Adiponectin regulates albuminuria and podocyte function in mice

Abstract

Increased albuminuria is associated with obesity and diabetes and is a risk factor for cardiovascular and renal disease. However, the link between early albuminuria and adiposity remains unclear. To determine whether adiponectin, an adipocyte-derived hormone, is a communication signal between adipocytes and the kidney, we performed studies in a cohort of patients at high risk for diabetes and kidney disease as well as in adiponectin-knockout (Ad(-/-)) mice. Albuminuria had a negative correlation with plasma adiponectin in obese patients, and Ad(-/-) mice exhibited increased albuminuria and fusion of podocyte foot processes. In cultured podocytes, adiponectin administration was associated with increased activity of AMPK, and both adiponectin and AMPK activation reduced podocyte permeability to albumin and podocyte dysfunction, as evidenced by zona occludens-1 translocation to the membrane. These effects seemed to be caused by reduction of oxidative stress, as adiponectin and AMPK activation both reduced protein levels of the NADPH oxidase Nox4 in podocytes. Ad(-/-) mice treated with adiponectin exhibited normalization of albuminuria, improvement of podocyte foot process effacement, increased glomerular AMPK activation, and reduced urinary and glomerular markers of oxidant stress. These results suggest that adiponectin is a key regulator of albuminuria, likely acting through the AMPK pathway to modulate oxidant stress in podocytes.

Figure 1. Negative correlation between albuminuria and plasma adiponectin levels in obese AAs.

Data show regression between adiponectin levels and urine ACRs. Confidence intervals, Spearman’s correlation coefficient, and P values for all variables tested are listed in Table 2.

Figure 2. Ad^–/– mice exhibit increased albuminuria, oxidant stress, and podocyte dysfunction.

(A) Urine ACR in Ad^–/– mice significantly increased compared with corresponding age-matched WT mice at 1, 2, 3, and 4 months of age (n = 10 per group). *P < 0.01 versus corresponding age-matched WT. (B) WT and Ad^–/– mice were made diabetic with low-dose streptozotocin, and urine ACR was measured before and at 2 and 4 months of diabetes. Albuminuria was significantly increased in Ad^–/– mice with diabetes compared with corresponding WT diabetic groups (n = 5–10 per group). DM, diabetes mellitus. *P < 0.05 versus WT control; **P < 0.05 versus WT DM at 2 mo; ***P < 0.05 versus WT DM at 4 mo. (C) Urinary hydrogen peroxide/creatinine levels significantly increased in Ad^–/– mice with and without diabetes (n = 10 per group). *P < 0.05 versus WT control; **P < 0.05 versus WT DM at 2 mo. Values are mean ± SEM. (D) Podocyte foot processes were segmentally effaced in Ad^–/– mouse kidneys by EM. Arrows denote areas of normal foot processes in WT kidneys and areas of foot process effacement in Ad^–/– glomeruli. Images are representative of 10 EM images per kidney from 2 mice per group. Original magnification, ×5,000.

Figure 3. Adiponectin inhibits permeability across a podocyte monolayer.

(A) Permeability of albumin across a podocyte monolayer was reduced by gAd or fAd at 3 μg/ml (n = 3 per group). Cells were treated as described in Methods, and permeability was assessed by albumin concentration across podocyte monolayer. Values (mean ± SEM) are presented as percent of control. *P < 0.01 versus control. (B and C) Expression of AdipoR1 (B) and AdipoR2 (C) by real-time PCR in WT mouse liver, kidney, and differentiated podocytes. Values (mean ± SEM) are presented relative to β-actin and expressed as 100% in mouse liver.

Figure 4. AMPK activity is increased by adiponectin and regulates podocyte permeability.

(A and B) Treatment of podocytes with 3 μg/ml gAd for 24 h increased AMPK activity in podocytes cultured in normal glucose (NG; 5.5 mM d-glucose) and high glucose (HG; 25 mM d-glucose), as demonstrated by confocal microscopy (A) and immunoblotting (B). AMPK activity was assessed with antibodies specific for the p-AMPKα subunit. Total AMPKα was measured with antibody for AMPKα as a loading control (B). Images are representative confocal photographs and immunoblots from 5 separate experiments. (C) Quantitation of P-AMPKα/AMPK from immunoblots in B (n = 5). Values are mean ± SEM. *P < 0.05 versus normal glucose; **P < 0.05 versus high glucose alone. (D) Albumin permeability was decreased by the AMPK activator AICAR (1 mM) and increased by the AMPK inhibitor ARA (n = 5 per group). The effect of adiponectin to reduce permeability was also blocked by ARA. Cells were treated as described in Figure 3A and Methods. Values (mean ± SEM) are presented as percent of control. *P < 0.01 versus control; **P < 0.001 versus gAd alone.

Figure 5. ZO-1 localization is regulated by adiponectin and AMPK in podocytes.

(A) Immunofluorescence microscopy demonstrated linear ZO-1 staining along the cell membranes of podocytes with normal glucose exposure, which was further enhanced by treatment with gAd and markedly reduced with inhibition of AMPK by ARA. (B) High glucose exposure–induced reduction of linear staining of ZO-1 was attenuated by adiponectin and increased with AMPK activation by AICAR. (C) Semiquantitation of the data from 30 cells per coverslip for each condition in A and B. Experiments were repeated 5 times and expressed as mean ± SEM of linear staining per condition. *P < 0.05 versus normal glucose; **P < 0.05 versus high glucose.

Figure 6. Adiponectin restores normoalbuminuria and increases AMPK activity.

(A) Ad^–/– mice at 4 months of age were treated with saline, gAd, fAd, or AICAR, and urine ACR was measured. In addition, Ad^–/– diabetic mice (2 months of diabetes) were treated with fAd. gAd, fAd, and AICAR treatment significantly decreased the urine ACR to the control values seen in WT mice (n = 7–10 per group). *P < 0.05 versus WT; ^#P < 0.05 versus Ad^–/–; ^##P < 0.05 versus Ad^–/– DM. (B) Podocyte foot process fusion in Ad^–/– mice was reduced with gAd treatment (compare with Figure 2D). (C) Semiquantitation of the degree of foot process effacement in WT mice, Ad^–/– mice, and Ad^–/– mice treated with gAd. Values represent percent foot process effacement of individual glomeruli. *P < 0.05 versus WT; **P < 0.05 versus Ad^–/–. (D) AMPK activity was demonstrated in normal glomerular podocytes by double labeling with P-AMPK antibody and podocyte-specific synaptopodin (Synpo) antibody. (E) AMPK activity was reduced in glomeruli of Ad^–/– mice and increased by adiponectin treatment. Mouse kidneys were immunostained by light microscopy with antibody specific for p-AMPKα as described Methods. Arrows denote p-AMPKα–positive podocytes. Insets show higher magnification of the same cells. Photomicrographs are representative of 50 glomeruli from each mouse kidney (n = 4 per group). (F) Quantitation of p-AMPKα–positive cells per glomerulus (n = 4 per group). *P < 0.05 versus WT; **P < 0.05 versus Ad^–/–. Values are mean ± SEM. Original magnification, ×5,000 (B); ×40 (D and E); ×100 (E, insets).

Figure 7. Regulation of oxidant stress and Nox4 by adiponectin.

(A) Urinary levels of hydrogen peroxide were reduced by gAd, fAd, or AICAR treatment in Ad^–/– mice (n = 7–10 per group). *P < 0.05 versus WT; ^#P < 0.05 versus Ad^–/–. (B) Glomerular 8-OHdG was increased in Ad^–/– kidneys and reduced with gAd, demonstrated by light microscopy immunostain and quantitation of 8-OHdG–positive cells per glomerulus (n = 4 per group). *P < 0.05 versus WT; **P < 0.05 versus Ad^–/–. (C) Glomerular nitrotyrosine was increased in Ad^–/– kidneys and reduced with gAd treatment, demonstrated by light microscopy immunostain and quantitation of nitrotyrosine staining per glomerulus (n = 4 per group). *P < 0.05 versus WT; **P < 0.05 versus Ad^–/–. (D) Nox4 was present in podocytes, as well as other glomerular cells and tubular cells, as demonstrated by double labeling with synaptopodin in WT kidney. Insets show representative background staining without primary antibody. (E) Light microscopy immunostain demonstrated that Nox4 protein was increased in glomerular cells of Ad^–/– kidneys and reduced with gAd treatment. Photomicrographs are representative of 50 glomeruli from each mouse kidney (n = 4 per group). (F) Quantitation of Nox4-positive cells per glomerulus (n = 4 per group). *P < 0.05 versus WT; **P < 0.05 versus Ad^–/–. Values are mean ± SEM. Original magnification, ×40 (B–E); ×10 (D, insets).

Figure 8. Podocyte Nox4 is increased by high glucose exposure and reduced by adiponectin or AICAR.

(A) Podocytes grown in the presence of 3 μg/ml gAd for 24 h showed suppression of Nox4 with normal or high glucose exposure. Transferred proteins were immunoblotted with antibody to Nox4 and β-actin. (B) Similar studies were performed with podocytes grown on coverslips, demonstrating reduction of Nox4 protein. (C) AMPK activation with AICAR demonstrated Nox4 protein reduction to a degree similar to that shown by gAd in podocytes grown with high glucose exposure. Shown are representative immunoblots and confocal images from 5 separate experiments. (D) Quantitation of Nox4 relative to β-actin from the immunoblots (n = 5). Values are mean ± SEM. *P < 0.05 versus normal glucose; **P < 0.05 versus high glucose.