Genetically increased angiotensin I-converting enzyme alters peripheral and renal vascular reactivity to angiotensin II and bradykinin in mice.

Angiotensin I-converting enzyme (ACE) levels in humans are under strong genetic influence. Genetic variation in ACE has been linked to risk for and progression of cardiovascular and renal diseases. Causality has been documented in genetically modified mice, but the mechanisms underlying causality are not completely elucidated. To further document the vascular and renal consequences of a moderate genetic increase in ACE synthesis, we studied genetically modified mice carrying three copies of the ACE gene (three-copy mice) and littermate wild-type animals (two-copy mice). We investigated peripheral and renal vascular reactivity to angiotensin II and bradykinin in vivo by measuring blood pressure and renal blood flow after intravenous administration and also reactivity of isolated glomerular arterioles by following intracellular Ca2+ mobilization. Carrying three copies of the ACE gene potentiated the systemic and renal vascular responses to angiotensin II over the whole range of peptide concentration tested. Consistently, the response of isolated glomerular afferent arterioles to angiotensin II was enhanced in three-copy mice. In these mice, signaling pathways triggered by endothelial activation by bradykinin or carbachol in glomerular arterioles were also altered. Although the nitric oxide (NO) synthase (NOS)/NO pathway was not functional in arterioles of two-copy mice, in muscular efferent arterioles of three-copy mice NOS3 gene expression was induced and NO mediated the effect of bradykinin or carbachol. These data document new and unexpected vascular consequences of a genetic increase in ACE synthesis. Enhanced vasoconstrictor effect of angiotensin II may contribute to the risk for cardiovascular and renal diseases linked to genetically high ACE levels. NEW & NOTEWORTHY A moderate genetic increase in angiotensin I-converting enzyme (ACE) in mice similar to the effect of the ACE gene D allele in humans unexpectedly potentiates the systemic and renal vasoconstrictor responses to angiotensin II. It also alters the endothelial signaling pathways triggered by bradykinin or carbachol in glomerular efferent arterioles.

Acute and chronic hyperglycemic effects of vasopressin in normal rats: involvement of V1A receptors.

Recent epidemiological studies have revealed novel relationships between low water intake or high vasopressin (AVP) and the risk of hyperglycemia and diabetes. AVP V1A and V1B receptors (R) are expressed in the liver and pancreatic islets, respectively. The present study was designed to determine the impact of different levels of circulating AVP on glucose homeostasis in normal Sprague-Dawley rats, as well as the respective roles of V1AR and V1BR. We showed that acute injection of AVP induces a dose-dependent increase in glycemia. Pretreatment with a selective V1AR antagonist, but not a V1BR antagonist, dose-dependently prevented the rise in glycemia. V1BR antagonism did not modify the hyperinsulinemic response, resulting from AVP-induced hyperglycemia, but enhanced the fall in glucagonemia. Acute administration of selective V1AR or V1BR agonists confirmed the involvement of V1AR in the hyperglycemic effect of AVP. In chronic experiments, AVP levels were altered in both directions. Sustained AVP infusion through implantable minipumps induced a time-dependent increase in fasting glycemia, whereas lowering endogenous AVP by increasing water intake had no effect. After 4 wk of AVP infusion, the rise in glycemia amounted to 1.1 mmol/l (P < 0.01) without significant change in insulinemia. This effect was attenuated by cotreatment with a V1AR antagonist. Similar results were observed in lean Zucker rats. These findings demonstrate for the first time a causal link between chronic high AVP and hyperglycemia through V1AR activation and, thus, provide a pathophysiological explanation for the relationship observed in human cohorts between the AVP-hydration axis and the risk of diabetes.

Improvement of skin wound healing in diabetic mice by kinin B2 receptor blockade.

Impaired skin wound healing is a major medical problem in diabetic subjects. Kinins exert a number of vascular and other actions limiting organ damage in ischaemia or diabetes, but their role in skin injury is unknown. We investigated, through pharmacological manipulation of bradykinin B1 and B2 receptors (B1R and B2R respectively), the role of kinins in wound healing in non-diabetic and diabetic mice. Using two mouse models of diabetes (streptozotocin-induced and db/db mice) and non-diabetic mice, we assessed the effect of kinin receptor activation or inhibition by subtype-selective pharmacological agonists (B1R and B2R) and antagonist (B2R) on healing of experimental skin wounds. We also studied effects of agonists and antagonist on keratinocytes and fibroblasts in vitro. Levels of Bdkrb1 (encoding B1R) and Bdkrb2 (encoding B2R) mRNAs increased 1-2-fold in healthy and wounded diabetic skin compared with in non-diabetic skin. Diabetes delayed wound healing. The B1R agonist had no effect on wound healing. In contrast, the B2R agonist impaired wound repair in both non-diabetic and diabetic mice, inducing skin disorganization and epidermis thickening. In vitro, B2R activation unbalanced fibroblast/keratinocyte proliferation and increased keratinocyte migration. These effects were abolished by co-administration of B2R antagonist. Interestingly, in the two mouse models of diabetes, the B2R antagonist administered alone normalized wound healing. This effect was associated with the induction of Ccl2 (encoding monocyte chemoattractant protein 1)/Tnf (encoding tumour necrosis factor α) mRNAs. Thus stimulation of kinin B2 receptor impairs skin wound healing in mice. B2R activation occurs in the diabetic skin and delays wound healing. B2R blockade improves skin wound healing in diabetic mice and is a potential therapeutic approach to diabetic ulcers.

Vasopressin and hydration play a major role in the development of glucose intolerance and hepatic steatosis in obese rats.

AIMS/HYPOTHESIS: High plasma copeptin, a marker of vasopressin (VP) secretion, has been shown to be associated with the metabolic syndrome and development of type 2 diabetes in humans. The present study was designed to determine the long-term influence of plasma VP concentration in a rodent model prone to metabolic dysfunction. METHODS: Obese Zucker rats and their lean counterparts were submitted for 4 weeks to one of three protocols inducing different levels of VP. Circulating VP was either reduced by increasing the daily water intake (low-VP), or increased by a chronic i.p. infusion of VP (high-VP). The control rats had normal VP levels that depended on their own regulation of water intake and VP secretion. RESULTS: Compared with controls with normal VP, lean rats with high-VP had a higher fasting glycaemia after 4 weeks. In obese rats, high-VP promoted hyperinsulinaemia, glucose intolerance, assessed by glucose and insulin tolerance tests, and an impaired response to a pyruvate challenge. Conversely, treatment with a selective arginine vasopressin receptor 1A (V1aR) antagonist reduced glucose intolerance. Low-VP obese rats had unchanged glucose tolerance but exhibited a drastic decrease in liver steatosis compared with control obese rats, associated with low hepatic triacylglycerol and cholesterol content, and reduced expression of hepatic lipogenic genes. These effects were independent of changes in body adiposity, and plasma sodium and osmolality did not differ among groups. CONCLUSION/INTERPRETATION: These findings show a causal relationship between the VP-hydration axis and the metabolic risk. Therapeutic perspectives include diet recommendations regarding hydration, but also potential pharmacological interventions targeting the VP V1aR.

Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice.

Limb ischemia is a major complication of thromboembolic diseases. Diabetes worsens prognosis by impairing neovascularization. Genetic or pharmacological inactivation of the kallikrein-kinin system aggravates limb ischemia in nondiabetic animals, whereas angiotensin I-converting enzyme/kininase II inhibition improves outcome. The role of kinins in limb ischemia in the setting of diabetes is not documented. We assessed whether selective activation of kinin receptors by pharmacological agonists can influence neovascularization in diabetic mice with limb ischemia and have a therapeutic effect. Selective pseudopeptide kinin B1 or B2 receptor agonists resistant to peptidase action were administered by osmotic minipumps at a nonhypotensive dosage for 14 days after unilateral femoral artery ligation in mice previously rendered diabetic by streptozotocin. Comparison was made with ligatured, nonagonist-treated nondiabetic and diabetic mice. Diabetes reduced neovascularization, assessed by microangiography and histologic capillary density analysis, by roughly 40%. B1 receptor agonist or B2 receptor agonist similarly restored neovascularization in diabetic mice. Neovascularization in agonist-treated diabetic mice was indistinguishable from nondiabetic mice. Both treatments restored blood flow in the ischemic hindfoot, measured by laser-Doppler perfusion imaging. Macrophage infiltration increased 3-fold in the ischemic gastrocnemius muscle during B1 receptor agonist or B2 receptor agonist treatment, and vascular endothelial growth factor (VEGF) level increased 2-fold. Both treatments increased, by 50-100%, circulating CD45/CD11b-positive monocytes and CD34(+)/VEGFR2(+) progenitor cells. Thus, selective pharmacological activation of B1 or B2 kinin receptor overcomes the effect of diabetes on postischemic neovascularization and restores tissue perfusion through monocyte/macrophage mobilization. Kinin receptors are potential therapeutic targets in limb ischemia in diabetes.

Selective kinin receptor agonists as cardioprotective agents in myocardial ischemia and diabetes.

Cardiac ischemia is a leading cause of death, especially in diabetic patients. The diabetic ischemic heart is resistant experimentally to established cardioprotective treatments. New pharmacological approaches to cardiac protection are warranted. The kallikrein-kinin system is involved in myocardial protection in ischemia. Respective roles of B1 (B1R) and B2 (B2R) receptors remain controversial. We tested whether pharmacological activation of kinin receptors may have therapeutic effect in cardiac ischemia-reperfusion in nondiabetic (NDiab) and diabetic (Diab) mice. We assessed effect on infarct size (IS) and signaling pathways involved in myocardial protection of potent selective pharmacological agonists of B1R or B2R given at reperfusion. In NDiab mice, a B2R agonist reduced IS significantly by 47%, similarly to ramiprilat or ischemic postconditioning, via activation of phosphoinositide 3 kinase/Akt pathway leading to inhibition of glycogen synthase kinase-3ß (GSK-3ß). B1R agonist had no effect on IS. In contrast, in Diab mice, the B2R agonist, ramiprilat, or ischemic postconditioning failed to reduce IS but a B1R agonist significantly reduced IS by 44% via activation of phosphoinositide 3 kinase/Akt and extracellular signal-regulated kinase 1/2, both leading to GSK-3ß inhibition. Differential effect of B2R or B1R agonists in NDiab and Diab mice can be linked to inactivation of B2R signaling and induction of B1R in heart of Diab mice. Thus, a pharmacological B2R agonist is cardioprotective in acute ischemia in nondiabetic animals. B1R agonist overcomes resistance of diabetic heart to cardioprotective treatments. Pharmacological activation of B1R and B2R may become a treatment for diabetic and nondiabetic patients, respectively, in acute coronary syndromes.

Adipocyte Reprogramming by the Transcriptional Coregulator GPS2 Impacts Beta Cell Insulin Secretion.

Glucose homeostasis is maintained through organ crosstalk that regulates secretion of insulin to keep blood glucose levels within a physiological range. In type 2 diabetes, this coordinated response is altered, leading to a deregulation of beta cell function and inadequate insulin secretion. Reprogramming of white adipose tissue has a central role in this deregulation, but the critical regulatory components remain unclear. Here, we demonstrate that expression of the transcriptional coregulator GPS2 in white adipose tissue is correlated with insulin secretion rate in humans. The causality of this relationship is confirmed using adipocyte-specific GPS2 knockout mice, in which inappropriate secretion of insulin promotes glucose intolerance. This phenotype is driven by adipose-tissue-secreted factors, which cause increased pancreatic islet inflammation and impaired beta cell function. Thus, our study suggests that, in mice and in humans, GPS2 controls the reprogramming of white adipocytes to influence pancreatic islet function and insulin secretion.

Kallikrein protects against microalbuminuria in experimental type I diabetes.

Tissue kallikrein is the main kinin-forming enzyme in mammals, and differences in kinin levels are thought to be a contributing factor to diabetic nephropathy. Here, we determined the role of the kallikrein-kinin system in the pathogenesis of streptozotocin-induced diabetic nephropathy in wild-type and tissue kallikrein-knockout mice. All diabetic mice developed similar hyperglycemia, but the knockout mice had a significant two-fold increase in albuminuria compared to the wild-type mice before and after blood pressure elevation. Ezrin mRNA, a podocyte protein potentially implicated in albuminuria, was downregulated in the kidney of knockout mice. One month after induction of diabetes, the mRNAs of kininogen, tissue kallikrein, kinin B1, and B2 receptors were all increased up to two-fold in the kidney in both genotypes. Diabetes caused a 50% decrease in renal angiotensin-converting enzyme expression and a 20-fold increase in kidney injury molecule-1 reflecting tubular dysfunction, but there was no genotype difference. Our study found an early activation of the kallikrein-kinin system in the kidney and that this has a protective role against the development of diabetic nephropathy. The effect of tissue kallikrein deficiency on microalbuminuria in diabetic mice is similar to the effect of genetically high angiotensin-converting enzyme levels, suggesting that both observations, in part, result from a deficiency in kinins.

GPS2 Deficiency Triggers Maladaptive White Adipose Tissue Expansion in Obesity via HIF1A Activation.

Hypertrophic white adipose tissue (WAT) represents a maladaptive mechanism linked to the risk for developing type 2 diabetes in humans. However, the molecular events that predispose WAT to hypertrophy are poorly defined. Here, we demonstrate that adipocyte hypertrophy is triggered by loss of the corepressor GPS2 during obesity. Adipocyte-specific GPS2 deficiency in mice (GPS2 AKO) causes adipocyte hypertrophy, inflammation, and mitochondrial dysfunction during surplus energy. This phenotype is driven by HIF1A activation that orchestrates inadequate WAT remodeling and disrupts mitochondrial activity, which can be reversed by pharmacological or genetic HIF1A inhibition. Correlation analysis of gene expression in human adipose tissue reveals a negative relationship between GPS2 and HIF1A, adipocyte hypertrophy, and insulin resistance. We propose therefore that the obesity-associated loss of GPS2 in adipocytes predisposes for a maladaptive WAT expansion and a pro-diabetic status in mice and humans.

Antagonism of vasopressin V2 receptor improves albuminuria at the early stage of diabetic nephropathy in a mouse model of type 2 diabetes.

AIMS: Vasopressin is increased in diabetes and was shown to contribute to development of diabetic nephropathy through V2 receptor (V2R) activation in an experimental model of type 1 diabetes. The role of V2R in type 2 diabetes remains undocumented. This study addresses the issue in a mouse model of type 2 diabetes. METHODS: Male obese diabetic db/db mice were treated for 12weeks with a selective V2R antagonist (SR121463) and compared to non-treated db/db and non-diabetic db/m mice. All animals were previously uninephrectomized. RESULTS: The V2R antagonist did not alter glycemia or glycosuria in db/db mice. It induced a two-fold increase in urine output and a 52% decrease in urine osmolality compared to non-treated db/db mice. After four weeks of treatment urinary albumin to creatinine ratio was 50% lower in treated mice compared to non-treated mice, and remained significantly lower until end of experiment. Glomerular filtration rate increased significantly over time in non-treated db/db mice but remained stable in treated mice. CONCLUSIONS: This study shows that vasopressin contributes to albuminuria and glomerular hyperfiltration via V2R in a mouse model of type 2 diabetes. It documents causality behind the association of vasopressin with renal disease observed in diabetic patients.

Tissue kallikrein deficiency, insulin resistance, and diabetes in mouse and man.

The kallikrein-kinin system has been suggested to participate in the control of glucose metabolism. Its role and the role of angiotensin-I-converting enzyme, a major kinin-inactivating enzyme, are however the subject of debate. We have evaluated the consequence of deficiency in tissue kallikrein (TK), the main kinin-forming enzyme, on the development of insulin resistance and diabetes in mice and man. Mice with inactivation of the TK gene were fed a high-fat diet (HFD) for 3 months, or crossed with obese, leptin-deficient (ob/ob) mice to generate double ob/ob-TK-deficient mutants. In man, a loss-of-function polymorphism of the TK gene (R53H) was studied in a large general population cohort tested for insulin resistance, the DESIR study (4843 participants, 9 year follow-up). Mice deficient in TK gained less weight on the HFD than their WT littermates. Fasting glucose level was increased and responses to glucose (GTT) and insulin (ITT) tolerance tests were altered at 10 and 16 weeks on the HFD compared with standard on the diet, but TK deficiency had no influence on these parameters. Likewise, ob-TK⁻/⁻ mice had similar GTT and ITT responses to those of ob-TK⁺/⁺ mice. TK deficiency had no effect on blood pressure in either model. In humans, changes over time in BMI, fasting plasma glucose, insulinemia, and blood pressure were not influenced by the defective 53H-coding TK allele. The incidence of diabetes was not influenced by this allele. These data do not support a role for the TK-kinin system, protective or deleterious, in the development of insulin resistance and diabetes.

Antihypertensive role of tissue kallikrein in hyperaldosteronism in the mouse.

Tissue kallikrein (TK) is synthesized in arteries and distal renal tubule, the main target of aldosterone. Urinary kallikrein excretion increases in hyperaldosteronism. We tested the hypothesis that TK is involved in the cardiovascular and renal effects of high aldosterone. Kallikrein-deficient mice (TK-/-), and wild-type (WT) littermates, studied on two different genetic backgrounds, were treated with aldosterone and high-NaCl diet for 1 month. Control mice received vehicle and standard NaCl diet. Treatment induced 5- to 7-fold increase in plasma aldosterone, suppressed renin secretion, and increased urinary TK activity. In 129SvJ-C57BL/6J mice, blood pressure monitored by radiotelemetry was not different between control TK-/- and WT mice. In TK-/- mice, aldosterone induced larger increases in blood pressure than in WT mice (+47 vs. +27 mm Hg; genotype-treatment interaction, P < 0.05). Night-day difference was also exacerbated in treated TK-/- mice (P < 0.01). Moderate cardiac septal hypertrophy was observed in hypertensive animals without major change in heart function. Aldosterone-salt increased kidney weight similarly in both genotypes but induced a 2-fold increase in renal mRNA abundance of epithelial sodium channel subunits only in TK-/- mice. The hypertensive effect of TK deficiency was also documented in treated C57BL/6J mice. In this strain, aldosterone-induced hypertension was only observed in TK-/- mice (+16 mm Hg, P < 0.01). These findings show that TK deficiency exacerbates aldosterone-salt-induced hypertension. This effect may be due at least in part to enhanced sodium reabsorption in the distal nephron aggravating sodium retention. The study suggests that kallikrein plays an antihypertensive role in hyperaldosteronism.

ACE and non-ACE mediated effect of angiotensin I on intracellular calcium mobilization in rat glomerular arterioles.

Because renin and angiotensin I (ANG I) level are high in the renal circulation, the conversion of ANG I is a critical step in the regulation of glomerular hemodynamics. We studied this conversion by investigating the effect of ANG I on intracellular Ca(2+) concentration ([Ca(2+)](i)) in rat juxtamedullary glomerular afferent and efferent arterioles (AA and EA, respectively). Two types of EA were considered, thin EA and muscular EA, terminating as peritubular capillaries and vasa rectae, respectively. In all arterioles, ANG I elicited [Ca(2+)](i) elevations. Maximal responses of 171 +/- 28 (AA), 183 +/- 7 (muscular EA), and 78 +/- 11 nM (thin EA) (n = 6), similar to those obtained with ANG II, were observed with 100 nM ANG I. The EC(50) values were 20 times higher for ANG I than for ANG II in AA (10.2 vs. 0.5) and muscular EA (6.8 vs. 0.4 nM) and 150 times higher in thin EA (15.2 vs. 0.1 nM). ANG I effect was blocked by losartan, indicating that AT(1) receptors were involved. The ANG-converting enyzme (ACE) inhibitor lisinopril inhibited the maximal response to ANG I in AA and muscular EA by 75 +/- 9% (n = 13) and 70 +/- 7% (n = 13), respectively, but had no effect in thin EA (n = 14). The serine protease inhibitor aprotinin, the chymase inhibitor chymostatin, and the cysteine protease inhibitors E64 and leupeptin had no effect on ANG I action. These data show that ANG I effects are mainly mediated by ACE in AA and muscular EA but not in thin EA. The lisinopril-insensitive response may be related to conversion by unknown enzyme(s) and/or to activation of AT(1) receptors by ANG I.

Angiotensin receptor subtypes in thin and muscular juxtamedullary efferent arterioles of rat kidney.

ANG II controls the vascular tone of pre- and postglomerular arterioles, and thereby glomerular filtration, through binding to either AT1A, AT1B, or AT2 receptors. AT1 receptors, which are coupled to intracellular Ca2+ signaling, have vasoconstricting effects, whereas AT2 receptors, whose signaling mechanism is unknown, induce vasodilatation. The angiotensin receptors have been characterized in afferent arterioles, which express the three types of receptors, but not in efferent arterioles. Two subpopulations of juxtamedullary efferent arterioles, muscular ones which terminate as vasa rectae and thin ones which terminate as peritubular capillaries, have been described. They display functional heterogeneity with regard to the ANG II response. To evaluate whether these differences are associated with differential expression of ANG II receptors, we examined the expression pattern of AT1A, AT1B, and AT2 receptor mRNAs by RT-PCR in these arterioles and studied the effect of valsartan, a specific AT1-receptor antagonist. Results indicate that muscular arterioles express AT1A, AT1B, and AT2 receptors, whereas thin arterioles only express the AT1A and AT2 types, and at a much lower level. Valsartan fully inhibited ANG II-induced increases in intracellular Ca2+ in both arteriolar types, but with different kinetics. In muscular arterioles, inhibition was monoexponential, whereas it displayed a marked positive cooperativity in thin arterioles. Finally, the apparent affinity for valsartan was higher in muscular than in thin arterioles. In conclusion, this study further documents the differences between muscular and thin efferent arterioles with regard to ANG II signalization in the rat kidney.