Effects of Angiotensin II Receptor Blockers - Diabetes - American ...

ORIGINAL ARTICLE

Expression of NG,NG-Dimethylarginine Dimethylaminohydrolase and Protein Arginine N-Methyltransferase Isoforms in Diabetic Rat Kidney Effects of Angiotensin II Receptor Blockers Maristela L. Onozato,1,2 Akihiro Tojo,1 James Leiper,3 Toshiro Fujita,1 Fredrik Palm,2 and Christopher S. Wilcox2

OBJECTIVE—The nitric oxide (NO) synthase inhibitor asymmetric dimethylarginine (ADMA) is generated by protein arginine N-methyltransferase (PRMT)-1 and is metabolized by NG,NGdimethylarginine dimethylaminohydrolase (DDAH). We tested the hypothesis that increased serum ADMA (SADMA) in the streptozotocin (STZ)-induced diabetic rat model of diabetes is mediated by an angiotensin receptor blocker–sensitive change in DDAH or PRMT expression. RESEARCH DESIGN AND METHODS—Data were compared from four groups of rats: sham-injected controls, untreated STZ-induced diabetic rats at 4 weeks, STZ-induced diabetic rats administered the angiotensin II (Ang II) receptor blocker telmisartan for 2 weeks, and control rats administered telmisartan for 2 weeks. RESULTS—Immunostaining and Western blotting of microdissected nephron segments localized DDAH I in the proximal tubules and DDAH II in the glomeruli, afferent arterioles, macula densa, and distal nephron. Renal Ang II and SADMA increased with diabetes but were normalized by 2 weeks of telmisartan. DDAH I expression was decreased in diabetic kidneys, while DDAH II expression was increased. These changes were reversed by telmisartan, which also reduced expression of PRMT-1 and -5. Telmisartan increased expressions of DDAH I but decreased DDAH II in Ang II-stimulated kidney slices ex vivo. CONCLUSIONS—Renal Ang II and SADMA are increased in insulinopenic diabetes. They are normalized by an Ang II receptor blocker, which increases the renal expression of DDAH I, decreases PRMT-1, and increases renal NO metabolites. Diabetes 57:172–180, 2008

From the 1Division of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan; 2Division of Nephrology and Hypertension, Cardiovascular Kidney Hypertension Institute, Georgetown University, Washington, DC; and the 3Centre for Clinical Pharmacology and Therapeutics, British Heart Foundation Laboratories, University College London, London, U.K. Address correspondence and reprint requests to Christopher S. Wilcox, MD, PhD, Division of Nephrology and Hypertension, Georgetown University Medical Center, 6PHC, Suite F6003, 3800 Reservoir Rd., NW, Washington, DC 20007. E-mail: [email protected]. Received for publication 22 December 2006 and accepted in revised form 26 September 2007. Published ahead of print at http://diabetes.diabetesjournals.org on 1 October 2007. DOI: 10.2337/db06-1772. ADMA, asymmetric dimethylarginine; Ang II, angiotensin II; ARB, Ang II receptor blocker; AT1-R, Ang II type 1 receptor; CCD, cortical collecting duct; DDAH, dimethylarginine dimethylaminohydrolase; eNOS, endothelial nitric oxide synthase; IMCD, inner medullary collecting duct; L-NMMA, NG-monomethyl-L-arginine; NOS, NO synthase; NOx, NO metabolite; PRMT, protein arginine N-methyltransferase; SDMA, symmetric dimethylarginine; STZ, streptozotocin; TBST, Tris-buffered saline containing 0.1% Tween 20. © 2008 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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A

nalogs of L-arginine, such as (NG-monomethylG G L-arginine (L-NMMA) and N ,N -dimethylarginine (asymmetric dimethylarginine [ADMA]), inhibit nitric oxide synthase (NOS). They originate from regular turnover of arginine residues within proteins that have been posttranslationally methylated by class 1 isoforms of protein arginine N-methyltransferase (PRMT) (1), notably PRMT-1 (2). NG,NG’-dimethylarginine (symmetric dimethylarginine [SDMA]) is an enantiomeric form of ADMA that is synthesized by class 2 isoforms of PRMT (notably PRMT-5) (2). SDMA does not inhibit NOS but, like the other methylarginine analogues, can compete with arginine for cellular uptake via system y⫹ (3,4). ADMA increases the tone of rat aortic rings (5), raises blood pressure in guinea pigs (6), and reduces resting forearm blood flow in humans (6). Therefore, ADMA, which has higher plasma concentrations than L-NMMA, has been considered an important endogenous regulator of the L-arginine/NO pathway (2,7). ADMA and SDMA are excreted in the urine, but ADMA specifically can be metabolized by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH) (8,9). Blockade of DDAH increases the concentration of ADMA in endothelial cells (10) and inhibits NO-mediated endothelium-dependent relaxation of blood vessels (10). Recently, Leiper et al. (11) identified a second DDAH isoform in humans, which was named DDAH II. Although they demonstrated that the RNAs for DDAH I and II are both found in the kidney, their localization in the nephron segments remains unknown. NO in the kidney can regulate vascular resistance, glomerular filtration rate (12), tubuloglomerular feedback (12,13), and tubular reabsorption of sodium and proton secretion (14,15). Therefore, a precise localization of the sites of DDAH isoforms expression and their intraorgan regulation could provide clues to the regulation of NO. We had previously reported that an antibody to DDAH located it at sites of NOS expression in the kidney (16). However, that report predated the discovery of different DDAH isoforms. Elevated concentrations of ADMA have been reported in hypercholesterolemia (17), and hypertension (18) and in animal models (19) and patients with insulinopenic diabetes, as well as those with type 2 diabetes (19,20) or insulin resistance syndromes (21–23). Elevated levels of ADMA have been related to poor cardiovascular outcomes (24,25). However, the effects of diabetes on the individual DIABETES, VOL. 57, JANUARY 2008

M.L. ONOZATO AND ASSOCIATES

TABLE 1 Physiological data of control and diabetic rats with/without telmisartan treatment

n Body weight (g) Blood glucose (mg/dl) Mean blood pressure (mmHg) Blood pH Urinary volume (ml/day) Creatinine clearance (ml 䡠 min⫺1 䡠 100 g body wt⫺1)

Control rats

Control rats with telmisartan

Diabetic rats

Diabetic rats with telmisartan

10 258 ⫾ 6 144 ⫾ 7 92 ⫾ 4 7.38 ⫾ 0.2 14 ⫾ 2

6 232 ⫾ 13 125 ⫾ 11 77 ⫾ 6 7.37 ⫾ 0.2 16 ⫾ 4

10 217 ⫾ 10* 415 ⫾ 28*† 83 ⫾ 6 7.39 ⫾ 0.3 91 ⫾ 27*

10 216 ⫾ 8* 431 ⫾ 28*† 78 ⫾ 5 7.37 ⫾ 0.4 94 ⫾ 22*†

0.85 ⫾ 0.12

1.23 ⫾ 0.28

1.40 ⫾ 0.13*

1.48 ⫾ 0.11*

Data are means ⫾ SE. *P ⬍ 0.01 vs. control rats; †P ⬍ 0.01 vs. control rats with telmisartan.

expression of each DDAH isoform and of PRMT class I and II in the kidney have not been reported. Studies in the streptozocin (STZ)-induced diabetic rat model of insulinopenic diabetes have shown generally decreased values for plasma renin activity and plasma angiotensinogen and angiotensin II (Ang II) concentrations but increased renal tissue levels of renin and angiotensinogen (26). This may be explained by an intrarenal system for renin (27), angiotensinogen (28), Ang II (29), and ACE (30) that is under regulation separate from juxtaglomerular renin release (28,30). Administration of an ACE inhibitor or an Ang II receptor blocker (ARB) to STZ-induced diabetic rats or patients with type 2 diabetes reduces markers of inflammation, oxidative stress, and albuminuria, independent of blood pressure or creatinine clearance (31,32). We used the STZ-induced model of diabetes, which we had characterized (31,33–37) to explore the regulation of DDAH and PRMT expression by Ang II type 1 receptors (AT1-Rs) in diabetes. The present work describes the localization of DDAH I and II in the rat kidney and evaluates the protein expression of DDAH and PRMT isoforms in a rat model of type 1 diabetes and their regulation by AT1-Rs. We have related these primary findings to blood measurements of ADMA and SDMA, kidney NO metabolites (NOxs), and renal expression of endothelial NOS (eNOS) to gain insight into the regulation of ADMA and NO in diabetes. The hypothesis that the studies are designed to test is that elevated levels of serum ADMA in insulinopenic diabetes can be

ascribed to AT1-R– dependent changes in renal expression of DDAH and PRMT enzymes. RESEARCH DESIGN AND METHODS All animal procedures were conducted in accordance with the Guide for Animal Experimentation of the Faculty of Medicine, University of Tokyo. Female Sprague-Dawley rats weighing 180 –200 g (Charles River Laboratories, Shizuoka, Japan) were housed in a temperature- and humidity-controlled room with a 12-h light/dark cycle and with free access to tap water and standard rat diet (Na⫹ content 0.3 g/100 g). Diabetes was induced by a single tail vein injection of STZ (60 mg/kg body wt; Sigma Chemical, St. Louis, MO) (diabetic rats, n ⫽ 20) diluted in citrate buffer, pH 4.5. Control rats (n ⫽ 16) were injected with an equal volume of citrate buffer. After 3 days, the induction of diabetes was confirmed by urinary glucose excretion. As in our prior studies (31,33–38) no attempt was made to treat the diabetes with insulin, since we wished to study the pathophysiology of insulinopenic diabetes. Two weeks after STZ injection, both control and diabetic rats were randomly divided into two groups matched for body weight and blood glucose. One group was untreated (control rats: n ⫽ 10, diabetic rats: n ⫽ 10), and one received the AT1-R blocker telmisartan (control rats plus telmisartan: n ⫽ 6, diabetic rats plus telmisartan: n ⫽ 10; 3 mg 䡠 kg⫺1 䡠 day⫺1 in the drinking water). Telmisartan (Boehringer Ingelheim, Biberach, Germany) was dissolved in 0.2 ml of 1 mol/l KOH and added to the drinking water in a dose previously reported to block angiotensin action (39,40). Blood pH was not affected by this procedure (Table 1). The drinking water was replaced daily. After 2 weeks of treatment and 4 weeks of diabetes, 24-h urine and blood were collected and rats were killed. Rats were allowed free access to food to permit ongoing hyperglycemia and hyperfiltration (31,33–38). The animals were anesthetized with pentobarbital (50 mg/kg body wt), the abdominal aorta was cannulated, blood pressure was measured with a pressure transducer, and the kidneys were perfused retrogradely with ice-cold PBS. The right kidney was

FIG. 1. Serum concentrations of ADMA and SDMA. Mean ⴞ SE values in control rats (C; n ⴝ 10), control rats given telmisartan (CⴙT; n ⴝ 6), diabetic rats (DM; n ⴝ 10), and diabetic rats given telmisartan (DMⴙT; n ⴝ 10). *P < 0.05 vs. control rats; ⴙP < 0.05; ⴙⴙP < 0.01 vs. control rats given telmisartan. DIABETES, VOL. 57, JANUARY 2008

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ANG II, DDAH, AND PRMT

FIG. 2. Immunohistochemistry of DDAH isoforms in the normal rat kidney. Immunostaining for DDAH I showed reactivity in the cytoplasm of the proximal convoluted tubular cells (A and B). DDAH II was localized in the glomeruli, distal convoluted tubule (DCT), CCD and IMCD (C–E), and macula densa (MD) and renal vasculature (E and F). Bars ⴝ 50 ␮m. taken and immediately frozen for Western blotting. The left kidney was perfused with periodate-lysine-paraformaldehyde solution and embedded in wax for immunohistochemical study. Microdissection of nephron segments. Microdissection was performed as reported previously (14). A separate set of four normal rats were anesthetized and prepared as described above to provide control tissue. Kidneys were perfused with 10 ml of cold dissection solution containing (in mmol/l) 135 NaCl, 5 KCl, 1 NaH2PO4, 1.2 MgSO4, 2 CaCl2, 6 L-alanine, 10 N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid, 5.5 glucose, and 0.1% BSA to rinse away the blood. This was followed by 10 ml of dissection solution containing 0.1% collagenase (Sigma Chemical). Thin sagittal slices were cut from the perfused kidneys and incubated in dissection solution with 0.1% collagenase at 37°C for 20 min. Microdissection of individual nephron segments was performed in cold dissection solution with a stereomicroscope. Fifteen glomeruli and 20 segments of specific renal tubules measuring between 0.5 and 1.0 mm were dissected from each animal and mixed with 10 ␮l SDS buffer (0.5 mol/l Tris-HCl, pH 6.8, 20% [vol/vol] glycerol, and 4.6% [wt/vol] SDS), sonicated, and processed for Western blot analysis. Western blotting. As described in detail previously (31,35), whole kidneys were homogenized with a tissue homogenizer in 3 ml of 20 mmol/l Tris, 174

followed by centrifugation at 4°C and 12,000 rpm for 20 min. Supernatants were diluted in the same volume of SDS buffer. Samples containing 50 ␮g of protein were resolved on a 4 –20% gradient gel (Daiichi Pure Chemicals, Tokyo, Japan) and electroblotted to polyvinylidene fluoride membranes, which were incubated with 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min, following overnight incubation with a polyclonal antibody for DDAH I or II (11) or eNOS (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal antibody for PRMT-1 (Abcam, Cambridge, U.K.) or rabbit polyclonal for PRMT-5 (Abcam) at 1:1,000 dilutions. After rinsing in TBST, membranes were incubated for 2 h with a horseradish peroxidase– conjugated secondary antibody against rabbit or mouse IgG (Dako, Glostrup, Denmark) in a 1:1,000 dilution and rinsed with TBST followed by 0.8 mmol/l diaminobenzidine with 0.01% H2O2 and 3 mmol/l NiCl2 for the detection of blots. The density of the bands was analyzed using National Institutes of Health Image software (version 1.63). Immunohistochemistry. The tissues embedded in wax were processed for immunohistochemistry using the labeled streptavidin biotin method as described previously (31,35). Sections (2 ␮m) were dewaxed and incubated with 3% H2O2 and blocking serum and thereafter with a polyclonal antibody against DDAH I or II in a 1:100 dilution. The sections were rinsed with TBST and a DIABETES, VOL. 57, JANUARY 2008


with normal distribution. For the Western blot densitometry data, a KruskalWallis nonparametrical test was used. P ⬎ 0.05 was required for statistical significance.

RESULTS

FIG. 3. Western blot of microdissected nephron segments from normal rat kidneys for DDAH I or II. Individual blots for glomerulus (GL), proximal tubule (PT), proximal straight tubule (PST), thick ascending limb of the loop of Henle (TAL), distal convoluted tubule (DCT), CCD, outer medullary collecting duct (OMCD), and IMCD. biotinylated secondary antibody against rabbit IgG (Dako) in a 1:400 dilution. After rinsing with TBST, the sections were incubated with horseradish peroxidase– conjugated streptavidin solution (Dako). Horseradish peroxidase labeling was detected using a peroxide substrate solution with 0.8 mmol/l diaminobenzidine and 0.01% H2O2. The sections were counterstained with hematoxylin before being examined under a light microscope. The antibodies to PRMT-1 and -5 were found not to be suitable for immunohistochemistry. Measurement of glucose, nitrite, ADMA, SDMA, and Ang II. Procedures for measurement of blood and urine glucose and kidney nitrate plus nitrite (NOx) were described in detail in our previous reports (31,41). Methylarginines in serum were measured with high-performance liquid chromatography as previously described (6). Renal tissue Ang II concentration was assessed by the radioimmunoassay method (SRL, Tokyo, Japan) and corrected by the amount of protein in the kidney homogenate. Tissue incubation with Ang II. Kidney slices (200 ␮m) containing cortex and medulla were isolated under sterile conditions in cultured growth medium consisting of Dulbecco’s Modified Eagle’s Medium with glutamine (Sigma Chemical) supplemented with 10% fetal bovine serum (Gibco, Paisley, U.K.) and antibiotic solution (100 units/ml penicillin G sodium, 100 ␮g/ml gentamicin, 100 ␮g/ml streptomycin, and 5 ␮g/ml amphotericin; Sigma Chemical). Each kidney slice was cultured in 1 ml of medium in a separate well at 37°C in a 5% CO2 incubator for 18 h in the presence or absence of Ang II (10⫺9, 10⫺7, 10⫺5 mol/l) and Ang II 10⫺7 with telmisartan (10⫺6 mol/l). Tissue was processed for Western blot analysis after rinsing with PBS. Statistics. Data are expressed as means ⫾ SE. An ANOVA with Bonferroni’s post hoc test was used for statistical comparisons of the physiological data DIABETES, VOL. 57, JANUARY 2008

Diabetes parameter. As reported in our previous studies in this STZ-induced diabetic rat model at 4 weeks (31,33– 38), rats with diabetes had a significant decrease in body weight, an increase in blood glucose to ⬃400 mg/dl, an increase in urinary volume, and an increase in creatinine clearance without changes in mean blood pressure measured under anesthesia (Table 1). These parameters were not changed significantly by 2 weeks of telmisartan administration in both control or diabetic rats (Table 1). ADMA and SDMA. Four weeks of diabetes increased SADMA but did not change SSDMA. Consequently, the serum ADMA-to-SDMA ratio increased significantly (Fig. 1). Two weeks of telmisartan normalized SADMA only in rats with diabetes and the serum ADMA/SDMA. DDAH I and II localization in the normal rat kidney. DDAH I immunoreactivity was observed in the proximal tubule, especially in the S3 segment (Fig. 2). This was confirmed by Western blot analysis of microdissected nephron segments (Fig. 3, upper panel). DDAH II immunoreactivity was observed in the glomeruli, the thick ascending limb of the loop of Henle, the distal convoluted tubule, the cortical collecting duct (CCD), the inner medullary collecting duct (IMCD), the macula densa, the renal afferent arteriole, the vascular smooth muscle cells, and the endothelial cells (Fig. 2). Western blot analysis of microdissected nephron segments confirmed DDAH II protein expression in the glomeruli, the thick ascending limb of the loop of Henle, the distal convoluted tubule, the CCD, and the IMCD (Fig. 3, lower panel). The renal expression of PRMT-1 and -5 were unaltered by diabetes, but both were significantly reduced after 2 weeks of telmisartan administration to rats with diabetes, whereas telmisartan was ineffective in control rats (Fig. 4). Immunohistochemistry of DDAH I in diabetic rats showed decreased reactivity in the proximal tubules (Fig. 5), which was confirmed by Western blot analysis of whole-kidney homogenates (Fig. 6). Telmisartan increased renal DDAH I expression above control levels in diabetes, but it did not change DDAH I in the kidney from control rats (Fig. 6). In contrast, DDAH II expression in the kidney was increased during this phase of diabetes, as shown by immunohistochemistry (Fig. 5) and confirmed by Western blot (Fig. 6). Telmisartan reduced DDAH II expression in the kidney of diabetes but not in controls (Fig. 6). Ang II levels in the kidney were increased in diabetes (962 ⫾ 55 vs. 637 ⫾ 125 pg/g protein; P ⬍ 0.05). Telmisartan treatment reduced Ang II levels in the kidney of diabetes (651 ⫾ 107; P ⬍ 0.05 vs. diabetic rats) but not in control rats (869 ⫾ 44). Ang II regulation of DDAH expression in the kidney. Incubation of slices of tissue from normal whole kidneys for 18 h with graded concentrations of Ang II did not change renal DDAH I expression significantly. However, incubation with telmisartan in slices incubated with 10⫺7 mol/l Ang II increased DDAH I expression significantly above values with 10⫺7 mol/l Ang II alone (Fig. 7A). On the other hand, incubation of the kidney with Ang II increased DDAH II expression dose dependently. Incubation with telmisartan in the presence of 10⫺7 mol/l Ang II reduced DDAH II expression significantly (Fig. 7B). 175


FIG. 4. Western blot of PRMT-1 and PRMT-5 in the whole kidney. Control rats (C; n ⴝ 10), control rats given telmisartan (CⴙT; n ⴝ 6), diabetic rats (DM; n ⴝ 10), and diabetic rats given telmisartan (DMⴙT; n ⴝ 10). *P < 0.05; **P < 0.01; ***P < 0.001 vs. control rats; ⴙP < 0.05; ⴙⴙP < 0.01; ⴙⴙⴙP < 0.001 vs. control rats given telmisartan.

Kidney NOxs and eNOS expression. Kidney NOx production was not changed significantly by diabetes but was increased in diabetic rats by telmisartan (Fig. 8). eNOS expression was increased at 4 weeks of diabetes, confirming our previous report (30) that it was reduced by telmisartan. DISCUSSION

These studies have confirmed some reports that have shown that plasma ADMA is elevated in insulinopenia or 176

diabetes (19 –23), although we found normal levels in a Wistar-Furth STZ-induced diabetic model (36). They have shown further that the elevated levels can be normalized by 2 weeks of ARB administration. These studies disclose distinct sites of expression and regulation of DDAH I and II proteins in the adult rat kidney. Induction of 4 weeks of diabetes decreases the renal expression of DDAH I but increases the expression of DDAH II. These effects apparently depend on AT1-Rs, since they are reversed by an ARB that also reduces PRMT-1 and -5 expression in the kidneys of diabetic rats below levels of normal rats. These changes are independent of blood pressure, which was not altered by diabetes or ARB administration in this, or previous, studies with the model (31,33,34,36). Distinct localization of DDAH isoforms. The finding that plasma ADMA does not always correlate with DDAH protein expression (2,19) led to studies by Leiper et al. (11) that demonstrated a second DDAH isoform, termed DDAH II. We confirm our previous report (16) that DDAH is expressed in the kidney at sites of NOS expression. We now localize DDAH I in the proximal tubules, especially in the S3 segment, and DDAH II in the glomerulus, macula densa, renal vasculature, and distal segments of the nephron. Since the proximal tubules comprise ⬃70% of the renal cortex, DDAH I may be especially important for renal metabolism of ADMA. This is consistent with the finding that serum ADMA was increased in diabetic rats that had a decreased renal DDAH I expression, and both of these changes were reversed by an ARB. In contrast, the localization of DDAH II in glomeruli, macula densa, and renal vasculature suggests that it may regulate kidney hemodynamics, which has been implicated in the development of diabetic nephropathy (42– 44). Distinct regulation of DDAH and PRMT isoform expression in diabetic kidney. The decrease in DDAH I expression and the increase in DDAH II expression in the kidneys of diabetic rats can be ascribed largely to Ang II acting on AT1-Rs, since Ang II levels are increased in the kidney of these diabetic rats and these changes in DDAH I and II are reversed by 2 weeks of telmisartan administration. The liver (45) and the kidney (6) contribute to the systemic clearance of ADMA, but only ADMA is metabolized by DDAH. Despite no change in PRMT-1, serum ADMA was increased, but serum SDMA was unchanged, in rats with diabetes, leading to an elevation in the serum ADMA-to-SDMA ratio. This is consistent with the finding of a reduction in renal DDAH I expression in diabetes, which could account for decreased ADMA metabolism. Administration of an ARB to rats with diabetes normalized the elevated level of serum ADMA and reduced serum SDMA below control values. The normalization of serum ADMA may relate to an increase in renal DDAH I expression and to a reduction in PRMT-1 expression below control values, both of which would be expected to reduce serum ADMA. The reduction in serum SDMA below control levels in diabetic rats given telmisartan may relate to their accompanying reduction in renal expression of PRMT-1 below control levels, which would be anticipated to reduce SDMA generation. However, a limitation of these studies is that we did not assess DDAH and PRMT expression in the liver and did not assess the enzyme activities directly. Oxidative stress is increased in the kidneys and renal afferent arterioles of rats or rabbits with STZ-induced diabetes (31,33,37,38). Oxidative stress in the diabetic rat DIABETES, VOL. 57, JANUARY 2008


FIG. 5. Immunohistochemical expression of DDAH I or DDAH II in the kidney of control rats, control rats given the ARB telmisartan (ControlⴙT), diabetic rats (DM), and diabetic rats treated with telmisartan (DMⴙT). Bar 200 ␮m.

kidney is reversed by administration of an ARB (31) or by apocynin to inhibit NADPH oxidase (37). Both DDAH and PRMT enzymes are redox sensitive (2,46) and may thereby be subject to posttranscriptional modification by oxidative stress in addition to the transcriptional changes described in the study. Kidney concentrations of NOxs were unaffected by diabetes, despite increased eNOS expression. One explanation would be reduced blood levels of L-arginine (36), but this was not assessed in this study. We do not discard that it could be due to uncoupling of NOS during oxidative stress, for example by oxidation of tetrahydrobiopterin (BH4) to dihydrobiopterin (BH2), which can convert con-

stitutive NOS from generating NO to generating predominantly superoxide (47). Such a mechanism could explain the increase in kidney NOxs in diabetic animals treated with telmisartan, despite reduced eNOS expression, since prolonged administration of an ARB reduces p47phox expression in kidney tubules and reduces renal reactive oxygen species generation in this model of diabetes (31). Additionally the changes in renal NOxs are in line with our findings of ADMA. Thus, an increase in ADMA and eNOS in diabetes might explain the unchanged level of renal NOxs, whereas a reduction in ADMA, but an unchanged eNOS, might explain an increase in renal NOxs with telmisartan in diabetic rats.

FIG. 6. Western blot of DDAH I or DDAH II in the whole kidney. Mean ⴞ SE values for renal protein expression for DDAH I (A) and DDAH II (B) in kidneys from control rats (C; n ⴝ 10), control rats given telmisartan (CⴙT; n ⴝ 6), diabetic rats (DM; n ⴝ 10), and diabetic rats given telmisartan (DMⴙT; n ⴝ 10). *P < 0.05; **P < 0.01; ***P < 0.001 vs. control rats; ⴙⴙⴙP < 0.001 vs. control rats given telmisartan. DIABETES, VOL. 57, JANUARY 2008

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FIG. 7. Western blot analysis of DDAH I (A) or DDAH II (B) protein expression in kidney slices. The slices were incubated with different concentrations of Ang II for 18 h alone or with telmisartan (10ⴚ7 mol/l). n ⴝ 10 in each group. *P < 0.05; ** P < 0.01; *** P < 0.001 vs. control rats; †P < 0.05 vs. Ang II 10ⴚ9 mol/l; ‡P < 0.05 vs. Ang II 10ⴚ7 mol/l; §P < 0.001 vs. Ang II 10ⴚ5 mol/l.

Ang II regulation of DDAH isoforms. A new finding was that kidneys of diabetic rats had increased concentrations of Ang II that were reduced by telmisartan. The administration of telmisartan increased DDAH I expression in the kidneys of diabetic rats. DDAH I expression was not changed by direct incubation of the kidney slices with Ang II but was increased significantly by telmisartan in Ang II–stimulated kidney slices. This suggests either that AT1-Rs decrease DDAH expression or that AT2-R activation increases DDAH I, but this was not studied further in the present report. The upregulation of DDAH I expression in Ang II–treated kidney slices by telmisartan is consistent with the finding that ACE inhibitors or ARBs reduce plasma ADMA in human subjects with essential hypertension (48) or end-stage renal disease (49). The finding that renal DDAH II expression is reduced both by telmisartan administration to diabetic rats and by telmisartan administration to Ang II–stimulated kidney slices indicates that AT1-R signaling increases DDAH II expression in the kidneys. This may explain our previous report that Ang II infusion, or a low-salt diet that also increases Ang II levels, increases DDAH expression in the rat kidney and that these effects are prevented by losartan treatment (50). In summary, DDAH I is expressed in the proximal 178

tubule, whereas DDAH II is expressed in the glomerulus, the thick ascending limb of the loop of Henle, the macula densa, the distal convoluted tubule, and the collecting duct and arterioles. Kidneys from rats with STZ-induced diabetes have enhanced tissue levels of Ang II and downregulated DDAH I and upregulated DDAH II protein levels. These can be ascribed to increased intrarenal Ang II signaling, since AT1-R blocker treatment normalizes the expression of these proteins in the diabetic rat kidney and also reduces the renal expression of PRMT-1 and -5 and the elevated serum level of ADMA. Perspective. The findings of differential regulation and location of DDAH I and II suggest site-specific regulation of ADMA, which might be involved in the development of diabetes-induced alterations in kidney function. Both eNOS and DDAH II are expressed in renal vascular cells (13) and neuronal NOS and DDAH II in the macula densa (12,13). An upregulation of macula densa DDAH II in diabetic rats, despite a downregulation of DDAH I in proximal tubular cells, could contribute to site-specific alterations in NO production and action within the diabetic kidney. This provides a potential explanation for the seemingly paradoxical findings in STZ-induced diabetic rats and patients with diabetes that renal blood flow is dependent on NO (51) that is largely derived from neuroDIABETES, VOL. 57, JANUARY 2008


FIG. 8. Kidney tissue NOxs and kidney eNOS expression. Mean ⴞ SE values in control rats (C; n ⴝ 10), control rats given telmisartan (CⴙT; n ⴝ 6), diabetic rats (DM; n ⴝ 10), and diabetic rat given telmisartan (DMⴙT; n ⴝ 10). Compared with control: *P < 0.05; **P < 0.01; ***P < 0.001.

nal NOS in the macula densa (42– 44,52), yet the bioavailability of NO in the renal cortex is reduced (36,42,44). Thus, downregulation of DDAH I in the proximal tubule might contribute to increased renal tissue levels of ADMA that inhibit renal cortical NOS and thereby reduce NO bioavailability, while upregulation of DDAH II in the macula densa may protect neuronal NOS at that site from inhibition by cortical ADMA and permit NO-dependent afferent arteriolar vasodilatation. Further studies will be required to explore these speculations. ACKNOWLEDGMENTS

This work was supported by grants from the Japanese Society for the Promotion of Science to M.L.O. (17-05229); by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (C2-14571014 and C216590780) and a grant-in-aid for scientific research from Japan Society for the Promotion of Science to A.T. (C19590938); by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-36079 and DK-49870) and the National Heart, Lung, and Blood Institute to C.S.W. (HL-68686); and by funds from the George E. Schreiner Chair of Nephrology. We are grateful to Patrick Vallance for providing the antibodies to DDAH I and II used in these studies and to Margaret Brierton and Emily Chan for preparation of the manuscript. DIABETES, VOL. 57, JANUARY 2008

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