Kidney intercalated cells and the transcription factor FOXi1 drive cystogenesis in tuberous sclerosis complex

Abstract

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H⁺-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl^-/H⁺ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H⁺-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H⁺-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H⁺-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H⁺-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

Keywords: H+-ATPase; cortical collecting duct; cysts; intercalated cells; prorenin receptor.

Fig. 1.

Generation of principal cell-specific Tsc1 KO mice. (A) Mice with principal cell-specific deletion of the Tsc1 gene were generated as described (Results). Kidneys of Tsc1 KO mice are larger than WT mice. (B) H&E staining with low magnification (global view; Upper) and higher magnifications (Lower) shows kidneys from age-matched 6-wk-old WT (Left) and Tsc1 KO mice (Right), respectively. There are multiple cysts with varying sizes (identified with the letter C) in 6-wk-old Tsc1 KO mice (Right).

Fig. 2.

Characterization of cystic epithelium in kidneys of Tsc1 KO mice. (A) Northern hybridizations of Foxi1 expression in kidneys of Tsc1 KO mice (Left) vs. Pkd1 mutant mice (Right) show enhanced Foxi1 expression in Tsc1 KO but not in Pkd1 KO mice. (B) Double-label immunofluorescence images with polyclonal H⁺-ATPase B1-subunit and monoclonal AQP-2 antibodies in 6-wk-old Tsc1 KO (Upper) and WT mice (Lower). Arrows indicate the few AQP-2–positive principal cells that are present in Tsc1 KO cysts. SI Appendix, Fig. S1B shows additional images. (C) Double-immunofluorescence labeling images with ATP6AP2 (PRR) and monoclonal AQP-2 antibodies in Tsc1 KO (Upper) and WT mice (Lower). (D). Double-labeling images with CAII and monoclonal H⁺-ATPase (E subunit) antibodies in age-matched Tsc1 KO (Upper) and WT mice (Lower). (E) Double-labeling images with AE-1 and monoclonal H⁺-ATPase E antibodies in Tsc1 KO (Upper) and WT mice (Lower). C represents cysts, and G represents glomerulus. Information regarding antibody concentrations can be found in SI Appendix, Table S1. Additional immunofluorescence images with polyclonal H⁺-ATPase B1 and monoclonal AQP-2 antibodies in human kidney cysts (SI Appendix, Fig. S2A) and in kidneys of heterozygote Tsc2^+/− mice (SI Appendix, Fig. S2B) are provided. Kidneys from Pkd1 KO (Pkhd1-Cre) mice are shown for comparison (SI Appendix, Fig. S2C). rRNA, ribosomal RNA.

Fig. 3.

mTORC1 activation in cyst epithelium is mainly detected in V-H⁺-ATPase–expressing IC cells that have intact Tsc1 and are proliferating. (A) Levels of phospho-S6 expression in Tsc1 KO (Right) vs. WT mice (Left). As indicated, IC cells that line the cysts in Tsc1 KO mice display enhanced mTORC1 activation. (B) Expression of Tsc1 (Hamartin) in kidneys of Tsc1 KO mice illustrated by double-label immunofluorescence using polyclonal H⁺-ATPase B1 and monoclonal Tsc1 antibodies. The IC cells lining the cysts display normal expression of Tsc1 (hamartin) in non–AQP-2–expressing cells. (C) Proliferation of IC cells in cyst epithelia of Tsc1 KO mice. Double labeling with V-H⁺-ATPase (B1 subunit; Right) and monoclonal PCNA (Left) antibodies, with merged images in Center performed on 6-wk-old Tsc1 KO mice (Upper) and WT mice (Lower). C represents cysts. G represents glomerulus. Arrows point to PCNA and H⁺-ATPase positive cells.

Fig. 4.

Generation and examination of Foxi1/Tsc1 dKO and CAII/Tsc1 dKO mice: effects on kidney cystogenesis. (A) Generation of Foxi1/Tsc1 dKO mice. Mice with Foxi1 deletion were crossed with Tsc1 KO mice. Northern hybridizations verified the generation of Foxi1/Tsc1 dKO mice (Left). H&E staining showed that compared with Tsc1 single-mutant mice (Middle Left and Bottom Left), Foxi1/Tsc1 dKO mice had complete absence of cyst generation in their kidneys vs. numerous cysts in kidneys of Tsc1 single-mutant mice (Middle Right and Bottom Right). (B) MRI examination of kidneys of Tsc1 and Foxi1/Tsc1 dKO mice. Live anesthetized WT, Tsc1 KO, and Foxi1/Tsc1 dKO mice were subjected to kidney MRI procedure (described in Materials and Methods). As demonstrated, Foxi1 deletion completely abrogated the cyst burden in Foxi/Tsc1 dKO mice. (C) Expression of H⁺-ATPase in kidneys of Foxi1/Tsc1 dKO mice. (Left) Northern hybridization of the H⁺-ATPase D2 subunit. The kidney mRNA expression of H⁺-ATPase D2 subunit is shown in WT, Tsc1 KO, Foxi1/Tsc1 dKO, and Foxi1 KO mice. (Right) Double-label images with polyclonal H⁺-ATPase (B1 subunit) and monoclonal AQP-2 are displayed in kidneys of Foxi1/Tsc1 dKO (Lower) vs. Tsc1 KO mice (Upper). SI Appendix, Fig. S4 shows additional images. (D) Generation of CAII/Tsc1 dKO mice. CAII KO mice were crossed with Tsc1 KO mice. H&E staining showed that compared with Tsc1 KO (Left), CAII/Tsc1-dKO had a significant reduction in the number and size of cysts (Right). C represents cysts. rRNA, ribosomal RNA. Arrows in D point to small cysts in kidneys of CAII/Tsc1 dKO mice.

Fig. 5.

Localization of CLC-5 in kidneys of Tsc1 KO mice. Double-labeling images with monoclonal H⁺-ATPase E-subunit (Left) and polyclonal CLC-5 (Right) antibodies, with the merged image in Center on 6-wk-old Tsc1 KO mice (Upper). White arrows in Upper point to the localization of H⁺-ATPase and CLC-5 on the apical membrane of cyst epithelia. The double-labeling studies in WT mice are shown in Lower. Blue arrows point to proximal tubule cells, while the yellow arrows highlight IC cells. C represents cysts.