Mice provide an experimental model of unparalleled flexibility for studying mammalian diseases. Inbred strains of mice exhibit substantial differences in their susceptibility to the renal complications of diabetes. Much remains to be established regarding the course of diabetic nephropathy (DN) in mice as well as defining those strains and/or mutants that are most susceptible to renal injury from diabetes. Through the use of the unique genetic reagents available in mice (including knockouts and transgenics), the validation of a mouse model reproducing human DN should significantly facilitate the understanding of the underlying genetic mechanisms that contribute to the development of DN. Establishment of an authentic mouse model of DN will undoubtedly facilitate testing of translational diagnostic and therapeutic interventions in mice before testing in humans.

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Mouse Models of Diabetic Nephropathy

Nitric oxide (NO) is synthesized by at least three distinct isoforms of NO synthase (NOS). Their substrate and cofactor requirements are very similar. All three isoforms have some implications, physiological or pathophysiological, in the cardiovascular system. The endothelial NOS III is physiologically important for vascular homeostasis, keeping the vasculature dilated, protecting the intima from platelet aggregates and leukocyte adhesion, and preventing smooth muscle proliferation. Central and peripheral neuronal NOS I may also contribute to blood pressure regulation. Vascular disease associated with hypercholesterolaemia, diabetes, and hypertension is characterized by endothelial dysfunction and reduced endothelium-mediated vasodilation. Oxidative stress and the inactivation of NO by superoxide anions play an important role in these disease states. Supplementation of the NOS substrate L-arginine can improve endothelial dysfunction in animals and man. Also, the addition of the NOS cofactor (6R)-5,6,7, 8-tetrahydrobiopterin improves endothelium-mediated vasodilation in certain disease states. In cerebrovascular stroke, neuronal NOS I and cytokine-inducible NOS II play a key role in neurodegeneration, whereas endothelial NOS III is important for maintaining cerebral blood flow and preventing neuronal injury. In sepsis, NOS II is induced in the vascular wall by bacterial endotoxin and/or cytokines. NOS II produces large amounts of NO, which is an important mediator of endotoxin-induced arteriolar vasodilatation, hypotension, and shock.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/%28SICI%291096-9896%28200002%29190%3A3%3C244%3A%3AAID-PATH575%3E3.0.CO%3B2-8

Nitric oxide in the pathogenesis of vascular disease

An endothelial nitric oxide synthase (eNOS) polymorphism in exon 7 (894 G/T) resulting in glutamate or aspartate, respectively, at position 298 on the protein is correlated with severity of cardiopulmonary diseases. Because glutamate and aspartate are considered to be conservative replacements, the polymorphism was thought to be a marker for a functional locus elsewhere in the gene. We now show in transfected cells, primary human endothelial cells, and human hearts, that eNOS with aspartate, but not glutamate, at position 298 is cleaved, resulting in the generation of 100-kDa and 35-kDa products. Recombinant or native eNOS was examined by immunoblotting either in lysates (COS7) or after partial purification over 2′,5′-ADP-Sepharose and calmodulin-Sepharose. Immunoblotting after SDS/PAGE with a carboxyl-terminal antibody showed a single major protein band in the predicted position for eNOS at 135 kDa. An additional band at approximately 100 kDa was present only in the recombinant 298Asp eNOS and in the eNOS synthesized by primary cells and heart tissue with a G/T genotype. Using an eNOS amino-terminal-specific antibody, an immunoreactive band at approximately 35 kDa, corresponding to the residual N-terminal cleavage fragment, was observed in those cells with a T genotype. Thus, eNOS with aspartate but not glutamate at position 298 is cleaved, resulting in the generation of N-terminal 35-kDa and C-terminal 100-kDa fragments. Thus, the eNOS gene with polymorphisms at nucleotide 894 generates protein products with differing susceptibility to cleavage, suggesting that, in contrast to prior predictions, this polymorphism has a functional effect on the eNOS protein.

https://www.pnas.org/content/pnas/97/6/2832.full.pdf

Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: Cleavage of proteins with aspartate vs. glutamate at position 298

Background Insulin resistance and arterial hypertension are related, but the underlying mechanism is unknown. Endothelial nitric oxide synthase (eNOS) is expressed in skeletal muscle, where it may govern metabolic processes, and in the vascular endothelium, where it regulates arterial pressure. Methods and Results To study the role of eNOS in the control of the metabolic action of insulin, we assessed insulin sensitivity in conscious mice with disruption of the gene encoding for eNOS. eNOS−/− mice were hypertensive and had fasting hyperinsulinemia, hyperlipidemia, and a 40% lower insulin-stimulated glucose uptake than control mice. Insulin resistance in eNOS−/− mice was related specifically to impaired NO synthesis, because in equally hypertensive 1-kidney/1-clip mice (a model of renovascular hypertension), insulin-stimulated glucose uptake was normal. Conclusions These results indicate that eNOS is important for the control not only of arterial pressure but also of glucose and lipid homeostasis. A single...

Insulin resistance, hyperlipidemia, and hypertension in mice lacking endothelial nitric oxide synthase.

The role of nitric oxide in cardiovascular diseases.

Essential hypertension has a genetic basis. Accumulating evidence, including findings of elevation of arterial blood pressure in mice lacking the endothelial nitric oxide synthase (eNOS) gene, strongly suggests that alteration in NO metabolism is implicated in hypertension. There are, however, no reports indicating that polymorphism in the eNOS gene is associated with essential hypertension. We have identified a missense variant, Glu298Asp, in exon 7 of the eNOS gene and demonstrated that it is associated with both coronary spastic angina and myocardial infarction. To explore the genetic involvement of the eNOS gene in essential hypertension, we examined the possible association between essential hypertension and several polymorphisms including the Glu298Asp variant, variable number tandem repeats in intron 4 (eNOS4b/4a), and two polymorphisms in introns 18 and 23. We performed a large-scale study of genetic association using two independent populations from Kyoto (n=458; 240 normotensive versus 218 hypertensive subjects) and Kumamoto (n=421; 223 normotensive versus 187 hypertensive subjects), Japan. In both groups, a new coding variant, Glu298Asp, showed a strong association with essential hypertension (Kyoto: odds ratio, 2.3 [95% confidence interval, 1.4 to 3.9]; Kumamoto: odds ratio, 2.4 [95% confidence interval, 1.4 to 4.0]). The allele frequencies of 298Asp in hypertensive subjects were significantly higher than those in normotensive subjects in both groups (Kyoto: 0.103 versus 0.050, P<0.0017; Kumamoto: 0.120 versus 0.058, P<0.0013, respectively). No such disequilibrium between genotypes was significantly associated with any other polymorphisms we examined; the Glu298Asp variant was also not linked to any other polymorphisms. In conclusion, the Glu298Asp missense variant was significantly associated with essential hypertension, which suggests that it is a genetic susceptibility factor for essential hypertension.

/pdf/endothelial-nitric-oxide-synthase-gene-is-positively-1jnckl1ia0.pdf

Hiroshi Teraoka

Papers

Endothelial Nitric Oxide Synthase Gene Is Positively Associated With Essential Hypertension