Renal expression of arachidonic acid metabolizing ... - Springer Link

Mol Cell Biochem (2010) 333:203–209 DOI 10.1007/s11010-009-0220-4

Renal expression of arachidonic acid metabolizing enzymes and RhoA/Rho kinases in fructose insulin resistant hypertensive rats Sally Mustafa Æ Harish Vasudevan Æ Violet G. Yuen Æ John H. McNeill

Received: 25 March 2009 / Accepted: 9 July 2009 / Published online: 25 July 2009 Ó Springer Science+Business Media, LLC. 2009

Abstract Fructose feeding has been shown to induce insulin resistance and hypertension. Renal protein expression for the cytochrome P (CYP) 450 arachidonic acid metabolizing enzymes has been shown to be altered in other models of diet-induced hypertension. Of special interest is CYP4A, which produces the potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid and CYP2C, which catalyzes the formation of the potent dilators epoxyeicosatrienoic acids as well as soluble epoxide hydrolase (sEH) which metabolizes the latter to dihydroxyeicosatrienoic acids. The RhoA/Rho kinase (ROCK) signaling pathway is downstream of arachidonic acid and is reported to mediate metabolic-cardio-renal dysfunctions in some experimental models of insulin resistance and diabetes. The aim of the present study was to determine the expression of CYP4A, CYP2C23, CYP2C11, sEH, RhoA, ROCK-1, ROCK-2, and phospho-Lin-11/Isl-1/Mec-3 kinase (LIMK) in kidneys of fructose-fed (F) rats. Male Wistar rats were fed a high fructose diet for 8 weeks. Body weight, systolic blood pressure, insulin sensitivity, and renal expression of the aforementioned proteins were assessed. No change was observed in the body weight of F rats; however, euglycemia and hyperinsulinemia implicating impaired glucose tolerance and significant elevation in systolic blood pressure were observed. Renal expression of CYP4A and CYP2C23 was significantly increased while that of CYP2C11 and sEH was not changed in F rats. Equal expression for RhoA in both control and F rats and an enhanced level of ROCK-1 and

S. Mustafa H. Vasudevan V. G. Yuen J. H. McNeill (&) Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, BC V6T 1Z3, Canada e-mail: [email protected]

ROCK-2 constitutively activate 130 kDa cleavage fragments as well as phospho-LIMK. These data suggest that the kidneys could be actively participating in the pathogenesis of insulin resistance-induced hypertension through the arachidonic acid CYP 450-RhoA/Rho kinase pathway(s). Keywords Insulin Blood pressure Cytochrome P450 Rho kinase Kidney

Introduction Type 2 diabetes is occurring at alarmingly increasing rates and it is considered to be an epidemic of the 21st century [1]. It is a complex disorder associated with a collection of abnormalities including obesity, dyslipidemia, impaired glucose tolerance, insulin resistance, and hypertension, collectively referred to as the metabolic syndrome [2]. Insulin resistance and hypertension represent two coexisting hallmarks of the metabolic syndrome [3]. Rats fed a high fructose diet resemble this syndrome with a decrease in insulin sensitivity and an increase in blood pressure [4]. The molecular/biochemical mechanism(s) underlying the contribution of insulin resistance to the pathogenesis of hypertension have been the focus of intense research; however, they are not yet fully elucidated. Arachidonic acid and its cytochrome P (CYP) 450 metabolites as well as the RhoA/Rho kinase (ROCK) signaling pathway are among the potential candidates assumed to play a role in fructoseinduced hypertension. The kidneys have an established role in long term regulation of blood pressure. Fructose-induced metabolic syndrome was found to produce considerable disturbances in renal hemodynamics such as glomerular hypertension and cortical vasoconstriction [5]. The arachidonic acid CYP 450 monooxygenase metabolites,

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20-hydroxyeicosatetraenoic acid (20-HETE), and epoxyeicosatrienoic acids (EETs) are the major arachidonic acid products of CYP 450 metabolism in the kidney and they play major roles in both renal and vascular functions [6]. 20-HETE is produced by the CYP4A x-hydroxylases, CYP4A1, CYP4A2, CYP4A3, and CYP4A8 [7] while EETs are produced by the CYP2C and CYP2J epoxygenases. The three major CYP2C isoforms expressed in the rat kidney are the CYP2C23, CYP2C24, and CYP2C11 with CYP2C23 being the predominant one [8, 9]. EETs are metabolized to their corresponding dihydroxyderivatives, the dihydroxyeicosatrienoic acids (DHETs) by the enzyme soluble epoxide hydrolase (sEH) [10]. Regulation of the renal CYP 450 pathway appears to occur in the metabolic syndrome. In male rats fed a high fat diet, a model of obesity-induced hypertension, blood pressure was elevated, eicosanoid production was decreased and the expression of CYP4A and CYP2C23 in the proximal tubules was downregulated [11]. Transient receptor potassium channels, the TRPC6 channels, a downstream effector of EETs, were also downregulated by high glucose in human glomerular mesangial cells [12]. In addition to the renal CYP 450 pathway, mesenteric arteries of obese Zucker rats, which represent a genetic model of the metabolic syndrome, showed decreased mRNA and protein expressions of the CYP epoxygenases CYP2C11 and CYP2J as well as increased mRNA and protein expressions of sEH [13]. A close association between the sEH gene G860A (Arg287Gln) polymorphism and insulin resistance in type 2 diabetic patients has been found suggesting the involvement of EETs as well as their hydroxylation products, the DHETs, in the pathogenesis of insulin resistance found in type 2 diabetes [14]. RhoA is a small GTPase involved in a number of cell functions cycling between an active GTP-bound and an inactive GDP-bound states [15]. The downstream effector of RhoA, Rho kinase called ROCK, is a serine/threonine kinase that has two isoforms in mammals namely, ROCK-1 and ROCK-2 [16]. In obese Zucker rats, the ROCK pathway showed an enhanced role in alpha1 adrenergic vasoconstriction [17]. RhoA/Rho kinase was activated in skeletal muscles and aortic tissues of Zucker obese rats and was responsible for both the insulin resistance and vascular dysfunctions. Rho kinase inhibition reduced blood pressure, corrected glucose and lipid metabolism, and improved serine phosphorylation of insulin receptor substrate-1 and insulin signaling in skeletal muscles [18]. RhoA is highly expressed in renal cortex and its translocation from cytosol to membrane and hence activation was increased in the renal cortex of streptozotocin-diabetic rats which suggests its involvement in diabetic renal injury [19]. In db/db mice, increased ROCK activity was observed in the kidney cortex. RhoA/ROCK inhibition improved the symptoms of diabetic nephropathy implying the active

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participation of the RhoA/ROCK pathway in the pathogenesis of diabetic nephropathy and proposing RhoA/ ROCK blockade to be an attractive approach toward managing diabetic nephropathy [20]. Cross talk between arachidonic acid and its metabolites and the Rho/Rho kinase pathway exists. Rho kinase was shown to mediate the release of arachidonic acid in lung epithelial cells [21] and arachidonic acid was shown to directly activate Rho kinase 5 to 6 fold independent of Rho A [22]. Moreover, 20-HETE-induced contraction in small porcine coronary arteries was mediated through the sensitization of the contractile apparatus to Ca2? by activating Rho kinase [23]. In the fructose hypertensive rat there is evidence that arachidonic acid metabolites of the cyclooxygenase-2 pathway are involved in the vascular changes reported [24]. The kidney is known to produce and release all arachidonic acid metabolites. The purpose of this study was to determine what effect fructose feeding has on the CYP 450 arm of the arachidonic acid cascade and its potential downstream targets, RhoA/ROCK, and to determine if the kidney is an active organ involved in fructose-induced hypertension. This study provides novel findings that renal CYP 450 enzymes as well as Rho kinases are upregulated by high fructose diet treatment in rats.

Materials and methods Animals and experimental design Twenty (20), 6 weeks old, male Wistar rats were obtained from Charles River Laboratories, Montreal, Canada. Rats were cared for in accordance with the guidelines set out by the Canadian Council on Animal Care. All protocols were approved by the University of British Columbia Animal Care Ethics Committee. The rats were randomly divided into two experimental groups: control (C, n = 10) and fructose-fed (F, n = 10). Blood was collected for determination of basal fasted (5 h) plasma glucose and insulin levels prior to the start of fructose feeding. Basal systolic blood pressure was also measured in all animals. For a total of 8 weeks, control rats were maintained on standard laboratory rat chow containing 30% carbohydrate in the form of starch while rats in the F group were started on a 60% fructose diet (Teklad Laboratory Diets, Madison, WI). This fructose-enriched diet has been previously shown to induce insulin resistance and hypertension [4, 24]. Rats were housed on a 12-h light–dark cycle and received food and water ad libitum. At the end of study week 7, systolic blood pressure and insulin sensitivity were determined in both groups. At week 8, rats were weighed and terminated with an intraperitoneal injection of pentobarbital (SomnotolTM;


100 mg/kg). Kidneys were isolated, cleaned, snap frozen in liquid nitrogen, and stored at -80°C until assayed. Blood pressure measurement Systolic blood pressure was measured in conscious rats by using the indirect noninvasive tail-cuff method using the model 179 semiautomatic BP analyzer, IITC Inc. as previously described [4, 25]. Insulin sensitivity Previous studies in our laboratory [26] have shown that following 7 weeks of fructose feeding, animals are euglycemic, hyperinsulinemic, and insulin resistant. In order to confirm a decrease in insulin sensitivity, animals were fasted overnight (15 h), and blood was collected at 0 and 90 min following a 1 g/kg glucose challenge. Blood was centrifuged at 10,0009g for 25 min, plasma was collected and stored at -20°C until analyzed for glucose and insulin levels. Biochemical measurements Plasma glucose levels were determined using a Beckman Glucose Analyzer II (Beckman, Fullerton, CA). Plasma insulin levels were determined using a radioimmunoassay kit from Linco Research (St. Charles, MO).

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Signaling Technology Inc., MA, USA). Membranes were then washed, incubated with goat anti-mouse or anti-rabbit horse-radish peroxidase-conjugated secondary antibodies (1:10,000, Santa Cruz Biotechnology Inc., CA, USA) for 1 h then exposed to chemiluminescence reagents (Amersham Inc., Que´bec, Canada), and developed on photographic film. Glyceraldehyde-3-phosphate dehydrogenase (rabbit polyclonal; 1:5,000; Santa Cruz Biotechnology Inc., CA, USA) or b-actin (mouse monoclonal; 1:5,000; Santa Cruz Biotechnology Inc., CA, USA) were used as internal standards to normalize band intensities of the proteins of interest. Densitometric analysis was performed to quantify band optical densities. Chemicals and reagents All chemicals were of reagent grade and were purchased from Sigma (St. Louis, MO).

Statistical analysis All data are expressed as mean ± standard error of the mean (SEM). Statistical analysis of all data was performed using the Number Cruncher Statistical Software 2000 (NCSS, Kaysville, UT). Data were analyzed using unpaired t-test for comparisons between 2 groups. A value of P \ 0.05 was taken as the level of significance.

Western blot analysis Results Frozen kidney was powdered and whole tissue homogenate was prepared using lysis (RIPA) buffer. The homogenate was spun at 10,0009g at 4°C for 15 min, and the protein content of the supernatant was determined by the Bradford protein assay. Equal amounts of protein (50 lg) from each sample were subjected to SDS-PAGE and transferred to PVDF or nitro-cellulose membranes. The membranes were blocked for 1 h in a solution of 5% skim milk and then incubated overnight at 4°C with primary antibodies against: CYP4A1, 2/3 (rabbit polyclonal; 1:3,000; Acris Gmbh, Germany), CYP2C23 (rabbit polyclonal; 1:500; Abcam Inc., Cambridge, MA), CYP2C11 (rabbit polyclonal; 1:400; prepared as described by Chang et al. 1992; generously provided by Dr Bandiera, University of British Columbia, Vancouver, BC), sEH (rabbit polyclonal; 1:10,000; a gift from Dr Bruce Hammock, University of California, Davis, CA), RhoA (mouse monoclonal; 1:500; Santa Cruz Biotechnology Inc., CA, USA), ROCK-1 (mouse monoclonal; 1:1,000; Santa Cruz Biotechnology Inc., CA, USA), ROCK-2 (rabbit polyclonal; 1:500; Santa Cruz Biotechnology Inc., CA, USA), and phospho-Lin-11/ Isl-1/Mec-3 kinase (LIMK) (rabbit polyclonal; 1:500; Cell

As previously reported [26], there was no effect of fructose feeding on body weight (C = 661 ± 20 vs. F = 639 ± 20 g). Plasma glucose and insulin levels at 90 min following a 1 g/kg glucose load showed no difference in plasma glucose levels (C = 7.3 ± 0.3 vs. F = 8.0 ± 0.4 mM, P = 0.3); however, plasma insulin levels were significantly elevated in the fructose-fed group (C = 1.2 ± 0.2 vs. F = 1.8 ± 0.2 ng/ml, P = 0.03). These data confirm that the fructose-fed animals were hyperinsulinemic and would indicate impairment in glucose handling. Systolic blood pressure measured after 7 weeks of fructose feeding was significantly higher in fructose-fed rats compared to control rats (C = 108 ± 2 vs. F = 137 ± 1 mmHg, P = 0.0001). The protein expression of CYP4A (Fig. 1A) as well as CYP2C23 (Fig. 1B) was elevated in whole kidney homogenate of fructose-fed animals compared to controls. However, no change was observed regarding the renal expression of CYP2C11 (Fig. 1C) or sEH (Fig. 1D) following fructose treatment. RhoA (Fig. 2A) was expressed to the same level in kidneys of control and fructose-fed rats. Western blot analysis detected a ROCK-1 as well as a

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A

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18 Kidney CYP2C23 protein abundance (relative optical density)

2.5 Kidney CYP4A protein abundance (relative optical density)

Fig. 1 Effect of high fructose diet on the expression of A CYP4A, B CYP2C23, C CYP2C11 or D sEH in the kidney of rats. Data are expressed as mean ± SEM. * Significantly different from control values at P \ 0.05


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ROCK-2 fragment of 130 kDa in all samples, in addition to the 160 kDa native protein. Thus, fructose feeding was accompanied by increased cleavage of ROCK-1 and ROCK-2 and the reciprocal appearance of a ROCK-1 and ROCK-2 subspecies that corresponded to the cleavage product. Statistical analysis showed a significant difference between fructose-treated and normal kidney samples for the presence of the cleaved subspecies of both ROCK-1 and ROCK-2 (Fig. 3A (ii), B (ii), respectively). These are constitutively active ROCK-1 and ROCK-2 generated by cleavage. Consistent with that, phosphorylation of LIMK, the downstream target of ROCK, was significantly higher in the fructose-fed group compared to the control group (Fig. 2B). Total ROCK-2 expression, calculated by the summation of the optical densities of both the 130 and the 160 kDa bands, showed a significant increase in the fructose-fed rats as compared to the normal-fed rats (Fig. 3B (i)). On the other hand, total ROCK-1 expression was not affected by fructose diet (Fig. 3A (i)). Measurement of ROCK-1 160 kDa native protein band optical density showed a significantly lower value in the fructose-fed rats than in control (1.78 ± 0.30 vs. 4.47 ± 1.18, respectively). It could be speculated that rats both higher expression and higher cleavage, and hence activation, of ROCK-2 takes place in fructose-fed rats. On the other hand, fructose-fed rats showed only increased cleavage in ROCK-1.

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Discussion In the present study, fructose treatment led to a state of impaired glucose tolerance in addition to elevated blood pressure as has been previously demonstrated both in our laboratory [27] and others [4]. It is agreed upon that insulin resistance is linked to hypertension [3]. The mechanisms involved are being extensively studied in our laboratory as well as others but still are far from full elucidation. It has been proposed that there is an increase in peripheral resistance and arachidonic acid metabolites of the cyclooxygenase-2 pathway have been shown to play a role [24]. Recently, the CYP 450 arm of the aracidonic acid cascade has received special attention regarding its role in regulating vascular tone. 20-HETE is a vasoconstrictor produced through omega hydroxylation of arachidonic acid by CYP4A isoforms while the EETs are vasodilators synthesized through arachidonic acid epoxidation by CYP2C isoforms [6]. In the current study, we attempted to further study the mechanisms underlying the contribution of insulin resistance to hypertension. Our results show increased protein expression of CYP4A and CYP2C23 in kidneys of fructose-fed rats as compared to normal controls. Previous studies have shown a change in the renal expression of these two enzymes in another diet-induced hypertensive animal model, the high fat fed rat [28]. However, in those studies


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A 140

Kidney phospho-LIMK protein abundance (relative optical density)

Kidney RhoA protein abundance (relative optical density)

Fig. 2 Effect of high fructose diet on the expression of A RhoA, or B on the phosphorylation of LIMK in the kidney of rats. Data are expressed as mean ± SEM. * Significantly different from control values at P \ 0.05

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expression of CYP4A and CYP2C23 was decreased in the renal tubules but not in the renal microvessels. One would expect CYP2C23 expression to decrease in our model in order to account for the increase in blood pressure. However, the increased expression could constitute a compensatory mechanism as EETs have been previously shown to compensate for nitric oxide deficiency in cardiovascular disease conditions [29]. The difference between our results and those of Wang et al. [28] could be explained by the diet treatments, duration of feeding (8 weeks of high fructose vs. 10 weeks of high fat diets), and/or tissues examined (whole kidney homogenate in our study vs. renal tubules and renal microvessels in Wang et al. [28]). Despite the increase in CYP2C23, renal expression of CYP2C11 was not observed in our study. The reason could

Kidney ROCK-2 130 kDa fragment protein abundance (relative optical density)

A i

Kidney total ROCK-1 protein abundance (relative optical density)

Fig. 3 Effect of high fructose diet on the expression of A i total ROCK-1, ii ROCK-1 130 kDa fragment, B i total ROCK-2, or ii ROCK-2 130 kDa fragment in the kidney of rats. Data are expressed as mean ± SEM. * Significantly different from control values at P \ 0.05

Fructose

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be tissue specificity since CYP2C23 is the most specific isoform to renal microvessels [8, 9]. Similarly, sEH did not significantly change in our kidney homogenates, which is not in agreement with Zhao et al. [13] who found increased expression of sEH in the mesenteric artery of obese Zucker rats. These different findings could be due to different experimental models: the one that Zhao et al. [13] used the obese Zucker rat which represents a genetic model of obesity and type 2 diabetes and the fructose hypertensive rat, a model of diet-induced hypertension and insulin resistance, used in this study. Also, Zhao et al. [13] used the mesenteric artery while we used the kidney, which could offer a second explanation to the difference between the results. The presence of high CYP2C23 level in our kidney samples would imply a high condition of EET

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synthesis. Therefore, it is not surprising to have the degrading machinery, sEH, in check. One of the downstream effectors of 20-HETE is ROCK [23]. In this study, we detected two bands for ROCK. One band is a 160-kDa fragement and it represents native ROCK protein expression. Another band is a 130-kDa fragment and it represents a cleavage product of ROCK, which has been reported to be constitutively active [30]. Interestingly, we observed a convincing increase in protein expression levels of cleaved activated subspecies for both isoforms of ROCK, 1 and 2, in kidney samples of fructosefed rats. In agreement with that, phosphorylation of LIMK, which is used as an index of the RhoA/ROCK pathway activity, was increased. Moreover, total protein expression of ROCK-2, but not ROCK-1 was higher in fructose-fed rats as compared to control. However, the expression of RhoA, the upstream activator of ROCK did not show any difference between our groups. In light of the aforementioned findings, we speculate that, following the onset of insulin resistance, renal CYP4A is activated, potentially producing 20-HETE, a potent vasoconstrictor that leads to an increase in peripheral resistance and therefore an increase in blood pressure. 20-HETE causes vasoconstriction through sensitizing the vasoconstrictor machinery to calcium and this is through activating Rho kinase. In compensation, CYP2C23 is upregulated to produce the vasodilatory EETs, which maintain renal blood flow. This study is the first to demonstrate the possible involvement of renal CYP 450 and RhoA/Rho kinase pathways in fructose-induced hypertension. However, detailed studies of the potential roles of these molecules in this model through the use of inhibitors and drugs acting at different points of these pathways remains to be carried out. Acknowledgments We thank Hesham Soliman, Dr Guorong Lin, and Dr Vijay Sharma for their continuous assistance with this study. Sally Mustafa was a visiting scientist from the University of Lahore, Pakistan and was supported by a Post Doctoral Fellowship from Higher Education Commission, the Government of Pakistan. This study was supported by a grant from the Heart and Stroke Foundation of BC and Yukon. Harish Vasudevan received funding from Heart and Stroke Foundation of Canada and the Michael Smith Foundation.

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