Estrogen-Related Receptor Agonism Reverses Mitochondrial Dysfunction and Inflammation in the Aging Kidney

Abstract

A gradual decline in renal function occurs even in healthy aging individuals. In addition to aging, per se, concurrent metabolic syndrome and hypertension, which are common in the aging population, can induce mitochondrial dysfunction and inflammation, which collectively contribute to age-related kidney dysfunction and disease. This study examined the role of the nuclear hormone receptors, the estrogen-related receptors (ERRs), in regulation of age-related mitochondrial dysfunction and inflammation. The ERRs were decreased in both aging human and mouse kidneys and were preserved in aging mice with lifelong caloric restriction (CR). A pan-ERR agonist, SLU-PP-332, was used to treat 21-month-old mice for 8 weeks. In addition, 21-month-old mice were treated with a stimulator of interferon genes (STING) inhibitor, C-176, for 3 weeks. Remarkably, similar to CR, an 8-week treatment with a pan-ERR agonist reversed the age-related increases in albuminuria, podocyte loss, mitochondrial dysfunction, and inflammatory cytokines, via the cyclic GMP-AMP synthase-STING and STAT3 signaling pathways. A 3-week treatment of 21-month-old mice with a STING inhibitor reversed the increases in inflammatory cytokines and the senescence marker, p21/cyclin dependent kinase inhibitor 1A (Cdkn1a), but also unexpectedly reversed the age-related decreases in PPARG coactivator (PGC)-1α, ERRα, mitochondrial complexes, and medium chain acyl coenzyme A dehydrogenase (MCAD) expression. These studies identified ERRs as CR mimetics and as important modulators of age-related mitochondrial dysfunction and inflammation. These findings highlight novel druggable pathways that can be further evaluated to prevent progression of age-related kidney disease.

Figure 1

Estrogen-related receptor (ERR) α, ERRγ, and pyruvate dehydrogenase (PDH) expression is decreased in the aging human kidney. A: ERRα and ERRγ immunohistochemistry of kidney sections from young and old human subjects. Insets: Magnified images of boxed areas. B: PDH immunohistochemistry of kidney sections from young and old human subjects. C: Renal transforming growth factor (TGF)-β mRNA expression in young and old mice. mRNA expression of the ERRs, PPARG coactivator (PGC)1α, medium chain acyl coenzyme A dehydrogenase (MCAD), superoxide dismutase 2 (SOD2), and uncoupling protein 2 (UCP2) in TGF-β1–treated human primary proximal tubule cells. D: Renal tumor necrosis factor (TNF)-α protein expression in young and old mice. mRNA expression of the ERRs, PGC1α, MCAD, SOD2, and UCP2 in TNF-α treatment of human primary proximal tubule cells. N = 3 for each group (A–D). ∗P < 0.05, ∗∗∗P < 0.001. Scale bar = 100 μm (A and B). AU, arbitrary unit.

Figure 2

Single-nuclei RNA sequencing of young and old kidneys. A: The t-distributed stochastic neighbor embedding of young and old mouse kidneys, with 100,000 read depth and 3000 to 5000 nuclei sequenced. A total of 12 clusters were identified and assigned to major cell types known in the mouse kidney. B: Estrogen-related receptor (ERR) α expression (purple) in young and old kidney as a function of total cell count. Top panels: The highest expression seen in proximal tubules, intercalated cells, and podocytes. ERRγ expression (purple) in young and old kidney as a function of total cell count. Bottom panels: The highest expression seen in proximal tubules and intercalated cells expressed most strongly. AL, ascending limb; CD-PC, collecting duct–principal cell; DCT, distal convoluted tubule; DL, descending limb; EC: endothelial cell; IC: intercalated cell; LH, loop of Henle; Mφ: macrophage; MC, mesangial cell; Pod: podocyte; PT, proximal tubule.

Figure 3

Pan–estrogen-related receptor (ERR) agonist improves age-related renal injury. A: Albuminuria and kidney weight (normalized by body weight) in young and old mice, with or without SLU-PP-332 (ERR) treatment. B: Left panels: NPHS2 (podocin) immunohistochemistry of kidney sections, labeling podocytes, in young and old mice, with or without SLU-PP-332 treatment. Right panel: The mean intensity of NPHS2 staining per glomeruli (glom) is also shown. C: Real-time quantitative PCR mRNA expression of kidney injury markers transforming growth factor (TGF)-β, plasminogen activator inhibitor 1 (PAI-1), collagen 4 A1 (Col4a1), F4/80, and neutrophil gelatinase-associated lipocalin (NGAL) in young and old mice, with and without SLU-PP-332 treatment. D: Cytokine array. Four major spots that correspond to kidney injury markers Ngal, kidney injury marker (Kim)-1, osteopontin, and CC-motif ligand chemokine-21 (CCL21) are highlighted (left panels), with relative changes in protein level, as assessed by densitometry (right panels). Each bar graph represented one sample pooled from four animals per group. N = 5 to 6 for each group (A, B, left panels, and C). ∗P < 0.05, ∗∗P < 0.01. Scale bar = 50 μm (B). ACR, albumin creatinine ratio.

Figure 4

RNA sequencing and proteomics of kidney from old mice treated with vehicle or the pan–estrogen-related receptor (ERR) agonist. A: ERRα, ERRβ, and ERRγ mRNA expression in young and old mouse kidneys, with and without SLU-PP-332 treatment. B: Heat map showing expression patterns of genes differentially expressed in kidneys of old mice treated with vehicle compared with kidneys of old mice treated with SLU-PP-332 treatment. The heat map indicates up-regulation (green), down-regulation (red), and unaltered gene expression (black). The columns represent individual samples. C: Functional pathway enrichment analysis of differentially expressed proteins in kidneys of old mice treated with vehicle compared with kidneys of old mice treated with pan-ERR agonist. The y axis shows significantly enriched pathways. The x axis indicates P value of enrichment of the given pathway. D: Heat map showing expression patterns of proteins differentially expressed in kidneys of old mice treated with vehicle compared with kidneys of old mice treated with pan-ERR agonist. The heat map indicates up-regulation (green), down-regulation (red), and unaltered gene expression (black). The columns represent individual samples. E: Functional pathway enrichment analysis of differentially expressed proteins in old mice treated with vehicle compared with kidneys of old mice treated with pan-ERR agonist. The y axis shows significantly enriched pathways. The x axis indicates P value of enrichment of the given pathway. F: Functional pathway enrichment analysis of subset of genes and proteins identified with two-way orthogonal partial least square (O2PLS) analysis as up-regulated in kidneys of old mice compared with kidneys of young mice and that were down-regulated by ERR treatment in old mice. The y axis shows significantly enriched pathways. The x axis indicates P value of enrichment of the given pathway. G: Functional pathway enrichment analysis of subset of genes and proteins identified with O2PLS analysis as down-regulated in kidneys of old mice compared with kidneys of young mice and that were up-regulated by ERR treatment in old mice. The y axis shows significantly enriched pathways. The x axis indicates P value of enrichment of the given pathway. N = 5 to 6 for each group (A). ∗P < 0.05. TNF, tumor necrosis factor.

Figure 5

Pan–estrogen-related receptor (ERR) agonist treatment restores mitochondrial function in aging kidneys. A: mRNA expression of PGC1α, PGC1β, and transcription factor A mitochondrial (Tfam1), coregulators of ERRs and mediators of mitochondrial biogenesis, and mitochondria/nuclear DNA ratios, in young and old mouse kidneys, with and without SLU-PP-332 treatment. B: mRNA expression levels of subunits of the mitochondrial electron chain complex (ETC). C: mRNA expression levels of tricarboxylic acid (TCA) cycle enzymes. D: Interrelationship of the TCA cycle and ETC. E: Maximum respiration capacity in mitochondria isolated from the kidneys. F: mRNA expression of the fatty acid β-oxidation enzymes, Cpt1a and Mcad. N = 5 to 6 for each group (A–C, E, and F). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗∗P < 0.0001.

Figure 6

Pan–estrogen-related receptor (ERR) agonist treatment alters mitochondrial dynamics in aging kidneys. A: Transmission electron microscopy of alterations in the mitochondria in young and old mouse kidneys, with and without SLU-PP-332 treatment. a: Normally distributed and structured mitochondria in young mice. b: Mitochondria in normal structure, perpendicularly oriented to the basolateral plasma membrane in young mice treated with SLU-PP-332. c: Chaotically distributed damaged and degraded mitochondria with cristae condensation (black arrow) or loss (red arrow) in old mice. Electron-dense lipofuscin granules are abundant in the cytoplasm (blue arrow). d: The structure of mitochondria preserved in old mice treated with SLU-PP-332. e–g: Quantification of area, perimeter, and minimum Feret diameter of mitochondria. B: Mitofusin-2 (Mfn2) protein abundance in young and old mouse kidneys, with and without SLU-PP-332 treatment (left panels), and protein levels of OPA1 mitochondrial dynamin like GTPase (Opa1) in mouse kidneys (right panels). C: Changes in mitoguardin 2 and mitoPLD (phospholipase D) mRNA levels in the mouse kidneys. D: Changes in dynamin related protein 1 (Drp1) and phosphorylated Drp1 (p-Drp1) protein in mouse kidneys. N = 3 to 4 for each group (A, e–g); N = 5 to 6 for each group in mRNA-level analysis (D); N = 4 for each group in protein analysis (D). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. Original magnification, ×5500 (A).

Figure 7

Pan–estrogen-related receptor (ERR) agonist treatment decreases inflammation in aging kidneys. A: Changes in cyclic GMP-AMP synthase (cGAS; left panels) and stimulator of interferon genes (STING; right panels) mRNA and protein levels in young and old mouse kidneys, with and without SLU-PP-332 treatment. B: RIG-I–like receptor [retinoic acid inducible gene 1 (RIG-I), melanoma differentiation associated protein 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2; alias RIG-I-like receptor 3) and toll-like receptor (TLR; 3, 7, and 9) mRNA levels in mouse kidneys. C:Rel, Relb, and Nfkb2 mRNA, and total p65 protein expression in the kidneys of mice. D: Changes in Stat3 mRNA, and phosphorylated Tyr705-STAT3 and total STAT3 protein expression. E: Changes in cellular senescence and senescence-associated secretory phenotype markers. N = 5 to 6 for each group in mRNA-level analysis (A and D); N = 4 for each group in protein analysis (A); N = 5 to 6 for each group (B and E); N = 3 to 4 for each group in protein analysis (D). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. ICAM1, intercellular adhesion molecular 1; NS, nonsignificant; Timp1, TIMP metallopeptisase inhibitor 1; TNF-α, tumor necrosis factor-α.

Figure 8

Treatment of aging mice with stimulator of interferon genes (STING) inhibitor (SI) C-176. A: Effect of the SI, C-176, on IL-1β, Stat3, phosphorylated Stat3 (p-Stat3), and p21 expression in mouse kidneys. B: Effect of STING inhibition on expression of PGC1α, PGC1β, estrogen-related receptor (ERR) α, mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit (Ndufs1) (complex I), mitochondrial cytochrome c oxidase subunit 6A2 (Cox6a2) (complex IV), Atp5e (complex V), and MCAD in mouse kidneys. N = 4 for each group (A and B). ∗P < 0.05, ∗∗P < 0.01.