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. 2011 Oct;121(10):4003-14.
doi: 10.1172/JCI58662. Epub 2011 Sep 1.

PGC-1α promotes recovery after acute kidney injury during systemic inflammation in mice

Mei Tran  1 , Denise Tam, Amit Bardia, Manoj Bhasin, Glenn C Rowe, Ajay Kher, Zsuzsanna K Zsengeller, M Reza Akhavan-Sharif, Eliyahu V Khankin, Magali Saintgeniez, Sascha David, Deborah Burstein, S Ananth Karumanchi, Isaac E Stillman, Zoltan Arany, Samir M Parikh
Affiliations

PGC-1α promotes recovery after acute kidney injury during systemic inflammation in mice

Mei Tran et al. J Clin Invest. 2011 Oct.

Abstract

Sepsis-associated acute kidney injury (AKI) is a common and morbid condition that is distinguishable from typical ischemic renal injury by its paucity of tubular cell death. The mechanisms underlying renal dysfunction in individuals with sepsis-associated AKI are therefore less clear. Here we have shown that endotoxemia reduces oxygen delivery to the kidney, without changing tissue oxygen levels, suggesting reduced oxygen consumption by the kidney cells. Tubular mitochondria were swollen, and their function was impaired. Expression profiling showed that oxidative phosphorylation genes were selectively suppressed during sepsis-associated AKI and reactivated when global function was normalized. PPARγ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis and metabolism, not only followed this pattern but was proportionally suppressed with the degree of renal impairment. Furthermore, tubular cells had reduced PGC-1α expression and oxygen consumption in response to TNF-α; however, excess PGC-1α reversed the latter effect. Both global and tubule-specific PGC-1α-knockout mice had normal basal renal function but suffered persistent injury following endotoxemia. Our results demonstrate what we believe to be a novel mechanism for sepsis-associated AKI and suggest that PGC-1α induction may be necessary for recovery from this disorder, identifying a potential new target for future therapeutic studies.

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Figures

Figure 1
Figure 1. Characterization of the endotoxemia model.
(A) 10 mg/kg of i.p. LPS triggers a rise in BUN and serum creatinine (Cr) at 18 hours; n = 4–11 per group. (B) Histological signs of injury in the LPS model are scarce. Left: Yellow arrows indicate individual proximal tubule cells with vacuolar changes; middle: yellow arrow points to a single necrotic cortical tubule; right: immunohistochemistry for cleaved caspase-3 (black arrows indicate positive cells) identifies scant apoptosis (representative images of n = 6 mice). Original magnification, ×40. (C) Color Doppler images (red indicates arterial flow; blue, venous flow) and renal artery blood flow (RBF) in n = 5 mice before and 18 hours after LPS administration. (D) BOLD MRI images for serial measurement of intrarenal oxygenation. Upper left: Axial R2* map of mouse with partial ligation of the left kidney and non-ligated contralateral kidney (R2* scale clipped at 80 Hz); upper right: axial R2* maps of non-ligated, pre-LPS, and 18 hours post-LPS kidney showing no major changes in overall or regional R2* values (R2* scale 0–60 Hz); bottom panel: overall R2* values for n = 5 mice before and 18 hours after LPS treatment showing no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001 versus control or pre-LPS condition.
Figure 2
Figure 2. Regional distribution and severity of mitochondrial dysfunction and injury.
(A) Staining (brown) for cytochrome c oxidase enzyme activity on slices from snap-frozen kidneys 18 hours after vehicle or LPS treatment. Cortex (C) and inner stripe of the outer medulla (ISOM) have the most intense staining, followed by outer stripe of the outer medulla (OSOM), with the weakest activity in the inner medulla (IM). Enzymatic activity is greatly reduced following LPS. Original magnification, ×4 (overview); ×40 (cortex, corticomedullary [CM] junction). (B) Western blot on whole kidney lysates for cytochrome c oxidase. (C) Transmission EM of proximal tubules demonstrating swelling of mitochondria and rarefaction of cristae. n = 3–6 mice per condition. Original magnification, ×3,000 (left); ×20,000 (right).
Figure 3
Figure 3. Similarities in expression profiles between early and persistent injury and between baseline and recovery states.
(A) Eighteen hours after LPS, mice received 10 ml/kg saline, and by 42 hours after LPS, the majority had recovered from AKI. Kidneys from n = 3 animals per indicated condition (baseline, early injury, persistent injury, recovery; red circles) were studied by expression profiling. Dashed horizontal lines indicate mean values. (B) PCA of the transcriptional data from each condition. The percentage values indicate the proportion of total variance described by the corresponding principal component. (C) Genes were selected using supervised analysis on the basis of 1-way ANOVA of the 4 conditions — baseline, early injury, persistent injury, and recovery. Columns represent the samples, and rows represent the genes, with expression demonstrated by pseudocolor scale (–3 = green, +3 = red).
Figure 4
Figure 4. SOM and pathway analyses.
(A) Array results were analyzed by a SOM algorithm for 50 target groups. Shown are the 6 strongest clusters of genes with similar relative expression profiles across conditions. Number of transcripts per cluster is indicated above each graph. (B) Analysis of pathways enriched in SOM clusters identified oxidative phosphorylation as the most strongly over-represented biological group. Each bar represents a significantly enriched pathway as determined using the Benjamini-Hochberg hypothesis (B-H) corrected P value (shown on x axis).
Figure 5
Figure 5. Coordinated changes in the expression of PGC-1α and downstream genes.
(A) Expression level for PGC-1α and other genes related to mitochondrial biogenesis and function was measured in RNA prepared from kidneys harvested at the indicated time points after LPS (reported as abundance relative to β-actin). P values were determined by ANOVA. Note that PGC-1β does not significantly vary over time, unlike PGC-1α. n = 35–37 data points per gene studied. MCAD, medium-chain acyl-CoA dehydrogenase; ERRα, estrogen related receptor α; Nrf1, nuclear respiratory factor 1. (B) In situ hybridization for PGC-1α (antisense) or sense probe performed on snap-frozen kidney sections. Representative images shown (overview; original magnification, ×4) with higher-power image of the cortex and outer medulla (original magnification, ×10) and high-power image of the cortex (original magnification, ×40), where the absence of glomerular staining and strong positivity of proximal tubules are evident.
Figure 6
Figure 6. Effects of TNF-α and heterologous PGC-1α expression on oxygen consumption and mitochondrial gene expression in human proximal tubular epithelial cells.
(A) Relative mRNA expression of PGC-1α and downstream electron transport components following the indicated treatments. (B) TNF-α dose- and time-dependently reduces oxygen consumption (pmol/min of O2 consumed) of confluent human renal proximal tubule cells. After measuring basal respiration, we applied oligomycin (OL) to block complex V, then 2,4-dinitrophenol (2,4-DNP) to uncouple mitochondria, then rotenone to inhibit complex I. (C) TNF-α–induced changes in basal respiration and uncoupled respiration. (D) Compared with backbone control adenovirus (Control), adenovirus encoding murine PGC-1α (Ad-PGC-1α) increases the expression of PGC-1α in the setting of TNF-α treatment (left) and prevents TNF-α–induced reductions in basal (middle) and uncoupled respiration (right; reported as pmol/min of O2 consumed). (E) Ad-PGC-1α restores the expression of downstream electron transport enzymes to non-TNF-α levels. Note that the PGC-1α measured is human, not mouse. n = 9 replicates per condition. **P < 0.01, ***P < 0.001.
Figure 7
Figure 7. Persistent endotoxemic AKI in global and proximal tubule–specific PGC-1α–knockout mice.
(A) Eight- to 10-week-old male global knockouts for PGC-1α (–/–) or their wild-type littermates (+/+) were given 10 mg/kg LPS, then 10 ml/kg saline 18 hours after LPS. Renal function was measured 42 hours after LPS (i.e., 24 hours after saline resuscitation). BUN and Cr were significantly elevated in knockouts versus wild-type littermates. (B) Schematic for generating proximal tubule–specific knockouts of PGC-1α. Mice expressing Cre recombinase under the control of the Sglt2 promoter (Sglt2-Cre) were crossed with mice bearing loxP sites surrounding exons 3–5 of PGC-1α (floxed PGC-1α), and progeny were genotyped as shown. (C) Eight- to 10-week-old male Sglt2Cre/+PGC-1αfl/fl (Cre/+ fl/fl) mice experienced persistent AKI in the saline-resuscitated model compared with age- and sex-matched Cre/+ or fl/fl controls. n ≥ 14 per group. *P < 0.05, **P < 0.01.
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