Selective renal overexpression of human heat shock protein 27 reduces renal ischemia-reperfusion injury in mice

Abstract

We have previously shown that exogenous and endogenous A(1) adenosine receptor (A(1)AR) activation protected against renal ischemia-reperfusion (IR) injury in mice by induction and phosphorylation of heat shock protein 27 (HSP27). With global overexpression of HSP27 in mice, however, there was a paradoxical increase in systemic inflammation with increased renal injury after an ischemic insult due to increased NK1.1 cytotoxicity. In this study, we hypothesized that selective renal expression of HSP27 in mice would improve renal function and reduce injury after IR. Mice were subjected to renal IR injury 2 days after intrarenal injection of saline or a lentiviral construct encoding enhanced green fluorescent protein (EGFP) or human HSP27 coexpressing EGFP (EGFP-huHSP27). Mice with kidney-specific reconstitution of huHSP27 had significantly lower plasma creatinine, renal necrosis, apoptosis, and inflammation as demonstrated by decreased proinflammatory cytokine mRNA induction and neutrophil infiltration. In addition, there was better preservation of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in the huHSP27-reconstituted groups than in the control groups. Furthermore, huHSP27 overexpression led to increased colocalization with F-actin in renal proximal tubules. Taken together, these findings have important clinical implications, as they imply that kidney-specific expression of HSP27 through lentiviral delivery is a viable therapeutic option in attenuating the effects of renal IR.

Fig. 1.

Expression and colocalization of human heat shock protein 27 (huHSP27) and enhanced green fluorescent protein (EGFP) in the kidney after lentiviral gene delivery. A: representation of 4 immunohistochemistry fluorescence photomicrographs for (top) EGFP expression (green) and (middle) huHSP27 (red) in C57BL/6 mice renally injected with either (left) an EGFP or (right) EGFP-huHSP27 lentivirus 48 h earlier. With 0.5-μm thickness z-sections, we were able to show that huHSP27 (red) and EGFP (green) colocalize together (yellow; bottom). B: representative gel images of RT-PCR results from 4 experiments for GAPDH and EGFP from mice renal cortices. Mouse kidneys were injected with the EGFP-huHSP27-encoding lentivirus 48 h before RT-PCR. Representative results from injected and contralateral (noninjected) kidneys are shown.

Fig. 2.

Plasma creatinine (Cr; in mg/dl) from C57BL/6 mice injected with saline, an EGFP-encoding lentivirus, or an EGFP-huHSP27-encoding lentivirus and subjected to sham operation (Sham) or ischemia-reperfusion (IR) injury. Mice were subjected to sham surgery (n = 5 each) or renal IR 48 h after renal injection with saline (n = 8), EGFP (n = 9)- or EGFP-huHSP27 (n = 7)-encoding lentivirus, and plasma creatinine was measured 24 h after reperfusion. *P < 0.01 vs. appropriate sham-operated mice. #P < 0.01 vs. saline-injected or EGFP-injected mice subjected to renal IR. Values are means ± SE and analyzed with 1-way ANOVA plus Tukey's post hoc multiple comparison test.

Fig. 3.

Representative photomicrographs of 6 experiments (hematoxylin and eosin staining, original magnification ×200) with C57BL/6 mice renally injected with saline, an EGFP-encoding lentivirus, or an EGFP-huHSP27-encoding lentivirus and subjected to sham operation or to renal IR. Pictures of the outer medulla of the kidneys of sham-operated mice and mice subjected to renal IR injury 24 h earlier are shown. Asterisks indicate areas of tubular dilatation, arrows indicate areas of tubular swelling and necrosis and medullary luminal congestion, and arrowheads indicate areas of hemorrhagic necrosis.

Fig. 4.

Lentivirus-mediated expression of huHSP27 reduces proinflammatory gene expression after renal IR injury. A: representative gel images of semiquantitative RT-PCR of mouse and human HSP27 as well as the proinflammatory markers keratinocyte-derived cytokine (KC), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-α (TNF-α) from renal cortices of C57BL/6 mouse kidneys renally injected with an EGFP-encoding lentivirus or an EGFP-huHSP27-encoding lentivirus and subjected to sham operation or to renal ischemia and 24 h of reperfusion. B: densitometric quantification of relative band intensities normalized to GAPDH from RT-PCR reactions for each indicated mRNA (n = 5). Values are means ± SE and analyzed with 1-way ANOVA plus Tukey's post hoc multiple comparison test. *P < 0.05 vs. EGFP lentivirus sham mice. #P < 0.05 vs. EGFP-encoding lentivirus-injected mice subjected to renal IR.

Fig. 5.

Specific renal expression of huHSP27 does not upregulate whole-blood KC mRNA. Representative gel images (of 4 experiments) of semiquantitative RT-PCR of whole-blood KC of huHSP27 wild-type (WT; lacking transgene encoding huHSP27) and huHSP27-overexpressing (OE) mice subjected to sham operation or to renal ischemia and 24 h reperfusion (A) and C57BL/6 mice renally injected with an EGFP-encoding lentivirus or an EGFP-huHSP27-encoding lentivirus 48 h before sham operation or to renal ischemia and 24 h reperfusion (B).

Fig. 6.

Reduced neutrophil infiltration with lentiviral gene huHSP27 delivery after renal IR injury. Representative photomicrographs (×400) of 4 experiments of immunohistochemistry for neutrophil infiltration in the outer medulla of the kidneys of C57BL/6 mice renally injected with saline, an EGFP-encoding lentivirus, or an EGFP-huHSP27-encoding lentivirus and subjected to renal ischemia and 24 h of reperfusion. Secondary antibody conjugated to horseradish peroxidase was developed with diaminobenzidine to stain neutrophils dark brown (arrows). PMN, polymorphonuclear neutrophils.

Fig. 7.

Representative fluorescence photomicrographs (of 4 experiments) of kidney sections illustrating apoptotic nuclei [terminal deoxynucleotidyl transferase biotin-dUTP nick end-labeling (TUNEL) fluorescence staining, ×100] in the outer medulla of the kidneys. C57BL/6 mouse kidneys were renally injected with saline, an EGFP-encoding lentivirus, or an EGFP-huHSP27-encoding lentivirus and subjected to sham operation or to renal ischemia and 24 h of reperfusion.

Fig. 8.

Representative gel images showing DNA laddering as an index of DNA fragmentation in kidney tissues. C57BL/6 mice were subjected to sham operation (sham) or renal IR 48 h after intrarenal injection with saline, an EGFP-encoding lentivirus, or an EGFP-huHSP27-encoding lentivirus. Kidney tissues were obtained 24 h after reperfusion. Images are representative of 4 independent experiments.

Fig. 9.

Reduction in F-actin disruption with lentiviral gene huHSP27 delivery after renal IR injury. A: representative (of 5 experiments) fluorescent photomicrographs of phalloidin labeling (red) to visualize F-actin in renal proximal tubules from C57BL/6 mouse kidneys. Sham kidneys demonstrate intact F-actin architecture. Mouse kidneys were renally injected with either an EGFP- or EGFP-huHSP27-encoding lentivirus 48 h earlier and subjected to 30-min renal ischemia and 24-h reperfusion. #, Proximal tubules with disrupted F-actin staining; *, intact F-actin cytoskeleton. We show that proximal tubules from kidneys injected with the EGFP-huHSP27-encoding lentivirus show better preserved F-actin (red) than the proximal tubules from kidneys injected with the EGFP-encoding lentivirus. B: quantification of mean fluorescent proximal tubule F-actin intensity as a measure of F-actin preservation in kidney sections (n = 5). Values are means ± SE and analyzed with 1-way ANOVA plus Tukey's post hoc multiple comparison test. #P < 0.001 vs. sham group. *P < 0.001 vs. EGFP IR group.

Fig. 10.

Lentivirus-mediated expression of huHSP27 promotes colocalization of F-actin and huHSP27. Representative fluorescent photomicrographs of phalloidin labeling (blue) to visualize F-actin (C and D) and huHSP27 (A and B) protein immunocytochemistry (red) in renal proximal tubules from C57BL/6 mouse kidneys. Mouse kidneys were renally injected with an EGFP-encoding lentivirus (A, C, and E) or EGFP-huHSP27-encoding lentivirus (B, D, and F). HSP27 and F-actin colocalize together (E and F; magenta), shown with 0.5-μm-thick z-sections. Images are representative of 4 independent experiments.