The Molecular Mechanism of Leptin Secretion and Expression Induced by Aristolochic Acid in Kidney Fibroblast

Tsung-Chieh Lin,Chung-Shi Yang,Tien-Chiang Lee,Shih-Lan Hsu,Anton Wutz

doi:10.1371/journal.pone.0016654

Tsung-Chieh Lin, Chung-Shi Yang + Show 3 more

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https://doi.org/10.1371/journal.pone.0016654

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Abstract

BackgroundLeptin is a peptide hormone playing pivotal role in regulating food intake and energy expenditure. Growing evidence has suggested the pro-inflammatory and fibrogenic properties of leptin. In addition, patients with renal fibrosis have higher level of plasma leptin, which was due to the increased leptin production. Aristolochic acid (AA) is a botanical toxin characterized to associate with the development of renal fibrosis including tubulointerstitial fibrosis. However, whether leptin is upregulated to participate in AA-induced kidney fibrosis remain completely unknown.Methodology/Principal FindingsIn this study, leptin expression was increased by sublethal dose of AA in kidney fibroblast NRK49f determined by enzyme-linked immunosorbent assay and Western blot. Data from real-time reverse transcriptase-polymerase chain reaction revealed that leptin was upregulated by AA at transcriptional level. DNA binding activity of CCAAT enhancer binding protein α (C/EBP α), one of the transcription factors for leptin gene, was enhanced in DNA affinity precipitation assay and chromatin immunoprecipitation experiments. Knockdown of C/EBP α expression by small interfering RNA markedly reduced AA-induced leptin expression. Moreover, AA promoted Akt interaction with p-PDK1, and increased phosphorylated activation of Akt. Akt knockdown, and inhibition of Akt signaling by LY294002 and mTOR inhibitor rapamycin reduced leptin expression. Furthermore, treatment of LY294002 or rapamycin significantly suppressed AA-induced C/EBP α DNA-binding activity. These results suggest that Akt and C/EBP α activation were involved in AA-regulated leptin expression.Conclusions/SignificanceOur findings demonstrate the first that AA could induce secretion and expression of fibrogenic leptin in kidney fibroblasts, which reveal potential involvement of leptin in the progression of kidney fibrosis in aristolochic acid nephropathy.

Highlights

Leptin, an obese gene product initially identified in 1994, was named from the Greek word leptos, which means thin [1]
Renal interstitial fibrosis is the process of renal fibroblasts activation and accumulation
To address whether the Aristolochic acid (AA)-induced leptin production might result from a transcriptional regulation, NRK-49f cells were stimulated without or with AA for 12 and 24 h, and the level of cellular leptin mRNA was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR (Fig. 2C & 2D)

Summary

Introduction

An obese gene (ob) product initially identified in 1994, was named from the Greek word leptos, which means thin [1]. Leptin is a 16 kDa peptide hormone of cytokine family mainly secreted by adipocyte into blood stream and functions as a central mediator that negatively regulates satiety in hypothalamus, while its deficiency is associated with the development of obesity and metabolic syndrome [2]. Growing evidences suggest the fibrogenic property of leptin. In rodent model of renal interstitial fibrosis, elevated TGF-b mRNA level, phosphorylated activation of Smad 2/3 and the up-regulated downstream target genes are significantly reduced in leptin deficient ob/ob mice [8]. Leptin was further considered as a cofactor of TGF-b activation, which enhanced TGF-b signaling in normal rat kidney fibroblasts [8]. These results implicate the regulatory role of leptin in renal interstitial fibrosis. Whether leptin is upregulated to participate in AA-induced kidney fibrosis remain completely unknown

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