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Renal Epithelial Mir-205 Expression Correlates with Disease Severity in a Mouse Model of Congenital Obstructive Nephropathy

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Renal Epithelial Mir-205 Expression Correlates with Disease Severity in a Mouse Model of Congenital Obstructive Nephropathy nature publishing group Articles Basic Science Investigation Renal epithelial miR-205 expression correlates with disease severity in a mouse model of congenital obstructive nephropathy Michael E. Wilhide1, James D. Feller1, Birong Li1, Ahmad Z. Mohamed2, Brian Becknell1,3, Ashley R. Jackson4, Kirk M. McHugh4 and Susan E. Ingraham1,3 BACKGROUND: Congenital obstructive nephropathy (CON) 20 y of age, resulting in a 10-y survival rate of approximately is a leading cause of pediatric chronic kidney disease (CKD). 80% and an age-specific mortality rate 30 times that seen in Despite optimal surgical and medical care, there is a high rate children without end-stage renal disease (4). The direct link of CKD progression. Better understanding of molecular and cel- between obstructed urine flow beginning in utero and abnor- lular changes is needed to facilitate development of improved mal renal development and function represents a central para- biomarkers and novel therapeutic approaches in CON. digm of urogenital pathogenesis that has far-reaching clinical METHODS: The megabladder (mgb) mouse is an animal implications. model of CKD with impaired bladder emptying, hydronephro- Animal models of obstructive nephropathy have utilized both sis, and progressive renal injury. In this study, we characterize a surgical and genetic approaches. We have previously character- particular microRNA, miR-205, whose expression changes with ized the pathogenesis of CON in the megabladder (mgb) mouse the degree of hydronephrosis in the mgb−/− kidney. (5,6). The mgb mouse is a unique transgene-induced mutant RESULTS: Expression of miR-205 is progressively increased in mouse line in which impaired bladder emptying and hydrone- the adult mgb−/− mouse with worsening severity of hydrone- phrosis are prominent features (7,8). The CKD course followed phrosis. miR-205 expression is correlated with altered expres- by male mgb−/− mice, from in utero voiding impairment to end- sion of cytokeratins and uroplakins, which are markers of stage renal disease and death at sexual maturation, provides an cellular differentiation in urothelium. We describe the spatial excellent opportunity to investigate the molecular and cellular pattern of miR-205 expression, including increased expression factors that drive the onset and progression of CON. in renal urothelium and novel miR-205 expression in medul- A rapidly expanding body of literature documents the criti- lary collecting duct epithelium in the congenitally obstructed cal role that microRNAs (miRs) play in renal physiology and kidney. pathology (9–12). miRs are short endogenous noncoding CONCLUSION: miR-205 is increased with severity of CON RNA molecules that regulate the expression of genes through and CKD in the mgb−/− mouse and may regulate urothelial mRNA cleavage, translational repression, or translational acti- differentiation. vation. miRs are extremely important gene regulators, con- trolling key cell functions such as developmental switching, cell differentiation, cell proliferation, and apoptosis. miRs are hronic kidney disease (CKD) is characterized by the known to play a role in the regulation of kidney homeostasis, Cgradual loss of kidney function over time. It is the com- kidney development, and in various kidney disease, including mon end result of a wide range of diseases and processes fibrotic kidney disease (10), although they have not previously that cause permanent damage and scarring to the kidney. been investigated in CON. Congenital obstructive nephropathy (CON), a leading cause miR-205 has been identified as an epithelial-specific master of pediatric CKD, is a complex disease process characterized regulator in multiple tissues, involved in both normal develop- by pathological changes in kidney development and function ment and pathological states including cancer (13–16). Studies resulting from restriction of antegrade urine flow beginning have suggested a critical role for miR-205 in several types of in utero (1,2). Pre- or postnatal surgical interventions may kidney diseases, including both glomerular and tubular dis- alter the rate of decline in renal function, but in many patients orders (17–19). miR-205 is also the most-upregulated miR in CKD progression is inevitable (3). Up to 70% of children with the unilateral ureteral obstruction mouse model of obstructive CKD due to CON will progress to end-stage renal disease by renal fibrosis (20). 1Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio; 2Department of Urology, University of Louisville, Louisville, Kentucky; 3Department of Pediatrics, The Ohio State University, Columbus, Ohio; 4Center for Molecular and Human Genetics, The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio. Correspondence: Susan E. Ingraham ([email protected]) Received 9 March 2016; accepted 7 April 2016; advance online publication 6 July 2016. doi:10.1038/pr.2016.121 602 Pediatric ReSearcH Volume 80 | Number 4 | October 2016 Copyright © 2016 International Pediatric Research Foundation, Inc. miR-205 in obstructive nephropathy Articles In this study, we examine the association of miR-205 expression patterns with the degree of hydronephrosis in the ac mgb−/− kidney, describe spatial and cellular patterns of miR- 205 expression in the normal and the obstructed kidney, iden- tify altered patterns of expression of urothelial differentiation markers that correlate with miR-205, and discuss potential roles of miR-205 in the pathogenesis of CON and CKD in this Moderate established animal model. b d RESULTS miR-205 Expression Increases During Maturation and Progression of Hydronephrosis in mgb−/− Mice miR-205 is present during kidney development from embry- Mild Severe onic day 15.5 (E15.5) through birth (day 0 to day 1), but the −/− differences in expression levels between mgb and wild-type e 10 (WT) kidneys are either small (no ˃1.3-fold) or not statistically 9 ** significant (Figure 1). Quantitative reverse transcription PCR 8 (RT-PCR) demonstrated that relative expression of mature miR-205 is significantly upregulated in weanling (3.1-fold) 7 and adult (3.5-fold) mgb−/− kidneys compared with WT. In 6 the adults, the level of miR-205 expression correlates with the 5 degree of hydronephrosis (Figure 2). We found a significant 4 † * proportional increase in relative miR-205 expression when the 3 Fold change vs. WT data were stratified by severity of hydronephrosis (Figure 2e: 2 mild, 1.3-fold increase relative to WT, not statistically signifi- 1 cant; moderate, 2.4-fold increase, P = 0.02; severe: 5.3-fold, P = 0 0.004; Kruskal–Wallis equality-of-populations rank test across WT Mild Mod Severe Degree of hydronephrosis in mgb −/− † f 6 10 ‡ 5 WT . 4 3 5 2 old change vs F * * ** 1 Relative miR-205 expression 0 n D0 n D1 0 E15.5 E16.5 E17.5 E18.5 Adult eanling wbor wbor W 0204060 80 100 Ne Ne % Hydronephrosis Figure 1. Timeline of renal miR-205 expression during development and Figure 2. miR-205 expression correlates with degree of hydronephrosis in progression of hydronephrosis in the mgb−/− mouse. Reverse transcrip- mgb−/− adults. (a–d) Kidney cross-sectional ultrasound showing represen- tion PCR (RT-PCR) results from a panel of male megabladder (mgb−/−) tative examples of hydronephrosis stratification in adult mgb−/− kidneys: kidney samples spanning developmental and chronic kidney disease (a) no hydronephrosis (0% of anterior–posterior line crosses fluid in the progression stages from embryonic day 15.5 (E15.5) through adulthood. renal pelvis), (b) mild hydronephrosis (8%), (c) moderate hydronephrosis miR-205 expression is indicated relative to mean miR-205 expression in (45%), and (d) severe hydronephrosis (81%). (e) Stratification of the adult age-matched wild-type (WT) kidneys. Time points include embryonic day mgb−/− population from Figure 1 by severity of hydronephrosis com- 15.5 (E15.5), E16.5, E17.5, E18.5, newborn day 0 (D0), day 1 (D1), weanling pared with wild type (WT). Pairwise comparisons of group means were (day 20–22), and adult (4–5 wk including all degrees of hydronephrosis). performed using the Mann–Whitney test, with resulting P values shown Fold change is relative to WT and represents the mean ± SD with three by crosses for comparison with WT and asterisks for comparison to severe independent RT-PCR runs of three technical replicates per run, normal- group. †P = 0.01; *P = 0.02; **P = 0.004. If no symbol is shown, result was ized to sno234. White, WT; black, mgb−/−. Number of animals (n) per group, not statistically significant (P > 0.05). Fold change is relative to WT kidneys E15.5: WT n = 4, mgb−/− n = 5. E16.5: WT n = 5, mgb−/− n = 5. E17.5: WT n = 4, and represents the mean ± SD with three independent reverse transcrip- mgb−/− n = 5. E18.5: WT n = 5, mgb−/− n = 5. D0: WT n = 9, mgb−/− n = 4. D1: tion PCR runs of three technical replicates per run, normalized to sno234. WT n = 4, mgb−/− n = 5. Weanling: WT n = 11, mgb−/− n = 9. Adult: WT n = 6, Number of animals (n) per group, WT n = 6, Mild n = 7, Mod n = 5, Severe mgb−/− n = 18. The P values resulting from Mann–Whitney test are indi- n = 6. (f) Correlation of relative miR-205 expression with measurement of cated: *P = 0.05; **P = 0.02; †P = 0.01; ‡P = 0.006. If no symbol is shown, degree of hydronephrosis in kidney cross-sectional 2D ultrasound images result was not statistically significant (P > 0.05). using the method of Carpenter et al. (40). Spearman rho = 0.78, P = 0.0003. Copyright © 2016 International Pediatric Research Foundation, Inc. Volume 80 | Number 4 | October 2016 Pediatric ReSearcH 603 Articles Wilhide et al. all three mgb−/− severity groups, P = 0.003). High positive corre- as measured in kidney cross-sectional 2D ultrasound images lation (Spearman rho = 0.78) was found between relative miR- (Figure 2f). Stratification by severity of hydronephrosis at the 205 expression by RT-PCR and the degree of hydronephrosis weanling stage was not possible due to small numbers of ani- mals with greater than mild hydronephrosis.
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