Mitochondrial biogenesis induced by the β2-adrenergic receptor agonist formoterol accelerates podocyte recovery from glomerular injury

Abstract

Podocytes have limited ability to recover from injury. Here, we demonstrate that increased mitochondrial biogenesis, to meet the metabolic and energy demand of a cell, accelerates podocyte recovery from injury. Analysis of events induced during podocyte injury and recovery showed marked upregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a transcriptional co-activator of mitochondrial biogenesis, and key components of the mitochondrial electron transport chain. To evaluate our hypothesis that increasing mitochondrial biogenesis enhanced podocyte recovery from injury, we treated injured podocytes with formoterol, a potent, specific, and long-acting β2-adrenergic receptor agonist that induces mitochondrial biogenesis in vitro and in vivo. Formoterol increased mitochondrial biogenesis and restored mitochondrial morphology and the injury-induced changes to the organization of the actin cytoskeleton in podocytes. Importantly, β2-adrenergic receptors were found to be present on podocyte membranes. Their knockdown attenuated formoterol-induced mitochondrial biogenesis. To determine the potential clinical relevance of these findings, mouse models of acute nephrotoxic serum nephritis and chronic (Adriamycin [doxorubicin]) glomerulopathy were used. Mice were treated with formoterol post-injury when glomerular dysfunction was established. Strikingly, formoterol accelerated the recovery of glomerular function by reducing proteinuria and ameliorating kidney pathology. Furthermore, formoterol treatment reduced cellular apoptosis and increased the expression of the mitochondrial biogenesis marker PGC-1α and multiple electron transport chain proteins. Thus, our results support β2-adrenergic receptors as novel therapeutic targets and formoterol as a therapeutic compound for treating podocytopathies.

Keywords: albuminuria; focal segmental glomerulosclerosis; glomerulonephritis; glomerulus; podocyte.

Figure 1:. MB is induced during recovery of podocytes from injury:

(A) Podocytes treated with PS showed actin cytoskeleton (Green) disorganization with accumulation of actin stress fibers at the cell periphery, and reduced Neph1 (Red) at cell-cell junctions. Recovery induced with supplementation of serum (serum induced recovery) restored actin cytoskeleton organization and localization of Neph1 at the cell junctions. Scale bar 20μm. (B) The quantitative analysis showed significant increase in the number of healthy podocytes (>40%) during recovery. (C) The quantitative assessment of Neph1 localization showed significant relocalization of Neph1 (~40%) at the cell membrane during recovery. (D) Analysis of mtDNA copy number showed significant enhancement during recovery of podocytes from injuries with NTS or PS. (E) Upregulation of β₂-AR2 and various other mitochondrial components as evaluated by qPCR. All experiments were performed at least in triplicates. Data are presented in mean±SEM and p-values were calculated using a 2-tailed t-test. *P≤0.05, **P≤0.01, control vs. injury; ^aP≤0.05, PS vs. PS+recovery; ^bP≤0.05 NTS vs. NTS+recovery.

Figure 2:. Formoterol induced MB in podocytes.

(A & B) FCCP uncoupled maximum OCR measurements were made in cultured human podocytes treated with 30 nM formoterol for 24 h and showed significantly enhanced mitochondrial oxygen consumption. *P≤0.05, 2-tailed t-test. (C) qPCR analysis of podocytes treated with either control vehicle or 30nM formoterol showed relative fold change in the expression of PGC-1a, ATPase 6, CYTB, FN1 and CO1 genes that are involved in MB. All experiments were performed at least in triplicates. Data are presented in mean±SEM and p-values were calculated using a 2-tailed t-test. *P≤0.05, **P≤0.01, ***p≤001 vs. control. (D) Rat glomeruli were stained with β₂-AR (Green) and Synaptopodin (Red) antibodies (arrows mark the presence of β₂-AR in podocytes). Scale bar 10μm (E) Cultured human podocytes immuno-stained with phalloidin (Green) and β₂-AR (Red) showed membrane and nuclear staining of β₂-AR. Scale bar 20μm (F) Western blot analysis of β₂-AR expression in podocyte cell lysate. (G) β₂-AR knockdown (β₂-AR-KD) podocytes were generated using five different sets of shRNA and the qPCR analysis showed maximal (~80%) knockdown using shRNA # 83, which was subsequently used for all experiments. (H) mtDNA copy number analysis showed induction of mtDNA by formoterol, whereas, β₂-AR-KD blunted formoterol-induced increase in mtDNA copy number. Data are presented in mean±SEM. *P<0.05, control-KD+vehicle vs control-KD+formoterol; #p<0.05, control-KD+vehicle vs β₂-AR-KD+ vehicle.

Figure 3:. Formoterol restored injury-induced changes to mitochondrial morphology, OCR and ATP:

(A) MitoTracker staining was used to assess mitochondrial morphology in cultured human podocytes. Elongated mitochondria were observed in healthy untreated podocytes (arrows), but shortened fragmented mitochondria were noted in podocytes injured by PS. Treatment with formoterol restored elongated morphology to a larger extent. Scale bar 10μm. (B) The quantitative analysis showed more than 70% recovery of mitochondrial morphology upon formoterol treatment of PS-injured cells. (C–D) Formoterol treatment along with PS-injury significantly enhanced the basal as well as uncoupled OCR in cultured podocytes. *P≤0.05 vs. untreated control (2-tailed t-test). (E) ATP measurements showed significant enhancement of ATP production in PS+formoterol treated cells. *P≤0.05 vs. untreated control.

Figure 4:. Formoterol treatment enhanced recovery of podocytes from PS and NTS induced injuries:

(A) Podocytes treated with NTS show damaged actin cytoskeleton (Green) with accumulation of actin stress fibers at cell periphery, and loss of Neph1 (Red) at the cell-cell junctions. In contrast, significant recovery of actin cytoskeletal organization was noted in formoterol treated cells along with restoration of Neph1 staining at cell-cell junctions (arrows). Scale bar 20μm. (B) The quantitative analysis showed ~27% increase in the number of healthy podocytes with a concomitant decrease in NTS injured podocytes upon formoterol treatment, while the vehicle treated podocytes recovered minimally. (C) Quantitation further showed complete relocalization of Neph1 at cell membrane upon formoterol treatment of NTS injured podocytes. Data are presented in mean±SEM, One-way ANOVA, ^###P≤0.0001 vs. untreated control; ***P≤0.0001 NTS+vehicle vs. NTS+formoterol. (D) PS injured podocytes showed damaged actin cytoskeleton (Green) with accumulation of actin stress fibers at cell periphery, and loss of Neph1 (Red) at the cell-cell junctions. Recovery of actin cytoskeletal organization was noted in formoterol treated cells with restoration of Neph1 staining at the cell junctions (arrows). Scale bar 20μm. (E) The quantitative analysis of actin cytoskeleton reorganization showed significant increase in the number of healthy podocytes (~40%), with a concomitant decrease in PS injured podocytes upon treatment with formoterol, whereas the recovery in vehicle treated podocytes was minimal. (F) The quantitative assessment of Neph1 relocalization at podocyte cell membrane shows, complete relocalization of Neph1 at cell membrane in formoterol treated PS injured podocytes. Data are presented in mean±SEM, One-way ANOVA, ^###P≤0.0001 vs. untreated control; *** P≤0.0001 PS+vehicle vs. PS+formoterol.

Figure 5:. Formoterol accelerated glomerular recovery from acute and chronic models of glomerular injury:

The schematic of experimental plan to evaluate in-vivo significance of formoterol using NTS (A) and ADR (B) induced glomerular injury models. (C) Urine samples were analyzed by SDS-PAGE and Coomassie blue staining and showed significant reduction in albuminuria starting at day3, which extended to day 7 in NTS+formoterol treated mice group, whereas minimal reduction was noted in the control NTS+vehicle treated mice group. (D) Similar urine analysis showed significant reduction in albuminuria at day 10 post formoterol treatment in ADR+formoterol treated mice, whereas minimal reduction was noted in the control ADR+vehicle mice. (E) Measurement of urine albumin/creatinine ratios by ELISA showed initiation of albuminuria at 4h post NTS injection, which further increased at days 1 and 2 in both groups. The ratios dropped to preinjection levels in NTS+formoterol treated mice group but remained significantly elevated in control mice group at days 5–7. 2-tailed t-test. n=5 mice per group. Data are presented in means±SEM. *P≤0.01, **P≤0.001 vs. control. (F) Measurements of urine albumin/creatinine ratios were made at 1 week post ADR-injection. The ratios remained significantly elevated in both the groups at day 5, but significant reduction was noted in ADR+formoterol mice at day 10 post formoterol treatment. 2-tailed t-test (*p<0.05, ADR+vehicle vs ADR+formoterol). n=5 mice per group. Data are presented as mean±SEM. In-vivo results were reproduced in three independent experiments with n=5, each group, each experiments.

Figure 6:. Formoterol restored injury-induced glomerular damage.

(A) Histological analysis of mice (sacrificed at day 7 post NTS injection) kidney sections shows that formoterol treatment (NTS+formoterol) reduced focal atrophy, proteinaceous tubular caste and tubular dilation as compared to control (vehicle) mice. Scale bar 50μm. (B) Major histological features were individually scored, and the comparative analysis showed reduction in loss of brush borders, degeneration of proximal tubules and reduced proteinaceous casts, tubular dilation and fibrosis in NTS+formoterol treated mice. 2-tailed t-test, ***P≤0.0001 NTS+vehicle vs. NTS+formoterol. (C) Glomerular scores demonstrating varying levels of glomerulosclerosis in control and NTS+formoterol treated mice are presented and show increased number of normal glomeruli in NTS+formoterol treated mice, along with reduction in sclerotic glomeruli. 2-tailed t-test, **P≤0.001, ***P≤0.0001 NTS+vehicle vs. NTS+formoterol. n=5 mice each group (both kidneys were scored) (D) Representative images from the TEM analysis show significant damage to podocyte foot processes in control NTS+vehicle treated mice, whereas, normal foot processes were present in the NTS+formoterol treated mice. Scale bar 2μm. (E) Histological analysis of mice (sacrificed at day 10 post formoterol injection) kidney sections showed that formoterol treatment (ADR+formoterol) reduced ADR-induced glomerular sclerosis, focal atrophy, proteinaceous tubular caste and tubular dilation. Scale bar 50μm. (F) Glomerulosclerosis severity score was calculated and was significantly reduced for ADR+formoterol mice. (**P≤0.001, NTS+vehicle vs. NTS+formoterol). n=5 mice each group.

Figure 7:. Formoterol treatment restored Neph1 localization at the podocyte cell membrane and reduced cellular apoptosis:

(A) Mice kidney sections were immunostained with Neph1 (Green) and Synaptopodin (Red) antibodies and DAPI (Blue). NTS induced mislocalization of Neph1 was largely restored by formoterol treatment, where increased Neph1 localization at the podocytes membrane and colocalization with synaptopodin was visible. (B) The Pearson’s correlation coefficient (Rr) analysis showed increased colocalization of Neph1 and Synaptopodin in NTS+formoterol treated mice. Data are presented in mean±SEM. One-way ANOVA, ***P≤0.001 NTS+vehicle vs. NTS+formoterol. (C & D) Apoptosis was measured in the kidney sections using TUNEL assay. Significant amounts of TUNEL positive (Green) nuclei (blue DAPI) were present in NTS+vehicle treated control mice and positive control, whereas they were largely absent in the NTS+formoterol treated mice (white arrows). Data are presented in mean±SEM. One-way ANOVA, ^###P≤0.001 Control vs NTS+vehicle; ***P≤0.001 NTS+vehicle vs. NTS+formoterol.

Figure 8:. Formoterol treatment induced the expression of mitochondrial proteins:

(A) Markers of MB in mice kidney lysates were evaluated by western blotting using PGC-1α, OXPHOS (cocktail antibodies of mitochondrial ETC complex I to V) and GAPDH antibodies. (B–C) Densitometric analysis of immunoblots showed increased expression of PGC-1α (One-way ANOVA, *P≤0.05 NTS+vehicle vs. NTS+formoterol; ^##P≤0.01 vehicle vs NTS+formoterol) and OXPHOS (mitochondrial complex I, III and V) proteins in NTS+formoterol treated mice (One-way ANOVA, **P≤0.01, ***P≤0.001 NTS+vehicle vs. NTS+formoterol; ^##P≤0.01 vehicle vs NTS+formoterol). Data are presented in mean±SEM. (D) The qPCR analysis showed that formoterol treatment upregulated the expression of PGC-α, NDUFB8, ND6, COXIII and NRF1 genes that are involved in MB. Data are presented in mean±SEM. One-way ANOVA, **P≤0.05, **P≤0.01, ***P≤0.001 NTS+vehicle vs. NTS+formoterol; ^aaP≤0.01 control vs NTS+vehicle; ^#P≤0.05, ^###P≤0.05 control vs NTS+formoterol. (E) mtDNA copy number analysis of kidney samples from NTS injured mice showed that mtDNA copy number significantly increased (~2fold) upon formoterol treatment. *P≤0.05 control vs NTS+formoterol.

Figure 9:. Formoterol treatment induced the expression of mitochondrial genes in mice glomeruli:

(A) Immunostaining analysis of kidney sections using PGC-1α (Green) and Synaptopodin (Red) antibodies and DAPI (Blue) showed increased PGC-1α staining in the NTS+formoterol treated mice. (B) The quantitative analysis of mean pixel intensity showed increased PGC-1α expression in the glomeruli of NTS+formoterol treated mice. Data are presented in mean±SEM. One-way ANOVA, ***P≤0.001 NTS+vehicle vs. NTS+formoterol. (C) Immunostaining with PGC-1α (Green) and Synaptopodin (Red) antibodies and DAPI (Blue) showed increased PGC-1α staining in the glomeruli of ADR+formoterol treated mice. (D) The quantitative analysis of mean pixel intensity showed increased PGC-1α expression in the glomeruli of ADR+formoterol treated mice. Data are presented in mean±SEM. One-way ANOVA, ***P≤0.001 NTS+vehicle vs. NTS+formoterol.