Efficacy of aliskiren, compared with angiotensin II blockade, in slowing the progression of diabetic nephropathy in db/db mice: should the combination therapy be a focus?

Abstract

Although the intensive use of angiotensin II blockade (ACEI or ARB), progression of diabetic nephropathy is common. A feedback increase in renin production often accompanies angiotensin II blockade. We therefore examined whether aliskiren, a direct renin inhibitor, confers better renoprotection than angiotensin II blockade and whether the addition of aliskiren to an ACEI or ARB would enhance the efficacy in slowing the progression of glomerulosclerosis in diabetes. Untreated db/db mice developed progressive mesangial matrix expansion and albuminuria between weeks 18 and 22, associated with reduction of WT-1 immunopositive podocytes and nephrin and podocin production and induction of desmin and B7-1 generation and renal expression of TGFß1, PAI-1, fibronectin and type IV collagen. Treatment with aliskiren at 30 mg/kg/d inhibited the increases in albuminuria and markers of renal fibrosis and the changes that are indicative of podocyte injury seen in the db/db mice. Notably, the therapeutic effect of aliskiren was similar to that of either enalapril or valsartan given alone at maximally effective doses. Combined therapy caused the loss of 10% ~ 16.6% of db/db mice, yielded no further reduction in renal fibrosis and podocyte injury but further reduced albuminuria and renal production of TNFα, Nox2 and p47phox and urine MCP-1 and malondialdehyde levels, the markers of renal inflammation and oxidative stress. These results suggest that aliskiren, enalapril and valsartan are equally effective in slowing the progression of diabetic nephropathy. The use of combination therapy with aliskiren and ACEI/ARB may not be strongly supported.

Keywords: Direct renin inhibitor (DRI); albuminuria; angiotensin II receptor blocker (ARB); angiotensin converting enzyme inhibitor (ACEI); podocyte; renal fibrosis.

Figure 1

Effect of aliskiren, enalapril, valsartan and dual therapy on glomerular matrix protein accumulation in diabetic db/db mice from weeks 18 to 22. The histological sections stained with PAS (A) and glomerular immunofluorescent staining for fibronectin (FN) (B) and type IV collagen (Col IV) (C) are presented at 400 x magnification. Representative photomicrographs of glomeruli from normal control mice (db/m), diabetic db/db mice without treatment at week 18 (DC18) or at week 22 (DC22) and diabetic db/db mice treated with enalapril (Ena), valsartan (Val), aliskiren (Ali), combination of aliskiren and enalapril (A+E) or aliskiren and valsartan (A+V). Graphic representations of glomerular matrix score (D), FN staining score (E) and Col IV staining score (F) are shown below. *P<0.05, vs. db/m. #P<0.05, vs. DC18. §P<0.05, vs. DC22.

Figure 2

Effect of aliskiren, enalapril, valsartan and dual therapy on renal production of fibrotic markers in diabetic db/db mice from weeks 18 to 22. (A) Total TGFß1 protein levels in renal cortical tissues were measured by ELISA. (B) Representative western blots illustrating PAI-1, FN and ß-actin protein expression in renal cortical tissue tissues. The graphs summarize the results of band density measurements for PAI-1 (C) and FN (D). *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22).

Figure 3

Effect of aliskiren, enalapril, valsartan and dual therapy on renal mRNA expression of fibrotic markers in diabetic db/db mice from weeks 18 to 22. Expression of mRNA was determined by real-time RT/PCR. Changes in mRNA levels were determined by first correcting the amplification of ß-actin for each sample. For comparison, this ratio was set at unity for normal control (db/m) samples and other groups were expressed as fold-increase over this value. A. Expression of TGF-ß1mRNA. B. Expression of PAI-1 mRNA. C. Expression of FN mRNA. D. Expression of type α1(IV) collagen mRNA. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22).

Figure 4

Effect of aliskiren, enalapril, valsartan and dual therapy on glomerular immunofluorescent staining for nephrin (A), podocin (B) and WT-1 (C) in diabetic db/db mice from weeks 18 to 22. Representative photomicrographs (at original magnification × 400) of glomeruli from normal control mice (db/m), diabetic db/db mice without treatment at week 18 (DC18) or at week 22 (DC22) and diabetic db/db mice treated with enalapril (Ena), valsartan (Val), aliskiren (Ali), combination of aliskiren and enalapril (A+E) or aliskiren and valsartan (A+V). Graphic representation of glomerular nephrin (D), podocin (E) staining scores or glomerular number of WT-1 positive podocytes (F) was shown below. *P<0.05, vs. db/m. #P<0.05, vs. DC18. §P<0.05, vs. DC22.

Figure 5

Effect of aliskiren, enalapril, valsartan and dual therapy on mRNA expression of podocyte protein markers in diabetic db/db mice from weeks 18 to 22. Expression of mRNA was determined by real-time RT/PCR. Changes in mRNA levels were determined by first correcting the amplification of ß-actin for each sample. For comparison, this ratio was set at unity for normal control samples and other groups were expressed as fold-increase over this value. A. Expression of nephrin mRNA. B. Expression of podocin mRNA. C. Expression of WT-1 mRNA. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22).

Figure 6

Effect of aliskiren, enalapril, valsartan and dual therapy on desmin and B7-1 production in renal cortical tissue. (A) Representative western blots showing desmin and B7-1 protein expression. ß-actin was included in the blot for normalization. Graphic representation of the mean band intensity of desmin (B) and B7-1 (C) normalized against the band intensity of ß-actin. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22). Diabetic db/db mice were treated with enalapril (Ena), valsartan (Val), aliskiren (Ali), combination of aliskiren and enalapril (A+E) or aliskiren and valsartan (A+V) from weeks 18 to 22.

Figure 7

Effect of aliskiren, enalapril, valsartan and dual therapy on protein production of renal TNFα in renal cortical tissue and urine MCP-1 levels. A. Representative western blots illustrating TNFα and ß-actin protein expression. The lower respective graph summarizes the results of band density measurements. B. Urinary MCP-1 levels were measured by ELISA. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22). Diabetic db/db mice were treated with enalapril (Ena), valsartan (Val), aliskiren (Ali), combination of aliskiren and enalapril (A+E) and aliskiren and valsartan (A+V) from weeks 18 to 22.

Figure 8

Effect of aliskiren, enalapril, valsartan and dual therapy on protein production of renal NAPDH oxidases and the MDA levels in urine. Representative western blots illustrating Nox2, p47phox and ß-actin protein expression (A). Graphic representation of the mean band intensity of Nox2 (B) and p47phox (C) normalized against the band intensity of ß-actin. (D) Urinary MDA levels were detected by a colorimetric assay. *P<0.05, vs. db/m. #P<0.05, vs. db/db-18wk (DC18). §P<0.05, vs. db/db-22wk (DC22). Diabetic db/db mice were treated with enalapril (Ena), valsartan (Val), aliskiren (Ali), combination of aliskiren and enalapril (A+E) or aliskiren and valsartan (A+V) from weeks 18 to 22.