Pioglitazone Enhances the Beneficial Effects of Glucocorticoids in Experimental Nephrotic Syndrome

Abstract

Glucocorticoids are the primary therapy for nephrotic syndrome (NS), but have serious side effects and are ineffective in ~20-50% of patients. Thiazolidinediones have recently been suggested to be renoprotective, and to modulate podocyte glucocorticoid-mediated nuclear receptor signaling. We hypothesized that thiazolidinediones could enhance glucocorticoid efficacy in NS. We found that puromycin aminonucleoside-induced proteinuria in rats was significantly reduced by both high-dose glucocorticoids (79%) and pioglitazone (61%), but not low-dose glucocorticoids (25%). Remarkably, pioglitazone + low-dose glucocorticoids also reduced proteinuria (63%) comparably to high-dose glucocorticoids, whereas pioglitazone + high-dose glucocorticoids reduced proteinuria to almost control levels (97%). Molecular analysis revealed that both glucocorticoids and pioglitazone enhanced glomerular synaptopodin and nephrin expression, and reduced COX-2 expression, after injury. Furthermore, the glomerular phosphorylation of glucocorticoid receptor and Akt, but not PPARγ, correlated with treatment-induced reductions in proteinuria. Notably, clinical translation of these findings to a child with refractory NS by the addition of pioglitazone to the treatment correlated with marked reductions in both proteinuria (80%) and overall immunosuppression (64%). These findings together suggest that repurposing pioglitazone could potentially enhance the proteinuria-reducing effects of glucocorticoids during NS treatment.

Figure 1. Pioglitazone Reduces PAN-Induced Proteinuria and Enhances the Protective Effects of Glucocorticoids.

Proteinuria was induced in male Wistar rats by single intravenous PAN injections (50 mg/kg) on Day 0. Treatment groups also received: (A) low-dose GC (5 mg/kg; n = 13), high-dose GC (15 mg/kg; n = 13), and pioglitazone (Pio; 10 mg/kg; n = 13); and (B) high-dose GC (15 mg/kg; n = 13) and combinations of pioglitazone with low-dose GC (Pio + low-dose GC; n = 13) or high-dose GC (Pio + high-dose GC; n = 4) for 10 days. Urinary protein/creatinine ratios (UPC) are plotted through the course of the experiment (Mean ± SEM; *P < 0.05, PAN vs. control; ^#P < 0.05, treatment vs. PAN; ^nsP > 0.05; ^$P < 0.05; determined by Two-way ANOVA tukey’s multiple comparison test). (C) Representative gels depicting proteinuria induction after PAN injection and reduction after treatments with GC and pioglitazone. Massive amounts of urinary albumin after PAN injection were reduced by treatment with high-dose GC and pioglitazone, and even more notably reduced by treatment with pioglitazone combined with low-dose GC and high-dose GC (Pio + low-dose GC and Pio + high-dose GC). Urines were collected daily and equal volumes (4 μl) from selected days were analyzed by SDS–polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining.

Figure 2. Pioglitazone and Glucocorticoids Restore Synaptopodin Expression in PAN-Injured Glomeruli.

The panels show representative images of the immunofluorescence staining for synaptopodin in the glomeruli of kidneys from control, PAN-injured and PAN-injured rats treated with high-dose GC, Pio, and Pio + low-dose GC.

Figure 3. Pioglitazone and Glucocorticoids Enhance the Glomerular Expression of Podocyte Markers and Reduce COX-2 Expression.

Total RNA was extracted from glomeruli isolated from control, PAN-injected, and PAN-injected rats treated with high-dose GC, Pio, and Pio + low-dose GC (n = 3 per group). Relative mRNA levels of (A) Synpo, (B) Nphs1 and (C) Ptgs2 were measured by quantitative reverse transcription–polymerase chain reaction and normalized to Gapdh. Data are presented as Mean ± SEM (*P < 0.05 vs. control; ^#P < 0.05 vs. PAN; determined by t-test).

Figure 4. Phosphorylation of the Glomerular Glucocorticoid Receptor and Akt, but not PPARγ, Correlate with Glucocorticoid- and Pioglitazone-Induced Reductions in Proteinuria.

Total protein was extracted from glomeruli isolated from control, PAN-injected, and PAN-injected rats treated with low-dose GC, high-dose GC, Pio and Pio + low-dose GC (n = 4 per group). Relative percentage phosphorylation of glucocorticoid receptor (GR), PPARγ and Akt was measured by western blotting and densitometry analyses. Values were plotted as Mean ± SEM (*P < 0.05 vs. control; ^#P < 0.05 vs. PAN; determined by t-test) distributed by treatment groups (A–C). Percentage phosphorylation was also plotted against the individual proteinuria values and correlations were computed by Pearson correlation coefficients (D–F).