Enrichment of Acidification Pathways in Tubules at Early Cystogenesis Stage of mTOR-Overactivated, Polycystic Kidney Disease Model

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary diseases and is primarily caused by mutations in the PKD1 or PKD2 gene, encoding polycystin 1 (PC1) and 2 (PC2). The PC1/2 forms a cell-surface complex in the primary cilia of renal tubular cells. mTOR is a master regulator of cell proliferation and nutrition that is localized in the lysosome membrane. The mTOR signal is primarily regulated by the TSC1 and 2 and is associated with PC1/2 function and localization. Dysregulation of mTOR is implicated in cystogenesis of the human ADPKD and Pkd1-KO mice. However, the functional consequence of mTOR signal in PKD remains elusive. This study aimed to clarify alterations of biological process and molecular function in the tubules developing early cysts by the omics approach.   

We used a Cd79a-Cre Tsc1 KO mice, in which mTORC1 is conditionally activated via Tsc1 ablation along the distal nephron (Linh N, 2023). The bulk RNA sequencing was done for total RNA from Cd79a-Tsc1 KO kidney (n=3 male, age, P9-10) and matched control (n=3). Differential expressed genes were selected for the enrichment and pathway analyses (GO or KEGG) by the DAVID and STRINGS. Ingenuity pathway analysis (IPA, Qiagen) was used for the canonical and upstream regulator analyses.         

Cd79a-Tsc1 KO kidney developed early cysts in the distal nephron from PO 9-10 and displayed definite PKD by 4 weeks. During P7-11, pre-cystic Cd79a-Tsc1 KO tubule cells showed cilia elongation, aberrant cell intercalation, and mitotic division, suggesting the defective cell planar polarity (PCP). 

To gain insight into the mechanisms implicated in the early cystogenesis, we compared the transcriptomes of Cd79a-Tsc1 KO kidneys with those of control. The enrichment analysis revealed that top 10 most significantly enriched GO or KEGG terms in PKD kidneys converge into the membrane transport process. The pathway analysis suggests the activation of collecting duct acid secretion (K004530). The upregulated genes included vacuolar (V) H+-ATPase subunits (V1:a1,b1,c2,e1,g3 and V0:a4,d2), carbonic anhydrase (Car2), anion exchanger (Slc4a1, Slc9a2), Cl channel (Clcnkb), with their regulatory GTPase (Rragd). The results agreed with those of other Tsc1/2 KO PKD model, showing that the cyst lining, A-intercalated cells abundantly express the V- ATPase. Upstream regulator analysis revealed that potentially activators for these altercations are Lhx1, Pkd1, Hnf1a, Hnf4a and Foxi1, while inhibitors include Tsc1, 2, Flcn, Cbx5, Rictor, and Tgfb2. 

Conditional mTOR-activation in the distal nephron triggers the cystogenesis with the concomitant upregulation of membrane transport processes. The enrichment of acid secretion pathways involving the V-ATPase as well as the cooperating anion exchangers may reflect selective proliferation of the A-intercalated cells. The V-ATPase acts as a component of an amino acid–sensing complex as well as co-activator of mTOR pathway by directly recruiting mTORC1 to the lysosome under sufficient amino acid supply. Understanding the V-ATPase-mTORC1 axis helps develop new therapeutic approach that suppress the early cystogenesis through modulation of nutrient signaling.