Novel urinary tract obstruction marker discovery by multi-marker profiling of urinary extracellular vesicles derived from a rat UTO model
Nora Marie Haney, MD/MBA, Chi-Ju Kim, PhD, Morgan D. Kuczler, BA, Cheng-Fan Lee, PhD, Kara Lombardo, BS, Trinity Bivalacqua, MD/PhD, Kenneth J. Pienta, MD, Sarah R. Amend, PhD.
Johns Hopkins Hospital, Baltimore, MD, USA.
BACKGROUND: Congenital urinary obstruction is a common cause of end-stage renal disease in the pediatric population. However, non-invasive diagnostics to predict which patients will benefit from early intervention are lacking. No study to date has evaluated sEVs as related to obstructive bladder injury exclusively or compared differences in the patterns sEVs cargo in upper compared to lower tract urinary obstruction. Therefore, the objective of this study was to evaluate the mRNA cargo of urinary sEVs and mRNA expression patterns of kidney and bladder tissues from rats with lower UTO (bad bladder, good kidney) or upper UTO (good bladder, bad kidney).
Methods: Using a rat model of upper and lower urinary tract partial obstruction and the Nanostring nCounter Fibrosis V2 Panel, we evaluated the mRNA cargo of urinary small extracellular vesicles (sEVs) and mRNA expression patterns of kidney and bladder tissues from rats with lower tract urinary obstruction and upper tract urinary obstruction.
Results: Histology confirmed remodeling of bladder and renal tissue with increased fibrosis in left renal specimens of UUTO and increased SMC/Collagen ratio in bladders of LUTO (Figure 1). Across all samples, 730 of the 770 genes probed were detected. 640 genes were detected in kidney tissues, 701 genes in bladder tissues, and 510 genes in urinary sEV samples (Figure 2). mRNA abundance was lower in urinary sEVs than in tissue samples, despite pre-amplification for sEV. While mRNA hierarchical clustering of urinary sEVs was unable to differentiate upper compared to lower tract urinary obstruction, clustering was able to detect overall disease state (UUTO or LUTO) versus healthy controls (Figure 3). Urinary sEVs carried genes unique to each treatment group (UUTO: 59 genes, LUTO: 17 genes), while only one gene was uniquely carried in the control group (Figure 3). Notable genes of interest found in urinary sEVs were VCAM-1 and NOS1 for UUTO, Egfr for LUTO, and Pck1 for healthy controls (Table 1).
Conclusions: This study provides support that differential gene expression of urinary sEV mRNA has potential to act as biomarkers in the diagnosis and prognosis of UTO. Urinary sEVs demonstrated higher numbers of unique genes representative of injury to the kidney than that of injury to the bladder. Importantly, there were genes unique to UUTO sEVs, indicating the extent and reversibility of renal damage can be independent of the function, damage, and architecture of the bladder.
| Treatment groups | mRNAs |
| UUTO (59) | Acaa2, Adam9, Adipor1, Angptl4, App, Arhgef6, Atg101, Brpf3, 3Ccr2, 3Cd209e, Col4a1, Csnk2a1, Cxcl16, Cyp27a1, Dusp8, Ehmt1, 3Eomes, Fzd2, Got2, Gpx3, 2, 4H2-Q2, Irf1, Irs1, Itgb1, Jag2, Kansl1, Kat6a, Kif3a, Lpar5, Lrp6, Maml3, Mapk1, Mmp3, Mtmr4, Mylk, Ncor2, Nid1, Nos1, Pde2a, Peli2, Pik3ca, Prf1, 3Prkacb, Ptger4, Ptk2, Rora, Rps6kb2, Sec61b, Seh1l, Serpinf1, Sirt1, Smad2, Sorbs1, Stat3, Tgfbr2, Tlr4, 3Tpsab1, Trrap, 1Vcam1 |
| LUTO (17) | Bcap31, Casp6, Egfr, Ep400, 3Il18r1, Ilk, Klrk1, Lamtor2, Ndufb8, Pik3cb, Ppara, Sec24c, Smad6, Stat1, Stk4, Tbl1xr1, Xiap |
| Control (1) | Pck1 |
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