High glucose augments angiotensinogen in human renal proximal tubular cells through hepatocyte nuclear factor-5.

Juan Wang,Zhiyu Wang,Akira Nishiyama,Ya Liu,Tsutomu Masaki,Hiroyuki Kobori,Hideki Kobara,Yuki Shibayama,Hideharu Abe

doi:10.1371/journal.pone.0185600

Abstract

High glucose has been demonstrated to induce angiotensinogen (AGT) synthesis in the renal proximal tubular cells (RPTCs) of rats, which may further activate the intrarenal renin-angiotensin system (RAS) and contribute to diabetic nephropathy. This study aimed to investigate the effects of high glucose on AGT in the RPTCs of human origin and identify the glucose-responsive transcriptional factor(s) that bind(s) to the DNA sequences of AGT promoter in human RPTCs. Human kidney (HK)-2 cells were treated with normal glucose (5.5 mM) and high glucose (15.0 mM), respectively. Levels of AGT mRNA and AGT secretion of HK-2 cells were measured by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Consecutive 5’-end deletion mutant constructs and different site-directed mutagenesis products of human AGT promoter sequences were respectively transfected into HK-2 cells, followed by AGT promoter activity measurement through dual luciferase assay. High glucose significantly augmented the levels of AGT mRNA and AGT secretion of HK-2 cells, compared with normal glucose treatment. High glucose also significantly augmented AGT promoter activity in HK-2 cells transfected with the constructs of human AGT promoter sequences, compared with normal glucose treatment. Hepatocyte nuclear factor (HNF)-5 was found to be one of the glucose-responsive transcriptional factors of AGT in human RPTCs, since the mutation of its binding sites within AGT promoter sequences abolished the above effects of high glucose on AGT promoter activity as well as levels of AGT mRNA and its secretion. The present study has demonstrated, for the first time, that high glucose augments AGT in human RPTCs through HNF-5, which provides a potential therapeutic target for diabetic nephropathy.

Highlights

Intrarenal renin-angiotensin system (RAS) plays a crucial role in development of diabetic nephropathy (DN) [1], which is the major renal complication of diabetes mellitus [2] as well as the main cause of end-stage renal disease [3]
Liu et al [11] have demonstrated that high glucose treatment induced much higher levels of apoptosis and caspase-3 activity in rat renal proximal tubular cells (RPTCs) overexpressing AGT in comparison to control RPTCs in vitro, and induction of diabetes in transgenic mice that overexpressing AGT in RPTCs led to significant increases in apoptosis of RPTCs compared with diabetic nontransgenic littermates, the above effects were markedly attenuated by insulin and/or RAS blockers
High glucose augments the levels of AGT mRNA and AGT secretion of human RPTCs

Summary

Introduction

Intrarenal renin-angiotensin system (RAS) plays a crucial role in development of diabetic nephropathy (DN) [1], which is the major renal complication of diabetes mellitus [2] as well as the main cause of end-stage renal disease [3]. Previous studies reported that intrarenal angiotensinogen (AGT) mRNA and angiotensin (Ang) II levels were enhanced in type 2 diabetic rats [4, 5]. This could further lead to renal proximal tubular hypertrophy and interstitial fibrosis by stimulating reactive oxygen species and eventually result in diabetic nephropathy [6, 7]. By using human kidney biopsied samples, Kamiyama et al [8] have demonstrated that AGT mRNA and protein levels in human renal cortex proximal tubules were significantly augmented in patients with type 2 diabetes. The level of AGT in RPTCs plays an important role in the pathogenesis of DN

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