Human heat shock protein 27-overexpressing mice are protected against acute kidney injury after hepatic ischemia and reperfusion

Abstract

Liver ischemia-reperfusion injury (IRI) causes acute kidney injury (AKI) in mice characterized by renal endothelial cell apoptosis, renal tubular necrosis, inflammation, and filamentous (F)-actin disruption. Since heat shock protein 27 (HSP27) protects against apoptosis, necrosis, and stabilizes F-actin, we questioned whether overexpression of human HSP27 (huHSP27 OE) in mice would attenuate AKI after liver IRI. Twenty-four hours after hepatic IRI, HSP27 wild-type (WT) mice developed acute liver and kidney injury with elevated plasma alanine aminotransferase and creatinine, a reduced glomerular filtration rate, and histological evidence of renal endothelial cell apoptosis and tubular injury (necrosis, vacuolization, and F-actin disruption). The huHSP27 OE mice, however, were significantly protected against both liver and kidney injury after hepatic IRI. The huHSP27 OE mice also showed less induction of several proinflammatory mRNAs (TNF-alpha, MIP-2, and keratinocyte-derived cytokine), neutrophil infiltration, and reduction in apoptosis (terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling assay and DNA laddering) in the kidney compared with the HSP27 WT mice. Moreover, the huHSP27 OE mice showed significantly less disruption of F-actin in renal proximal tubules and better preserved vascular endothelial cell integrity compared with the huHSP27 OE mice. Finally, the kidney plays a major role in the hepatoprotective effects of huHSP27 overexpression as the hepatoprotection was reduced or abolished in mice subjected to unilateral or bilateral nephrectomy, respectively. Our results show that overexpression of huHSP27 protects against hepatic injury and AKI associated with liver IRI in vivo. Harnessing the mechanisms of cytoprotection with renal HSP27 may lead to new therapies for the perioperative AKI and liver injury associated with liver IRI.

Fig. 1.

A: plasma alanine aminotransferase (ALT) and creatinine (Cr) levels in heat shock protein 27 wild-type (HSP27 WT) and human HSP27-overexpressing (huHSP27 OE) mice subjected to sham operation (sham) and 45-, 60-, or 75-min liver ischemia and 24 h of reperfusion injury (IRI). *P < 0.01 vs. sham group. ^#P < 0.01 vs. IRI group. +P < 0.05 vs. 45-min liver ischemia group. ++P < 0.05 vs. 60-min liver ischemia group. B: plasma ALT and Cr levels in HSP27 WT and huHSP27 OE mice subjected to sham operation (sham) and 60-min liver IRI. The blood samples were collected at 4 and 24 h after reperfusion. Values are means ± SE. *P < 0.01 vs. sham group. ^#P < 0.01 vs. IRI group. ++P < 0.05 vs. 4-h reperfusion group.

Fig. 2.

Plasma ALT levels in HSP27 WT and huHSP27 OE mice subjected to sham operation (sham) and 45-min liver ischemia and 24 h of reperfusion (IRI). Some mice were subjected to concomitant unilateral (Uni Nx) or bilateral nephrectomy (Bi Nx) during liver ischemia. The blood samples were collected at 24 h after reperfusion. Values are means ± SE. *P < 0.01 vs. sham group. ^#P < 0.01 vs. HSP27 WT IRI group.

Fig. 3.

Representative (of 6 slides) hematoxylin- and eosin-stained photomicrographs in kidney sections for HSP27 WT and huHSP27 OE mice subjected to sham operation or to liver IRI (magnification ×200). Hypereosinophilic (long arrow) and vacuolization (short arrow) of proximal tubules were prominent in HSP27 WT but not in huHSP27 OE mice subjected to liver IRI.

Fig. 4.

Renal histological assessment after liver IRI in HSP27 WT and huHSP27 OE mice. Mice were subjected to sham operation (n = 4) or to liver IRI (n = 6 for HSP27 WT and n = 7 for huHSP27 OE). Twenty-four hours later, HSP27 WT mice subjected to liver IRI showed an increased percentage of cortical vacuolization, peritubular capillary leukocyte margination, and proximal tubule hypereosinophilia. Values are means ± SE.*P < 0.01 vs. sham-operated mice. #P < 0.05 vs. HSP27 WT IRI group.

Fig. 5.

Densitometric quantifications of relative band intensities from RT-PCR reactions (normalized to GAPDH). Values are means ± SE. *P < 0.05 vs. HSP27 WT sham group. #P < 0.05 vs. HSP27 WT IRI group. ++P < 0.05 vs. 4-h reperfusion group.

Fig. 6.

Representative photomicrographs of neutrophil accumulation (dark brown punctate stain, arrows) in kidney sections (magnification ×400). HSP27 WT mice subjected to 60 min of liver ischemia and 24 h of reperfusion injury exhibited neutrophil accumulation whereas the huHSP27 OE mice showed very scant neutrophil infiltration after liver IRI (representative of 6–7 experiments).

Fig. 7.

A: representative fluorescence photomicrographs of kidney sections illustrating apoptotic nuclei [terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) fluorescence staining, ×100] at 24 h after 60 min of liver IRI. TUNEL-positive cells were mainly peritubular capillary endothelial cells. Photographs are representative of 5 independent experiments. Inset: higher magnification of image showing TUNEL-positive cells. In the kidney, endothelial cells not proximal tubule cells underwent apoptosis. B: representative DNA ladder (of 4 experiments). Apoptotic DNA fragments were separated from intact chromatin and loaded onto the agarose gel.

Fig. 8.

A: representative (of 5 experiments) fluorescent photomicrographs of FITC-phalloidin labeling to visualize filamentous (F)-actin in the kidneys from HSP27 WT or huHSP27 OE mice subjected to sham operation or to 60-min liver ischemia and 24 h of reperfusion. The F-actin stains of proximal tubular epithelial cells are prominent in the brush border from sham-operated mice (arrows), which is severely degraded in the kidneys of HSP27 WT mice subjected to liver IRI (*). B: quantification of mean fluorescent proximal tubule F-actin intensity as a measure of F-actin preservation in liver sections (n = 5). *P < 0.05 vs. HSP27 WT sham group. #P < 0.01 vs. HSP27 WT IRI group.

Fig. 9.

Evans blue dye (EBD) extravasation as an index of renal vascular permeability. HSP27 WT or huHSP27 OE mice were subjected to sham operation (n = 4) or to 60-min liver ischemia and 24 h of reperfusion (n = 6 for HSP27 WT mice and n = 7 for huHSP27 OE mice). Kidney EBD was extracted in formamide, and the amount of extravasated EBD concentration in the kidney was calculated against a standard curve. *P < 0.05 vs. HSP27 WT sham group. #P < 0.01 vs. HSP27 WT IRI group.