An insertion polymorphism of the angiotensin-I converting enzyme gene (ACE) is common in humans and the higher expressing allele is associated with an increased risk of diabetic complications. The ACE polymorphism does not significantly affect blood pressure or angiotensin II levels, suggesting that the kallikrein-kinin system partly mediates the effects of the polymorphism. We have therefore explored the influence of lack of both bradykinin receptors (B1R and B2R) on diabetic nephropathy, neuropathy, and osteopathy in male mice heterozygous for the Akita diabetogenic mutation in the insulin 2 gene (Ins2). We find that all of the detrimental phenotypes observed in Akita diabetes are enhanced by lack of both B1R and B2R, including urinary albumin excretion, glomerulosclerosis, glomerular basement membrane thickening, mitochondrial DNA deletions, reduction of nerve conduction velocities and of heat sensation, and bone mineral loss. Absence of the bradykinin receptors also enhances the diabetes-associated increases in plasma thiobarbituric acid-reactive substances, mitochondrial DNA deletions, and renal expression of fibrogenic genes, including transforming growth factor beta1, connective tissue growth factor, and endothelin-1. Thus, lack of B1R and B2R exacerbates diabetic complications. The enhanced renal injury in diabetic mice caused by lack of B1R and B2R may be mediated by a combination of increases in oxidative stress, mitochondrial DNA damage and over expression of fibrogenic genes.diabetes mellitus complications | kinins T he angiotensin-I converting enzyme (ACE) is a dipeptidyl carboxypeptidase, named because it removes two amino acids from the carboxyl terminus of the inactive peptide angiotensin I and converts it into the active blood pressure-raising peptide, angiotensin II. However, ACE is also a kininase and converts the active vasodilatory kinins into inactive metabolites by removing two amino acids from their carboxyl termini (1). Prior experimental findings (2) and computer simulations (3) show that modest changes in ACE levels affect the levels of its substrates much more than its products, indicating that relatively small changes in the levels of ACE affect kinin levels more than angiotensin II levels.The common insertion/deletion (I/D) polymorphism of the ACE gene in humans is due to the presence or absence of an Alu retrotransposon in the 16th intron of the gene. The polymorphism is associated with up to a twofold difference in relative plasma ACE levels (4), but the polymorphism does not significantly affect blood pressure or angiotensin II or aldosterone levels (5). Nevertheless, the I and D human ACE alleles are associated with different risks for developing diabetic complications including nephropathy (6

Nephropathy develops in many but not all patients with longstanding type 1 diabetes. Substantial efforts to identify genotypic differences explaining this differential susceptibility have been made, with limited success. Here, we show that the expression of the transforming growth factor β1 gene (Tgfb1) affects the development of diabetic nephropathy in mice. To do this we genetically varied Tgfb1 expression in five steps, 10%, 60%, 100%, 150%, and 300% of normal, in mice with type 1 diabetes caused by the Akita mutation in the insulin gene (Ins2 Akita ). Although plasma glucose levels were not affected by Tgfb1 genotype, many features of diabetic nephropathy (mesangial expansion, elevated plasma creatinine and urea, decreased creatinine clearance and albuminuria) were progressively ameliorated as Tgfb1 expression decreased and were progressively exacerbated when expression was increased. The diabetic 10% hypomorphs had comparable creatinine clearance and albumin excretion to wild-type mice and no harmful changes in renal morphology. The diabetic 300% hypermorphs had ∼1/3 the creatinine clearance of wild-type mice, >20× their albumin excretion, ∼3× thicker glomerular basement membranes and severe podocyte effacement, matching human diabetic nephropathy. Switching Tgfb1 expression from low to high in the tubules of the hypomorphs increased their albumin excretion more than 10-fold but creatinine clearance remained high. Switching Tgfb1 expression from low to high in the podocytes markedly decreased creatinine clearance, but minimally increased albumin excretion. Decreasing expression of Tgfb1 could be a promising option for preventing loss of renal function in diabetes.aldosterone | glomerular filtration rate | glomerulosclerosis | megalin | nephrin D iabetes is the number one cause of end-stage renal disease in the United States and many other developed countries. However, despite having similar levels of blood glucose only 20-40% of all diabetic patients develop diabetic nephropathy. In diabetic nephropathy, increased expression of transforming growth factor β1 (TGFβ1) has been demonstrated to promote accumulation of extracellular matrix components (1), apoptosis (2), dedifferentiation of podocytes (3), and epithelial-mesenchymal transition of proximal tubules (4), all of which are thought to facilitate a decline in nephron number and renal function.Tgfb1-null mice on a mixed genetic background show severe multiorgan inflammation with massive infiltration of lymphocytes and macrophages that culminates in death by 3-4 wk of age (5, 6). Their death effectively prevents determining whether absence of TGFβ1 influences the development of nephropathy. To overcome this problem and also to allow the study of the effects of above-normal TGFβ1, we have generated mice with five genetically graded levels of TGFβ1, and have made them diabetic with the Ins2 Akita mutation, which causes pancreatic beta-cell dysfunction and type 1 diabetes.Here we show that the features characteristic of diabetic nephropathy are progressively ...

Human genome-wide association studies have demonstrated that polymorphisms in the engulfment and cell motility protein 1 gene (ELMO1) are strongly associated with susceptibility to diabetic nephropathy. However, proof of causation is lacking. To test whether modest changes in its expression alter the severity of the renal phenotype in diabetic mice, we have generated mice that are type 1 diabetic because they have the Ins2Akita gene, and also have genetically graded expression of Elmo1 in all tissues ranging in five steps from ∼30% to ∼200% normal. We here show that the Elmo1 hypermorphs have albuminuria, glomerulosclerosis, and changes in the ultrastructure of the glomerular basement membrane that increase in severity in parallel with the expression of Elmo 1. Progressive changes in renal mRNA expression of transforming growth factor β1 (TGFβ1), endothelin-1, and NAD(P)H oxidase 4 also occur in parallel with Elmo1, as do the plasma levels of cystatin C, lipid peroxides, and TGFβ1, and erythrocyte levels of reduced glutathione. In contrast, Akita type 1 diabetic mice with below-normal Elmo1 expression have reduced expression of these various factors and less severe diabetic complications. Remarkably, the reduced Elmo1 expression in the 30% hypomorphs almost abolishes the pathological features of diabetic nephropathy, although it does not affect the hyperglycemia caused by the Akita mutation. Thus, ELMO1 plays an important role in the development of type 1 diabetic nephropathy, and its inhibition could be a promising option for slowing or preventing progression of the condition to end-stage renal disease.reactive oxygen species | 3′-untranslated region | fibrosis

The authors wish to note the following: "The contrast of our final projection map was inverted, so that we interpreted the background density rather than the actual protein density in terms of structural features of the potassium channel-Fv complex. In addition, we indexed the 2D crystals with unit cell parameters of a = b = 175 Å, while the correct indexing would be a = b = 124 Å. Given these analysis errors, the resulting density map and our interpretation of the structural features are not correct. Accordingly, we would like to retract this paper. We acknowledge Yoshinori Fujiyoshi, Rod MacKinnon, Kazutoshi Tani, and Tom Walz for identifying the errors and pointing them out to us."

We have generated low-expressing and high-expressing endothelin-1 genes (L and H) and have bred mice with four levels of expression: L/L, ∼20%; L/+, ∼65%; +/+ (wild type), 100%; and H/+, ∼350%. The hypomorphic L allele can be spatiotemporally switched to the hypermorphic H allele by Cre-loxP recombination. Young adult L/L and L/+ mice have dilated cardiomyopathy, hypertension, and increased plasma volumes, together with increased ventricular superoxide levels, increased matrix metalloproteinase 9 (Mmp9) expression, and reduced ventricular stiffness. H/+ mice have decreased plasma volumes and significantly heavy stiff hearts. Global or cardiomyocyte-specific switching expression from L to H normalized the abnormalities already present in young adult L/L mice. An epithelial sodium channel antagonist normalized plasma volume and blood pressure, but only partially corrected the cardiomyopathy. A superoxide dismutase mimetic made superoxide levels subnormal, reduced Mmp9 overexpression, and substantially improved cardiac function. Genetic absence of Mmp9 also improved cardiac function, but increased superoxide remained. We conclude that endothelin-1 is critical for maintaining normal contractile function, for controlling superoxide and Mmp9 levels, and for ensuring that the myocardium has sufficient collagen to prevent overstretching. Even a modest (∼35%) decrease in endothelin-1 gene (Edn1) expression is sufficient to cause cardiac dysfunction.amiloride | extracellular matrix | reactive oxygen species | sodium retention | tempol P revious studies have demonstrated that individuals with dilated cardiomyopathy have increased plasma levels of endothelin-1 (1) and elevated endothelin-1 mRNA levels in the heart (2). In addition, two polymorphisms in the endothelin type A receptor gene (EDNRA), G231A and C1363T, are associated with differences in the risk for pathogenesis and mortality in patients with idiopathic dilated cardiomyopathy (3, 4). These findings suggest endothelin-1 plays a causative and/or compensatory role in dilated cardiomyopathy.In animal studies, mice completely lacking endothelin-1 have severe anomalies in the heart and aorta with craniofacial abnormalities and die at age 10-12 d post coitum (5). Mice with a cardiomyocyte-specific deletion of endothelin-1 develop dilated cardiomyopathy as they age or if they are subjected to aortic banding when young (6). In the opposite direction, mice having a conditional ∼10-fold overexpression of endothelin-1 in the heart also develop dilated cardiomyopathy associated with increases in the expression of inflammatory cytokines (7).To gain a better understanding of the physiological role of endothelin-1 in mammals, we have used a method of altering the 3′ untranslated region (UTR) of a gene of interest without changing its 5′ transcriptional regulatory elements (8) to generate mice having four step-wise levels of expression of endothelin-1 covering physiologically likely ranges (from ∼20% normal to ∼350%). We here report that plasma volumes and blood pressure increas...

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthineguanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.hypertension | lactation | protein kinase B/Akt P rolactin, a 23-kDa polypeptide hormone, is a potent multifunctional cytokine with a broad range of biological effects, including water and salt balance, lactogenesis, cell proliferation and differentiation, testicular Leydig cell function, T-cell immunity, pancreatic β-cell function, hematopoiesis, and adipogenesis (1). Prolactin is physiologically secreted mainly from the anterior lobe of pituitary gland. The secretion is negatively regulated by dopamine and positively regulated by prolactin-releasing peptide (2) synthesized in the hypothalamus. The levels of prolactin in the serum, urine, and amniotic fluids are significantly higher in patients with preeclampsia than in subjects with normal pregnancy (3-5), suggesting that prolactin is involved in the pathogenesis of pregnancy-associated hypertension. However, animal experiments have shown inconsistent effects of prolactin on blood pressure (BP). Thus, acute i.v. infusion of prolactin increased BP in rabbits (6), but, when ovine prolactin was chronically given i.p. via an osmotic minipump in rats, BP was decreased (7). It has also been reported that chronic prolactin infusion caused an increase in urinary sodium, potassium, and water excretion, but no significant changes in arterial pressure, in rats (8).To study the chronic effects of different plasma concentrations of prolactin on BP and general well-being, we have generated male mice with a si...

No abstract

Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/HIns2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling.