Inhibition of Angiotensin-Converting Enzyme Ameliorates Renal Fibrosis by Mitigating DPP-4 Level and Restoring Antifibrotic MicroRNAs

Abstract

Two class of drugs 1) angiotensin-converting enzyme inhibitors (ACEis) and 2) angiotensin II receptor blockers (ARBs) are well-known conventional drugs that can retard the progression of chronic nephropathies to end-stage renal disease. However, there is a lack of comparative studies on the effects of ACEi versus ARB on renal fibrosis. Here, we observed that ACEi ameliorated renal fibrosis by mitigating DPP-4 and TGFβ signaling, whereas, ARB did not show. Moreover, the combination of N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), one of the substrates of ACE, with ACEi slightly enhanced the inhibitory effects of ACEi on DPP-4 and associated-TGFβ signaling. Further, the comprehensive miRome analysis in kidneys of ACEi+AcSDKP (combination) treatment revealed the emergence of miR-29s and miR-let-7s as key antifibrotic players. Treatment of cultured cells with ACEi alone or in combination with AcSDKP prevented the downregulated expression of miR-29s and miR-let-7s induced by TGFβ stimulation. Interestingly, ACEi also restored miR-29 and miR-let-7 family cross-talk in endothelial cells, an effect that is shared by AcSDKP suggesting that AcSDKP may be partially involved in the anti-mesenchymal action of ACEi. The results of the present study promise to advance our understanding of how ACEi regulates antifibrotic microRNAs crosstalk and DPP-4 associated-fibrogenic processes which is a critical event in the development of diabetic kidney disease.

Keywords: ACE; ARBs; AcSDKP; DPP-4; EMT; EndMT; diabetic nephropathy; kidney fibrosis.

Figure 1

Inhibition of ACE suppresses DPP-4 and associated TGFβ signaling in diabetic kidneys (A) Quantitative analysis of DPP-4 mRNA expression by real time PCR using specific primers in the kidney of control, DM, DM+ACEi, DM+combination and DM+ARB treated mice. N = 6 were analyzed in each group. 18S was used as internal control to normalize the expression data. (B) Western blot analysis of DPP-4, TGFβR1, p-smad3, smad3, FSP-1, αSMA, Colla1a and fibronectin (FN) in the kidney of control, DM, DM + ACEi, DM + combination treatment and DM + ARB treated diabetic mice. Representative blots are shown. Quantification of DPP-4, TGFβR1, smad3 phosphorylation, FSP-1, αSMA, Colla1a and FN by densitometry. The data were normalized by β-actin. N = 5 were analyzed in each group. (C) Co-immunofloroscence analysis of DPP-4/CD31 and DPP-4/ αSMA in the kidney of control, DM, DM + ACEi, DM + ACEi + AcSDKP and DM + ARB, the representative pictures are shown. Scale bar 50 µm. DPP-4 FITC (green) labeled whereas, CD31 and αSMA are rhodamine labeled and DAPI blue. N = 5 were analyzed in each group. (D) DPP activity analysis by fluorimeter in kidney homogenate of control, DM, DM + ACEi, DM + combination and DM + ARB treated mice. N = 6 were analyzed in each group. (E) DPP activity analysis in the plasma of control, DM, DM + ACEi, DM + combination and DM + ARB treated mice. N = 6 were analyzed in each group. Data in the graph are presented as mean ± SEM. One-way Anova Tukey test was performed for calculation of statistical significance. C = control (non-diabetic), DM = diabetic group, combination = (ACEi + AcSDKP), Colla1 = collagen I, FN = fibronectin.

Figure 2

miRome analysis reveal up-regulated expression of miR-29 and miR-let-7 family members in the kidneys of ACE inhibitor or combination treated diabetic mice. (A) microRNA-array analysis in the diabetic group vs ACEi + AcSDKP treatment in diabetic mice revealed alteration in the expression level of pro and antifibrotic microRNAs. N = 3 were analyzed in each group. (B) miR-29 and miR-let-7 family members emerged as important regulatory antifibrotic molecules and validation by the real time PCR using specific primers in the kidney of control, DM, ACEi, combination treatment and ARB group. N = 6 were analyzed in each group. Hs_RNU6 was used as internal control to normalize the expression data. Data in the graph are presented as mean±SEM. One-way Anova Tukey test was performed for calculation of statistical significance.

Figure 3

Inhibition of ACE inhibit DPP-4 level and TGFβ signaling in endothelial cells. (A) Western blot analysis in the ACEi and ACEi+AcSDKP treated HMVECs in presence and absence of TGFβ2. Quantification of DPP-4, TGFβR1, FSP-1, p-smad3 and smad3 respectively by densitometry. Representative blots are shown. The data was normalized by β-actin. N = 3 were analyzed in each group. (B) Western blot analysis in the ARB treated HMVECs in presence and absence of TGFβ2. Quantification of DPP-4, TGFβR1, FSP-1, p-smad3 and smad3 respectively by densitometry. Representative blots are shown. The data was normalized by β-actin. N = 3 were analyzed in each group. Data in the graph are presented as mean ± SEM. One-way Anova Tukey test was performed for calculation of statistical significance.

Figure 4

ACEi and combination treatment restore the downregulated level of miR-29 and miR-let-7 family members in the TGFβ2-stimulated HMVECs. (A) qPCR analysis of miR-29 and miR-let-7 family members in the control, ACEi, and ACEi+AcSDKP in the presence and absence of TGFβ2 in the HMVECs. N = 4 were analyzed in each group. Hs_RNU6 was used as internal control to normalize the expression data. (B) qPCR analysis of miR-29 and miR-let-7 family members in the control and ARB stimulation in the presence and absence of TGFβ2. N = 4 were analyzed in each group. Hs_RNU6 was used as internal control to normalize the expression data. Data in the graph are presented as mean±SEM. One-way Anova Tukey test was performed for calculation of statistical significance.

Figure 5

Gene expression analysis of DPP-4 and antifibrotic microRNAs in the combination treatments (ACEi + AcSDKP and ARB + AcSDKP). (A) Gene expression analysis of DPP-4 mRNA. (B–C) Gene expression analysis of miR-29 family members and miR-let-7 family member in the TGFβ2-stimulated HMVECs. N = 5 were analyzed in each group Data in the graph are presented as mean±SEM. One-way Anova Tukey test was performed for calculation of statistical significance.

Figure 6

Inhibition of ACE restored the TGFβ2-associated disruption of cross-talk regulation between miR-29 and miR-let-7 family members in the endothelial cells. (A) Gene expression analysis of miR-29 family members in the anti-miR-let-7b transfected HMVECs, ACEi+anti-miR-let-7b transfected, and ARB+anti-miR-let-7b transfected HMVECs. N = 4 were analyzed in each group. (B) Gene expression studies of miR-let-7b and miR-let-7c in the anti-miR-29b transfected HMVECs, ACEi+anti-miR-29b transfected, and ARB+anti-miR-29b transfected HMVECs. N = 4 were analyzed in each group. Data in the graph are presented as mean ± SEM. One-way Anova Tukey test was performed for calculation of statistical significance.

Figure 7

Working hypothesis for ACEi action on the suppression of DPP-4 associated fibrogenic program and restoration of antifibrotic microRNAs.