Accelerated recovery of renal mitochondrial and tubule homeostasis with SIRT1/PGC-1α activation following ischemia-reperfusion injury

Abstract

Kidney ischemia-reperfusion (I/R) injury elicits cellular injury in the proximal tubule, and mitochondrial dysfunction is a pathological consequence of I/R. Promoting mitochondrial biogenesis (MB) as a repair mechanism after injury may offer a unique strategy to restore both mitochondrial and organ function. Rats subjected to bilateral renal pedicle ligation for 22 min were treated once daily with the SIRT1 activator SRT1720 (5mg/kg) starting 24h after reperfusion until 72h-144 h. SIRT1 expression was elevated in the renal cortex of rats after I/R+vehicle treatment (IRV), but was associated with less nuclear localization. SIRT1 expression was even further augmented and nuclear localization was restored in the kidneys of rats after I/R+SRT1720 treatment (IRS). PGC-1α was elevated at 72 h-144 h in IRV and IRS kidneys; however, SRT1720 treatment induced deacetylation of PGC-1α, a marker of activation. Mitochondrial proteins ATP synthase β, COX I, and NDUFB8, as well as mitochondrial respiration, were diminished 24h-144 h in IRV rats, but were partially or fully restored in IRS rats. Urinary kidney injury molecule-1 (KIM-1) was persistently elevated in both IRV and IRS rats; however, KIM-1 tissue expression was attenuated in IRS rats. Additionally, sustained loss of Na(+),K(+)-ATPase expression and basolateral localization and elevated vimentin in IRV rats was normalized in IRS rats, suggesting restoration of a differentiated, polarized tubule epithelium. The results suggest that SRT1720 treatment expedited recovery of mitochondrial protein expression and function by enhancing MB, which was associated with faster proximal tubule repair. Targeting MB may offer unique therapeutic strategy following ischemic injury.

Keywords: ATP synthase subunit β; ATPβ; Acute kidney injury; COX I; Drp1; Ischemia–reperfusion; KIM-1; Mfn2; Mitochondrial biogenesis; Mitochondrial dysfunction; NADH dehydrogenase (ubiquinone) 1 beta subcomplex 8; NDUFB8; PGC-1α; Proximal tubule; SIRT1; SRT1720; cytochrome c oxidase subunit I; dynamin related protein 1; kidney injury molecule 1; mitofusin-2; peroxisome proliferator activated receptor γ coactivator-1α; sCr; serum creatinine; sirtuin 1.

Figure 1. SIRT1 expression and localization after I/R and SRT1720 treatment

pAMPK and total AMPK (A) and SIRT1 (B) were examined in the renal cortex at 144 h by immunoblot analysis. (C) SIRT1 expression was quantified from immunoblot images, and normalized to GAPDH (n = 3). (D) SIRT1 expression was examined by IHC (40X objective, DAB chromogen, hematoxylin counterstain). In sham animals, nuclear SIRT1 staining was noticeable in tubule epithelial cells (arrowheads). Nuclear localization was lost after I/R, and many tubules exhibited intense cytoplasmic staining with less nuclear staining (asterisks) or complete loss of SIRT1 staining (arrows). Nuclear localization was restored with SRT1720 treatment after I/R (arrowheads). (E) The percentage of SIRT1-positive nuclei per field for each group was quantified (n = 3 – 6, 8 fields per n, Mean % +/− SEM). In (C) and (E), asterisks (*) indicate data are significantly different from V group, and pound signs (#) indicate IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 2. SRT1720-induced deacetylation of PGC-1α after I/R

(A) PGC-1α protein in the renal cortex at 72 h and 144 h after I/R by immunoblot analysis. (B) Nuclear PGC-1α from the renal cortex at 144 h after I/R by immunoblot analysis. (C) Acetylation of PGC-1α at 72 h and 144 h after I/R by immunoprecipitation with an anti-acetylated-lysine antibody, followed by immunoblot analysis of PGC-1α, with densitometric quantification of (C) at the 144 h time point in the graph to the right (n = 3). Asterisk (*) indicates data are significantly different from V group, and pound sign (#) indicates IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 3. Mitochondrial protein expression after I/R and SRT1720 treatment

Mitochondrial proteins (A) ATP synthase β, cytochrome c oxidase subunit I, NDUFB8 and (B) Drp1 And Mfn2 were examined in the renal cortex at 24 h, 72 h and 144 h by immunoblot analysis, with densitometric quantification (normalized to GAPDH) of the 144 h time point in the graph to the right of each representative blot (n = 3 – 6, Mean fold change +/− SEM). Asterisks (*) indicate data are significantly different from V group, and pound signs (#) indicate IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 4. Oxygen consumption and protein expression in isolated mitochondria

(A) Basal and FCCP-uncoupled respiration were measured in mitochondria isolated from the renal cortex at 144 h (n = 4, Mean fold change +/− SEM). (B) ATP synthase β (ATPβ), cytochrome c oxidase subunit I (COX I), and NDUFB8, and the fusion/fission proteins Mfn2 and Drp1 were examined in mitochondria isolated from the renal cortex at 144 h, with densitometric quantification (normalized to VDAC) in (C) and (D) (n = 3). Asterisks (*) indicate data are significantly different from V group, and pound signs (#) indicate IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 5. Histopathology and renal function after I/R

(A) Renal histology was examined by hematoxylin and eosin (H&E) staining at 144 h (10X objective). (B) Histological grades were determined by evaluating H&E and PAS sections to generate a composite severity score from individual scores (0 – 4) for factors including: loss of brush border, casts, inflammatory cells, tubular dilation, degeneration, interstitial edema, and wide interstitium (n = 5, Mean +/− SEM). (C) Serum creatinine was monitored in vehicle and SRT1720-treated I/R and sham rats from 24h to 144h after injury (n = 6, Mean mg/dl +/− SEM). Asterisks (*) indicate data are significantly different from V group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 6. Markers of proximal tubule injury and homeostasis

(A) Urinary kidney injury molecule-1 (KIM-1) measured by ELISA in urine collected from IRV and IRS rats (n = 6, Mean ng/ml +/− SEM). (B) KIM-1 in the renal corex at 72 h and 144 h after injury by immunoblot analysis, with (D) densitometric quantification (normalized to GAPDH) at the 144 h time point (n = 5, Mean +/− SEM). (C) Na⁺,K⁺-ATPase and vimentin were examined in the renal cortex at 72 h and 144 h after injury by immunoblot analysis, with (E) densitometric quantification (normalized to GAPDH) at the 144 h time point (n = 4, Mean +/− SEM). Asterisks (*) indicate data are significantly different from V group, and pound signs (#) indicate IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).

Figure 7. Immunohistochemical analysis of proximal tubule injury/recovery after I/R

(A) KIM-1, Na⁺,K⁺-ATPase, and PCNA were examined by IHC after I/R with SRT1720 or vehicle treatment (DAB chromogen, hematoxylin counterstain). Injury groups were evaluated separately (moderate or severe) based on extent of initial injury at 24 h post-reperfusion. KIM-1 was not detected in sham animals (40X objective). In IRV and IRS kidneys, KIM-1 was localized to the apical membrane of injured tubules (asterisks), and was associated with cellular debris in the luminal space (arrows). Na⁺,K⁺-ATPase was ubiquitously expressed at the basolateral membrane of tubules throughout the renal cortex in sham animals (20X objective). In IRV kidneys, there was an overall loss of Na⁺,K⁺-ATPase localization and expression. In IRS kidneys, Na⁺,K⁺-ATPase expression was mostly restored in both the moderate and the severe injury groups, and localization to the basolateral membrane was more evident in the moderate injury group, but was also present in the severe injury group (arrows). In sham kidneys, PCNA was sporadically detected in the nucleus of isolated tubule cells throughout the renal cortex (arrow, 40X objective). After injury, the number of PCNA-positive (PCNA+) cells increased, and could be detected in groups of tubule cells throughout the renal cortex (arrows). (B) The number of KIM-1 positive tubules per field was quantified in IRV and IRS kidneys from the moderate and the severe injury groups (n = 3, 8 fields per n, Mean +/− SEM). (C) The number of PCNA+ cells per field from IRV and IRS kidneys in the moderate and the severe injury groups was quantified (n = 3, 8 fields per n, Mean +/− SEM). Pound signs (#) indicate IRS was significantly different from the IRV group (ANOVA followed by Newman-Keuls, p<0.05).