Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury - PubMed (original) (raw)

Increased podocyte Sirtuin-1 function attenuates diabetic kidney injury

Quan Hong et al. Kidney Int. 2018 Jun.

Abstract

Podocyte injury and loss contribute to the progression of glomerular diseases, including diabetic kidney disease. We previously found that the glomerular expression of Sirtuin-1 (SIRT1) is reduced in human diabetic glomeruli and that the podocyte-specific loss of SIRT1 aggravated albuminuria and worsened kidney disease progression in diabetic mice. SIRT1 encodes an NAD-dependent deacetylase that modifies the activity of key transcriptional regulators affected in diabetic kidneys, including NF-κB, STAT3, p53, FOXO4, and PGC1-α. However, whether the increased glomerular SIRT1 activity is sufficient to ameliorate the pathogenesis of diabetic kidney disease has not been explored. We addressed this by inducible podocyte-specific SIRT1 overexpression in diabetic OVE26 mice. The induction of SIRT1 overexpression in podocytes for six weeks in OVE26 mice with established albuminuria attenuated the progression of diabetic glomerulopathy. To further validate the therapeutic potential of increased SIRT1 activity against diabetic kidney disease, we developed a new, potent and selective SIRT1 agonist, BF175. In cultured podocytes BF175 increased SIRT1-mediated activation of PGC1-α and protected against high glucose-mediated mitochondrial injury. In vivo, administration of BF175 for six weeks in OVE26 mice resulted in a marked reduction in albuminuria and in glomerular injury in a manner similar to podocyte-specific SIRT1 overexpression. Both podocyte-specific SIRT1 overexpression and BT175 treatment attenuated diabetes-induced podocyte loss and reduced oxidative stress in glomeruli of OVE26 mice. Thus, increased SIRT1 activity protects against diabetes-induced podocyte injury and effectively mitigates the progression of diabetic kidney disease.

Keywords: PGC1α; SIRT1; diabetic kidney disease; podocytes.

Published by Elsevier Inc.

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Conflict of interest statement

COMPETING INTEREST STATEMENT

The authors declare that they have no competing financial interests.

Figures

Figure 1

Figure 1

Podocyte-specific SIRT1 overexpression attenuates albuminuria in diabetic OVE26 mice. (A) Schema of study design. Blood glucose and urinary albumin of healthy nondiabetic littermate controls, OVE26;WT and OVE26;Pod-SIRT1OV mice were monitored starting at 8 weeks of age. Dox-supplemented chow was administered starting at 16 weeks of age, and all mice were sacrificed at 22 weeks of age. (B) Fasting blood glucose levels of nondiabetic healthy littermate controls (Healthy Ctr), OVE26;WT, and OVE26;Pod-SIRT1 mice. Both diabetic OVE26 mouse groups showed highly elevated blood glucose levels at 8 weeks in comparison to healthy controls, which was sustained throughout the duration of the study. (C) Body weight of mice did not differ between three groups at 22 weeks of age. (D) Kidney-to-body weight (K/BW) was markedly elevated in OVE26;WT compared to healthy control mice, but was significantly reduced in OVE26;Pod-SIRT1OV mice. (E) Urinary albumin-to-creatinine ratio (UACR) of spot urine collected during the duration of the study is shown. (F) Urine albumin excretion (UAE) was measured from the 24-hour urine collected from mice at 22 weeks of age. n=5 to 9 in each group; P-values between groups from one-way or two-way ANOVA analyses are as indicated. n.s., not significant.

Figure 2

Figure 2

Podocyte-specific SIRT1 overexpression attenuates glomerular injury in diabetic OVE26 mice. (A) Representative images of periodic acid-Schiff (PAS)-stained mouse kidneys at 22-weeks of age are shown (top panels: original magnification x200, scale bar: 50μm; bottom panels: original magnification x400, scale bar: 20μm). (B–C) Quantification of glomerular volume (B) and fraction of mesangial area (C) are shown. (D) Electron microscopy was performed to assess ultra-structural changes in podocyte morphology (top panels: original magnification ×12,000, scale bar: 2μm; bottom panels: original magnification ×40,000, scale bar: 500nm). Representative images are shown. (E) Semi-quantification of podocyte effacement (n=5) (F) Measurements of glomerular basement membrane (GBM) thickness (n=5). P-values between groups from one-way ANOVA analyses are as indicated.

Figure 3

Figure 3

BF175 is a novel agonist of SIRT1. Immortalized human podocytes were stimulated with high glucose (HG, 30mM) with or without BF175 (10μg/ml) for 24 hours. Treatment with high mannitol (HM, 5mM glucose+25mM mannitol) served as a control. (A) Total cell extracts were subjected to immunoprecipitation (IP) using an anti-PGC-1α antibody and immunoblotting with anti-acetyl lysine (Ac-Lys) or anti-PGC-1α antibodies. Representative image of 3 independent experiments are shown. (B) Densitometric analysis shows the levels of acetylated PGC-1α normalized to total PGC-1α in each group. P-values between groups from one-way ANOVA analyses are indicated. (C) Quantitative PCR analysis of mitochondrial biogenesis genes TFAM (left panel) and NRF-1 (right panel). (D–E) BF175 reduces the oxidative stress under high glucose conditions. Podocytes cultured in high glucose or high mannitol conditions were incubated with MitoSOX red for 15 minutes. Cells were then counterstained with DAPI and imaged (D, scale bar: 25μm) or dissociated and fluorescence intensity of MitoSOX were quantified with fluorescence activated cell sorting (FACS) (E). (F) Quantitative PCR analysis of mitochondrial DNA (mtDNA). P-values between groups from one-way or two-way ANOVA analyses are as indicated. n=3 in each group.

Figure 4

Figure 4

BF175 treatment attenuates albuminuria in diabetic OVE26 mice. (A) Schema of study design. Healthy controls and diabetic OVE26 mice were monitored starting at 8 weeks of age. Vehicle or BF175 (0.4mg/kg) was administered intraperitoneally each day starting at 16 weeks of age, and all mice were sacrificed at 22 weeks of age. (B) Fasting blood glucose levels of normal healthy controls (Ctr), vehicle-treated (OVE26+vehicle), and BF175-treated (OVE26+BF175) mice were monitored starting at 8 to 22 weeks of age. Both diabetic OVE26 mouse groups showed highly elevated blood glucose levels at 8 weeks of age in comparison to healthy controls, which was sustained throughout the duration of the study. (C) Body weight of mice did not differ between three groups at 22 weeks of age. (D) Kidney-to-body weight (K/BW) was markedly elevated in OVE26+vehicle compared to healthy control mice, but was significantly reduced in OVE26+BF175 mice. (E) Urinary albumin-to-creatinine ratio (UACR) of spot urine collected during the duration of the study is shown. Black arrow notes the time when BF175 or vehicle treatment commenced. (F) Urine albumin excretion (UAE) was measured from the 24-hour urine collected from mice at 22 weeks of age. n=5 to 9 in each group; P-values between groups from one-way or two-way ANOVA analyses are indicated. n.s., not significant.

Figure 5

Figure 5

BF175 treatment attenuates glomerular injury in diabetic OVE26 mice. (A) Representative images of PAS-stained mouse kidneys at 22-weeks of age are shown (top panels: original magnification x200, scale bar: 50μm; bottom panels: original magnification x400, scale bar: 20μm). (B–C) Quantification of glomerular volume (B) and fraction of mesangial area (C) are shown. (D) Electron microscopy was performed to assess ultra-structural changes in podocyte morphology (top panels: original magnification ×12,000, scale bar: 2μm; bottom panels: original magnification ×40,000, scale bar: 500nm). Representative images are shown. (E) Semi-quantification of podocyte effacement (n=5) (F) Measurements of glomerular basement membrane (GBM) thickness (n=5). P-values between groups from one-way ANOVA analyses are indicated.

Figure 6

Figure 6

Podocyte-specific SIRT1 overexpression or BF175 treatment increases podocyte marker expression and podocyte number in diabetic OVE26 mice. (A) Representative images of glomerular expression of SIRT1 (red) and podocin (green) are shown of healthy control, OVE26 with or without podocyte SIRT1 overexpression, and OVE26 with or without BF175 treatment. Scale bar: 20μm. (B–C) Representative images of glomerular p57 expression (B) and their quantification (C) are shown. Scale bar: 20μm. P-values between groups from one-way ANOVA analyses are as indicated. n=5 in each group.

Figure 7

Figure 7

Podocyte-specific SIRT1 overexpression or BF175 treatment reduces oxidative stress in glomeruli of diabetic OVE26 mice. Representative images of nitro-tyrosine immunostaining in paraffin-embedded kidney sections. Scale bar: 20μm.

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