Abstract: FR-PO386
Functional Impact of Endogenous ATRAP, a Novel Angiotensin II Type 1 Receptor-Associated Protein, on Human Renal Proximal Tubule Cells
Session Information
- CKD: Mechanisms - II
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2103 CKD (Non-Dialysis): Mechanisms
Authors
- Yamaji, Takahiro, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Urate, Shingo, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Abe, Eriko, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Suzuki, Toru, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Yamada, Takayuki, Mount Sinai Beth Israel, New York, New York, United States
- Tanaka, Shohei, Yokohama City University Graduate School of Medicine, Yokohama, Japan
- Kinguchi, Sho, Yokohama City University, Kanagawa, Yokohama city, Japan
- Azushima, Kengo, Duke-NUS Medical School, Singapore, Singapore
- Kanaoka, Tomohiko, Yokohama City University, Kanagawa, Yokohama city, Japan
- Wakui, Hiromichi, Yokohama City University, Kanagawa, Yokohama city, Japan
- Toya, Yoshiyuki, Yokohama City University, Kanagawa, Yokohama city, Japan
- Tamura, Kouichi, Yokohama City University Graduate School of Medicine, Yokohama, Japan
Background
The proximal tubule is a particularly important site for aging-related kidney damage. Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase in the proximal tubule, is involved in renal damage associated with aging. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that ATRAP-deficient mice displayed an exacerbation of age-associated renal function decline and tubulointerstitial fibrosis. Although the molecular mechanisms by which ATRAP deficiency exacerbates age-associated tubulointerstitial fibrosis has not yet been defined, renal SIRT1 protein expression was more decreased in aged ATRAP-deficient mice compared with aged wild type mice. Further investigations of ATRAP-dependent SIRT1 protein expression are important to resolve aging-associated kidney dysfunction. in this study we aimed to establish an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression.
Methods
We established an immortalised RPTEC line by expressing hTERT and shRNA-targeted CDKN2A. Next, we cloned this immortalized RPTEC and then characterised the cells based on the expression of a proximal tubular marker (SGLT2, DPP4). We call this cloned immotarised RPTEC, ciRPTEC. To analyze the ATRAP function in the proximal tubule cells, we use siRNA-mediated ATRAP knockdown in ciRPTEC and CRISPR-CAS9 mediated ATRAP knockout.
Results
SIRT1 mRNA expression, which was induced by serum starvation, was unaffected by transient ATRAP knockdown. On the other hand, SIRT1 protein expression was not induced by serum starvation in our ciRPTEC cells, although transient ATRAP knockdown reduced the expression of SIRT1 protein under both normal and serum-starved conditions. Like ATRAP knockdown, ATRAP knockout did not affect SIRT1 mRNA expression under either the normal or serum-starved condition. However, SIRT1 protein expression was significantly decreased by serum starvation in ATRAP knockout cells, while no significant reduction in SIRT1 protein was observed in control cells.
Conclusion
These results indicate that ATRAP mediates SIRT1 protein abundance in ciRPTEC by regulating its synthesis and/or stability but does not affect the level of SIRT1 mRNA transcription or stability.