BACKGROUND: The intestinal microbiome is a promising tool for prognostics, diagnostics, and interventions in patients with congenital anomalies of the kidney and urinary system. The goal of this study was to characterize the neonatal rat intestinal microbiome in response to partial urinary tract upper and lower obstruction using 16S rRNA Illumina sequencing and quantitative polymerase chain reaction (qPCR).
METHODS: Male and female neonatal Sprague Dawley rats were operated on day three of life. Partial left ureteral obstruction was performed in UUTO via suture ligation over a wire. Partial urethral obstruction was created in LUTO via modified clip applier technique. Kidney, bladder, and colonic fecal samples were obtained at the time of sacrifice. The intestinal microbiome was evaluated via 16S rRNA gene amplicon sequencing. Alpha diversity (diversity within the sample or richness) and beta diversity (dissimilarity of the comparisons within a sample), single taxa abundance, and unique indicator species analysis was performed using R Statistical Software 4.2.0 (p<0.05).
RESULTS: Preoperative body mass was not significantly different between groups (C: 12.2+/-0.3 g, LUTO: 11.9+/-0.5 g, UUTO:11.8+/-0.6 g, p=0.4861). At 21 days post-operatively, LUTO was significantly heavier than UUTO (C: 62.8+/-3.4 g, LUTO:72.7+/-5.2 g, UUTO:53.9 +/-3.2 g, p=0.0037; LUTO vs UUTO, p=0.0030). Serum creatinine was not different across groups (C: 0.25 +/-0.08 mg/dL, LUTO: 0.23 +/-0.06 mg/dL, UUTO: 0.16 +/-0.04 mg/dL, p=0.0654). Alpha diversity using the Observed (C vs LUTO, p=0.695; C vs UUTO, p=0.097, LUTO vs UUTO, p=0.068) and Shannon (C vs LUTO, p=0.799; C vs UUTO, p=0.203; LUTO vs UUTO p=0.171) methods were not statistically significant. Beta diversity analysis demonstrated significantly different clustering between the 3 groups (C vs LUTO, p=0.016; C vs UUTO, p=0.004; LUTO vs UUTO, p=0.012). Subgroup analysis demonstrated sex was not a confounder (C vs LUTO p=0.418; C vs UUTO, p=0.151, LUTO vs UUTO p=0.381). Stool microbiome demonstrated elevated Firmicutes:Bacteroidota ratio in LUTO compared to UUTO (C: 2.31, LUTO: 4.16, UUTO:2.05, LUTO vs UUTO 0.0146; Figure 1). A unique LUTO indicator was the absence of CAG-302 (p=0.014). Lactobacillus_B was significantly downregulated in the UUTO group (C vs UUTO, p=0.028; LUTO vs UUTO, p=0.033). All unique indicators of the UUTO group derived from the Lachnospiraceae family (Genus: Clostridium_Q, p=0.013; 28-4, p=0.013; CAG-81, p=0.017).
CONCLUSIONS: This is the first study of its kind to evaluate the neonatal response of the gastrointestinal microbiome to upper and lower urinary tract obstruction. LUTO resulted in high-risk intestinal microbiome profiles such as increased Firmicutes:Bacteriodota ratios and absence of CAG_302, consistent with obesity and metabolic syndrome. UUTO rats had gut microbiome profiles consistent with CKD models such as upregulation of the Lachnospiracea family, known modulators of short chain fatty acids and uremic toxins. Such patterns in the intestinal microbiome may be used as a prognostic model to noninvasively detect children at high-risk for end stage organ damage of the genitourinary system secondary to obstructive uropathy.
