The Effect of Treatment With Aminoguanidine, an Advanced Glycation End Product Inhibitor, on Streptozotocin-Induced Diabetic Rats and Its Effects on Physiological and Renal Functions.

BACKGROUND/AIM: Diabetes is a multifactorial syndrome that affects the functioning of the renin-angiotensin system (RAS). The role of advanced glycation end products (AGEs) in diabetes is well known. In the present study, we hypothesized that the prevention of AGE accumulation or abrogation of AGE synthesis using an AGE inhibitor, aminoguanidine (AG), in streptozotocin (STZ)-induced diabetic animal models would affect the progression of diabetes and its related complications. We determined the effects of aminoguanidine (AG), an AGE inhibitor, in STZ-induced diabetic rats by determining various indices of RAS and renal functions. Additionally, we also investigated the effect of the drug, AG, on various hemodynamic and physiological functions in the body of the animals. METHODS: Male Sprague Dawley rats weighing 200-250 g were assigned to four groups (n = 4-6): Vehicle, Vehicle+AG, STZ-induced, and STZ-induced+AG rats. Type 1 diabetes was induced by a single intraperitoneal (IP) injection of streptozotocin (55 mg/kg) dissolved in sodium citrate buffer. The control groups (Vehicle) were injected with buffer. The blood glucose levels were measured after 48 hours, and animals with blood glucose levels > 300 mg/dL were included in the study. Blood glucose levels in the vehicle rats were also determined to ensure non-diabetic conditions. After confirmation, AG was administrated at a dose of 1 g/L in drinking water for two weeks. Urine was collected to measure the glomerular filtration rate (GFR), and the immune reactivity for AT1 and AT2 proteins was analyzed by immunoblotting. Data were expressed as mean ± standard error of the mean (SEM), and a p-value < 0.05 was considered statistically significant. RESULTS: Diabetic rats had a significant drop in body weight, accompanied by increased food and water consumption. The diabetic rats exhibited significantly increased urine flow and GFR. These phenotypes were significantly or considerately reversed by AG treatment in the STZ+AG-treated diabetic rats. Aminoguanidine prevented the increase in blood sugar levels compared to STZ-induced diabetic rats alone (295.9 ± 50.69 versus 462.3 ± 18.6 mg/dL (p < 0.05)). However, it did not affect the glomerular filtration rate (GFR) and glomerular damage, as assessed by the renal histopathological studies. The STZ-induced diabetic rats had an increased sodium excretion (3.24 ± 0.40 mmol) and significantly increased expression of the AT2 receptor and that of the AT1 receptor, which was slightly reversed by the treatment with AG. Treatment with AG decreased sodium excretion (2.12 ± 0.63, as compared to the diabetic rats). These rats also had modestly decreased expression of the AT2 receptor (0.99 ± 0.07 versus 1.12 ± 0.08, as compared to the STZ-induced diabetic rats), while the AT1 receptors showed a slight increase in the STZ+AG-treated rats compared to the STZ-induced diabetic rats (1.1 ± 0.19 versus 1.08 ± 0.12). CONCLUSION: This study highlights the action of the drug AG in not exacerbating any damage in diabetic rats. Employing AG as a pharmacological intervention to prevent an increase in blood sugar adds a new dimension to controlling increased blood sugar and preventing diabetic complications. The employability and pharmacological intervention of the drug AG, in diabetes, therefore, need a renewed and further investigation.

Biochemical markers in the pathogenesis of preeclampsia: novel link between placental growth factor and interleukin-6.

Preeclampsia (PE) is a multisystem disorder of pregnancy characterized by sudden onset of hypertension and proteinuria. The appearance and diagnosis of the disease remain elusive and the only treatment is the termination of pregnancy. The onset of the disease is preceded by a shift in the balance of the angiogenic and antiangiogenic proteins in the maternal circulation. We surmised that the assessment of the levels of these proteins during pregnancy could lead to a proper diagnosis of the disease. In this study, we determined the levels of angiogenic and antiangiogenic proteins and IL-6 in maternal circulation during normotensive and hypertensive pregnancy, including PE. Serum isolated from pregnant women during antenatal visits was used to determine the concentrations of these proteins. The levels of antiangiogenic proteins, sFlt-1 and sEng, were higher in hypertensive disorders [gestational hypertension (GH), mild PE, and PE] of pregnancy and were significantly higher for PE than for GH. The levels of sFlt-1 and sEng were higher in PE samples compared to those in GH and NT samples. These proteins may have contributed to increased blood pressure. The levels of PlGF were decreased in pregnant women having GH, mild PE, and PE. The levels of the inflammatory intermediate, IL-6, were increased in PE samples compared to those in the GH and normotensive samples. The evaluation of the altered levels of antiangiogenic and angiogenic proteins can be useful for diagnosis of PE.

Neurokinin 3 receptor and phosphocholine transferase: missing factors for pathogenesis of C-reactive protein in preeclampsia.

C-reactive protein (CRP), an innate immune mediator, is elevated in the circulation before symptoms in patients with preeclampsia, a severe hypertensive pregnancy disorder with high mortality and morbidity. However, the specific sources underlying increased CRP and the role of elevated CRP in preeclampsia are undefined. Here, we report that circulating CRP levels are significantly increased in a large cohort of normotensive pregnant individuals when compared with nulligravid women and is further increased in patients with preeclampsia. These findings led us to discover further that placental syncytiotrophoblasts are previously unrecognized cellular sources of CRP and underlie elevated CRP in normotensive pregnant women and the additional increase in patients with preeclampsia. Next, we demonstrated that injection of CRP induces preeclampsia features, including hypertension (157 mm Hg CRP treated versus 119 mm Hg control), proteinuria (35.0 mg/µg CRP treated versus 14.1 mg/µg control), kidney, and placental damage and increased levels of sFlt-1 in pregnant mice but not in nonpregnant mice. Our study implicates that phosphocholine transferase, a placental-specific enzyme post-translationally modifying neurokinin B, is essential for the pathogenic role of CRP in preeclampsia through activation of the neurokinin 3 receptor. Overall, our studies have provided significant new insight on the pathogenic role of CRP in preeclampsia and highlighted innovative therapeutic strategies.

Angiotensin receptor agonistic autoantibody-mediated soluble fms-like tyrosine kinase-1 induction contributes to impaired adrenal vasculature and decreased aldosterone production in preeclampsia.

Preeclampsia (PE) is a life-threatening hypertensive disorder during pregnancy associated with decreased circulating aldosterone levels. However, the molecular mechanisms underlying aldosterone reduction in PE remain unidentified. Here we demonstrate that reduced circulating aldosterone levels in preeclamptic women are associated with the presence of angiotensin II type 1 receptor agonistic autoantibody and elevated soluble Fms-like tyrosine kinase-1, 2 prominent pathogenic factors in PE. Using an adoptive transfer animal model of PE, we provide in vivo evidence that the injection of IgG from women with PE, but not IgG from normotensive individuals, resulted in hypertension, proteinuria, and a reduction in aldosterone production from 1377 ± 272 pg/mL to 544 ± 92 pg/mL (P<0.05) in pregnant mice. These features were prevented by coinjection with an epitope peptide that blocks antibody-mediated angiotensin type 1 receptor activation. In contrast, injection of IgG from preeclamptic women into nonpregnant mice induced aldosterone levels from 213 ± 24 pg/mL to 615 ± 48 pg/mL (P<0.05). These results indicate that maternal circulating autoantibody in preeclamptic women is a detrimental factor causing decreased aldosterone production via angiotensin type 1 receptor activation in a pregnancy-dependent manner. Next, we found that circulating soluble Fms-like tyrosine kinase-1 was only induced in autoantibody-injected pregnant mice but not nonpregnant mice. As such, we further observed vascular impairment in adrenal glands of pregnant mice. Finally, we demonstrated that infusion of vascular endothelial growth factor(121) attenuated autoantibody-induced adrenal gland vascular impairment resulting in a recovery in circulating aldosterone (from 544 ± 92 to 1110 ± 269 pg/mL; P<0.05). Overall, we revealed that angiotensin II type 1 receptor agonistic autoantibody-induced soluble Fms-like tyrosine kinase-1 elevation is a novel pathogenic mechanism underlying decreased aldosterone production in PE.

Recombinant vascular endothelial growth factor 121 attenuates autoantibody-induced features of pre-eclampsia in pregnant mice.

BACKGROUND: Pre-eclampsia (PE) is a serious hypertensive disorder of pregnancy characterized by excessive production of a soluble form of the vascular endothelial growth factor (VEGF) receptor-1, termed soluble fms-like tyrosine kinase-1 (sFlt-1). This placental-derived factor is believed to be a key contributor to the clinical features of PE. Women with PE are also characterized by the presence of autoantibodies, termed angiotensin type 1 receptor activating autoantibody (AT(1)-AA), that activate the major angiotensin receptor, AT(1). These autoantibodies cause clinical features of PE and elevated sFlt-1 when injected into pregnant mice. The research reported here used this autoantibody-injection model of PE to assess the therapeutic potential of recombinant VEGF(121), a relatively stable form of the natural ligand. METHODS: Immunoglobulin G (IgG) from women with PE was injected into pregnant mice with or without continuous infusion of recombinant VEGF(121). Injected mice were monitored for symptoms of PE. RESULTS: As a result of infusion of recombinant VEGF(121) autoantibody-induced hypertension (systolic blood pressure) was reduced from 159 ± 5 to 124 ± 5 mm Hg, proteinuria from 111 ± 16 to 40 ± 5 mg protein/mg creatinine and blood urea nitrogen levels from 31 ± 1 mg/dl to 18 ± 2 mg/dl, P < 0.05. Histological analysis revealed that autoantibody-induced glomerular damage including the narrowing of Bowman's space and occlusion of capillary loop spaces was largely prevented by VEGF(121) infusion. Finally, impaired placental angiogenesis resulting from AT(1)-AA injection was significantly improved by VEGF(121) infusion. CONCLUSIONS: The infusion of recombinant VEGF(121) significantly attenuated autoantibody-induced features of PE.

Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity.

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT(1)-AA). However, the prevalence of AT(1)-AA in PE remains unknown, and the correlation of AT(1)-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT(1)-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT(1)-AA is highly prevalent in PE ( approximately 95%). Next, by comparing the levels of AT(1)-AA among women with mild and severe PE, we found that the titer of AT(1)-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT(1)-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT(1)-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT(1)-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

Protective role of angiotensin II subtype 2 receptor in blood pressure increase in obese Zucker rats.

Earlier, we reported that there was an increase in angiotensin II type 2 (AT(2)) receptor expression in the renal proximal tubule, and selective activation of the AT(2) receptor by AT(2) agonist inhibits Na,K-ATPase activity in the proximal tubules and increases urinary Na excretion in obese Zucker rats. We hypothesized that the AT(2) receptor has a protective role against blood pressure increase in obese Zucker rats. To test this hypothesis, we treated obese Zucker rats with the AT(2) receptor antagonist PD123319 (PD; 30 microg/kg per minute) using osmotic pumps. Age-matched lean rats and vehicle-treated obese Zucker rats served as controls. On day 15 of the treatment with PD, arterial blood pressure was measured by cannulation of the left carotid artery under anesthesia. Control obese rats exhibited higher mean arterial pressure (122.0+/-3.4 mm Hg) compared with lean control rats (97.0+/-4.8 mm Hg). The PD treatment of obese rats raised mean arterial pressure further by 13 mm Hg. The plasma renin activity was significantly increased in the PD-treated obese compared with control-obese or lean rats. Western blot analysis revealed that the PD treatment in obese rats caused an approximately 3-fold increase in the renin expression in the kidney cortex but had no effect on the expression of the cortical angiotensin II type 1 and AT(2) receptors. The present study suggests that the renal AT(2) receptors provide a protective role against blood pressure increase in obese Zucker rats, and this protective effect, in part, could be because of the ability of the AT(2) receptors to keep the kidney renin expression low in obese rats.

Impaired angiotensin II AT(1) receptor function and enhanced Na, K-ATPase affinity for sodium in proximal tubule of streptozotocin-treated diabetic rats.

We determined angiotensin II (Ang II) AT(1) receptor function in terms of Na-K-ATPAse (NKA) stimulation in the proximal tubule (PTs) of streptozotocin-induced diabetic rats. Ang II (10 pM) stimulated NKA activity in PTs of control rats but not diabetic rats. The AT(1) receptor expression was similar, but the expression of G-proteins (G(i)alpha2 and G(i)alpha3) in the PTs was decreased in diabetic compared with control rats. Kinetic studies revealed an increase in NKA affinity, low K(0.5,) for Na, with no changes in V(max) of the enzyme in diabetic compared with control rats. Basal Ser-phosphorylation of NKA alpha1-subunit was lower in diabetic compared with control rats. This data suggest that the higher basal NKA affinity for Na, possibly due to lower Ser-phosphorylaion of alpha1-subunit and not the AT(1) receptor function, in the PTs may be responsible for increased renal Na reabsorption associated with early stage of streptozotocin-induced diabetes.

Enhanced AT1 receptor-mediated vasocontractile response to ANG II in endothelium-denuded aorta of obese Zucker rats.

In the present study, we tested the hypothesis that ANG II causes a greater vasoconstriction in obese Zucker rats, a model of type 2 diabetes, with mild hypertension. Measurement of isometric tension in isolated aortic rings with intact endothelium revealed a modest but not significantly greater ANG II-induced contraction in obese than lean rats. Removal of endothelium or inhibition of nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester (L-NAME) enhanced 1) ANG II-induced contraction in both lean and obese rats, being significantly greater in obese rats (E(max) g/g tissue, denuded: lean 572 +/- 40 vs. obese 664 +/- 16; L-NAME: lean 535 +/- 14 vs. obese 818 +/- 23) and 2) ANG II sensitivity in obese compared with lean rats, as revealed by the pD(2) values. Endothelin-1 and KCl elicited similar contractions in the aortic rings of lean and obese rats. ACh, a NO-dependent relaxing hormone, produced greater relaxation in the aortic rings of obese than lean rats, whereas sodium nitroprusside, an NO donor, elicited similar relaxations in both rat strains. The expression of the ANG type 1 (AT(1)) receptor protein and mRNA in the endothelium-intact aorta was significantly greater in obese than lean rats, whereas the endothelium-denuded rings expressed modest but not significantly greater levels of AT(1) receptors in obese than lean rats. The endothelial NO synthase protein and mRNA expression levels were higher in the aorta of obese than lean animals. We conclude that, although ANG II produces greater vasoconstriction in obese rat aortic rings, enhanced endothelial AT(1) receptor-mediated NO production appears to counteract the increased ANG II-induced vasoconstriction, suggesting that arterial AT(1) receptor may not be a contributing factor to hypertension in this model of obesity.

Renal angiotensin II AT2 receptors promote natriuresis in streptozotocin-induced diabetic rats.

Angiotensin II AT2 receptors have been implicated to play a role in the regulation of renal/cardiovascular functions under pathological conditions. The present study is designed to investigate the function of the AT2 receptors on renal sodium excretion and AT(2) receptor expression in the cortical membranes of streptozotocin (STZ)-induced diabetic rats. The STZ treatment led to a significant weight loss, hyperglycemia, and decrease in plasma insulin levels compared with control rats. STZ-induced diabetic rats had significantly elevated basal urine flow, urinary sodium excretion rate (U(Na)V), urinary fractional sodium excretion, and urinary cGMP compared with control rats. Infusion of PD-123319, an AT2 receptor antagonist, caused a significant decrease in U(Na)V (mumol/min) in STZ-induced diabetic rats (1 +/- 0.09 vs. 0.45 +/- 0.1) but not in control rats (0.35 +/- 0.05 vs. 0.4 +/- 0.07). The decrease in U(Na)V was associated with a significant decrease in urinary cGMP levels (pmol/min) in STZ-induced diabetic rats (21 +/- 2 vs. 10 +/- 0.8) but not in control rats (11.75 +/- 3 vs. 12.6 +/- 2). The infusion of PD-123319 did not alter glomerular filtration rate (STZ: 0.3 +/- 0.02 vs. 0.25 +/- 0.03; control: 1.4 +/- 0.05 vs. 1.5 +/- 0.09 ml/min) or mean arterial pressure (STZ: 82 +/- 3 vs. 79 +/- 3.5; control: 90 +/- 4 vs. 89 +/- 4 mmHg), suggesting a tubular effect of the drug. Western blot analysis using an AT2 receptor antibody revealed a significantly enhanced expression of the AT2 receptor protein ( approximately 45 kDa) in brush-border ( approximately 50-fold) and basolateral membranes ( approximately 80-fold) of STZ-induced diabetic compared with control rats. In conclusion, our data suggest that the tubular AT2 receptors in diabetic rats are profoundly enhanced and possibly via a cGMP pathway promote sodium excretion in this model of diabetes.

Insulin treatment enhances AT1 receptor function in OK cells.

Increased renal sodium retention is considered a major risk factor contributing to hypertension associated with chronic hyperinsulinemia and obesity. However, the molecular mechanism involved is not understood. The present study investigates the effect of insulin treatment on AT1 receptor expression and ANG II-induced stimulation of Na/H exchanger (NHE) and Na-K-ATPase (NKA) in opossum kidney (OK) cells, a proximal tubule cell line. The presence of the AT1 receptors in OK cells was confirmed by the specific binding of 125I-sar-ANG II and by detecting approximately 43-kDa protein on Western blot analysis with AT1 receptor antibody and blocking peptide as well as by expression of AT1 receptor mRNA as determined by RT-PCR. Insulin treatment (100 nM for 24 h) caused an increase in 125I-sar-ANG II binding, AT1 receptor protein content, and mRNA levels. The whole cell lysate and membrane showed similar insulin-induced increase in the AT1 receptor protein expression, which was blocked by genistein (100 nM), a tyrosine kinase inhibitor, and cycloheximide (1.5 microg/ml), a protein synthesis inhibitor. Determination of ethyl isopropyl amiloride-sensitive 22Na+ uptake, a measure of the NHE activity, revealed that ANG II (1-100 pM)-induced stimulation of NHE in insulin-treated cells was significantly greater than in the control cells. Similarly, ANG II (1-100 pM)-induced stimulation of ouabain-sensitive 86Rb+ uptake, a measure of NKA activity in insulin-treated cells, was significantly greater than in the control cells. ANG II stimulation of both the transporters was blocked by AT1 receptor antagonist losartan, suggesting the involvement of AT1 receptors. Thus chronic insulin treatment causes upregulation of AT1 receptors, which evoked ANG II-induced stimulation of NHE and NKA. We propose that insulin-induced increase in the renal AT1 receptor function serves as a mechanism responsible for the increased renal sodium reabsorption and thus may contribute to development of hypertension in conditions associated with hyperinsulinemia.