Protection of glucagon-like peptide-1 in cisplatin-induced renal injury elucidates gut-kidney connection

Abstract

Accumulating evidence of the beyond-glucose lowering effects of a gut-released hormone, glucagon-like peptide-1 (GLP-1), has been reported in the context of remote organ connections of the cardiovascular system. Specifically, GLP-1 appears to prevent apoptosis, and inhibition of dipeptidyl peptidase-4 (DPP-4), which cleaves GLP-1, is renoprotective in rodent ischemia-reperfusion injury models. Whether this renoprotection involves enhanced GLP-1 signaling is unclear, however, because DPP-4 cleaves other molecules as well. Thus, we investigated whether modulation of GLP-1 signaling attenuates cisplatin (CP)-induced AKI. Mice injected with 15 mg/kg CP had increased BUN and serum creatinine and CP caused remarkable pathologic renal injury, including tubular necrosis. Apoptosis was also detected in the tubular epithelial cells of CP-treated mice using immunoassays for single-stranded DNA and activated caspase-3. Treatment with a DPP-4 inhibitor, alogliptin (AG), significantly reduced CP-induced renal injury and reduced the renal mRNA expression ratios of Bax/Bcl-2 and Bim/Bcl-2. AG treatment increased the blood levels of GLP-1, but reversed the CP-induced increase in the levels of other DPP-4 substrates such as stromal cell-derived factor-1 and neuropeptide Y. Furthermore, the GLP-1 receptor agonist exendin-4 reduced CP-induced renal injury and apoptosis, and suppression of renal GLP-1 receptor expression in vivo by small interfering RNA reversed the renoprotective effects of AG. These data suggest that enhancing GLP-1 signaling ameliorates CP-induced AKI via antiapoptotic effects and that this gut-kidney axis could be a new therapeutic target in AKI.

Figures

Figure 1.

Time course of BUN, serum creatinine, body weight, and blood glucose. Animals are given 10 mg/kg of AG once daily from 7 days before and 96 hours after CP or saline injection (n=5–6 per group). (A and B) Seven days of AG pretreatment does not influence BUN or serum creatinine. After exposure to CP, BUN and serum creatinine concentrations are significantly higher 72 hours and 96 hours after CP injection. These higher levels are suppressed by AG treatment. (C and D) No significant difference is observed in body weight or blood glucose. (E and F) Delayed treatment started 6 hours after CP injection also significantly reduces BUN and serum creatinine (n=5–6 per group). *P<0.05; **P<0.001. Data are expressed as the mean ± SEM.

Figure 2.

Morphologic analysis of the kidney at 96 hours after CP administration. (A) Exposure to CP causes a loss of brush border, necrosis of tubular cells, and cast formation at 96 hours after injection (n=5–6 per group). These changes are significantly attenuated in the AG pretreated mice (CP+AG). (B) A semiquantitative assessment of histologic damage shows a significant beneficial effect of AG pretreatment in CP-AKI (n=5–6 per group). (C) Images show 4-HHE and 8-OHdG staining. CP administration causes severe accumulation of 4-HHE and 8-OHdG in damaged tubules, although their accumulation is reduced remarkably by AG treatment. (D) 4-HHE– and 8-OHdG–positive areas per ×400 visual field at 96 hours after CP administration are increased, but are reduced significantly by AG treatment (n=5–6 per group). Data are expressed as the mean ± SEM. *P<0.05; **P<0.001. Scale bar, 75 μm in A; 50 μm in C.

Figure 3.

Renal tubular apoptosis evaluated by ssDNA and cleaved caspase-3 staining, Quantitative analysis of renal Bax, Bim, Bcl-2, and Bcl-xL mRNA expression. (A and B) Representative images show ssDNA and cleaved caspase-3 staining (n=5–6 per group). The fold increase of the renal mRNA expression ratio of Bax/Bcl-2, Bim/Bcl-2, Bax/Bcl-xL, and Bim/Bcl-xL is quantified using quantitative real-time RT-PCR analysis (n=5–6 per group). (C and D) AG treatment reduces the Bax/Bcl-2 and Bim/Bcl-2 mRNA expression ratio compared with the CP treatment group. (E and F) The increased renal Bax/Bcl-xL and Bim/Bcl-xL mRNA ratio after CP injection is attenuated by AG treatment. Data are expressed as the mean ± SEM. *P<0.05.

Figure 4.

Plasma and renal DPP-4 activity, plasma GLP-1 levels, SDF-1, and NPY. (A and B) DPP-4 activity is evaluated in mouse plasma and kidney homogenates (n=6–7 per group). Plasma and renal DPP-4 activity is suppressed significantly by AG at 96 hours after CP administration. (C) The bioactive GLP-1 level does not differ between AG-treated and untreated mice before CP administration. The CP+AG–treated mice show a remarkably higher GLP-1 level 48 hours after CP administration compared with controls (n=6–7 per group). (D and E) The other DPP-4 substrates, SDF-1 and NPY, are also evaluated. Plasma SDF-1 and NPY levels in AG-treated mice are lower than untreated mice (n=5–6 per group). *P<0.05; **P<0.001. Data are expressed as the mean ± SEM.

Figure 5.

Effects of the GLP-1R agonist on CP-AKI and morphologic analysis. (A) Electrophoresis shows RT-PCR amplification products of GLP-1R from kidney homogenates. (B and C) Ex-4 treatment attenuates the increase in BUN and serum creatinine induced by CP administration (n=5–6 per group). (D) CP-induced renal pathologic injuries are significantly attenuated in Ex-4 pretreated mice. Data are expressed as the mean ± SEM. *P<0.05. cont, control; G3PDH, glyceraldehyde-3-phosphate dehydrogenase. Scale bar, 75 μm in D.

Figure 6.

Renal tubular apoptosis is attenuated by Ex-4. (A and B) Representative images show ssDNA and cleaved caspase-3 staining. (C and D) Treatment by Ex-4 reduces the Bax/Bcl-2 and Bim/Bcl-2 mRNA expression ratio compared with the CP treatment group. (E and F) The increased renal Bax/Bcl-xL and Bim/Bcl-xL mRNA ratio after CP injection is attenuated by Ex-4 treatment. Data are expressed as the mean ± SEM (n=5–6 per group). *P<0.05.

Figure 7.

Renal expression of the GLP-1R and in vivo effects of siRNA on CP-AKI. (A) Western blot analysis shows GLP-1R expression in protein extracted from kidney homogenate. GLP-1R evaluated by immunohistochemistry shows positive staining mainly on proximal tubular cells. (B) GLP-1R knockdown efficacy in the kidney is determined using quantitative real-time RT-PCR. The control group receives scrambled siRNA by injection. The 18S rRNA is used to normalize the relative expression of the GLP-1R (n=5–6 per group). (C) Western blot analysis of GLP-1R protein derived from siRNA-treated kidneys. Fold decrease of GLP-1R protein corrected using actin (n=5–6 per group). (D–F) GLP-1R siRNA cancels the renal-protective effect of AG, which is evaluated using BUN, serum creatinine, and pathologic analysis (n=5–6 per group). Data are expressed as the mean ± SEM. *P<0.05.