Effects of systemic inhibition of Rho kinase on blood pressure and renal haemodynamics in diabetic rats.

BACKGROUND AND PURPOSE: The RhoA/Rho associated kinases (ROCK) pathway has been implicated in the pathophysiology of diabetic nephropathy (DN). Early stages of diabetes are associated with renal haemodynamic changes, contributing to later development of DN. However, the role of RhoA/ROCK, known regulators of vascular tone, in this process has not been studied. EXPERIMENTAL APPROACH: Blood pressure (BP), glomerular filtration (GFR), effective renal plasma flow and filtration fraction (FF) in response to the ROCK inhibitors Y27632 (0.1 and 0.5 mg·kg(-1) ) and fasudil (0.3 and 1.5 mg·kg(-1) ) were examined in streptozotocin-diabetic rats and non-diabetic controls. KEY RESULTS: Diabetic rats demonstrated baseline increases in GFR and FF. In contrast to similar decreases in BP in diabetic and control rats, renal vasodilator effects and a decrease in FF, following ROCK inhibition were observed only in diabetic rats. The vasodilator effects of Y27632 and a further decrease in FF, were also detected in diabetic rats pretreated with the angiotensin antagonist losartan. The effects of ROCK inhibitors in diabetic rats were modulated by prior protein kinase C (PKC)ß inhibition with ruboxistaurin, which abolished their effects on FF. Consistent with the renal vasodilator effects, the ROCK inhibitors reduced phosphorylation of myosin light chain in diabetic kidneys. CONCLUSIONS AND IMPLICATIONS: The results indicate greater dependence of renal haemodynamics on RhoA/ROCK and beneficial haemodynamic effects of ROCK inhibitors in diabetes, which were additive to the effects of losartan. In this process, the RhoA/ROCK pathway operated downstream of or interacted with, PKCß in some segments of the renal vascular tree.

A polymorphism in the cyclooxygenase 2 gene in type 1 diabetic patients with nephropathy.

Diabetic nephropathy (DN), the most serious complication of Type 1 diabetes (DM1), has a strong genetic component. Cyclooxygenase-2 (COX-2), an inducible enzyme by a number of stimuli, has been implicated in pathophysiology of cardiovascular and renal disease, including DN. The allele -765C, of the -765G > C polymorphism (rs20417) in the COX-2 promoter has lower promoter activity compared with the -765G allele and protective effects in cardiovascular disease. This polymorphism was not investigated in patients with DM1 and nephropathy. The study was conducted in 779 Caucasian patients with DM1 and compared to a representative sample of healthy Czech population. The patients demonstrated lower frequencies of the CC genotype (P = 0.005). From the DM1 cohort, 153 patients met the criteria for low risk of the development of DN (LRDN, duration of DM1 > 10 years, normoalbuminuria, normotension) and 139 patients had manifest DN. There were no differences in -765G > C polymorphisms between LRDN and DN patients. Moreover, the C/G allele frequencies did not also differ between the groups. In conclusion, patients with DM1 display lower freqencies of the protective CC genotype as compared to healthy subjects. However, the study did not reveal associations of -765G > C polymorphism with the risk of DN.

Determination of caveolin-1 in renal caveolar and non-caveolar fractions in experimental type 1 diabetes.

Caveolin-1 (CAV-1) is the main structural component of caveolae, acting as a modulator of signal transduction. CAV-1 might be involved in the pathophysiology of microvascular complications in Type 1 diabetes (DM). We sought to determine whether fractionation on sucrose gradient (SF), a method routinely utilized for isolation of caveolar fractions in homogenous cell lines, is applicable for CAV-1-related studies in tissues with multiple cell types, such as the normal rat kidney cortex (C). Using this method, we also determined whether streptozotocin-induced DM in rats (4-week duration) leads to changes in renal subcellular targeting of CAV-1, and evaluated the effects of tight metabolic control (insulin, 12 IU/day) and angiotensin receptor blocker, losartan (4 weeks, 20 mg/kg/day). Immunoblotting of individual fractions obtained from C revealed CAV-1 expression in fractions 4-6 that corresponded to light scattering band that typically forms after separating cellular fractions on SF. These fractions were considered to be caveolar fractions. In C, CAV-1 was also detectable in fractions 8-10. These and all other fractions except caveolar fractions were considered to be non-caveolar fractions. A ratio of caveolar/non-caveolar expression of CAV-1 (CNCR) was computed for each renal cortex allowing comparisons of CAV-1 subcellular distribution in C and DM rats, and effects of treatments. Using this approach, DM was characterized by marked increases in CNCR as compared to C (5.54+/-1.56 vs. 2.65+/-1.33, p<0.05) that were reduced by treatment with insulin (0.78+/-0.24, p<0.01 vs. DM) or losartan (0.84+/-0.06, p<0.01 vs. DM). In summary, analysis of CAV-1 following the SF of renal cortex detected similar distribution of the protein as in homogenous cell lines, DM-induced changes in CAV-1 targeting, and the effects of pharmacological treatments. This suggests applicability of SF in studies focusing on CAV-1 targeting in organs with various cell lines in vivo.

Renal activity of Akt kinase in experimental Type 1 diabetes.

Akt kinase regulates numerous cell functions including glucose metabolism, cell growth, survival, protein synthesis, and control of local hemodynamics. mTOR is one of down-stream effectors of Akt involved in the initiation of protein translation. However, renal Akt signaling in Type 1 diabetes (DM) in vivo, in particular under the conditions reflecting differences in metabolic control, has received less attention. Renal cortical activity and expression of Akt and mTOR (kinase assay, western blotting) were determined in streptozotocin-diabetic rats (D) with different levels of glycemic control (blood glucose 22.0+/-1.0, 13.4+/-1.5, 8.1+/-0.4 mmol/l, p<0.05 between the groups), achieved by varying insulin treatment (0, 4 and 12 IU/day), and in control rats with (C4) or without (C) chronic insulin administration. Renal Akt activity was reduced in D rats without insulin treatment and severe hyperglycemia (D-0, -62 %, p<0.01 vs. C), partially restored in moderately hyperglycemic rats (D-4, -30 %, p<0.05 vs. C), and normalized in D rats with intensive insulin and tight metabolic control (D-12). Expression of active mTOR paralleled Akt activity in D-0 (-51 %, p<0.01 vs. C), but not in D-4 and D-12 that demonstrated increases in active mTOR (+55 %, +80 % resp., p<0.05) as compared to C. Moreover, insulin activated renal Akt (+82 %, p<0.01), but not mTOR in C4. In conclusion, glycemic control and intensity of insulin treatment are important modulators of renal Akt and mTOR activity in diabetes. While Akt activity is reversible by tight metabolic control, combination of hyperglycemia and insulin treatment resulted in enhancement of mTOR activity. In addition to Akt, other signaling pathways likely contribute to regulation of renal mTOR activity in diabetes.

[Effects of specific cycloxygenase-2 inhibition on the renal functions of ederly patients with renal function impairment]. / Vliv specifické inhibice cyklooxygenázy 2 na ledvinové funkce u starsích pacientu s poruchou ledvinové funkce.

BACKGROUND: Elderly patients suffering from nociceptive pain of locomotive organs and concomitantly from renal impairment represent a target population for painkilling drugs. That is why they are predisposed to nephrotoxic effects non-steroidal anti-inflammatory drugs. The aim of our study was to evaluate cycloxygenase-2 (COX-2) inhibition effect on renal function in elderly with moderate impairment of renal function. METHODS AND RESULTS: Based on 24-h urine collection we assessed creatinine clearance (C(Cr), fractional excretion of sodium (FE(Na)), potassium (FE(K)), chloride (FE(Cl)), osmotic active solutes (FE(OSM)) and 24h urinary excretion of prostaglandin PGE2 and PGF(2 alpha). Under conditions of sub-maximal water load fractional excretion of electrolytes, inulin clearance (C(in)), serum cystatin C (S(cyst)) were assessed. In addition basal and stimulated plasma renin activity (PRA) and plasma aldosteron (P(aldo)) were examined. Using comparison of parameters before and at the end of 7-days rofecoxib treatment we found out C(in) 0,82 +/- 0,34 vs 0,74 +/- 0,18 ml/s/l,73 m2, FE(Na) 1,0 +/- 0,3 vs 1,2 +/- 0.4 (p=0,02), FE(OSM) 2.9 +/- 0,7 vs 3,7 +/- 1,2% (p=0,03), U(PGE2 alpha),V 663 +/- 528 vs 414 +/- 195 (p=0,059), U(PGD2) V (559 +/- 625) vs 205 +/- 174 eta g/24h (p=0,02), stimulated PRA 0.94 +/- 0,73 vs 0,4 +/- 0,27 +/- pg/l/h (p=0,019), P(aldo) 104,56 +/- 50,15 vs 56,94 +/- 27,08 eta g/l/h (p=0,008). CONCLUSIONS: Short-term COX-2 inhibition in patients with moderate renal impairment was associated with significant decrease of tubular transport of sodium, without changing GFR and water excretion.

Effect of rofecoxib on the glomerular filtration rate, proteinuria and the renin-angiotensin-aldosterone system in elderly subjects with chronic renal impairment.

UNLABELLED: Considering the increasing popularity and prescribing of specific COX-2 inhibitors, a new class of NSAIDs lacking gastrointestinal side effects, the evaluation of their effects on renal function has become very important. OBJECTIVE: The aim of the study was to evaluate the effect of rofecoxib on GFR, proteinuria and the renin-angiotensin-aldosterone system (RAAS) in elderly patients with chronic renal impairment under controlled conditions of water and salt intake. SUBJECTS: There were ten patients (average age 67 years, range 53 - 80 years) with analgesic or vascular nephropathy (average GFR 54 ml/min/1.73 m2, range 30 - 79 ml/min/ 1.73 m2) given 25 mg rofecoxib daily for seven days under balanced conditions of water and sodium metabolism (salt intake 6 - 8 g/24 hours). METHODS: The effect of rofecoxib on GFR measured using inulin clearance (C(in)), creatinine clearance (C(Cr)) serum cystatin C concentration (S(cystatin)), tubular creatinine secretion (using the ratio C(Cr)/C(in)), 24-hour urinary excretion of albumin (U(alb)V) and prostaglandins (U(PGE2)V and U(PGF2alpha)V), basal and stimulated plasma renin activity (PRA) and serum aldosterone concentration S(aldosterone) was evaluated before and on Day 7 during rofecoxib treatment. RESULTS: Rofecoxib did not significantly change C(in), C(Cr), S(cystatin), C(Cr)/C(in) and U(alb)V. However, U(PGE2)V and U(PGF2alpha)V were decreased during rofecoxib administration (p = 0.059 and p = 0.024, respectively). Rofecoxib attenuated the stimulated rise of PRA and S(aldosterone) (p = 0.019 and p = 0.008, respectively). CONCLUSIONS: Short-term rofecoxib administration was not associated with significant change in GFR in elderly patients with moderate chronic renal impairment under conditions of balanced salt and water metabolism despite significant attenuation of RAAS activity. Since the C(Cr)/C(in) ratio did not change in our study, we assume rofecoxib to have no influence on creatinine tubular secretion.

Emerging role of Akt kinase/protein kinase B signaling in pathophysiology of diabetes and its complications.

In addition to a number of deleterious effects on cellular integrity and functions, diabetic metabolic milieu has been implicated in a rapidly growing number of alterations in signal transduction. In this review we focus on Akt kinase physiology, its alterations in diabetes mellitus (DM), and on the emerging role of this signaling system in the pathophysiology of diabetic microvascular complications. Studies focusing on Akt in diabetes suggest both decrease and increase of Akt activity in DM. Alterations of Akt activity have been found in various tissues and cells in diabetes depending on experimental and clinical contexts. There is convincing evidence suggesting defective Akt signaling in the development of insulin resistance. Similar defects, as in insulin-sensitive tissues, have been reported in endothelia of DM Type 2 models, possibly contributing to the development of endothelial dysfunction under these conditions. In contrast, Akt activity is increased in some tissues and vascular beds affected by complications in DM Type 1. Identification of the role of this phenomenon in DM-induced growth and hemodynamic alterations in affected vascular beds remains one of the major challenges for future research in this area. Future studies should include the evaluation of therapeutical benefits of pharmacological modulators of Akt activity.

Renal and cardiovascular effects of selective cyclooxygenase-2 inhibitors.

Selective inhibition of cyclooxygenase-2 (COX-2) was proposed as a novel anti-inflammatory and analgesic treatment with a reduced profile of gastrointestinal side effects compared with conventional nonsteroidal anti-inflammatory drugs (NSAIDs). Although perceived as an inducible enzyme by inflammatory and other stimuli, COX-2 is constitutively expressed in the kidney. In this review, we focus on renal and cardiovascular (CV) physiological and pathophysiological characteristics of COX-2 and renal and CV aspects of treatment with selective COX-2 inhibitors. Both clinical and experimental studies have shown that renal and CV effects of COX-2 inhibitors are similar to those of NSAIDs. These effects include sodium, potassium, and water retention and decreases in renal function, as well as mild to modest increases in blood pressure (BP) and edema. These deleterious effects are amplified in patients with volume and/or sodium depletion. The concomitant administration of COX-2 inhibitors may destabilize BP control in hypertensive patients treated with antihypertensive agents. In contrast to the normal kidney, which could constitute a target for adverse actions of COX-2 inhibitors, recent experimental studies showed increased renal COX-2 expression in several models of renal injury, such as the remnant kidney, renovascular hypertension, and diabetes, and implicated COX-2 in the progression of renal failure. This suggests that COX-2 inhibitors may confer a renoprotective effect in diverse renal disorders. These intriguing formulations must be delineated further in appropriately designed prospective clinical trials.

Immunohistochemical and functional correlations of renal cyclooxygenase-2 in experimental diabetes.

Prostaglandins (PGs) generated by the enzyme cyclooxygenase (COX) have been implicated in the pathological renal hemodynamics and structural alterations in diabetes mellitus, but the role of individual COX isoenzymes in diabetic nephropathy remains unknown. We explored COX-1 and COX-2 expression and hemodynamic responses to the COX-1 inhibitor valeryl salicylate (VS) or the COX-2 inhibitor NS398 in moderately hyperglycemic, streptozotocin-diabetic (D) and control (C) rats. Immunoreactive COX-2 was increased in D rats compared with C rats and normalized by improved glycemic control. Acute systemic administration of NS398 induced no significant changes in mean arterial pressure and renal plasma flow in either C or D rats but reduced glomerular filtration rate in D rats, resulting in a decrease in filtration fraction. VS had no effect on renal hemodynamics in D rats. Both inhibitors decreased urinary excretion of PGE(2). However, only NS398 reduced excretion of thromboxane A(2). In conclusion, we documented an increase in renal cortical COX-2 protein expression associated with a different renal hemodynamic response to selective systemic COX-2 inhibition in D as compared with C animals, indicating a role of COX-2-derived PG in pathological renal hemodynamic changes in diabetes.

Treatment of hypertension in diabetic patients with nephropathy.

In type 1 diabetes, hypertension is closely linked to the development of nephropathy. An association of hypertension and the impact of hypertension on the clinical course of type 2 diabetes, including the development of vascular complications, has been well established. However, the association with nephropathy in type 2 diabetes is less clear. Despite that, antihypertensive treatment has a crucial impact on the course of nephropathy in both types of diabetes. In this article, we discuss recent evidence focusing on the nephroprotective potential of various classes of antihypertensive agents and confront it with current recommendations for the treatment of hypertension in diabetic patients with nephropathy. Unlike type 1 diabetes, where the nephroprotection could be a good sole measure for assessing the efficiency of a particular agent or their combination, defining of the optimal antihypertensive agent or agents in type 2 diabetes requires consideration of both cardiovascular, cerebrovascular, and nephroprotective potentials of such a treatment. In both types of diabetes, recent data support the use of inhibitors of the renin-angiotensin system with or without diuretics as the initial therapy. In type 1 diabetes, additional beneficial effect can be expected from calcium channel blockers (CCBs). In type 2 diabetic patients, combining more agents may be necessary early in the course of nephropathy to affect both micro- and macrovascular targets. beta blockers should be applied early to enhance cardioprotectivity, followed by CCBs to achieve goal blood pressure. Although not supported by all recent data, aggressive blood pressure control (< 130/75 mm Hg) is warranted. Furthermore, multifactorial intervention targeting metabolic derangements and lifestyle, is a necessary complimentary measure that must accompany antihypertensive treatment.

Therapeutic potential of ACE inhibitors for the treatment of hypertension in Type 2 diabetes.

Type 2 diabetes mellitus is associated with hypertension. If untreated, hypertension has a major impact on the clinical course of Type 2 diabetes and its vascular complications. In this review, we discuss rationale for the use of ACE inhibitors (ACEI) in hypertensive Type 2 diabetic patients and compare those theoretical assumptions with results of recent major clinical trials. Furthermore, possible directions for future clinical and experimental research are outlined. The RAS and its effector angiotensin II are important players in a number of cardiovascular and renal disorders. Recent evidence suggests that RAS and factors functionally linked to RAS are activated in Type 2 diabetes. Therefore, there is a theoretical basis for the use of ACEI in the treatment of hypertension in diabetic patients. Some recent studies reported superior outcome in patients treated with ACEI-based antihypertensive regimens compared with non-ACEI based treatments in reducing the risk of macrovascular disease (CAPPP, FACET, ABCD) or both micro- and macrovascular complications in Type 2 diabetes (HOPE). However, at least two of the large prospective studies discussed in this review (UKPDS 38, HOT), supported by results from previously published SHEP study, have recently suggested that the degree of reduction of blood pressure, rather than the choice of a particular class of antihypertensive agent, is associated with decreased risk of cardiovascular events. Studies focusing on renal end-points suggest that ACEI have a superior antiproteinuric effect than the other agents. However, whether ACEI are more nephroprotective, as assessed by the rate of decline in renal function, still remains to be elucidated. Despite promising results from recent trials, large numbers of patients progress despite ACEI treatment. Incomplete inhibition of the RAS may underlie this phenomenon. Treatment strategies that could enhance the degree of RAS inhibition represent one possible direction for clinical research in the near future. However, it is unlikely that the course of such a complex syndrome as Type 2 diabetes could be dramatically changed by just one class of antihypertensive agents. This goal is more likely to be achieved by multifactorial intervention, reflecting the complexity of metabolic syndrome. ACEI should be viewed as an important, but not the only, part of this complex approach.

Optimal strategies for preventing progression of renal disease: should angiotensin converting enzyme inhibitors and angiotensin receptor blockers be used together?

Interruption of the renin-angiotensin system (RAS) with angiotensin converting enzyme (ACE) inhibitors or angiotensin AT(1) receptor blockers has been shown to delay progression in a variety of renal diseases, suggesting that the RAS, and its major effector molecule, angiotensin II, are important players in renal pathophysiology. Both antagonists combine inhibition of deleterious effects of angiotensin II with activation of potentially beneficial pathways mediated by nitric oxide and prostaglandins. Some concerns have been raised about the completeness of the RAS blockade achieved by these agents. ACE-independent pathways can generate angiotensin II, whereas increases in angiotensin II levels may compete with the AT(1) receptor blocker at the receptor site. It has been suggested that an ACE inhibitor/AT(1) receptor blocker combination offers a better therapeutic effect than treatment with either agent alone. In this review, we focus on mechanisms of actions of ACE inhibitors and AT(1) receptor blockers, implicate them in the rationale for the use of an ACE inhibitor/AT(1) receptor blocker combination, and discuss evidence evaluating the renal effects of the combination as compared to the effects of a single agent. There is a surprising lack of information about the nephroprotective potential of the combination, allowing no consistent conclusions about the superiority of the combination over the single agent. Several experimental and clinical reports suggest that in some conditions, the combination may be beneficial. Rather than providing unequivocal evidence for the use of combination treatment in the renal disease, these studies should be considered as stimuli for more detailed exploration of this issue.

Role of neuronal nitric oxide synthase (NOS1) in the pathogenesis of renal hemodynamic changes in diabetes.

Nitric oxide (NO) has been implicated in the pathogenesis of renal hemodynamic changes in diabetes mellitus. However, the contribution of nitric oxide synthase (NOS) isoforms to intrarenal production of NO in diabetes remains unknown. To explore the role of NOS1 in the control of renal hemodynamics in diabetes, we assessed renal responses to inhibition of NOS1 with S-methyl-L-thiocitrulline (SMTC; administered into the abdominal aorta) in moderately hyperglycemic streptozotocin-diabetic rats (D) and their nondiabetic (C) and normoglycemic diabetic counterparts. The contribution of other NOS isoforms was also evaluated by assessing the responses to nonspecific NOS inhibition [N(G)-nitro-L-arginine methyl ester (L-NAME)] in SMTC-treated diabetic rats. The number of NOS1-positive cells in macula densa of D and C kidneys was also evaluated by immunohistochemistry. D rats demonstrated elevated glomerular filtration rate (GFR) compared with C. SMTC (0.05 mg/kg) normalized GFR in D but had no effect in C. SMTC-induced reduction of renal plasma flow (RPF) was similar in C and D. Normoglycemic diabetic rats demonstrated blunted renal hemodynamic responses to NOS1 inhibition compared with hyperglycemic animals. Mean arterial pressure was stable in all groups. L-NAME induced a further decrease in RPF, but not in GFR, in D rats treated with SMTC. Immunohistochemistry revealed increased numbers of NOS1-positive cells in D. These observations suggest that NOS1-derived NO plays a major role in the pathogenesis of renal hemodynamic changes early in the course of diabetes. NOS1 appears to be the most important isoform in the generation of hemodynamically active NO in this condition.

Are angiotensin-converting enzyme inhibitors the best treatment for hypertension in type 2 diabetes?

The influence of hypertension on the clinical course and complications of type 2 diabetes is well established. With a special focus on angiotensin-converting enzyme inhibitors, this paper will review recently published results of prospective studies addressing two important aspects: the degree of blood pressure control, and the choice of antihypertensive regimen, in the prevention of complications in hypertensive type 2 diabetic patients. None of the recent studies have shown worse outcomes in patients treated with angiotensin-converting enzyme inhibitor-based regimens compared with alternative treatments. Some studies have suggested that angiotensin-converting enzyme inhibitor-based antihypertensive regimens may be superior to alternative treatments in reducing the risk of micro- and macrovascular complications, whereas other studies found similar effects for beta-blockers or calcium antagonists. Several trials showed beneficial effects of angiotensin-converting enzyme inhibitors over calcium antagonists, and have raised concerns about the use of dihydropyridine calcium antagonists in these patients. However, it remains to be determined whether there should be more reserved use of calcium antagonists in such patients, in the light of more major trials showing the safety and efficacy of calcium antagonists in preventing cardiovascular and renal endpoints. The degree of reduction of blood pressure rather than the choice of a particular drug may be the most important factor. Studies focusing on renal endpoints suggest that angiotensin-converting enzyme inhibitors have a better antiproteinuric effect than other agents, but this phenomenon is not always reflected by a more beneficial effect of angiotensin-converting enzyme inhibitors on the decline in glomerular filtration rate. In many ways, the question of whether angiotensin-converting enzyme inhibitors are the best class of agent in these patients is academic. Angiotensin-converting enzyme inhibitors are sufficiently safe, and, according to recent evidence, equally or more effective than other classes of agents. Tight blood pressure control is usually achievable only with a combination of agents. On the basis of available evidence, it appears that angiotensin-converting enzyme inhibitors, together with a low-dose cardioselective beta-blocker and a diuretic, should be used in most hypertensive type 2 diabetes patients, with calcium antagonists serving as reserve drugs in case of insufficient blood pressure control.

Effect of ACE inhibition and angiotensin AT1 receptor blockade on renal and blood pressure response to L-arginine in humans.

OBJECTIVE: Nitric oxide (NO) may contribute to the actions of angiotensin converting enzyme (ACE) inhibitors. In contrast, angiotensin type 1 (AT1) receptor blockers (AT1B) have been considered to act exclusively by inhibiting angiotensin II actions. However, recent experimental findings suggest that AT1B actions may be also partly mediated by NO. In this study, we explored whether ACE inhibitors and AT1B modulate hemodynamic responses to L-arginine (L-arg), a NO precursor. METHODS: Systemic (Finapres) and renal hemodynamic responses to L-arg (200 mg/kg body weight), associated with markers of systemic and renal NO production, were assessed before (control) and after 3 weeks of randomized pretreatment with the ACE inhibitor ramipril (5 mg/day for 3 weeks) or the AT1B losartan (50 mg/day for 3 weeks) in nine healthy male subjects (33 +/- 2 years; body mass index 25.5 +/- 0.5 kg/m2). RESULTS: Control L-arg did not influence mean arterial pressure (MAP) (92 +/- 5 versus 90 +/- 5 mmHg; not significant). In contrast, L-arg decreased MAP when administered after pretreatment with ramipril (89 +/- 5 versus 83 +/- 4 mmHg; P< 0.01) or losartan (90 +/- 44 versus 86 +/- 4; P< 0.05). Control L-arg infusion had no effect on renal plasma flow (RPF) (paraminohippuric acid clearance) and renal vascular resistance (RVR), whereas the glomerular filtration rate (GFR) (inulin clearance) decreased (98 +/- 4 versus 89 +/- 5 ml/min; P< 0.05), resulting in a decrease in filtration fraction (P< 0.05). After ramipril, L-arg induced renal vasodilation as indicated by significant changes in RPF (576 +/- 41 versus 669 +/- 21 ml/min; P< 0.01) and RVR (P< 0.05). The GFR did not change statistically after ramipril pretreatment (91 +/- 3 versus 97 +/- 4 ml/min; not significant); however, the trend was different as compared with control (F= 5.7, P < 0.05). L-Arg-induced renal vasodilation was also observed after losartan (RPF, 637 +/- 34 versus 706 +/- 40 ml/min; P< 0.05). Enhanced renal and systemic responses to L-arg after ACE inhibitor and AT1B were associated with a rise in plasma L-citrulline levels, which was greater than after control L-arg (P < 0.05). However, other indicators of NO activity such as plasma and urinary cyclic guanosine 3',5'-monophosphate, and nitrates, remained unchanged throughout all experiments. CONCLUSION: The results indicate that ACE inhibitors and AT1B have a potential to enhance L-arg-induced vasodilation both in systemic and renal vascular beds. However, these hemodynamic responses were not associated with convincing changes in indicators of systemic or renal NO activity, suggesting a contribution of NO-independent vasodilator mechanisms.

Effects of systemic inhibition of neuronal nitric oxide synthase in diabetic rats.

Diabetes is associated with alterations in nitric oxide-mediated vasomotor function. The role of nitric oxide generated via the neuronal nitric oxide synthase pathway in the control of systemic and renal hemodynamics in diabetes has not been studied. To explore the hypothesis that diabetic vascular dysfunction is in part caused by altered neuronal nitric oxide synthase activity, systemic and renal hemodynamics were assessed before and after acute inhibition of this enzyme with a specific inhibitor, S-methyl-L-thiocitrulline, in control and diabetic rats. The interaction of this pathway and the renin-angiotensin system was studied in separate groups of rats pretreated with the angiotensin II receptor blocker losartan; these rats were compared with rats treated with losartan alone. Diabetic animals demonstrated higher baseline glomerular filtration rates and filtration fractions. At a low dose, the neuronal nitric oxide synthase inhibitor induced similar dose-dependent pressor responses in control and diabetic rats. Losartan abolished the pressor response in both groups. No changes in renal plasma flow or renal vascular resistance occurred in control rats. In contrast, diabetic rats responded with significant renal vasoconstriction. At a high dose, the renal vasoconstriction was similar in both groups and was not affected by losartan. In conclusion, neuronal nitric oxide synthase-derived nitric oxide plays a role in the control of systemic and renal hemodynamics in normal and diabetic rats. Diabetic rats are more sensitive to the inhibitor, suggesting increased activity of this pathway in the diabetic kidney. Furthermore, renal responses in diabetic rats were attenuated by angiotensin II receptor blockade, whereas losartan alone induced hemodynamic changes that were opposite those seen with neuronal nitric oxide synthase inhibition. This observation implicates angiotensin II as an important modulator of this nitric oxide pathway in diabetes.

Effect of hyperinsulinaemia on renal function and nitrate/nitrite excretion in healthy subjects.

1. In addition to its metabolic actions, insulin acts as a vasodilator in certain vascular beds, such as skeletal muscle. It has been shown that this effect is mediated by endothelium-derived nitric oxide (NO). Unlike in the skeletal muscle, insulin-NO interactions in the kidney, another major site of insulin action, have been less studied. The aim of the present study was to explore the role of NO in renal effects of hyperinsulinaemia in healthy subjects. 2. Changes in renal function and urinary nitrate/nitrite (NO2-/NO3-; Griess method) levels as a marker of renal production of NO were assessed during euglycaemic hyperinsulinaemic clamp and compared with normoinsulinaemic isovolaemic conditions (administration of the same amount of insulin/glucose-free vehicle) in 10 healthy male volunteers. 3. Hyperinsulinaemia was associated with a decrease in renal excretion of stable metabolites of NO (mean (+/- SEM) 0.56 +/- 0.12 vs 0.38 +/- 0.05 mumol/min, respectively; P < 0.05). In contrast, administration of the same volume of insulin-free vehicle resulted in elevation of urinary NO2-/NO3- (P < 0.05). The changes in renal sodium handling followed a similar pattern as changes in the renal excretion of NO2-/NO3- with a significantly different response to hyperinsulinaemia when compared with normoinsulinaemia (F = 12.2; P < 0.001). The mean arterial pressure, blood levels of low-density lipoprotein-cholesterol and free fatty acids, possible factors influencing renal and systemic NO production, remained constant throughout both experiments. 4. These results suggest that hyperinsulinaemia is associated, in healthy males, with a decrease in renal generation of NO. In contrast, mild volume expansion with insulin-free vehicle resulted in increased excretion of NO metabolites. This insulin-induced attenuation of renal NO synthesis may contribute to the anti-natriuretic actions of insulin.

Thiazolidinediones--tools for the research of metabolic syndrome X.

The resistance to insulin (insulin resistance, IR) is a common feature and a possible link between such frequent disorders as non-insulin dependent diabetes mellitus (NIDDM), hypertension and obesity. Pharmacological amelioration of IR and understanding its pathophysiology are therefore essential for successful management of these disorders. In this review, we will discuss the mechanisms of action of thiazolidinediones (TDs), a new family of insulin-sensitizing agents. Experimental studies of various models of IR and an increasing number of clinical studies have shown that TDs normalize a wide range of metabolic abnormalities associated with IR. By improving insulin sensitivity in skeletal muscles, the adipose tissue and hepatocytes, TDs reduce fasting hyperglycaemia and insulinaemia. Furthermore, TDs markedly influence lipid metabolism--they decrease plasma triglyceride, free fatty acid and LDL-cholesterol levels, and increase plasma HDL-cholesterol concentrations. Although TDs do not stimulate insulin secretion, they improve the secretory response of beta cells to insulin secretagogues. TDs act at various levels of glucose and lipid metabolism--ameliorate some defects in the signalling cascade distal to the insulin receptor and improve glucose uptake in insulin-resistant tissues via increased expression of glucose transporters GLUT1 and GLUT4. TDs also activate glycolysis in hepatocytes, oppose intracellular actions of cyclic AMP, and increase intracellular magnesium levels. TDs bind to peroxisome proliferator activating receptors gamma (PPAR gamma), members of the steroid/thyroid hormone nuclear receptor superfamily of transcription factors involved in adipocyte differentiation and glucose and lipid homeostasis. Activation of PPAR gamma results in the expression of adipocyte-specific genes and differentiation of various cell types in mature adipocytes capable of active glucose uptake and energy storage in the form of lipids. Furthermore, TDs inhibit the pathophysiological effects exerted by tumour-necrosis factor (TNF alpha), a cytokine involved in the pathogenesis of IR. These effects are most likely also mediated by stimulation of PPAR gamma. In mature adipocytes, PPAR gamma stimulation inhibits stearoyl-CoA desaturase 1 (SCD1) enzyme activity resulting in a change of cell membrane fatty acid composition. Apart from their metabolic actions, TDs modulate cardiovascular function and morphology independently of the insulin-sensitizing effects. TDs decrease blood pressure in various models of hypertension as well as in hypertensive insulin-resistant patients, and inhibit proliferation, hypertrophy and migration of vascular smooth muscle cells (VSMC) induced by growth factors. These processes are considered to be crucial in the development of vascular remodelling, atherosclerosis and diabetic organ complications. TDs induce vasodilation by blockade of Ca2+ mobilisation from intracellular stores and by inhibition of extracellular calcium uptake via L-channels. Furthermore, TDs interfere with pressor systems (catecholamines, renin-angiotensin system) and enhance endothelium-dependent vasodilation. A key role of TDs effects in vascular remodelling is played by inhibition of the mitogen-activated protein (MAP) kinase pathway. This signalling pathway is important for VSMC growth and migration in response to stimulation with tyrosine-kinase dependent growth factors. In addition to the vasoprotective mechanisms mentioned above, troglitazone, the latest representative of this pharmacological group, possesses antioxidant actions comparable to vitamin E. In summary, TDs have the unique ability to attack mechanisms responsible for metabolic alterations as well as for vascular abnormalities characteristic for IR. Therefore, TDs represent a powerful research tool in attempts to find a common denominator underlying the pathophysiology of the metabolic syndrome X. A recently reported link between MAP kinase signalling pathway and PPAR gamma

Increased density of renal amylin binding sites in experimental hypertension.

High-affinity binding sites for the pancreatic beta-cell hormone amylin have been reported in the kidney, and it has been postulated that these sites may be involved in the genesis of hypertension. In the present study, we have used in vivo injection of 125I-amylin and in vitro autoradiographic techniques to assess renal amylin binding in both a genetic and a surgically induced model of hypertension. In the spontaneously hypertensive rat (SHR) at 6 weeks of age, before the rise in systolic blood pressure, there was a 36% increase in density of amylin binding compared with their normotensive counterpart, the Wistar-Kyoto rat (WKY). In SHR, there was a further increase in the density of amylin binding (to 53% greater) as the systolic blood pressure rose between 6 and 12 weeks of age. Histological examination of kidneys from SHR at 12 weeks of age revealed staining for a brush border glycoprotein, normally restricted to the proximal tubules, extending from the urinary pole into half of the epithelial lining of the glomerular capsule. In contrast to WKY, these cells also bound 125I-amylin with high density in SHR. In a rat model of renal ablation and hypertension, systolic blood pressure correlated with the density of 125I-amylin binding in the renal cortex (r=.54, P=.003, n=28). The changes in amylin binding reported here suggest a possible role for this peptide and/or activation of its receptor in the genesis as well as the maintenance of hypertension.

Renal sodium handling in experimental diabetes: role of NO.

Recent studies have suggested that diabetes is a state of increased renal nitric oxide (NO) activity as assessed by urinary excretion of nitrites and nitrates (NOx), and that NO synthase inhibitors reverse the increased glomerular filtration rate (GFR) observed in experimental diabetes. In addition to being a potent vasodilator in the renal vasculature, NO also plays a role in modulation of renal sodium excretion. To explore the role of NO in diabetes-associated alterations in renal excretory function, renal haemodynamic and sodium handling parameters were evaluated in conscious control (C) and streptozotocin diabetic rats (D) and correlated to the renal activity of NO, as assessed by urinary excretion of its metabolites NOx. To further explore this issue, the changes in renal haemodynamics and sodium handling were also assessed after NO synthase inhibition with a non-pressor dose of L-nitro-arginine-methyl-ester (L-NAME) and after administration of the NO donor, glyceryl trinitrate (GTN). Systolic blood pressure was not different between C and D rats. D rats exhibited marked hyperglycaemia (P < 0.001), and increases in GFR (P < 0.001), renal plasma flow, filtration fraction, urinary sodium excretion (UNaV, P < 0.001), filtered load of sodium (FLNa, P < 0.01), and a decrease in fractional reabsorption of sodium (FRNa, P < 0.0001). In contrast, total reabsorption of sodium (TRNa) was increased in D rats compared to C rats (P < 0.001). The urinary excretion of NOx was markedly increased in D rats (P < 0.01). Regression analyses performed in D rats revealed a close relationship between UNaV and GFR and a weaker correlation with urinary NOx. Although FRNa correlated only with urinary excretion of NOx, there was a strong relationship between TRNa and GFR. In contrast to D rats, control rats demonstrated only a relationship between TRNa and GFR and no other correlations were found. In D rats, NO inhibition with L-NAME (1 mg/kg body weight) resulted in a marked decrease in GFR and urinary NOx associated with decreases in FLNa and TRNa but did not influence FRNa. In contrast, in C rats the post-L-NAME decrease in NOx was not associated with significant changes in GFR and renal sodium handling. GTN-treated C rats exhibited a renal vasodilatory response and an increase in natriuresis and urinary NOx whereas no renal changes were observed in D rats during GTN administration. The present data indicate that changes in renal sodium handling before and after NO modulation in experimental diabetes are related to changes in GFR rather than to the renal activity of NO. Therefore, in contrast to the effects on renal haemodynamics, NO does not play an important role in the altered renal sodium handling observed in experimental diabetes.