than wt transport

CaCC Whole- Small High Similar to Significant Higher than Smaller than Significant Higher than Smaller cell current activation activation Pkd1 to control wt wt to control wt than wt

Higher than CaCC (Cl - Significant Higher Similar to Significant Higher than Smaller than Higher than Significant Pkd1/T16a permeability) Quenching than wt wt to control wt wt wt to control KO

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Cell line PrC PrC PrC PrC Cortex PrC PrC M1 Pkd1 M1 Pkd2 Medulla M1 wt Cortex Cortex Pkd1/T16a Medulla Medulla KO KO Pkd1/T16a WT Pkd1 KO KO WT Pkd1 KO Property KO

CFTR Significant Data not Data not Significant Smaller Similar to Significant Similar to Similar to (Electrolyte ISC available available to control than wt wt to control wt wt transport)

CFTR High Smaller Similar to Data not Data not Data not Data not Data not Data not (Whole- cell activation than wt Pkd1 available available available available available available current)

Higher than Higher CFTR (Cl - No Higher Similar to Significant Higher than Significant Higher than Pkd1/T16a than Pkd1 permeability) activation than WT WT to control wt to control wt KO ko

Higher than Basal whole- Smaller Higher Similar to Smaller Higher than Similar to Similar to Smaller Pkd1/T16A cell current current than wt Pkd1 KO baseline wt Pkd1 KO Pkd1 KO baseline KO

Basal Cl - Small Higher Similar to Small Similar to Smaller than Small Higher than Similar to permeability quenching quenching Pkd1 KO quenching wt wt baseline wt wt

Higher Higher Smaller Higher than Smaller than Small Higher than Similar to Basal [Ca 2+ ]i Small than Pkd2 than wt baseline Pkd1/T16a Pkd1 KO baseline wt wt KO

ATP- Higher Significant Higher Significant Higher than Similar to Significant Similar to Similar to stimulated than Pkd2 2+ to control than wt to control wt wt to control wt wt [Ca ]i KO

2+ Significant Higher Similar to Significant Higher than Similar to Significant Higher than Smaller [Ca ]ER to control than wt Pkd1 KO to control wt wt to control WT than wt

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