Susan E. Ingraham

LINKAGE MAPPING OF THE SPINAL MUSCULAR-ATROPHY GENE

Spinal muscular atrophy (SMA) is a common autosomal recessive disorder resulting in loss of motor neurons. We have performed linkage analysis on a panel of families using nine markers that are closely linked to the SMA gene. The highest lod score was obtained with the marker D5S351 (Zmax = 10.04 at θ = 0 excluding two unlinked families, and Zmax = 8.77 at θ = 0.007 with all families). One type III family did not show linkage to the 5q13 markers, and in one type I consanguineous family the affected individual did not show homozygosity except for the marker D5S435. Three recombinants were identified with the closest centromeric marker, D5S435, which position the gene telomeric of this marker. These recombinants will facilitate finer mapping of the location of the SMA gene. Lastly, two families provide strong evidence for a remarkable variability in presentation of the SMA phenotype, with the age at onset in one family varying from 17 months to 13 years.

Pathogenesis of Renal Injury in the Megabladder Mouse: A Genetic Model of Congenital Obstructive Nephropathy

Congenital obstructive nephropathy (CON) is the most common cause of chronic renal failure in children often leading to end-stage renal disease. The megabladder (mgb) mouse exhibits signs of urinary tract obstruction in utero resulting in the development of hydroureteronephrosis and progressive renal failure after birth. This study examined the development of progressive renal injury in homozygous mgb mice (mgb−/−). Renal ultrasound was used to stratify the disease state of mgb−/− mice, whereas surgical rescue was performed using vesicostomy. The progression of renal injury was characterized using a series of pathogenic markers including alpha smooth muscle isoactin (α-SMA), TGF-β1, connective tissue growth factor (CTGF), E-cadherin, F4/80, Wilms tumor (WT)-1, and paired box gene (Pax) 2. This analysis indicated that mgb−/− mice are born with pathologic changes in kidney development that progressively worsen in direct correlation with the severity of hydronephrosis. The initiation and pattern of fibrotic development observed in mgb−/− kidneys appeared distinctive from previous animal models of obstruction. These observations suggest that the mgb mouse represents a unique small animal model for the study of CON.

Current perspectives on congenital obstructive nephropathy.

Congenital obstructive nephropathy is the leading cause of chronic renal disease in children. As a result, it represents a tremendous societal burden in terms of morbidity and mortality, as well as in health care expenses of caring for children with chronic kidney disease and end-stage renal disease. The various diagnostic, prognostic, and therapeutic challenges associated with congenital obstructive nephropathy highlight the importance of developing effective experimental models for studying this disease process. In this review, we define the clinical entity that is congenital obstructive nephropathy, outline the current standards of diagnosis and care, and discuss the utilization of current experimental models designed to help clarify some of the clinical conundrums associated with this important disease.

Renin Angiotensin System Blocker Fetopathy: A Midwest Pediatric Nephrology Consortium Report

OBJECTIVES Fetuses continue to be exposed to renin angiotensin system (RAS) blockers despite their known teratogenicity and a black box warning. We hypothesized that fetopathy from in utero exposure to RAS blockers has a broader spectrum of clinical manifestations than described previously and that there are a variety of clinical scenarios leading to such exposures. STUDY DESIGN This was a retrospective study performed through the Midwest Pediatric Nephrology Consortium. Cases of RAS blocker fetopathy were identified, with determination of renal and extrarenal manifestations, timing of exposure, and the explanation for the fetal exposure. RESULTS Twenty-four cases were identified. RAS blocker exposure after the first trimester was associated with more severe renal manifestations. Chronic dialysis or kidney transplantation was required in 8 of 17 (47%) patients with RAS blocker exposure after the first trimester and 0 of 7 patients with exposure restricted to the first trimester (P = .05). Extrarenal manifestations, some not previously noted in the literature, included central nervous system anomalies (cystic encephalomalacia, cortical blindness, sensorineural hearing loss, arachnoid cysts) and pulmonary complications (pneumothorax, pneumomediastinum). RAS blocker exposure usually was secondary to absent or poor prenatal care or undiagnosed pregnancy. CONCLUSION RAS blocker fetopathy continues to be a cause of considerable morbidity, with more severe renal manifestations associated with exposure after the first trimester. A variety of significant extrarenal manifestations occur in these patients. Clinicians should emphasize the risk of fetopathy when prescribing RAS blockers to women of childbearing age.

Molecular Basis of Renal Adaptation in a Murine Model of Congenital Obstructive Nephropathy

Congenital obstructive nephropathy is a common cause of chronic kidney disease and a leading indication for renal transplant in children. The cellular and molecular responses of the kidney to congenital obstruction are incompletely characterized. In this study, we evaluated global transcription in kidneys with graded hydronephrosis in the megabladder (mgb −/−) mouse to better understand the pathophysiology of congenital obstructive nephropathy. Three primary pathways associated with kidney remodeling/repair were induced in mgb −/− kidneys independent of the degree of hydronephrosis. These pathways included retinoid signaling, steroid hormone metabolism, and renal response to injury. Urothelial proliferation and the expression of genes with roles in the integrity and maintenance of the renal urothelium were selectively increased in mgb −/− kidneys. Ngal/Lcn2, a marker of acute kidney injury, was elevated in 36% of kidneys with higher grades of hydronephrosis. Evaluation of Ngalhigh versus Ngallow kidneys identified the expression of several novel candidate markers of renal injury. This study indicates that the development of progressive hydronephrosis in mgb −/− mice results in renal adaptation that includes significant changes in the morphology and potential functionality of the renal urothelium. These observations will permit the development of novel biomarkers and therapeutic approaches to progressive renal injury in the context of congenital obstruction.

Ultrasound imaging of the murine kidney.

Ultrasound (US) is the most common and least invasive modality for clinical imaging of the kidney. One important application of US in nephrology is the detection and monitoring of structural changes in the kidney. Recent advances in US technology have facilitated the application of similar techniques to animal models of human disease. We have developed a simple US-based method of detection and quantitation of hydronephrosis in a mouse model of congenital obstructive nephropathy, the megabladder (mgb) mouse.

Struvite Urolithiasis and Chronic Urinary Tract Infection in a Murine Model of Urinary Diversion

OBJECTIVE To characterize the clinical course after cutaneous vesicostomy (CV) in megabladder (mgb(-/-)) mice with functional urinary bladder obstruction. MATERIALS AND METHODS A total of 45 mgb(-/-) male mice underwent CV at a median age of 25 days. The 34 mice that survived >3 days after CV were evaluated by serial observation and renal ultrasonography. The moribund mice were killed. The urinary bladders and kidneys were analyzed by histopathologic analysis, and urine biochemical studies were performed. RESULTS At a median duration of 11 weeks after CV, 35% of mgb(-/-) male mice (12 of 34) had become moribund with pelvic masses, which were identified as bladder stones at necropsy. The urine pH was alkaline, and microscopic examination demonstrated struvite crystals. The urine samples contained Gram-positive cocci, and the urine cultures were polymicrobial. The stone composition was chiefly struvite (88%-94%) admixed with calcium phosphate. In 40% of cases (2 of 5), retained intravesical polypropylene suture was identified as the presumed nidus. No stones were detected in >100 male mice before CV or in 25 cases when CV was performed using polydioxanone suture. The kidneys from 33% of the mice (4/12) with bladder stones contained staghorn calculi. The histopathologic findings from the mice with struvite stones demonstrated active cystitis, pyelitis, and chronic pyelonephritis. CONCLUSION These findings attest to the importance of the nidus in lithogenesis and provide a novel murine model for struvite urolithiasis and chronic infection of the diverted urinary tract.

Identification of Distinct Myocardin Splice Variants in the Bladder

PURPOSE Although the importance of myocardin in smooth muscle development is well established, many tissue specific intricacies of smooth muscle differentiation remain to be determined. We characterized myocardin expression in the developing and adult bladder to identify potential tissue specific differences that may have a role in detrusor smooth muscle development. MATERIALS AND METHODS Reverse transcriptase and quantitative polymerase chain reaction were done to determine myocardin expression in the mouse and human bladder vs various other tissues. Sequence analysis was done to confirm the genomic location of the various polymerase chain reaction products. RESULTS Exonic profiling of the mouse myocardin gene identified a series of unique myocardin splice variants derived from a novel 305 bp exon between exons 2 and 3 of the previously identified myocardin gene. Each variant showed a differential pattern of expression in the mouse and primary protein sequences suggested a unique function for each myocardin variant identified. Identical myocardin splice variants were also observed in the human bladder as well as a unique human specific exon 12 myocardin splice variant that was not observed in the mouse. CONCLUSIONS Identifying a series of unique myocardin splice variants that are differentially expressed in the bladder, and other muscle and nonmuscle tissues provides a potential molecular platform for mediating many unique tissue specific functions associated with the myocardin transcriptional program.

Renal epithelial miR-205 expression correlates with disease severity in a mouse model of congenital obstructive nephropathy.

Background:Congenital obstructive nephropathy (CON) is a leading cause of pediatric chronic kidney disease (CKD). Despite optimal surgical and medical care, there is a high rate of CKD progression. Better understanding of molecular and cellular changes is needed to facilitate development of improved biomarkers and novel therapeutic approaches in CON.Methods:The megabladder (mgb) mouse is an animal model of CKD with impaired bladder emptying, hydronephrosis, and progressive renal injury. In this study, we characterize a particular microRNA, miR-205, whose expression changes with the degree of hydronephrosis in the mgb−/− kidney.Results:Expression of miR-205 is progressively increased in the adult mgb−/− mouse with worsening severity of hydronephrosis. miR-205 expression is correlated with altered expression of cytokeratins and uroplakins, which are markers of cellular differentiation in urothelium. We describe the spatial pattern of miR-205 expression, including increased expression in renal urothelium and novel miR-205 expression in medullary collecting duct epithelium in the congenitally obstructed kidney.Conclusion:miR-205 is increased with severity of CON and CKD in the mgb−/− mouse and may regulate urothelial differentiation.

Urine Stasis Predisposes to Urinary Tract Infection by an Opportunistic Uropathogen in the Megabladder (Mgb) Mouse.

Purpose Urinary stasis is a risk factor for recurrent urinary tract infection (UTI). Homozygous mutant Megabladder (Mgb-/-) mice exhibit incomplete bladder emptying as a consequence of congenital detrusor aplasia. We hypothesize that this predisposes Mgb-/- mice to spontaneous and experimental UTI. Methods Mgb-/-, Mgb+/-, and wild-type female mice underwent serial ultrasound and urine cultures at 4, 6, and 8 weeks to detect spontaneous UTI. Urine bacterial isolates were analyzed by Gram stain and speciated. Bladder stones were analyzed by x-ray diffractometry. Bladders and kidneys were subject to histologic analysis. The pathogenicity of coagulase-negative Staphylococcus (CONS) isolated from Mgb-/- urine was tested by transurethral administration to culture-negative Mgb-/- or wild-type animals. The contribution of urinary stasis to CONS susceptibility was evaluated by cutaneous vesicostomy in Mgb-/- mice. Results Mgb-/- mice develop spontaneous bacteriuria (42%) and struvite bladder stones (31%) by 8 weeks, findings absent in Mgb+/- and wild-type controls. CONS was cultured as a solitary isolate from Mgb-/- bladder stones. Bladders and kidneys from mice with struvite stones exhibit mucosal injury, inflammation, and fibrosis. These pathologic features of cystitis and pyelonephritis are replicated by transurethral inoculation of CONS in culture-negative Mgb-/- females, whereas wild-type animals are less susceptible to CONS colonization and organ injury. Cutaneous vesicostomy prior to CONS inoculation significantly reduces the quantity of CONS recovered from Mgb-/- urine, bladders, and kidneys. Conclusions CONS is an opportunistic uropathogen in the setting of urinary stasis, leading to enhanced UTI incidence and severity in Mgb-/- mice.