The primary objective of this project is to define the role of ROBO2 deficiency in the pathogenesis of vesicoureteral reflux (VUR) and reflux nephropathy using a reflux mouse model with a conditional Robo2 knockout allele. VUR is one of the commonest genetic disorders found in children, with an incidence of about 1:100. It is characterized by reflux of urine from the bladder into the ureters and kidneys and leads to scarring of the kidney cortex. Patients with VUR may present later in life with reflux nephropathy, a condition characterized by proteinuria, hypertension and focal glomerulosclerosis, which accounts for about 10% of cases of end-stage renal failure. Despite the high incidence of VUR in the pediatric population, the molecular basis of VUR and reflux nephropathy remains unknown. ROBO2 is a transmembrane protein for SLIT ligand that controls axon elongation and arborization. We have shown that ROBO2 is also involved in urinary tract development and is mutated in a subset of patients with VUR. We have generated and studied a conditional Robo2 knockout mouse model, which exhibits striking urinary tract abnormalities closely resembling those in human VUR. We also found that Robo2 is expressed in developing mouse glomeruli in a pattern that suggests a location in podocytes. In addition, Robo2 deficient mice exhibit low nephron number and post-injury proteinuria as well as abnormal ureteric branching and defective elongation of the ureters. Thus, our studies have provided strong evidence for the involvement of ROBO2 mutations in human VUR and provided us with a viable reflux mouse model to further investigate the role of Robo deficiency in the etiology of VUR and determine if loss of glomerular Robo2 confers susceptibility to reflux nephropathy. To examine potentially unique pathogenic mechanisms of VUR and reflux nephropathy, we propose first to characterize the reflux and reflux nephropathy phenotype in Robo2 deficient mice and to determine if Robo2 deletion leads to abnormal branching morphogenesis and low nephron endowment, which could confer risk of reflux nephropathy. Second, we propose to investigate the normal localization of Robo2 during glomerulogenesis and the structural and functional effects of Robo2 deletion in developing podocytes and in mature kidney. This will test the hypothesis that a primary abnormality of ROBO2 in the podocyte may render the kidney susceptible to injury in the face of VUR or obstruction. Lastly, given the abnormal ureteric branching and ureter elongation defects in Robo2 deficient mice and substantial actions of Robo/Slit signaling in neural development, we will examine if Robo2 controls ureteral structure and function and urinary tract innervation. In sum, these experiments will rigorously define the role of ROBO2 in the pathogenesis of VUR and reflux nephropathy. They will yield considerable mechanistic insights in vivo and ex vivo on the role of Robo in normal and abnormal developmental processes of the kidney and urinary tract. Results from these studies will provide new knowledge of disease mechanisms underlying developmental antecedents of VUR, which may assist us to predict who is at risk of reflux nephropathy and identify novel therapeutic strategies.
Vesicoureteral reflux (VUR) is a common condition in childhood that causes substantial morbidity from recurrent urinary infection and scarring of the kidneys. A significant proportion of patients with VUR will develop progressive kidney damage leading to reflux nephropathy and end-stage kidney failure. Understanding the underlying pathogenic mechanism of VUR and reflux nephropathy will provide novel approaches to detect patients at risk and identify novel therapeutic strategies.
|Rasouly, Hila Milo; Kumar, Sudhir; Chan, Stefanie et al. (2016) Loss of Zeb2 in mesenchyme-derived nephrons causes primary glomerulocystic disease. Kidney Int 90:1262-1273|
|Havasi, Andrea; Lu, Weining; Cohen, Herbert T et al. (2016) Blocking Peptides & Molecular Mimicry as Treatment for Kidney Disease. Am J Physiol Renal Physiol :ajprenal.00601.2015|
|Fan, Xueping; Yang, Hongying; Kumar, Sudhir et al. (2016) SLIT2/ROBO2 signaling pathway inhibits nonmuscle myosin IIA activity and destabilizes kidney podocyte adhesion. JCI Insight 1:e86934|
|Vivante, Asaf; Kleppa, Marc-Jens; Schulz, Julian et al. (2015) Mutations in TBX18 Cause Dominant Urinary Tract Malformations via Transcriptional Dysregulation of Ureter Development. Am J Hum Genet 97:291-301|
|Hwang, Daw-Yang; Kohl, Stefan; Fan, Xueping et al. (2015) Mutations of the SLIT2-ROBO2 pathway genes SLIT2 and SRGAP1 confer risk for congenital anomalies of the kidney and urinary tract. Hum Genet 134:905-16|
|Rasouly, Hila Milo; Lu, Weining (2013) Lower urinary tract development and disease. Wiley Interdiscip Rev Syst Biol Med 5:307-42|
|Fan, Xueping; Li, Qinggang; Pisarek-Horowitz, Anna et al. (2012) Inhibitory effects of Robo2 on nephrin: a crosstalk between positive and negative signals regulating podocyte structure. Cell Rep 2:52-61|
|Wang, Hang; Li, Qinggang; Liu, Juan et al. (2011) Noninvasive assessment of antenatal hydronephrosis in mice reveals a critical role for Robo2 in maintaining anti-reflux mechanism. PLoS One 6:e24763|
|Paredes, Jose; Sims-Lucas, Sunder; Wang, Hang et al. (2011) Assessing vesicoureteral reflux in live inbred mice via ultrasound with a microbubble contrast agent. Am J Physiol Renal Physiol 300:F1262-5|
|Bonegio, Ramon G B; Beck, Laurence H; Kahlon, Roopkiranjot K et al. (2011) The fate of Notch-deficient nephrogenic progenitor cells during metanephric kidney development. Kidney Int 79:1099-112|