The Role of Intercalated Cells and Their Acid Base and Electrolyte Transport Machinery in Kidney Cystogenesis by Tuberous Sclerosis

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Abstract. Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder, which is caused by inactivating mutations in either the TSC1 or TSC2 gene and affects multiple organs, including kidney and lung. The disease affects over two million people worldwide, with a large proportion developing angiomyolipomata and cysts, which eventually lead to renal failure. While the initial genetic events of TSC have been delineated, little is known about the biological processes and factors that facilitate progression or expansion of cysts. We have developed mice with kidney principal (PC) cell-specific inactivation of Tsc1 or Tsc2 genes (Published and Prelim. Data) which recapitulate the human TSC cystic kidney disease. The cyst epithelia display few PC cells, but robust presence of A-intercalated (A-IC) cells. RNA-seq and confirmatory expression studies demonstrated a significant increase in the expression of Foxi1, a transcription factor critical to the development of IC cells and activation of H+-ATPase and carbonic anhydrase II (CAII) in Tsc1 KO mice. Double immunofluorescent labeling studies with antibodies against H+-ATPase and AQP2; H+-ATPase and PCNA (proliferative nuclear cell antigen, a marker of cell proliferation) and H+-ATPase and CAII demonstrated progressive loss of PC cells and hyperproliferation of A-IC cells in cyst epithelium in Tsc1 KO mice. In addition, the electrogenic 2Cl-/H+ exchanger CLC-5, which colocalizes with H+-ATPase in membranes of late endosomes and lysosomes under basal conditions, demonstrated remarkable co-localization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Further, our results indicated the co-localization of pro-renin receptor PRR, a critical player in Wnt signaling pathway, with H+-ATPase on the apical membrane of cyst epithelia. These changes are distinct from those in autosomal dominant PKD cysts (Prelim Data). Deletion of Foxi1 in Tsc1 KO mice resulted in complete abrogation of cyst burden in Foxi1/Tsc1 double mutant mice. In addition, deletion of CAII, which is regulated by Foxi1 and is critical to H+-ATPase activity, significantly blunted the cyst burden in CAII/Tsc1 double mutant mice. We propose that the robust proliferation of A-intercalated cells and their acid/base/electrolyte transport machinery are crucial to kidney cystogenesis in Tsc1 KO mice. We further propose that unlike cysts in PKD, which respond to AVP V2 receptor antagonism by reduction in their fluid secretion and size, TSC cysts, which have few principal cells, will be resistant to V2 receptor antagonists. To test our hypotheses, we propose to: Ascertain the role of A-IC cells and their acid base transport machinery (H+-ATPase and CAII) in the growth and expansion of cysts in TSC disease; Determine the role of CLC-5 and CFTR in chloride secretion into cyst lumen and cyst expansion in mice with TSC; and Examine the effect of inhibitors of CAII/H+-ATPase, PRR and mTOR on cyst growth in TSC mice. To this end, we will examine the effect of simultaneous deletion of Tsc1 (in principal cells) in combination with Foxi1, CAII, H+-ATPase B subunit, or CLC-5 on renal cystogenesis. Further, the effect of pharmacologic inhibitors of CFTR activation (V2 receptor antagonists), H+-ATPase/CAII (acetazolamide), PRR (the handle region peptide) or mTORC (everolimus) will be explored to determine their effect on cyst expansion in Tsc1 KO mice. Cyst growth and size, kidney function, systemic blood pressure, life expectancy, as well as mTORC1 activation in cyst epithelium will be determined in mutant mice. We strongly believe the proposed studies are novel and could lead to new therapies for this devastating disease as well as a number of other renal cystic diseases.