DEPLETION OF GUT MICROBIOTA ALLEVIATES PROTEINURIA IN PUROMYCIN AMINONUCLEOSIDE-INDUCED NEPHROSIS IN RATS

The gut–kidney axis has recently attracted attention as a bidirectional connection between organs. While its role in chronic kidney disease is increasingly recognized, the involvement of gut microbiota in idiopathic nephrotic syndrome (INS) remains unclear. Previous studies, including our own, suggest that gut dysbiosis—imbalances in intestinal microbiota composition and/or abundance—may contribute to INS. Dysbiosis in the intestine is typically characterized by a reduction in beneficial bacteria and an increase in harmful bacteria. However, whether a loss of beneficial bacteria or an increase in harmful bacteria plays a greater role in INS pathogenesis remains unknown. We hypothesized that depleting harmful gut bacteria would alleviate proteinuria and designed this study to clarify the role of dysbiosis in minimal change nephrotic syndrome (MCNS), the most common type of INS.

The Animal Research Committee of Kansai Medical University approved all experimental protocols. We used a puromycin aminonucleoside (PAN)-induced nephropathy rat model, which replicates human MCNS pathology. Rats were divided into three groups (n=6 each): a control group (oral PBS for 10 days followed by a single subcutaneous injection of PAN (50 mg/kg) on day 0, an antibiotic-treated group (oral amoxicillin, cefotaxime, vancomycin, and metronidazole) for 10 days, followed by PAN injection on day 0, and an indoxyl sulfate (IS)-treated group (antibiotics for 10 days followed by intraperitoneal IS and PAN injection on day 0). Spot urine was collected daily for 10 days from day 0 to measure protein and creatinine. Administration of antibiotics or PBS was maintained until the animals were sacrificed, following the PAN injection. On day 10, the kidneys were examined by transmission electron microscopy (TEM). Urinary IS and 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured in a separate cohort (n=10 per group). Fecal samples underwent 16S rRNA gene sequencing to assess microbial composition and diversity. To confirm causality, fecal microbiota transplantation (FMT) was also performed: antibiotic-pretreated rats received FMT from healthy donors (with-FMT group, n=7) or PBS (without-FMT group, n=6).

Antibiotic treatment markedly suppressed proteinuria after PAN injection: On day 8, urinary protein was significantly lower in the antibiotic-treated group (1.39 g/gCre) than in controls (16.8 g/gCre, p=0.0141). Conversely, the IS-treated group developed markedly higher proteinuria (117.3 g/gCre) compared with both control and antibiotic groups (p<0.01). TEM revealed severe podocyte foot process effacement in controls, whereas podocyte integrity was preserved in antibiotic-treated rats. The IS-treated group showed effacement comparable to controls. Antibiotic treatment significantly reduced urinary IS (842.8±66.0 µg/mgCre vs. 361.8±63.9 µg/mgCre; p<0.001) and urinary 8-OHdG (86.9±9.4 ng/mgCre vs. 36.3±4.6 ng/mgCre; p<0.001). Microbiota analysis showed a significant decrease in alpha-diversity (observed species p=0.0014; Shannon index p=0.0028) following antibiotics. LEfSe analysis identified a significant reduction in indole-producing genera, including Alistipes, Bacteroides, and Desulfovibrio. Notably, FMT abolished the protective effect of antibiotics: on day 8, proteinuria did not differ significantly between the with-FMT group (120.3 g/gCre) and the without-FMT group (110.8 g/gCre).

This study provides direct evidence that gut microbiota contributes to MCNS pathogenesis, because depletion of the gut microbiota by antibiotic treatment in a rat MCNS model alleviated proteinuria, which was reversed by FMT. The protective effects of antibiotics were associated with the loss of indole-producing bacteria and reduced urinary IS, suggesting that pathogenic gut-derived metabolites drive podocyte injury. Targeting indole-producing bacteria or their metabolites may represent a novel therapeutic approach for MCNS, potentially reducing reliance on corticosteroids.