Insights into the effects of apelin-13 on renal function and NHE3 activity following ischemia/reperfusion-induced acute kidney injury

Figures

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  FIGURE 1. Renal ischemia/reperfusion (I/R) upregulates apelin receptor expression in kidney tissue and decreases apelin gene expression. (A) Representative immunohistochemistry from 40× micrographs (4 µm thick) and (B) graphical presentation showing that the apelin receptor (APLNR) was significantly increased in the renal I/R mice group. The black arrows indicate positive staining for APLNR. (C) Apelin mRNA expression (Apln) in whole kidney tissue from sham or I/R mice was analyzed by quantitative PCR. The results were normalized to Gapdh expression (endogenous gene) and are shown as relative units compared to the sham group. Statistical analysis was performed as t-test with Welch correction. The p-value is inserted in the graph. Sham mice (filled circles); I/R, ischemia/reperfusion mice (filled squares); G, glomerulus; PT, proximal tubule; TALH, thick ascending loop of Henle; CD, collecting duct; SD, standard deviation. Scale bar: 10 µm.

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  FIGURE 2. Prior administration of apelin-13 did not improve tubular morphology or kidney function after renal I/R. (A) Plasma urea analysis. (B) Plasma creatinine analysis. (C) Representative 10% SDS-PAGE and (D) graphical presentation showing the abundance of urinary albumin, which was not attenuated by apelin-13 administration. A bovine serum albumin (BSA, 0.2 mg/mL) standard was loaded on the adjacent lane (left). The cropped gel is displayed, and the full-length gel is included in Supplementary Figure S2. (E) Representative 20× micrographs of hematoxylin–eosin staining of kidney sections (4 µm thick) and (F) graphical presentation showing that prior administration of exogenous apelin-13 sustained tubular injury in mice subjected to renal I/R. The red arrows show protein casts inside the tubules, and the black arrows indicate necrotic cells inside the tubular structures. The values are expressed as mean ± SD. Statistical analyses were performed as two-way ANOVA. The p-value is indicated in the graphs. Sham mice (filled circles); I/R, ischemia/reperfusion mice (filled squares); SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; G, glomerulus; PT, proximal tubule; TALH,…

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  FIGURE 3. Exogenous apelin-13 administration sustains kidney tubular injury after renal I/R. (A) Representative immunohistochemistry from 20× micrographs (4 µm thick) of KIM-1, NGAL, and megalin and (B, C, D) graphical presentations showing that previous administration of exogenous apelin-13 sustained tubular injury markers KIM-1 and NGAL as well as decreased the levels of proximal tubular megalin in mice subjected to renal I/R. The black arrows indicate positive staining. The values are expressed as mean ± SD. Statistical analyses were performed as two-way ANOVA. The p-value is indicated in the graphs. Sham mice (filled circles); I/R, ischemia/reperfusion mice (filled squares); G, glomerulus; PT, proximal tubule; DT, distal tubule; SD, standard deviation. Scale bar: 25 µm.

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  FIGURE 4. ERK 1/2-dependent tubular proliferation after renal I/R is impaired by apelin-13 administration. (A) Representative immunohistochemistry from 20× micrographs (4 µm thick) of Ki67 and phosphorylated ERK 1/2 and (B, C) graphical presentations showing that previous administration of exogenous apelin-13 decreased Ki67 and phospho ERK 1/2 staining in mice subjected to renal I/R. The black arrows indicate positive staining for each protein. (D) Representative immunoblotting of phosphorylated ERK 1/2, total ERK 1/2, and GAPDH, and (E) graphical presentation of the ratios. The values are expressed as mean ± SD. Statistical analyses were performed as two-way ANOVA. The p-value is indicated in the graphs. Sham mice (filled circles); I/R, ischemia/reperfusion mice (filled squares); G, glomerulus; PT, proximal tubule; TALH, thick ascending loop of Henle; SD, standard deviation. Scale bar: 25 µm.

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  FIGURE 5. Both apelin-13 and I/R reduce intracellular pH recovery rates in the proximal tubular cells in vitro. (A) Representative immunohistochemistry from 40× micrographs (4 µm thick) showing that renal I/R affected NHE3 staining patterns in the proximal tubules of vehicle + I/R mice when compared to the sham group. Moreover, prior administration of exogenous apelin-13 did not affect the apical distribution of NHE3 in the proximal tubules adjacent to the glomerulus in the apelin-13 + sham group. The black arrows indicate positive staining for proximal tubular NHE3. (B) Protein expression of NHE1 in the TKPTS control cells. (C) Protein expression of NHE3 in the TKPTS control cells. (D) Protein expression of APLNR in the TKPTS control cells. (E) Representative experiment of pHi recovery in which the TKPTS cells were initially exposed to a ringer solution containing Na+ (138 mM), acid loading with NH4Cl (20 mM), a Na+-free solution with NMDG (138 mM), followed by a ringer solution containing Na+ (138 mM). (F) Effects of S3226 (10 µM) or cariporide (10 µM) on the pHi recovery rates of TKPTS cells. (G) Effects of I/R or apelin-13 (100 nM) on the pHi recovery rates of TKPTS cells. (H) Effects…