Shali Zhang

Inhibition or Deletion of Soluble Epoxide Hydrolase Prevents Hyperglycemia, Promotes Insulin Secretion, and Reduces Islet Apoptosis

Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of endogenous inflammatory and antiapoptotic mediators. However, the roles of sEH in diabetes and the pancreas are unknown. Our aims were to determine whether sEH is involved in the regulation of hyperglycemia in diabetic mice and to investigate the reasons for the regulation of insulin secretion by sEH deletion or inhibition in islets. We used two separate approaches, targeted disruption of Ephx2 gene [sEH knockout (KO)] and a selective inhibitor of sEH [trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB)], to assess the role of sEH in glucose and insulin homeostasis in streptozotocin (STZ) mice. We also examined the effects of sEH KO or t-AUCB on glucose-stimulated insulin secretion (GSIS) and intracellular calcium levels in islets. Hyperglycemia in STZ mice was prevented by both sEH KO and t-AUCB. In addition, STZ mice with sEH KO had improved glucose tolerance. More important, when insulin levels were assessed by hyperglycemic clamp study, sEH KO was found to promote insulin secretion. In addition, sEH KO and t-AUCB treatment augmented islet GSIS. Islets with sEH KO had a greater intracellular calcium influx when challenged with high glucose or KCl in the presence of diazoxide. Moreover, sEH KO reduced islet cell apoptosis in STZ mice. These results show not only that sEH KO and its inhibition prevent hyperglycemia in diabetes, but also that sEH KO enhances islet GSIS through the amplifying pathway and decreases islet cell apoptosis in diabetes.

Impaired Ca2+ Signaling Attenuates P2X Receptor–Mediated Vasoconstriction of Afferent Arterioles in Angiotensin II Hypertension

This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca2+ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X1 agonist &bgr;,&ggr;–methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca2+ concentration ([Ca2+]i) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and &bgr;,&ggr;–methylene ATP (P<0.05). During exposure to ATP (1, 10, and 100 &mgr;mol/L), afferent diameter declined by 17±2%, 29±3%, and 30±2% in normal control rats and 8±3%, 7±3%, and 22±3% in kidneys of Ang II–infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or &bgr;,&ggr;–methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca2+]i averaged 107±21, 170±38, and 478±79 nmol/L at ATP concentrations of 1, 10, and 100 &mgr;mol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 &mgr;mol/L) to 65±13, 102±20, and 367±73 nmol/L, respectively. The peak increases in [Ca2+]i in response to &bgr;,&ggr;–methylene ATP were also reduced in Ang II hypertensive rats. However, angiotensin hypertension did not change the UTP-mediated vasoconstrictor responses or the myocyte calcium responses to UTP. These results indicate that the impaired autoregulatory response observed in Ang II–dependent hypertension can be attributed to impairment of P2X1 receptor–mediated signal transduction.

P2X1 receptor-mediated vasoconstriction of afferent arterioles in angiotensin II-infused hypertensive rats fed a high-salt diet.

Experiments tested the hypothesis that P2 receptor reactivity is impaired in angiotensin (Ang) II hypertensive rats fed an 8%NaCl diet (Ang II+HS). Juxtamedullary afferent arteriolar autoregulatory behavior was determined over a pressure range of 65 to 200 mm Hg. Arteriolar responsiveness to P2X1 (&bgr;,&ggr;-methylene ATP) or P2Y2 receptor (uridine triphosphate) activation was determined in vitro. Systolic blood pressure averaged 126±3 and 225±4 mm Hg in control and Ang II+HS rats, respectively (P<0.05). In control kidneys, &bgr;,&ggr;-methylene ATP (10−8 to 10−4 mol/L) reduced arteriolar diameter by 8±3%, 13±5%, 19±5%, 22±6%, and 24±9%, respectively, whereas uridine triphosphate reduced diameter by 2±1%, 2±2%, 9±3%, 37±7%, and 58±7%. Autoregulation was markedly blunted in Ang II+HS kidneys, with arteriolar diameter remaining essentially unchanged when perfusion pressure increased to 200 mm Hg compared with a 40±2% decline in diameter observed in normal kidneys over the same pressure range (P<0.05). P2X1 receptor–mediated vasoconstriction was significantly attenuated in Ang II+HS kidneys. &bgr;,&ggr;-Methylene ATP reduced arteriolar diameter by 1±1%, 3±2%, 6±1%, 9±3%, and 7±1%, respectively (P<0.05), versus control rats. Similar patterns were noted when hypertensive perfusion pressures were used. Uridine triphosphate–mediated responses were unchanged in Ang II+HS rats compared with control, indicating preservation of P2Y2 receptor function. Ang II+HS blunted P2X1-mediated increases in intracellular Ca2+ concentration in preglomerular smooth muscle cells. Therefore, Ang II+HS rats exhibit attenuated afferent arteriolar responses to P2X1 receptor stimulation. These data support the hypothesis that P2X1 receptors are important for pressure-mediated autoregulatory responses. Impairment of P2X1 receptor function may explain the hypertension-induced decline in renal autoregulatory capability.

High-salt diet blunts renal autoregulation by a reactive oxygen species-dependent mechanism

High dietary salt is common in Western countries and is an important contributor to increased cardiovascular disease. Autoregulation of renal blood flow (RBF) and glomerular filtration rate (GFR) is an essential function of the renal microcirculation that could be affected by excessive dietary salt. High salt (HS) increases renal ROS generation partly by the enzyme NADPH oxidase. We hypothesized that a HS diet would impair autoregulation via NADPH oxidase-dependent ROS generation. The role of NADPH-dependent ROS production on the blunted autoregulatory response with a HS diet was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. The increase in renal lipid peroxidation and p67(phox) expression induced by HS was prevented by apocynin treatment. Control afferent arterioles exhibited normal autoregulatory behavior in response to acute increases in renal perfusion pressure, whereas arterioles from HS rats exhibited a blunted response. Autoregulatory behavior in HS rats was restored in vitro by acute exposure to the NADPH oxidase inhibitor apocynin. At the whole kidney level, in vivo experiments showed that both RBF and GFR declined in HS rats when left kidney renal perfusion pressure was reduced from ambient to 95 mmHg, whereas control rats maintained stable GFR and RBF consistent with efficient autoregulatory behavior. Apocynin treatment improved in vivo autoregulatory behavior in HS rats and had no detectable effect in normal salt diet-fed rats. These data support the hypothesis that impaired renal autoregulatory behavior in rats fed a HS diet is mediated by NADPH oxidase-derived ROS.

Immunosuppression preserves renal autoregulatory function and microvascular P2X1 receptor reactivity in ANG II-hypertensive rats

Autoregulation is critical for protecting the kidney against arterial pressure elevation and is compromised in some forms of hypertension. Evidence indicates that activated lymphocytes contribute importantly to cardiovascular injury in hypertension. We hypothesized that activated lymphocytes contribute to renal vascular dysfunction by impairing autoregulation and P2X(1) receptor signaling in ANG II-infused hypertensive rats. Male Sprague-Dawley rats receiving ANG II infusion were treated with a lymphocyte proliferation inhibitor, mycophenolate mofetil (MMF) for 2 wk. Autoregulation was assessed in vitro and in vivo using the blood-perfused juxtamedullary nephron preparation and anesthetized rats, respectively. ANG II-treated rats exhibited impaired autoregulation. At the single vessel level, pressure-mediated afferent arteriolar vasoconstriction was significantly blunted (P < 0.05 vs. control rats). At the whole kidney level, renal blood flow passively decreased as renal perfusion pressure was reduced. MMF treatment did not alter the ANG II-induced hypertensive state; however, MMF did preserve autoregulation. The autoregulatory profiles in both in vitro or in vivo settings were similar to the responses from control rats despite persistent hypertension. Autoregulatory responses are linked to P2X(1) receptor activation. Accordingly, afferent arteriolar responses to ATP and the P2X(1) receptor agonist β,γ-methylene ATP were assessed. ATP- or β,γ-methylene ATP-induced vasoconstriction was significantly attenuated in ANG II-infused hypertensive rats but was normalized by MMF treatment. Moreover, MMF prevented elevation of plasma transforming growth factor-β1 concentration and lymphocyte and macrophage infiltration in ANG II-infused kidneys. These results suggest that anti-inflammatory treatment with MMF prevents lymphocyte infiltration and preserves autoregulation in ANG II-infused hypertensive rats, likely by normalizing P2X(1) receptor activation.

Clopidogrel, independent of the vascular P2Y12 receptor, improves arterial function in small mesenteric arteries from AngII-hypertensive rats.

The P2Y12 receptor antagonist clopidogrel blocks platelet aggregation, improves systemic endothelial nitric oxide bioavailability, and has anti-inflammatory effects. Since P2Y12 receptors have been identified in the vasculature, we hypothesized that clopidogrel ameliorates angiotensin II (Ang II) -induced vascular functional changes by blockade of P2Y12 receptors in the vasculature. Male Sprague Dawley rats were infused with Ang II (60 ng.min-1) or vehicle for 14 days. The animals were treated with clopidogrel (10mg*kg-1*day-1) or vehicle. Vascular reactivity was evaluated in second-order mesenteric arteries. Clopidogrel treatment did not change systolic blood pressure [(mmHg) control-vehicle, 117+/-7.1 vs. control- Clopidogrel, 125+/-4.2; AngII-vehicle, 197+/-10.7 vs. AngII-Clopidogrel, 198+/-5.2], but it normalized increased phenylephrine-induced vascular contractions [(%KCl) vehicle-treated, 182.2+/-18 vs. Clopidogrel, 133+/-14%), as well as impaired vasodilation to acetylcholine [(%) vehicle-treated, 71.7+/-2.2 vs. Clopidogrel, 85.3+/-2.8) in Ang II-treated animals. Vascular expression of P2Y12 receptor was determined by western blot. Pharmacological characterization of vascular P2Y12 was performed with the P2Y12 agonist 2-MeS-ADP. Although 2-MeSADP induced endothelium-dependent relaxation [(Emax %) = 71%+/-12), as well as contractile vascular responses (Emax %= 83+/-12) these actions are not mediated by P2Y12 receptor activation. 2-MeS-ADP produced similar vascular responses in control and Ang II rats. These results indicate potential effects of Clopidogrel, such as improvement of hypertension-related vascular functional changes that are not associated with direct actions of clopidogrel in the vasculature, supporting the concept that activated platelets contribute to endothelial dysfunction, possibly via impaired NO bioavailability.

Clopidogrel preserves whole kidney autoregulatory behavior in ANG II-induced hypertension.

This study tested the hypothesis that P2Y12 receptor blockade with clopidogrel preserves renal autoregulatory ability during ANG II-induced hypertension. Clopidogrel was administered orally to male Sprague-Dawley rats chronically infused with ANG II. After 14 days of treatment, whole kidney autoregulation of renal blood flow was assessed in vivo in pentobarbital-anesthetized rats using an ultrasonic flow probe placed around the left renal artery. In ANG II-vehicle-treated rats, decreasing arterial pressure over a range from 160 to 100 mmHg resulted in a 25 ± 5% decrease in renal blood flow, demonstrating a significant loss of autoregulation with an autoregulatory index of 0.66 ± 0.15. However, clopidogrel treatment preserved autoregulatory behavior in ANG II-treated rats to levels indistinguishable from normotensive sham-operated (sham) rats (autoregulatory index: 0.04 ± 0.14). Compared with normotensive sham-vehicle-treated rats, ANG II infusion increased renal CD3-positive T cell infiltration by 66 ± 6%, induced significant thickening of the preglomerular vessels and glomerular basement membrane and increased glomerular collagen I deposition, tubulointerstitial fibrosis, damage to the proximal tubular brush border, and protein excretion. Clopidogrel significantly reduced renal infiltration of T cells by 39 ± 9% and prevented interstitial artery thickening, ANG II-induced damage to the glomerular basement membrane, deposition of collagen type I, and tubulointerstitial fibrosis, despite the maintenance of hypertension. These data demonstrate that systemic P2Y12 receptor blockade with clopidogrel protects against impairment of autoregulatory behavior and renal vascular injury in ANG II-induced hypertension, possibly by reducing renal T cell infiltration.