Kidney-specific reconstitution of the A1 adenosine receptor in A1 adenosine receptor knockout mice reduces renal ischemia-reperfusion injury

Abstract

Genetic deletion of the adenosine A1 receptor (A1AR) increased renal injury following ischemia-reperfusion injury suggesting that receptor activation is protective in vivo. Here we tested this hypothesis by expressing the human-A(1)AR in A(1)AR knockout mice. Renal ischemia-reperfusion was induced in knockout mice 2 days after intrarenal injection of saline or a lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-human-A(1)AR. We found that the latter procedure induced a robust expression of the reporter protein in the kidneys of knockout mice. Mice with kidney-specific human-A(1)AR reconstitution had significantly lower plasma creatinine, tubular necrosis, apoptosis, and tubular inflammation as evidenced by decreased leukocyte infiltration, pro-inflammatory cytokine, and intercellular adhesion molecule-1 expression in the kidney following injury compared to mice injected with saline or the control lentivirus. Additionally, there were marked disruptions of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in both sets of control mice upon renal injury, whereas the reconstituted mice had better preservation of the renal tubule actin cytoskeleton, which co-localized with the human-A(1)ARs. Consistent with reduced renal injury, there was a significant increase in heat shock protein-27 expression, also co-localizing with the preserved F-actin cytoskeleton. Our findings suggest that selective expression of cytoprotective A(1)ARs in the kidney can attenuate renal injury.

Figure 1. Selective cortico-medullary expression of huA1AR and EGFP via lentiviral gene delivery

Representative kidney fluorescent photomicrographs of five experiments from A₁AR KO mice (original magnification of (a) × 40 and (b) × 200) demonstrating predominant localization of EGFP expression in the cortex and corticomedullary junction with relative sparing of the medullary areas. A₁AR KO mouse kidneys were renally injected with saline, EGFP encoding lentivirus, or EGFP-huA₁AR encoding lentivirus, and 48 h later, their kidneys were embedded in Oxytetracycline compound. (Top) A₁AR KO mouse kidney injected with saline only. (Middle) A₁AR KO mouse kidney injected with EGFP encoding lentivirus. (Bottom) A₁AR KO mouse kidney injected with EGFP-huA₁AR encoding lentivirus. (c) Representative gel images of RT–PCR results from six experiments for GAPDH and A₁AR from A₁AR KO mice renal cortices. A₁AR KO mouse kidneys were injected with EGFP encoding lentivirus or EGFP-huA₁AR encoding lentivirus 48 h before RT-PCR. Representative results from injected and contralateral kidneys are shown.

Figure 2. Co-localization of huA1ARs and EGFP in the kidney after lentiviral gene delivery

Representation of four immunohistochemistry fluorescence photomicrographs for (a, b) huA₁ARs (red) and (c, d) EGFP expression (green) in A₁AR KO mice renally injected with either (a, c, e) EGFP or (b, d, f) EGFP-huA₁AR lentivirus 48 h earlier, (e, f) With 0.25 µm thickness Z-sections, we were able to show that huA₁ARs (red) and EGFP (green) colocalize together (yellow).

Figure 3. Plasma creatinine (Cr in mg/100ml) from A₁AR KO mice injected with saline, EGFP encoding lentivirus, or EGFP-huA₁AR 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) or EGFP (N = 9) or EGFP-huA₁AR (N = 9) encoding lentivirus, and plasma creatinine was measured 24 h after reperfusion. *P < 0.05 vs appropriate sham-operated A₁AR KO mice. ^#P < 0.05 vs A₁AR KO saline-injected mice subjected to renal IR. Data presented as mean ± s.e.m.

Figure 4. Representative photomicrographs of six experiments (hematoxylin-and-eosin staining, original magnification × 200) with A₁AR KO mice renally injected with saline, EGFP lentivirus, or EGFP-huA₁AR 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 ischemia-reperfusion (IR) injury 24 h earlier are shown.

Figure 5. Lentivirus-mediated expression of huA1ARs reduces pro-inflammatory gene expression after renal IR injury

(a) Representative gel images of semiquantitative RT-PCR of the proinflammatory markers ICAM-1, KC, MCP-1, MIP-2, and TNF-α from renal cortices of A₁AR KO mouse kidneys renally injected with saline, EGFP lentivirus, or EGFP-huA₁AR 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). *P < 0.05 vs appropriate sham-operated A₁AR KO mice. ^#P < 0.05 vs A₁AR KO saline renally injected mice subjected to renal IR. Error bars represent 1 s.e.m.

Figure 6. Reduced neutrophil infiltration with lentiviral gene huA1AR delivery after renal IR injury

Representative photomicrographs (× 200) of four experiments of immunohistochemistry for neutrophil infiltration (top and middle panel) or ICAM-1 expression (bottom panel) in the outer medulla of the kidneys of A₁AR KO mice renally injected with saline, EGFP encoding lentivirus, or EGFP-huA₁AR encoding lentivirus and subjected to sham operation (top panel) or to renal ischemia and 24 h of reperfusion (middle and bottom panels).

Figure 7. Representative fluorescence photomicrographs 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

A₁AR KO mouse kidneys were renally injected with saline, EGFP encoding lentivirus, or EGFP-huA₁AR encoding lentivirus and subjected to sham operation or to renal ischemia and 24 h of reperfusion.

Figure 8. Reduction in renal apoptosis with lentiviral gene huA1AR delivery after renal IR injury

(a) Representative immunoblotting images from four separate experiments for uncleaved poly-ADP ribose polymerase (PARP) and caspase 3 in A₁AR KO mouse renal cortices. A₁AR KO mouse kidneys injected with saline, lentivirus encoding EGFP, or lentivirus encoding EGFP-huA₁AR were subjected to sham operation or 30 min renal ischemia and 24 h reperfusion, (b) Densitometrie quantifications of unfragmented PARP (top, N = 4) and caspase 3 (bottom, N = 4) from A₁AR KO mouse renal cortices injected with lentivirus encoding EGFP or lentivirus encoding EGFP-huA₁AR 24 h after sham operation or IR. *P < 0.05 vs sham-operated mice. ^#P < 0.05 vs A₁AR KO EGFP encoding lentivirus injected mice subjected to renal IR.

Figure 9. Reduction in F-actin disruption with lentiviral gene huA1AR delivery after renal IR injury

Representative fluorescent photomicrographs of (a, b) phalloidin labeling (red) to visualize (c, d) F-actin and EGFP expression (green) in renal proximal tubules from A₁AR KO mouse kidneys. A₁AR KO mouse kidneys were renally injected with either (a, c, e) EGFP (identical fields shown) or (b, d, f) EGFP-huA₁AR lentivirus (identical fields shown) 48 h earlier and subjected to 30 min renal ischemia and 24 h reperfusion. ^#Indicates proximal tubules with disrupted F-actin staining and *indicates intact F-actin cytoskeleton. We show that proximal tubules expressing EGFP (green) show better preserved F-actin (red) and (e, f) coexpress together (yellow). Representative images of six independent experiments.

Figure 10. Lentivirus-mediated expression of huA1ARs increases expression of HSP27 in the kidney

(a) Representative gel images of RT-PCR results from four experiments for GAPDH and HSP27 from A₁AR KO mouse renal cortices. A₁AR KO mouse kidneys were renally injected with EGFP encoding lentivirus or EGFP-huA₁AR encoding lentivirus 48 h before RT-PCR. Representative results from injected kidneys are shown. (b) Densitometric quantifications of relative band intensities from RT-PCR reactions (N = 4) for HSP27 mRNA (normalized to GAPDH). Data in bar graphs are means ± s.e.m. ^#P < 0.05 vs EGFP-injected mice.

Figure 11. Lentivirus-mediated expression of huA1ARs increases expression of HSP27 and promotes colocalization of F-actin and HSP27

Representative fluorescent photomicrographs of (a, d, g) phalloidin labeling (green) to visualize (b, e, h) F-actin and HSP27 protein immunocytochemistry (red) in renal proximal tubules from A₁AR KO mouse kidneys. A₁AR KO mice kidneys were renally injected with (a–c) saline, (d–f) EGFP encoding lentivirus (g–i) or EGFP-huA₁AR encoding lentivirus 48 h earlier and subjected to 30 min renal ischemia and 24 h reperfusion. ^#Indicates proximal tubules with disrupted F-actin staining and * indicates intact F-actin cytoskeleton. Note that increased HSP27 expression (red) correlated with better preserved F-actin structure (green). Moreover, (c, f, i) HSP27 and F-actin colocalize together (yellow) showed with 0.25 µm thickness Z-sections. Representative images of four independent experiments.