RAAS inhibitors directly reduce diabetes-induced renal fibrosis via growth factor inhibition - PubMed (original) (raw)

. 2019 Jan;597(1):193-209.

doi: 10.1113/JP277002. Epub 2018 Nov 2.

Agnes Molnar [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.") 2, Lilla Lenart [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary."), Judit Hodrea [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary."), Dora Bianka Balogh [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.") 2, Tamas Lakat [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary."), Edgar Szkibinszkij [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.") 3, Adam Hosszu [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.") 2, Nadja Sparding 4 5, Federica Genovese 4, Laszlo Wagner 3, Adam Vannay 6, Attila J Szabo 2 6, Andrea Fekete [ 1](#full-view-affiliation-1 "MTA-SE "Lendület" Diabetes Research Group, Hungarian Academy of Sciences and Semmelweis University, Budapest, Hungary.") 2

Affiliations

RAAS inhibitors directly reduce diabetes-induced renal fibrosis via growth factor inhibition

Sandor Koszegi et al. J Physiol. 2019 Jan.

Abstract

Key points: Increased activation of the renin-angiotensin-aldosterone system (RAAS) and elevated growth factor production are of crucial importance in the development of renal fibrosis leading to diabetic kidney disease. The aim of this study was to provide evidence for the antifibrotic potential of RAAS inhibitor (RAASi) treatment and to explore the exact mechanism of this protective effect. We found that RAASi ameliorate diabetes-induced renal interstitial fibrosis and decrease profibrotic growth factor production. RAASi prevents fibrosis by acting directly on proximal tubular cells, and inhibits hyperglycaemia-induced growth factor production and thereby fibroblast activation. These results suggest a novel therapeutic indication and potential of RAASi in the treatment of renal fibrosis.

Abstract: In diabetic kidney disease (DKD) increased activation of renin-angiotensin-aldosterone system (RAAS) contributes to renal fibrosis. Although RAAS inhibitors (RAASi) are the gold standard therapy in DKD, the mechanism of their antifibrotic effect is not yet clarified. Here we tested the antifibrotic and renoprotective action of RAASi in a rat model of streptozotocin-induced DKD. In vitro studies on proximal tubular cells and renal fibroblasts were also performed to further clarify the signal transduction pathways that are directly altered by hyperglycaemia. After 5 weeks of diabetes, male Wistar rats were treated for two more weeks per os with the RAASi ramipril, losartan, spironolactone or eplerenone. Proximal tubular cells were cultured in normal or high glucose (HG) medium and treated with RAASi. Platelet-derived growth factor (PDGF) or connective tissue growth factor (CTGF/CCN2)-induced renal fibroblasts were also treated with various RAASi. In diabetic rats, reduced renal function and interstitial fibrosis were ameliorated and elevated renal profibrotic factors (TGFβ1, PDGF, CTGF/CCN2, MMP2, TIMP1) and alpha-smooth muscle actin (αSMA) levels were decreased by RAASi. HG increased growth factor production of HK-2 cells, which in turn induced activation and αSMA production of fibroblasts. RAASi decreased tubular PDGF and CTGF expression and reduced production of extracellular matrix (ECM) components in fibroblasts. In proximal tubular cells, hyperglycaemia-induced growth factor production increased renal fibroblast transformation, contributing to the development of fibrosis. RAASi, even in non-antihypertensive doses, decreased the production of profibrotic factors and directly prevented fibroblast activation. All these findings suggest a novel therapeutic role for RAASi in the treatment of renal fibrosis.

Keywords: CTGF; PDGF; diabetic nephropathy; profibrotic growth factors; renin-angiotensin-aldosterone system inhibitors; tubulointerstitial fibrosis.

© 2018 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

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Figures

Figure 1

Figure 1. Renal functional parameters, kidney histology and urinary fibrotic markers of control, diabetic and renin‐angiotensin‐aldosterone system inhibitor‐treated diabetic rats

A, kidney‐to‐body weight ratio, creatinine clearance, serum creatinine, and blood urea nitrogen were measured in rats. B, urinary collagen type III degradation fragment (uC3M), tumstatin (TUM) and N‐terminal pro‐peptide of rodent type III collagen (rPRO‐C3) levels measured from rat urine. C, representative periodic acid‐Schiff (PAS)‐stained histological sections and quantitative evaluation of glomerular hypertrophy and mesangial matrix expansion. Arrows mark mesangial matrix deposition stained with purple. 400× magnification; scale bar = 50 μm. Values are presented as means ± 95% confidence intervals; n = 7–8 rats/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control; # P = 0.08, § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. D.

Figure 2

Figure 2. Fibrotic alterations in kidneys of control, diabetic and renin‐angiotensin‐aldosterone system inhibitor treated diabetic rats

A, representative Massonʹs trichrome‐stained histological sections and quantitative evaluation of renal tubulointerstitial fibrosis by Masson‐positive and glomerulus‐free vs. total areas in the kidney cortex. Arrows mark fibrotic tissue stained with blue. 200× magnification; scale bar = 100 μm. B, representative Picrosirius Red‐stained histological kidney sections and quantitative evaluation of collagen accumulation. Arrows mark red‐stained collagen. 100× magnification; scale bar = 200 μm. On each graph values are presented as means ± 95% confidence intervals; n = 7–8 rats/group; Kruskal‐Wallis ANOVA on ranks; *** P < 0.001 vs. control; §§§ P < 0.001 vs. D. C, representative fluorescent immunohistochemical staining of renal sections and Western blot analysis of alpha‐smooth muscle actin (αSMA) protein levels in the kidneys, with representative examples above. Samples might be from different gels but were derived at the same time and processed in parallel. 630× magnification; green, αSMA; blue, nucleus; scale bar = 20 μm; values are presented as means ± 95% confidence intervals; n = 7–8 rats/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; *** P < 0.001 vs. control; §§§ P < 0.001 vs. D.

Figure 3

Figure 3. mRNA expression of profibrotic factors and fibrotic marker extracellular matrix enzymes in control, diabetic and renin‐angiotensin‐aldosterone system inhibitor‐treated diabetic rats

A, transforming growth factor β 1 (Tgfb1), platelet‐derived growth factor B (Pdgfb) and connective tissue growth factor (Ctgf) mRNA expression measured in kidney. B, matrix metalloproteinase 2 (Mmp2) and tissue inhibitor of matrix metalloproteinase 1 (Timp1) mRNA expression measured in kidney. Values are presented as means ± 95% confidence intervals; n = 7–8 rats/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05, ** P < 0.01 vs. control; § P < 0.05, §§ P < 0.01, §§§ P < 0.001 vs. D.

Figure 4

Figure 4. Profibrotic factor production in human kidney 2 (HK‐2) cells on normal, high glucose (HG), mannitol and renin‐angiotensin‐aldosterone system inhibitor (RAASi) treatment

A, transforming growth factor β (TGFB1), platelet‐derived growth factor B (PDGFB), and connective tissue growth factor (CTGF) mRNA expression in HK‐2 proximal tubular epithelial cells on HG or osmotic control mannitol treatment. B, TGFB1, PDGFB, and CTGF mRNA expression in HK‐2 cells on HG and RAASi treatment. Values are presented as means ± 95% confidence intervals; n = 6 wells/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05, *** P < 0.001 vs. control; § P < 0.05, NS, not significant vs. HG

Figure 5

Figure 5. Extracellular matrix formation and degradation biomarkers measured in human kidney 2 (HK‐2) cells on high glucose (HG) and renin‐angiotensin‐aldosterone system inhibitor (RAASi) treatment and in normal rat kidney fibroblast (NRK‐49F) cells treated with profibrotic factors and RAASi

A, C‐terminal of fibronectin (FBN‐C) turnover marker secretion by HK‐2 proximal tubular epithelial cells on HG and RAASi treatment. B, type IV collagen formation biomarker (PRO‐C4) secretion by HK‐2 cells on HG and RAASi treatment. C and D, PRO‐C4 secretion by NRK‐49F cells treated with platelet‐derived growth factor (PDGF; C) or connective tissue growth factor (CTGF; D). Values are presented as means ± 95% confidence intervals; n = 6 wells/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05 vs. control; §§ P < 0.01 vs. HG.

Figure 6

Figure 6. Effect of profibrotic factors and renin‐angiotensin‐aldosterone system inhibitors (RAASi) on normal rat kidney fibroblast (NRK‐49F) cells

A, representative picture of platelet‐derived growth factor receptor β (PDGFR‐β) stained NRK‐49F cells. 1000× magnification; red, PDGFRβ; blue, nucleus; scale bar = 20 μm. B_–_D, representative pictures of phalloidin‐TRITC immunostained NRK‐49F cells (control, B) treated with platelet‐derived growth factor (PDGF; C) or connective tissue growth factor (CTGF/CCN2; D). 1000× magnification; red, F‐actin; blue, nucleus; scale bar = 20 μm. E and F, alpha‐smooth muscle actin (αSMA) protein levels in NRK‐49F cells treated with PDGF (E) or CTGF/CCN2r (F) and RAASi. Representative gel image examples shown above the panels. Samples might be from different gels but were derived at the same time and processed in parallel. On each graph values are presented as means ± 95% confidence intervals; n = 6 wells/group; one‐way ANOVA followed by Bonferroniʹs multiple‐comparison post hoc test; * P < 0.05, *** P < 0.001 vs. control; § P < 0.05, §§§ P < 0.001 vs. PDGF or CTGF.

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