**6.3 Genetic models**

Although numerous genes have been postulated to play a role in the normal and abnormal development of the urinary tract, none have been shown to be directly responsible for the primary lesions associated with congenital obstructive nephropathy in humans. Even so, several mutational or transgenic rodent models of obstructive nephropathy have been described.

### **6.3.1 Naturally-arising mutations associated with obstructive nephropathy**

Congenital progressive hydronephrosis, a hereditary condition in a mutant strain of *C57BL/6J* mice, results from a spontaneous point mutation in the aquaporin-2 gene. Affected mice produce excessive quantities of hypotonic urine, which is believed to exceed the capacity of the ureteral peristaltic machinery producing hydronephrosis, obstructive nephropathy, and death (McDill et al., 2006). Male C57BL/KsJ mice also have a high incidence of hydronephrosis, although the mechanism of this finding has not been identified (McDill, et al., 2006; Weide & Lacy, 1991). In both of these strains, hydronephrosis is not present at birth; therefore, the urological defect is hereditary, but not congenital. Genetic models that develop *in utero* obstruction include certain inbred lines of rats (Aoki et al., 2004; Fichtner et al., 1994; Miller et al., 2004) that develop unilateral UPJ obstruction. Some of these strains display minimal or no morphological change in the renal parenchyma, but one strain of Wistar rats has been shown to develop hydronephrosis, loss of renal parenchyma, tubular and ductal atrophy and dilation, and interstitial fibrosis (Seseke et al., 2000).

#### **6.3.2 Targeted models with complex phenotypes**

Mice with the targeted deletion of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), lysosomal membrane protein LIMP-2/LGP85, or calcineurin also develop urinary tract obstruction in the postnatal period (Chang et al., 2004; Gamp et al., 2003; Yokoyama et al., 2002). Transgenic mice over-expressing human chorionic gonadotropin develop functional urethral obstruction that is not apparent until adulthood (Rulli et al., 2003). Mice deficient in the transcription factor *Id2* (Aoki, et al., 2004; Fichtner, et al., 1994; Miller, et al., 2004) develop unilateral UPJ obstruction. Bilateral ureteral obstruction *in utero* has been reported in mice heterozygous for bone morphogenetic protein 4 (Miyazaki et al., 2000). However, each of these animal models exhibits complex urological phenotypes including renal hypoplasia, dysplasia, aplasia, ureteral tortuosity, or duplicated ureters, thereby confounding analysis due to the inextricability of the secondary effects of obstruction from primary renal maldevelopment.

Developmental urinary tract anomalies including hydronephrosis also arise from knockout of the uroplakin II or III genes and conditional knockout of homeobox gene Lim1 in the nephric duct epithelium (Hu et al., 2000; Kong et al., 2004; Pedersen et al., 2005). However, VUR is also a prominent feature in these CAKUT models, and it is not clear whether there is a true or a functional obstruction, nor what the relative contributions of reflux and obstruction to the renal phenotype are.

Mice lacking either of the two angiotensin receptors likewise develop abnormal renal phenotypes. The Agtr2 knockout has incomplete penetrance, with approximately 2-20% of mutant mice demonstrating a wide spectrum of renal and urological anomalies including UPJ stenosis or megaureter as well as multicystic dysplastic kidney, hypoplastic kidney, VUR, or duplicated ureter (Nishimura, et al., 1999). Deficiency of Agtr1 produces a renal phenotype with some features similar to that seen in complete UUO, including papillary atrophy, medullary thinning, calyceal enlargement, tubulointerstitial apoptosis, macrophage infiltration, and fibrosis. The Agtr1 mutant also demonstrates hypertrophy of the renal vasculature, a feature not seen in UUO models. There is some evidence supporting a role for Agtr1 in promoting growth and contractility of smooth muscle cells (Miyazaki & Ichikawa, 2001), but the relative contributions of primary effects of the mutation on renal development and secondary consequences of a possible functional obstruction in these mice remain unclear. Agtr1 knockout mice also display significant extrarenal abnormalities, including poor weight gain, marked hypotension, and increased frequency of ventral septal defects (Tsuchida et al., 1998).

#### **6.3.3 Megabladder mouse**

18 Novel Insights on Chronic Kidney Disease, Acute Kidney Injury and Polycystic Kidney Disease

fistula with esophageal atresia, and radial limb dysplasia, which complicates application of

Although numerous genes have been postulated to play a role in the normal and abnormal development of the urinary tract, none have been shown to be directly responsible for the primary lesions associated with congenital obstructive nephropathy in humans. Even so, several mutational or transgenic rodent models of obstructive nephropathy have been

Congenital progressive hydronephrosis, a hereditary condition in a mutant strain of *C57BL/6J* mice, results from a spontaneous point mutation in the aquaporin-2 gene. Affected mice produce excessive quantities of hypotonic urine, which is believed to exceed the capacity of the ureteral peristaltic machinery producing hydronephrosis, obstructive nephropathy, and death (McDill et al., 2006). Male C57BL/KsJ mice also have a high incidence of hydronephrosis, although the mechanism of this finding has not been identified (McDill, et al., 2006; Weide & Lacy, 1991). In both of these strains, hydronephrosis is not present at birth; therefore, the urological defect is hereditary, but not congenital. Genetic models that develop *in utero* obstruction include certain inbred lines of rats (Aoki et al., 2004; Fichtner et al., 1994; Miller et al., 2004) that develop unilateral UPJ obstruction. Some of these strains display minimal or no morphological change in the renal parenchyma, but one strain of Wistar rats has been shown to develop hydronephrosis, loss of renal parenchyma,

**6.3.1 Naturally-arising mutations associated with obstructive nephropathy** 

tubular and ductal atrophy and dilation, and interstitial fibrosis (Seseke et al., 2000).

Mice with the targeted deletion of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), lysosomal membrane protein LIMP-2/LGP85, or calcineurin also develop urinary tract obstruction in the postnatal period (Chang et al., 2004; Gamp et al., 2003; Yokoyama et al., 2002). Transgenic mice over-expressing human chorionic gonadotropin develop functional urethral obstruction that is not apparent until adulthood (Rulli et al., 2003). Mice deficient in the transcription factor *Id2* (Aoki, et al., 2004; Fichtner, et al., 1994; Miller, et al., 2004) develop unilateral UPJ obstruction. Bilateral ureteral obstruction *in utero* has been reported in mice heterozygous for bone morphogenetic protein 4 (Miyazaki et al., 2000). However, each of these animal models exhibits complex urological phenotypes including renal hypoplasia, dysplasia, aplasia, ureteral tortuosity, or duplicated ureters, thereby confounding analysis due to the inextricability of the secondary effects of

Developmental urinary tract anomalies including hydronephrosis also arise from knockout of the uroplakin II or III genes and conditional knockout of homeobox gene Lim1 in the nephric duct epithelium (Hu et al., 2000; Kong et al., 2004; Pedersen et al., 2005). However, VUR is also a prominent feature in these CAKUT models, and it is not clear whether there is a true or a functional obstruction, nor what the relative contributions of reflux and

**6.3.2 Targeted models with complex phenotypes** 

obstruction from primary renal maldevelopment.

obstruction to the renal phenotype are.

this approach to modeling congenital obstructive nephropathy.

**6.3 Genetic models** 

described.

Our laboratory has identified a unique transgenic mouse model of congenital obstructive nephropathy designated the megabladder (*mgb*) mouse (Ingraham et al., 2010; Singh et al., 2007). As shown in Figure 7, these mice develop a nonfunctional, over-distended bladder due to a bladder-specific defect in smooth muscle differentiation. This leads to a functional lower urinary tract obstruction, antenatal hydronephrosis, and signs of renal failure evident shortly after birth. Male *mgb* homozygotes develop early renal insufficiency and rarely survive beyond 4-6 weeks, whereas females may live a year or longer.

Megabladder mice closely mirror the pathophysiology associated with a lower urinary tract obstruction in several key respects (Ingraham, et al., 2010; Singh, et al., 2007). *Mgb-/-* mice develop a functional obstruction of the lower urinary tract that leads to hydroureteronephrosis during embryogenesis. *Mgb-/-* mice are born with histopathological evidence of renal injury, indicating that their kidneys possess preexisting pathological changes resulting from *in utero* obstruction. The obstruction and its renal consequences develop within the uterine and fetal environment, in contrast to the postnatal timing of obstruction in UUO and many genetic models of obstructive nephropathy. *Mgb-/-* mice preferentially develop right-sided hydronephrosis reminiscent of the "pop-off" mechanism theorized in children with PUV and secondary unilateral VUR (Greenfield, et al., 1983). *Mgb-/-* mice also exhibit a variable clinical course, in much the same way that children with seemingly identical obstructive lesions may have very different clinical outcomes. Male *mgb-/-* mice can be rescued from the complications of renal failure and early demise by cutaneous vesicostomy, but of the vesicostomized animals that survive the perioperative period, approximately 40% die within the first two weeks despite a patent stoma and no apparent surgical complications. This result is reminiscent of the fact that 27% to 70% of children with PUV will have progressive CKD despite surgery (Ansari, et al., 2010; Kousidis, et al., 2008; Parkhouse, et al., 1988; Roth, et al., 2001; Sanna-Cherchi, et al., 2009). Finally, *mgb-/-* mice possess no extrarenal features to complicate their utilization as a functional model of congenital obstructive nephropathy. Taken together, these observations suggest that *mgb-/-* mice represent an excellent experimental model for the study of the pathophysiological events associated with congenital obstructive nephropathy involving the lower urinary tract.

Congenital Obstructive Nephropathy: Clinical Perspectives and Animal Models 21

Morbidity and mortality remain very high for patients with congenital obstructive nephropathy, with few effective therapeutic options. Clearly, additional research is needed to illuminate the cellular and molecular changes that characterize congenital obstructive nephropathy, with particular emphasis on developing reliable biomarkers and new therapeutic approaches to reduce the impact of this devastating disease. Experimental animal models of obstructive nephropathy have provided valuable information regarding renal pathogenesis and function following surgical occlusion or genetic manipulation. The continued development of new animal models of congenital obstructive nephropathy, like the *mgb* mouse, will provide increasing opportunities to identify and manipulate the key molecular pathways associated with the development of chronic renal failure, while at the same time providing an experimental platform for biomarker development and the

We thank Dr. Andrew Schwaderer and Dr. Brian Becknell for valuable comments in drafting this manuscript, Ashley R. Carpenter for technical assistance in obtaining images of the *mgb*  mouse, and our clinical colleagues at Nationwide Children's Hospital for patient cases and

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**7. Conclusion** 

assessment of novel therapeutic strategies.

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**8. Acknowledgment** 

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**9. References** 

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In the kidneys of *mgb-/-* mice, fibrotic changes are observed in a distinctive pattern. Increased interstitial collagen deposition is first apparent in the renal parenchyma immediately subadjacent to the urothelium of the renal capsule, followed by the outer cortex near the renal capsule. In severe cases, fibrosis ultimately extends throughout the renal parenchyma. Altered patterns of -smooth muscle isoactin (-SMA), E-cadherin, TGF-1 and connective tissue growth factor expression are also observed in *mgb-/-* kidneys, supporting a role for these pathways in the development of fibrosis associated with congenital obstructive nephropathy (Ingraham, et al., 2010). Severely affected *mgb-/-* kidneys also display several dysplastic features including alteration in the developmental distribution of WT1 and PAX2. These observations are consistent with Edith Potter's classic work suggesting that the renal pathology associated with CAKUT includes varying degrees of renal hypodysplasia (Potter, 1972). In contrast to the well-characterized UUO model of upper urinary tract obstruction, inflammation does not appear to play a prominent or early role in the pathogenesis of renal injury in the megabladder model.

Fig. 7. Megabladder (*mgb*) mouse. **A.** Two *mgb-/-* mice, prior to (right) and immediately after (left) cutaneous vesicostomy. The mouse on the right demonstrates a massively distended abdomen secondary to the megabladder, whereas the mouse on the left demonstrates the flat belly attained with decompression of the bladder. **B.** Upon dissection and with the megabladder (MGB) reflected caudally, hydroureteronephrosis involving both kidneys (RK and LK) is apparent. **C.** Trichrome staining demonstrates a band of fibrosis (white arrows) underlying the urothelium in a mildly affected *mgb-/-* mouse. **D.** In a more severely affected kidney from a *mgb-/-* mouse, interstitial fibrosis (blue staining) extends throughout the renal medulla, and in a stripe along the outer cortex (yellow arrows) adjacent to the renal capsule.
