A Sensitized Screen of N-ethyl-N-nitrosourea–Mutagenized Mice Identifies Dominant Mutants Predisposed to Diabetic Nephropathy

Abstract

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2 Akita ). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2Akita controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2 Akita G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2 Akita G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Lack of A1 Adenosine Receptors Augments Diabetic Hyperfiltration and Glomerular Injury

“…Furthermore, as described by others in previous studies, we observed mesangial expansion as sign of early diabetic glomerular alterations by both light and electron microscopic examination. 10,17,32,33 In addition, an increase of urinary albumin excretion was noted, indicative of some abnormality of glomerular permselectivity, but as in other studies in Ins2 ϩ/Ϫ mice, the increase in albumin excretion was mild. 16,17,32,34 Our BASIC RESEARCH www.jasn.org study shows that the glomerular abnormalities observed in Ins2 ϩ/Ϫ mice are enhanced in these mice when they lack A1AR.…”

“…In a previous study in Akita mice that did not include comparison with WT controls, GFR was reported to be 520 Ϯ 120 l/min, similar to our value of 457 Ϯ 29 l/min and consistent with mild hyperfiltration. 10 Overall, the degree of hyperfiltration in various diabetic mouse models has been modest and seems to depend on both duration and strain. In this regard, hyperfiltration has been found at 3 mo of age in both OVE26 mice, a type 1 diabetic model, and in db/db mice, a model of type 2 diabetes [11][12][13] ; no hyperfiltration was observed in either strain when mice were older than 5 mo.…”

“…FITCinulin (5%), dialyzed overnight against 0.9% NaCl (final concentration approximately 3%), was injected at 3.7 l/g body wt into the retro-orbital plexus during brief isoflurane anesthesia (recovery within approximately 20 s). At 3,7,10,15,35,55, and 75 min, the mice were placed in a restrainer, the tail vein was punctured with a 30-G atraumatic needle, and approximately 2 l blood was collected by capillarity into heparinized 5-l microcaps (Drummond Scientific, Broomall, PA). A total of 500 nl of plasma was diluted 1:10 in 500 mmol of HEPES (pH 7.4) and measured against a standard curve (1 l of approximately 3% FITC-inulin diluted 1:50, 1:100, and 1:500 in 500 mmol of HEPES).…”

Systemic overexpression of growth hormone (GH) in transgenic FVB/N inbred mice: an optimized model for holistic studies of molecular mechanisms underlying GH-induced kidney pathology

“…This new strain is therefore ideal for holistic transcriptome and proteome studies aiming at the identification of molecular mechanisms underlying GH-induced alterations particularly of the kidney. Moreover, genetically defined cba-bGH mice facilitate random mutagenesis screens for modifier genes which influence the effects of chronic GH excess and associated pathological lesions (Rathkolb et al 2005;Tchekneva et al 2007).…”

Sensitized mutagenesis screen in Factor V Leiden mice identifies thrombosis suppressor loci

“…A dominant suppressor screen in Mecp2-deficient mice (Rett syndrome) identified a mutation in squalene epoxidase (Sqle) as a heritable suppressor, resulting in prolonged survival and amelioration of neurologic manifestations (19). Other successful sensitized screens include analysis of mouse mutants predisposed to diabetic nephropathy (20), a screen in Sox10 haploinsufficient mice identifying the Gli3 gene as a modifier of neurocristopathy Significance Venous thromboembolism (VTE) is a common disease characterized by the formation of inappropriate blood clots. Inheritance of specific genetic variants, such as the Factor V Leiden polymorphism, increases VTE susceptibility.…”