Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line

Abstract

The proximal tubule is a particularly important site for ageing-related kidney damage. Sirtuin 1 (SIRT1), an NAD+ (nicotinamide adenine dinucleotide)-dependent deacetylase in the proximal tubule, may be involved in renal injury associated with ageing. However, the mechanisms of SIRT1 regulation remain to be elucidated. We recently reported that angiotensin II type 1 receptor (AT1R)-associated protein (ATRAP)-deficient mice displayed age-associated renal function decline and tubulointerstitial fibrosis. Our data showed that SIRT1 protein expression was reduced in ATRAP-deficient mice, although the relationship between ATRAP deficiency and age-associated renal fibrosis is still not fully understood. It is, therefore, necessary to investigate how ATRAP affects SIRT1 protein expression to resolve ageing-associated kidney dysfunction. Here, since ageing studies are inherently lengthy, we used an ex vivo model of the proximal tubule to determine the role of ATRAP in SIRT1 protein expression. We first generated a clonal immortalised human renal proximal tubule epithelial cell line (ciRPTEC) expressing AT1R and ATRAP. Using this cell line, we demonstrated that ATRAP knockdown reduced SIRT1 protein expression in the ciRPTEC but did not alter SIRT1 mRNA expression. Thus, ATRAP likely mediates SIRT1 protein abundance in ciRPTEC.

Angiotensin II type-1 receptor-associated protein interacts with transferrin receptor-1 and promotes its internalization

“…Lentiviral supernatants were produced as described elsewhere 38 . Briefly, pLenti_TetOn_F-mATRAP or pLenti_HA-TfR1 plasmid (3 µg), pLP1, 3 µg of pLP2/VSVG (3 µg; Thermo Fisher Scientific), and pAdvantage (1.3 µg; Promega) were mixed in Opti-MEM medium (1.5 mL; Thermo Fisher Scientific) and added to Lipofectamine2000 (39.9 μL; Thermo Fisher Scientific) in Opti-MEM (1.5 mL).…”

“…Western blot analysis was performed as described elsewhere 38 . Briefly, total protein was extracted from cells using a sample buffer containing SDS (1%).…”

Angiotensin II Type 1 Receptor-Associated Protein Interacts With Transferrin Receptor 1 and Promotes Its Internalization

“…Lentiviral supernatants were produced as described elsewhere [37]. Brie y, pLenti_TetOn_F-mATRAP plasmid (3 µg), pLP1, 3 µg of pLP2/VSVG (3 µg), and pAdvantage (1.3 µg) were mixed in Opti-MEM medium (1.5 mL; Thermo Fisher Scienti c) and added to Lipofectamine2000 (39.9 µL; Thermo Fisher Scienti c) in Opti-MEM (1.5 mL).…”

“…Western blot analysis. Western blot analysis was performed as described elsewhere [37]. Brie y, total protein was extracted from cells using a sample buffer containing SDS (1%).…”