High circulatory leptin mediated NOX-2-peroxynitrite-miR21 axis activate mesangial cells and promotes renal inflammatory pathology in nonalcoholic fatty liver disease

Abstract

High circulatory insulin and leptin followed by underlying inflammation are often ascribed to the ectopic manifestations in non-alcoholic fatty liver disease (NAFLD) but the exact molecular pathways remain unclear. We have shown previously that CYP2E1-mediated oxidative stress and circulating leptin in NAFLD is associated with renal disease severity. Extending the studies, we hypothesized that high circulatory leptin in NAFLD causes renal mesangial cell activation and tubular inflammation via a NOX2 dependent pathway that upregulates proinflammatory miR21. High-fat diet (60% kcal) was used to induce fatty liver phenotype with parallel insulin and leptin resistance. The kidneys were probed for mesangial cell activation and tubular inflammation that showed accelerated NASH phenotype and oxidative stress in the liver. Results showed that NAFLD kidneys had significant increases in α-SMA, a marker of mesangial cell activation, miR21 levels, tyrosine nitration and renal inflammation while they were significantly decreased in leptin and p47 phox knockout mice. Micro RNA21 knockout mice showed decreased tubular immunotoxicity and proinflammatory mediator release. Mechanistically, use of NOX2 siRNA or apocynin,phenyl boronic acid (FBA), DMPO or miR21 antagomir inhibited leptin primed-miR21-mediated mesangial cell activation in vitro suggesting a direct role of leptin-mediated NOX-2 in miR21-mediated mesangial cell activation. Finally, JAK-STAT inhibitor completely abrogated the mesangial cell activation in leptin-primed cells suggesting that leptin signaling in the mesangial cells depended on the JAK-STAT pathway. Taken together the study reports a novel mechanistic pathway of leptin-mediated renal inflammation that is dependent on NOX-2-miR21 axis in ectopic manifestations underlying NAFLD-induced co-morbidities.

Keywords: JAK/STAT; Leptin; Mesangial cells; NADPH; NAFLD; NOX-2; Oxidative stress; miR21; siRNA.

Fig. 1

Increased circulatory leptin causes mesangial cell activation in the NAFLD-Kidney. A. Western blot analysis for serum leptin in NAFLD group and NAFLD group treated with BDCM. B. The morphometric analysis of the western blot normalized with ponceau S band. C. Immunoreactivity of alpha-smooth muscle actin (α- SMA, a marker for mesangial cell activation) as shown by immunohistochemistry in kidney slices from mice fed with high fat diet (60% kcal fat) NAFLD serve as a control, NAFLD mice exposed to BDCM (NAFLD + BDCM), Leptin gene-deficient mice fed with high fat diet (Leptin KO) and exposed to BDCM (Leptin KO+BDCM). Images were taken at 60×. D. Morphometric analysis of α- SMA immunoreactivity (mean data measured as arbitrary light units from three separate microscopic fields were plotted on y-axis) in NAFLD, NAFLD + BDCM, Leptin KO and Leptin KO+ BDCM groups (*P < 0.05).

Fig. 2

Leptin induces renal NOX2 activation and subsequent tyrosyl radical formation. A. Colocalization of GP91phox/P47phox (GP91/P47phox) as shown by immunofluorescence imaging in kidney slices from mice fed with high- fat diet (NAFLD), NAFLD mice exposed to BDCM, and Leptin gene-deficient mice fed with high-fat diet (Leptin KO) and exposed to BDCM (Leptin KO + BDCM). B Immunoreactivity of 3-nitrotyrosine (3-NT) as shown by immunofluorescence imaging in kidney slices from mice fed with high- fat diet (NAFLD), NAFLD mice exposed to BDCM, and P47phox gene-deficient mice fed with high-fat diet (P47phox KO) and exposed to BDCM (P47phox KO + BDCM). C Morphometric analysis of GP91/P47phox colocalized in NAFLD, NAFLD+BDCM, Leptin KO, Leptin KO + BDCM group of mice. All immunofluorescent Images were taken at 40× and mean colocalization events were measured as arbitrary light units from three separate microscopic fields(plotted on y-axis) (*P < 0.05). D Morphometric analysis of 3-NT immunoreactivity in NAFLD, NAFLD + BDCM, P47phox KO, and P47phox KO + BDCM group of mice. Morphometric analysis was performed as mean data (immunoreactivity measured as arbitrary light units) from three separate microscopic fields (plotted on y-axis) (*P < 0.05).

Fig. 3

NOX2 activation leads to mesangial cell priming and increased TLR4 expression in the renal tissue. A and B Immunoreactivity and morphometric analysis of α-SMA immunoreactivity as shown by immunohistochemistry in kidney slices from mice fed with high- fat diet (NAFLD), NAFLD mice exposed to BDCM (NAFLD + BDCM), and P47phox gene-deficient mice fed with high-fat diet (P47phox KO) and exposed to BDCM (P47phox KO + BDCM). All immunohistochemistry Images were taken at 60×. C. mRNA expression of TLR4 gene in kidney tissue of NAFLD, NAFLD + BDCM, P47phox KO + BDCM group of mice fed with high-fat diet. mRNA expression had been appraised by quantitative real-time PCR (qRTPCR) and expressions were normalized against NAFLD group (*P < 0.05). D and E Immunoreactivity and morphometric analysis of TLR4 as shown by immunohistochemistry in kidney slices from mice fed with high- fat diet (NAFLD), NAFLD mice exposed to BDCM (NAFLD + BDCM), and P47phox gene-deficient mice fed with high-fat diet (P47phox KO) and exposed to BDCM (P47phox KO + BDCM). All immunohistochemistry Images were taken at 60×. All morphometric analysis were carried out as mean data from three separate microscopic fields. (plotted on y-axis) in NAFLD, NAFLD + BDCM, P47phox KO, and P47phox KO + BDCM group of mice.

Fig. 4

NOX2 activation leads to a surge of proinflammatory mediators in NAFLD-Kidney A. mRNA expression analysis of IL-1β, IFN-γ, TNF-α, CD4, and CD8 genes in kidney tissue of NAFLD, NAFLD + BDCM, and P47phox KO mice fed with high-fat diet. All mRNA expression had been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against NAFLD group (*P < 0.05). B and C Kidney tissue slices were probed for IL-1β and TNF-α immunoreactivity as shown by immunohistochemistry in kidney slices from mice fed with high- fat diet NAFLD group (serve as a control), NAFLD+BDCM, and P47phox mice group. D and E Morphometric analysis of IL-1β and TNF-α immunoreactivity as observed in the region of interest (ROI) (*P < 0.05).

Fig. 5

Role of GP91phox in activation of leptin induced NADPH oxidase and oxidative stress. A. mRNA expression analysis of GP91phox and P47phox in mesangial cells exposed with leptin or siRNA or co-exposed with siRNA and Leptin. All mRNA expression have been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against Control (Cont) group (*^#P < 0.05). B and D. Colocalization and morphometric analysis of GP91phox/P47phox as shown by immunofluorescence imaging in mesangial cells exposed with leptin or siRNA or co-exposed with siRNA and Leptin.(*^#P < 0.05). C and E. Immunoreactivity and morphometric analysis of 3-nitrotyrosine (3-NT, red) as shown by Immunofluorescence microscopy in kidney mesangial cells exposed with leptin or siRNA or co-exposed with siRNA and Leptin (*^#P < 0.05).

Fig. 6

NOX2-derived peroxynitrite causes generation of tyrosyl radicals in mesangial cells. A. Immunoreactivity of 3-nitrotyrosin (3-NT) as shown by Immunofluorescence microscopy (red) in mesangial cell line. Mesangial cells were treated with vehicle only served as control and other sets of Mesangial cells were treated with leptin, leptin + Apocynin, leptin + DMPO, leptin + FBA and JAK/STAT inhibitor. B Morphometric analysis of 3-NT immunoreactivity in control, leptin, leptin + Apocynin, leptin + DMPO, leptin + FBA, and leptin + JAK/STAT inhibitor groups of mesangial cells (*P < 0.05).

Fig. 7

Leptin-NOX2-Peroxynitrite axis causes mesangial cell activation via miR21. A and B. mRNA expression analysis of miRNA 21 (miR21) gene in control (mesangial cells line (CRL-1927) treated with vehicle only served as control), mesangial cells treated with leptin (leptin), siRNA, or co-exposed with leptin and siRNA (leptin + siRNA), leptin and Apocynin (leptin + Apocynin), leptin and FBA (leptin + FBA), leptin and DMPO (leptin + DMPO), and leptin and JAK/STAT inhibitor (Leptin + JAK/STAT inh). miRNA expression had been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against control (*P < 0.05). C and D. mRNA expression analysis of α-SMA gene in mesangial cells in Control, Leptin, siRNA, Leptin + siRNA, Leptin + miRNA 21 inhibitor (miR21 inh) and leptin + JAK/STAT inhibitor. mRNA expression has been assessed by quantitative real-time PCR (qRTPCR) and expressions were normalized against mesangial cell control (*P < 0.05). E. Immunoreactivity of α-SMA as observed by immunohistochemistry in kidney slices from (NAFLD), NAFLD mice exposed to BDCM, and miRNA21 gene-deficient mice fed with high-fat diet (miR21 KO) and exposed to BDCM (miR21 KO + BDCM). All immunohistochemistry Images were taken at 60× oil. F. Morphometric analysis of α-SMA immunoreactivity in NAFLD, NAFLD + BDCM, miR21 KO, and miR21 KO + BDCM group of mice. All morphometric analysis was performed as mean data from three separate microscopic fields (designed on y-axis) (*P < 0.05). G. mRNA expression analysis of α-SMA gene in kidney tissue of NAFLD, NAFLD + BDCM, and miR21 KO + BDCM mice. mRNA expression has been assessed by qRTPCR and was normalized against NAFLD group (*P < 0.05).

Fig. 8

MiR21 increase causes kidney inflammation. A. mRNA expression of IL-1β, TNFα, and IFN-γ in kidney tissue from mice fed with high-fat diet. NAFLD serve as a control, NAFLD mice exposed to BDCM (NAFLD + BDCM), miRNA 21 gene-deficient mice exposed to BDCM (miR21 KO + BDCM) as assessed by quantitative real-time PCR, the expressions were normalized with 18S and compared to the NAFLD group (*P < 0.05). B. Immunoreactivity of IL-1β as shown by immunohistochemistry in kidney slices from. of NAFLD, NAFLD + BDCM, miR21 KO, and miR21 KO + BDCM group of mice Images were taken at 20×. C. Morphometric analysis of IL-1β immunoreactivity in NAFLD, NAFLD + BDCM, miR21 KO, and miR21 KO + BDCM group of mice (*P < 0.05).