Cre/loxP approach‐mediated downregulation of Pik3c3 inhibits the hypertrophic growth of renal proximal tubule cells. Issue 12 (31st May 2020)

• Record Type: Journal Article
• Authors: Liu, Ting; Yuan, Jialing; Dai, Caihong; Xu, Jinxian; Li, Shude; Humphreys, Benjamin D.; Kleven, Daniel T.; Chen, Jian‐Kang
• Is Part Of: Journal of cellular physiology. Volume 235:Issue 12(2020:Dec.)
• Page Start: 9958
• Page End: 9973
• Publication Date: 2020-05-31
• Subjects: class III phosphatidylinositol‐3 kinase (Pik3c3) -- compensatory nephron hypertrophy (CNH) -- mammalian target of rapamycin complex 1 (mTORC1) -- renal proximal tubule cells (RPTC) -- ribosomal protein S6 (rpS6) phosphorylation -- unilateral nephrectomy (UNX); Physiology -- Periodicals; Cell physiology -- Periodicals; 571.6
• DOI: 10.1002/jcp.29811
• Languages: English
• ISSNs: 0021-9541

Abstract

Abstract: Nephron loss stimulates residual functioning nephrons to undergo compensatory growth. Excessive nephron growth may be a maladaptive response that sets the stage for progressive nephron damage, leading to kidney failure. To date, however, the mechanism of nephron growth remains incompletely understood. Our previous study revealed that class III phosphatidylinositol‐3‐kinase (Pik3c3) is activated in the remaining kidney after unilateral nephrectomy (UNX)‐induced nephron loss, but previous studies failed to generate a Pik3c3 gene knockout animal model. Global Pik3c3 deletion results in embryonic lethality. Given that renal proximal tubule cells make up the bulk of the kidney and undergo the most prominent hypertrophic growth after UNX, in this study we used Cre‐loxP‐based approaches to demonstrate for the first time that tamoxifen‐inducible SLC34a1 promoter‐driven CreER T2 recombinase‐mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to inhibit UNX‐ or amino acid‐induced hypertrophic nephron growth. Furthermore, our mechanistic studies unveiled that the SLC34a1 ‐ CreER T2 recombinase‐mediated Pik3c3 downregulation inhibited UNX‐ or amino acid‐stimulated lysosomal localization and signaling activation of mechanistic target of rapamycin complex 1 (mTORC1) in the renal proximal tubules. Moreover, our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibited amino acid‐stimulated mTORC1 signaling and blunted cellular growth in primary cultures of renal proximal tubule cells. Together, both our in vivo and in vitro experimental results indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1–S6K1–rpS6 signaling pathway. Abstract : Using Cre‐loxP‐based approaches, we for the first time demonstrated that tamoxifen‐induced Cre‐recombinase‐mediated downregulation of Pik3c3 expression in renal proximal tubule cells alone is sufficient to significantly inhibit UNX‐ or amino acid‐stimulated mechanistic target of rapamycin complex 1 (mTORC1) signaling to phosphorylation of ribosomal protein S6 (rpS6) and, consequently, prevent UNX‐ or amino acid‐induced hypertrophic nephron growth in adult mice. Our additional cell culture experiments using RNAi confirmed that knocking down Pik3c3 expression inhibits amino acid‐stimulated mTORC1–S6K1 signaling activity and blunts cellular growth in the primary cultures of renal proximal tubule cells. Together, both in vivo and in vitro experimental results in the present study indicate that Pik3c3 is a major mechanistic mediator responsible for sensing amino acid availability and initiating hypertrophic growth of renal proximal tubule cells by activation of the mTORC1–S6K1–rpS6 signaling pathway through a mechanism of recruiting mTOR to its activation site—the lysosomal membranes.