Terry T. Oyama

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.

Rho kinase inhibition protects kidneys from diabetic nephropathy without reducing blood pressure

Rho-associated kinases (ROCK) are activated in the kidney as well as in cultured cells of diabetic models and have been implicated in renal pathophysiology. To explore whether inhibition of ROCK is protective, we studied its role in a model of accelerated diabetic nephropathy where uninephrectomized rats were made diabetic by streptozotocin. After establishing diabetes, rats were treated with the ROCK inhibitor fasudil continuously or for the final 6 weeks of an 18-week experimental period. The results were compared to similar rats given losartan, an established treatment of clinical and experimental diabetic nephropathy, or a combination of both agents. Vehicle-treated diabetic and non-diabetic uninephrectomized rats served as controls. Diabetes resulted in a rapid development of albuminuria, higher glomerulosclerosis and interstitial fibrosis scores, lower glomerular filtration rates, and increased expression of several molecular markers of diabetic nephropathy. Eighteen weeks of fasudil treatment reduced renal ROCK activity, and ameliorated diabetes-induced structural changes in the kidney and expression of the molecular markers in association with a modest anti-proteinuric effect but no change in blood pressure. Late intervention with fasudil reduced glomerulosclerosis, but did not influence proteinuria. Most effects of fasudil were comparable to those of losartan, although losartan lowered blood pressure and further lowered proteinuria. The combination of both treatments was no different than losartan alone. Thus, ROCK inhibition protected the kidney from diabetic nephropathy even though it did not reduce the blood pressure.

Altered Endothelial Nitric Oxide Synthase Targeting and Conformation and Caveolin-1 Expression in the Diabetic Kidney

Experimental diabetes is associated with complex changes in renal nitric oxide (NO) bioavailability. We explored the effect of diabetes on renal cortical protein expression of endothelial NO synthase (eNOS) with respect to several determinants of its enzymatic function, such as eNOS expression, membrane localization, phosphorylation, and dimerization, in moderately hyperglycemic streptozotocin-induced diabetic rats compared with nondiabetic control rats and diabetic rats with intensive insulin treatment to achieve near-normal metabolic control. We studied renal cortical expression and localization of caveolin-1 (CAV-1), an endogenous modulator of eNOS function. Despite similar whole-cell eNOS expression in all groups, eNOS monomer and dimer in membrane fractions were reduced in moderately hyperglycemic diabetic rats compared with control rats; the opposite trend was apparent in the cytosol. Stimulatory phosphorylation of eNOS (Ser1177) was also reduced in moderately hyperglycemic diabetic rats. eNOS colocalized and interacted with CAV-1 in endothelial cells throughout the renal vascular tree both in control and moderately hyperglycemic diabetic rats. However, the abundance of membrane-localized CAV-1 was decreased in diabetic kidneys. Intensive insulin treatment reversed the effects of diabetes on each of these parameters. In summary, we observed diabetes-mediated alterations in eNOS and CAV-1 expression that are consistent with the view of decreased bioavailability of renal eNOS-derived NO.

CYCLO‐OXYGENASE‐2 INHIBITION ATTENUATES THE PROGRESSION OF NEPHROPATHY IN UNINEPHRECTOMIZED DIABETIC RATS

1 Cyclo‐oxygenase (COX)‐2 is involved in constitutive production of prostanoids in the kidney and plays a role in the control of renal function and morphology. Renal cortical COX‐2 expression and function is increased in experimental models of diabetes (DM). However, pathophysiological roles of this phenomenon in the diabetic kidney have not been fully elucidated. To address this issue, we studied the nephroprotective potential of long‐term (16 weeks) COX‐2 inhibition in uninephrectomized streptozotocin‐diabetic rats (D). 2 Diabetic rats received either a low or high dose of the selective COX‐2 inhibitor MF‐tricyclic (MF; 1 or 5 mg/kg per day in chow). Another group of D rats received high‐dose MF as late intervention starting at 8 weeks of DM (D‐MFlate). The effects of treatments were compared with age‐matched uninephrectomized diabetic and non‐diabetic rats receiving drug‐free chow (D‐VE and C‐VE, respectively). 3 No differences in blood pressure and metabolic control were observed between groups of D rats throughout the study. The D‐VE group developed progressive albuminuria and glomerulosclerosis, associated with increased excretion of the thromboxane (TX) A2 metabolite TxB2. Treatment with MF attenuated albuminuria in diabetic rats with late intervention, but not in D rats treated with MF from the onset of DM. Moreover, D‐MFlate rats demonstrated a significant reduction in the development of glomerulosclerosis. These effects coincided with prevention of diabetes‐induced rise in urinary TxB2 excretion. 4 In conclusion, long‐term COX‐2 inhibition is associated with modest nephroprotection in uninephrectomized diabetic rats when administered as late intervention. These effects are independent of metabolic control and blood pressure.

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.

p21 is decreased in polycystic kidney disease and leads to increased epithelial cell cycle progression: roscovitine augments p21 levels

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease with few treatment options other than renal replacement therapy. p21, a cyclin kinase inhibitor which has pleiotropic effects on the cell cycle, in many cases acts to suppress cell cycle progression and to prevent apoptosis. Because defects in cell cycle arrest and apoptosis of renal tubular epithelial cells occur in PKD, and in light of earlier reports that polycystin-1 upregulates p21 and that the cyclin-dependent kinase inhibitor roscovitine arrests progression in a mouse model, we asked whether (1) p21 deficiency might underlie ADPKD and (2) the mechanism of the salutary roscovitine effect on PKD involves p21.Methodsp21 levels in human and animal tissue samples as well as cell lines were examined by immunoblotting and/or immunohistochemisty. Apoptosis was assessed by PARP cleavage. p21 expression was attenuated in a renal tubular epithelial cell line by antisense methods, and proliferation in response to p21 attenuation and to roscovitine was assessed by the MTT assay.ResultsWe show that p21 is decreased in human as well as a non-transgenic rat model of ADPKD. In addition, hepatocyte growth factor, which induces transition from a cystic to a tubular phenotype, increases p21 levels. Furthermore, attenuation of p21 results in augmentation of cell cycle transit in vitro. Thus, levels of p21 are inversely correlated with renal tubular epithelial cell proliferation. Roscovitine, which has been shown to arrest progression in a murine model of PKD, increases p21 levels and decreases renal tubular epithelial cell proliferation, with no affect on apoptosis.ConclusionThe novelty of our study is the demonstration in vivo in humans and rat models of a decrement of p21 in cystic kidneys as compared to non-cystic kidneys. Validation of a potential pathogenetic model of increased cyst formation due to enhanced epithelial proliferation and apoptosis mediated by p21 suggests a mechanism for the salutary effect of roscovitine in ADPKD and supports further investigation of p21 as a target for future therapy.

Renal p38 MAP kinase activity in experimental diabetes

Renal cell activity of p38 mitogen-activated protein kinase (p38) is increased in the diabetic milieu. p38 mediates signals relevant for the development of diabetic nephropathy (DN). However, renal p38 in Type 1 diabetes in vivo, particularly in conditions reflecting the differences in metabolic control, and its activity in advanced stages of DN, has received less attention. We examined the p38 pathway in renal cortex of rats with streptozotocin diabetes (4 weeks) with poor (DS), moderate (DM), and intensive (DII) metabolic control, achieved by varying doses of insulin therapy. Renal p38 was also studied in 12-month diabetic rats with established nephropathy (DM12) and compared with age-matched controls. p38 activity (in vitro kinase assay and expression of phosphorylated (active) p38 (P-p38)) was increased in DM and DS rats, as compared with non-diabetic controls, and attenuated by intensive insulin treatment. In all groups, P-p38 was predominantly localized in macula densa cells. Diabetic rats also demonstrated P-p38 immunoreactivity in the distal tubule and glomeruli. Enhanced p38 activity in DS and DM rats was not associated with increases in expression of active mitogen-activated protein kinase 3/6, an activator of p38, but paralleled with increased expression of scaffolding protein transforming growth factor-β-activated protein kinase 1-binding protein 1. Expression of mitogen-activated protein phosphatase-1 (MKP-1), one of the phosphatases involved in inactivation of mitogen-activated protein kinase signaling, was increased in all diabetic groups, irrespective of metabolic control. Renal p38 activation was also detectable in D12 rats with established albuminuria and glomerulosclerosis. In summary, renal cortical p38 activity was increased in diabetic rats at early and advanced stages of nephropathy, as compared with non-diabetic animals, and attenuated by improved metabolic control. p38 activation in diabetes is likely to occur via multiple pathways and cannot be explained by downregulation of MKP-1.

Enhanced phosphorylation of Na(+)-Cl- co-transporter in experimental metabolic syndrome: role of insulin.

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na(+)-Cl(-) co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr(53) NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

Interactions of the Kallikrein-Kinin and Renin-Angiotensin Systems in Experimental Diabetes

The kallikrein-kinin system (KKS) has been postulated to play a role in modulation of hemodynamic function in diabetes and to contribute to the hemodynamic effects of angiotensin-converting enzyme inhibition (CEI). To further explore the KKS and its interactions with the renin-angiotensin system (RAS), studies were conducted in nondiabetic control rats and in moderately hyperglycemic diabetic rats. In protocol 1, control and diabetic rats were studied before and after administration of one of two dissimilar B2 kinin receptor antagonists (BK2As), or vehicle. At a low dose (0.5 μg · kg−1 · min−1), the first generation antagonist D-Arg, [Hyp3,Thi5,8,D-Phe7]-bradykinin significantly reduced the glomerular filtration rate (GFR) and renal plasma flow rate in diabetic rats, despite variable effectiveness in blocking the hypotensive response to injected bradykinin. However, a similar hemodynamic effect occurred in nondiabetic rats, suggesting that the observed effect was not specific to diabetes. Higher doses (20 μg bolus, then 1 μg · kg−1 · min−1 infusion) did not affect hemodynamics in either group, perhaps because of partial agonist effect. The second BK2A tested was the newer compound, icatibant (Hoe 140; D-Arg,[Hyp3,Thi5,D-Tic7,Oic8]-bradykinin). Hoe 140 consistently blocked the vasodepressor action of injected bradykinin, but had no effect on systemic or renal hemodynamics in either control or diabetic rats. In protocol 2, control and diabetic rats were pretreated with the CEI ramipril for 1–2 weeks, after which renal function was studied before and after Hoe 140 (0.1 mg s.c. and i.v.) or vehicle. CEI lowered blood pressure in both groups. Hoe 140 did not affect renal function in control rats, but in diabetic rats pretreated with ramipril, it induced a modest but significant decline in GFR. Ramipril induced the predicted changes in the systemic and intrarenal RAS, while acute BK2A had no consistent effect on RAS parameters. These studies suggest that the endogenous KKS has only a minor role in modulation of renal hemodynamics in the euvolemic diabetic rat, in the absence of KKS stimulation by CEI. However, angiotensin-converting enzyme is also kininase II, which serves to increase endogenous kinin activity. The increased kinin activity resulting from CEI treatment may participate, to a modest degree, in hemodynamic regulation of the diabetic kidney.

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.