expression of ddah and prmt isoforms in the diabetic rat ... · 10/1/2007 · methylated by class...

Expression of DDAH and PRMT isoforms in the diabetic rat kidney; effects of angiotensin II receptor blocker

1Maristela L. Onozato, 1Akihiro Tojo, 3James Leiper, 1Toshiro Fujita, 2Frederik Palm and 2Christopher S. Wilcox

1Division of Nephrology and Endocrinology, University of Tokyo, Tokyo, Japan; 2

Division of Nephrology and Hypertension, Cardiovascular Kidney Institute, Georgetown University, Washington, DC, USA; 3Centre for Clinical Pharmacology and Therapeutics,

British Heart Foundation Laboratories, University College London, London, United Kingdom.

Short Title: Ang II, DDAH and PRMT

Address for correspondence: Christopher S. Wilcox, M.D., Ph.D.

Division of Nephrology and Hypertension, Georgetown University Medical Center 6PHC, Suite F6003

3800 Reservoir Road, NW Washington, DC 20007

E-mail:[email protected]

Received for publication 22 December 2006 and accepted in revised form 26 September 2007.

Diabetes Publish Ahead of Print, published online October 1, 2007

Copyright American Diabetes Association, Inc., 2007

Ang II, DDAH and PRMT

ABSTRACT Objective: The nitric oxide (NO) synthase inhibitor, asymmetric dimethylarginine (ADMA) is generated by protein arginine N-methyltransferase (PRMT)-1 and is metabolized by NG, NG-dimethylarginine dimethylaminohydrolase (DDAH). We tested the hypothesis that increased serum ADMA (SADMA) in the streptozotocin (STZ)-induced rat model of diabetes mellitus (DM) is mediated by an angiotensin receptor blocker– sensitive change in DDAH or PRMT expression. Research design and Methods: Data were compared from 4 groups of rats: sham injected controls; untreated STZ- DM at 4 weeks; STZ-DM rats administered the angiotensin II receptor blocker telmisartan for 2 weeks; 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 angiotensin II and SADMA increased with DM but were normalized by 2 weeks of telmisartan. DDAH I expression was decreased in DM 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. Conclusion: Renal angiotensin II and SADMA are increased in insulinopenic DM. They are normalized by an angiotensin II receptor blocker which increases the renal expression of DDAH I, decreases PRMT-1 and increases renal NO metabolites.

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Analogs of L-arginine such as NG-monomethyl-L-arginine (L-NMMA) and NG,NG-dimethylarginine (asymmetric dimethylarginine; ADMA) inhibit nitric oxide synthase (NOS). They originate from regular turnover of arginine residues within proteins that have been post-translationally 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 the 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 identified a second DDAH isoform in humans which was named DDAH II (11). Although they demonstrated that the RNAs for DDAH I and DDAH 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 (GFR) (12), tubuloglomerular feedback (TGF) (12, 13), tubular

reabsorption of sodium and proton secretion (14, 15). Therefore, a precise localization of the sites of DDAH isoforms expression, and their intra-organ 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), hypertension (18), and in animal models (19) and patients with insulinopenic as well as those with type 2 diabetes mellitus (DM) (19, 20) or insulin resistance syndromes (21-23). Elevated levels of ADMA have been related with poor cardiovascular outcomes (24, 25). However the effects of diabetes on the individual expression each DDAH isoform and of PRMT class I and II in the kidney have not been reported. Studies in the streptozocin (STZ) rat model of insulinopenic DM have shown generally decreased values for plasma renin activity (PRA), plasma angiotensinogen and angiotensin II (Ang II) concentrations, yet 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 angiotensin converting enzyme (ACE) (30), that is under separate regulation from juxtaglomerular renin release (28, 30). Administration of an ACE inhibitor or an angiotensin II receptor blocker (ARB) to STZ-diabetic rats or patients with type 2 DM reduces markers of inflammation, oxidative stress and albuminuria, independent of blood pressure or creatinine clearance (31, 32). We used the STZ model of DM which we had characterized (31, 33-37) to explore

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the regulation of DDAH and PRMT expression by Ang II type 1 receptor (AT1-R) in DM.

The present work describes the localization of DDAH I and DDAH II in the rat kidney, and evaluates the protein expression of DDAH and PRMT isoforms in 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 (NOx) and renal expression of endothelial nitric oxide synthase (eNOS) to gain insight into the regulation of ADMA in DM. The hypothesis that the studies are designed to test is that elevated level of serum ADMA in insulinopenic DM 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, The University of Tokyo. Female Sprague-Dawley rats weighing 180-200g (Charles River Laboratories, Shizuoka, Japan) were housed in a temperature-and humidity-controlled room with a 12-hour light/dark cycle and with free access to tap water and standard rat chow (Na+ content 0.3g·100g-1). Diabetes was induced by a single tail vein injection of streptozotocin (STZ, 60 mg/kg body weight; DM, n= 20; Sigma Chemical Co., St. Louis, MO, USA) diluted in citrate buffer, pH 4.5. Control rats (C, n= 16) were injected with an equal volume of citrate buffer. After three 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 DM with insulin since we wished to study the pathophysiology of insulinopenic DM.

Two weeks after STZ injection, both control and DM rats were each randomly divided into two groups matched for body weight and blood glucose. One group was untreated (C, n=10, DM, n=10) and one received the AT1 receptor-blocker, telmisartan (C+T, n=6, DM+T, n= 10; 3 mg/kg/day, in the drinking water). Telmisartan (Boehringer Ingelheim, Biberach, Germany) was dissolved in 0.2 ml of 1 M 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 two weeks of treatment and four weeks of DM, 24 hour urine and blood were collected and rats were sacrificed. 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 weight), the abdominal aorta was cannulated, blood pressure was measured with a pressure transducer and the kidneys were perfused retrogradely with ice-cold phosphate buffered saline (PBS). The right kidney was taken and immediately frozen for Western blotting. The left kidney was perfused with periodate-lysine-paraformaldehyde (PLP) 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 mM) 135 NaCl, 5 KCl, 1 NaH2PO4, 1.2 MgSO4, 2 CaCl2, 6 L-alanine, 10 N-2-hydroxy-ethylpiperazine-N’-2-ethanesulfonic acid, 5.5 glucose, and 0.1 % bovine serum albumin to rinse away the blood. This was followed by 10

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ml of dissection solution containing 0.1% collagenase (Sigma Chemical Co.). Thin sagittal slices were cut from the perfused kidneys and incubated in dissection solution with 0.1% collagenase at 37° C for 20 minutes. 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 to 1.0 mm were dissected from each animal and mixed with 10 µl of sodium dodecyl sulfate (SDS) buffer (0.5 M 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 mM Tris, 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 µg of protein were resolved on a 4-20% gradient gel (Daiichi Pure Chemicals Co., 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 minutes, following overnight incubation with a polyclonal antibody for DDAH I or DDAH II (11) or eNOS (Santa Cruz Biotechnology, Inc., Santa Cruz, USA) or mouse monoclonal antibody for PRMT-1 (Abcam, Cambridge, UK) or rabbit polyclonal for PRMT-5 (Abcam, Cambridge, UK) at 1:1,000 dilutions. After rinsing in TBST, membranes were incubated for 2 hours with a horseradish peroxidase (HRP)-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 mM diaminobenzidine (DAB) with

0.01% H2O2 and 3 mM NiCl2 for the detection of blots. The density of the bands was analyzed using NIH 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 µm) were dewaxed, incubated with 3% H2O2, and blocking serum, and thereafter with a polyclonal antibody against DDAH I or DDAH II in a 1:100 dilution. The sections were rinsed with TBST and a biotinylated secondary antibody against rabbit IgG (Dako) in a 1: 400 dilution. After rinsing with TBST, the sections were incubated with HRP-conjugated streptavidin solution (Dako). HRP labeling was detected using a peroxide substrate solution with 0.8 mM DAB 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+nitrite (NOx) were described in detail in our previous reports (31, 41). Methylarginines in serum were measured with high-performance liquid chromatography (HPLC) as previously described (6). Renal tissue angiotensin II concentration was assessed by RIA method (SRL, Tokyo, Japan) and corrected by the amount of protein in the kidney homogenate. Tissue incubation with angiotensin II

Kidney slices (200 µm) 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 Co.)

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supplemented with 10% fetal bovine serum (Gibco, UK) and antibiotic solution (100 U/mL penicillin G sodium, 100 µg/mL gentamicin, 100 µg/mL streptomycin, and 5 µg/mL amphotericin, Sigma Chemical Co.). Each kidney slice was cultured in 1 ml of medium in a separate well at 37 °C in a 5% CO2 incubator for 18 hours in the presence or absence of Ang II (10–9, 10–7, 10–5 M) and Ang II 10-7 with telmisartan (10–6 M). Tissue was processed for Western blot analysis after rinsing with PBS. Statistics Data are expressed as mean ± SEM. An analysis of variance with Bonferroni’s post hoc test was used for statistical comparisons of the physiological data with normal distribution. For the western blot densitometry data a non-parametrical test, Kruskal-Wallis test was used. P >0.05 was required for statistical significance. RESULTS Diabetes Parameter As reported in our previous studies in this STZ rat model at 4 weeks (31, 33-38) rats with DM had a significant decrease in body weight, increase in blood glucose to about 400 mg·dl-1, increased urinary volume and an increase in creatinine clearance without changes in mean blood pressure (MBP) measured under anesthesia (Table 1). These parameters were not changed significantly by two weeks of telmisartan administration in both either control or DM rats (Table 1). ADMA and SDMA

Four weeks of DM increased SADMA but did not change SSDMA. Consequently the serum ADMA/SDMA ratio increased significantly (Fig. 1). Two weeks of telmisartan normalized SADMA only in rats with DM and the serum ADMA/SDMA.

DDAH I and DDAH 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 (Gl), thick ascending limb of the loop of Henle (TAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), inner medullary collecting duct (IMCD), macula densa (MD), renal afferent arteriole (AA), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) (Fig. 2). Western blot analysis of microdissected nephron segments confirmed DDAH II protein expression in Gl, TAL, DCT, CCD and IMCD (Fig. 3 lower panel).

The renal expression of PRMT-1 and -5 were unaltered by DM but both were significantly reduced after 2 weeks of telmisartan administration to rats with DM 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 DM 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 DM as shown by immunohistochemistry (Fig. 5) and confirmed by Western blot (Fig. 6). Telmisartan reduced DDAH II expression in the kidney of DM but not in controls (Fig. 6).

Angiotensin II levels in the kidney were increased in DM (962±55

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vs. 637±125 pg/g protein, p<0.05). Telmisartan treatment reduced angiotensin II level in the kidney of DM (651±107, p<0.05 vs. DM) but not in control (869±44). Angiotensin II regulation of DDAH expression in the kidney

Incubation of slices of tissue from normal whole kidneys for 18 hours with graded concentrations of Ang II did not change renal DDAH I expression significantly. However, incubation with telmisartan in slices incubated with 10-7 Ang II increased DDAH I expression significantly above values with 10-7 M 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 M Ang II reduced DDAH II expression significantly (Fig. 7B). Kidney NOx and eNOS expression

Kidney NOx production was not changed significantly by DM but was increased in DM rats by telmisartan (Fig. 8). eNOS expression was increased at four weeks of diabetes confirming our previous report (30) it was reduced by telmisartan. DISCUSSION These studies have confirmed some reports that have shown that plasma ADMA is elevated in insulinopenia or DM (19-23) although we found normal levels in a Wistar-Furth STZ model (36). They have shown further that the elevated level can be normalized by two weeks of ARB administration. These studies disclose distinct sites of expression and regulation of DDAH I and DDAH II proteins in the adult rat kidney. Induction of four weeks of DM decreases the renal expression of DDAH I but increases the expression of DDAH II. These effects apparently depend on AT1-receptors since they are reversed by an ARB which also

reduces PRMT-1 and -5 expressions in the kidneys of DM rats below levels of normal rats. These changes are independent of blood pressure which was not altered by DM 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, that demonstrated a second DDAH isoform, termed DDAH II (11). We confirm our previous report that DDAH is expressed in the kidney at sites of NOS expression (16). 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 about 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 which 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 isoforms expression in diabetic kidney

The decrease in DDAH I expression and the increase in DDAH II expression in the kidneys of DM rats can be ascribed largely to Ang II acting on AT1-R since Ang II levels are increased in the kidney of these diabetic rats and these changes in DDAH I and II are reversed by two weeks of telmisartan administration.

The liver (45) and the kidney (6)

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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 DM, leading to an elevation in the serum ADMA: SDMA ratio. This is consistent with the finding of a reduction in renal DDAH I expression in DM which could account for decreased ADMA metabolism. Administration of an ARB to rats with DM 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 DM (31, 33, 37, 38). Oxidative stress in the diabetic rat 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 post-transcriptional modification by oxidative stress in addition to the transcriptional changes described in the study.

Kidney concentrations of NOx were unaffected by diabetes, despite increased eNOS expression. One explanation would be due to 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 constitutive NOS from generating NO to generating predominantly superoxide (47). Such a mechanism could explain the increase in kidney NOx in diabetic animals treated with telmisartan, despite reduced eNOS expression, since prolonged administration of an ARB reduces p47phox expression in the kidney tubules and reduces renal reactive oxygen species generation in this model of diabetes (31). Additionally the changes in renal NOx are in line with our findings of ADMA. Thus, an increase in ADMA and eNOS in diabetes might explain the unchanged level of renal NOx whereas a reduction in ADMA but an unchanged eNOS might explain an increase in renal NOx with telmisartan in diabetic rats Angiotensin 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 receptors decrease DDAH expression, or that AT2 receptors activation increase 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

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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 receptor 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 tubule, whereas DDAH II is expressed in the glomerulus, thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule, collecting duct and arterioles. Kidneys from rats with STZ-induced diabetes have enhanced tissue levels of Ang II and down-regulated DDAH I and up-regulated DDAH II protein levels. These can be ascribed to increased intrarenal Ang II signaling since AT1-receptor 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 nNOS 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-diabetic rats and patients with DM that renal blood flow is dependent on NO (51) that is largely derived from nNOS 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 PT 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 nNOS at that site from inhibition by cortical ADMA and permit NO-dependent afferent arteriolar vasodilatation. Further studies will be required to explore these speculations. ACKNOWLEDGEMENTS This work was supported by grants from the Japanese Society for the promotion of Science (JSPS) to Dr. Onozato (17-05229), the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant C2-14571014, C2-16590780) and Grant-in-Aid for Scientific Research from Japan Society for the Promotion of Science (C-19590938) to Dr. Tojo, and the NIDDK (DK-36079 and DK-49870) and the NHLBI (HL-68686) to Dr. Wilcox 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.

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

C (n=10) C+T (n=6) DM (n=10) DM+T (n=10)

Body weight (g) 258±6 232±13 217±10** 216±8**

Blood glucose 144±7 125±11 415±28**++ 431±28**++

(mg/dL)

MBP (mmHg) 92±4 77±6 83±6 78±5

Blood pH 7.38±0.2 7.37±0.2 7.39±0.3 7.37±0.4

Urinary volume 14±2 16±4 91±27** 94±22**

(ml/day)

Ccr 0.85±0.12 1.23±0.28 1.40±0.13** 1.48±0.11**

(ml/min/100gBW)

**p<0.01 vs C, ++p<0.01 vs C+T

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FIGURE LEGENDS Fig. 1. Serum concentrations of ADMA and SDMA. Mean ± SEM values, in control rat (C, n=10), control rat given telmisartan (C+T, n=6), diabetic rat (DM, n=10) and diabetic rat given telmisartan (DM+T, n=10). * p<0.05, vs C, +p<0.05, ++p<0.01 vs C+T. 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, B). DDAH II was localized in the glomeruli, distal convoluted tubule (DCT), cortical collecting duct (CCD) and inner medullary collecting duct (IMCD) (C, D, E), macula densa (MD) and renal vasculature (E, F). Bars, 50 µm. 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), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), inner medullary collecting duct (IMCD). Fig. 4. Western blot of PRMT-1 and PRMT-5 in the whole kidneys. Control rat (C, n=10), control rat given telmisartan (C+T; n=6), diabetic rat (DM, n=10) and diabetic rat given telmisartan (DM + T; n=10). *p<0.05, **p<0.01, ***p<0.001 vs C, +p<0.05, ++p<0.01, +++p<0.001 vs C+T. Fig. 5. Immunohistochemical expression of DDAH I or DDAH II in the kidney of control rat, control rat given the angiotensin receptor blocker telmisartan (C+T), diabetic (DM) rat and diabetic rat treated with telmisartan (DM+T). Bar, 200 µm. Fig. 6. Western blot of DDAH I or DDAH II in the whole kidneys. Mean ± SEM values for renal protein expression for DDAH I (Panel A) and DDAH II (Panel B) in kidneys from control rat (C, n=10), control rat given telmisartan (C+T, n=6), diabetic rat (DM, n=10) and diabetic rat given telmisartan (DM + T, n=10). *p<0.05, **p<0.01, ***p<0.001 vs C, +++p<0.001 vs C+T. 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 angiotensin II (Ang II) for 18 hours alone, or with telmisartan (10-7 M). N= 10 in each group. * P<0.05, ** p<0.01, *** p<0.001 vs. control; † p<0.05 vs. AngII 10-9M; ‡ p<0.05 vs. AngII 10-7M; § p<0.001 vs. AngII 10-5M. Fig. 8. Kidney tissue NOx and kidney eNOS expression. Mean ± SEM values in control rat (C, n=10), control rat given telmisartan (C+T, n=6) diabetic rat (DM, n=10) and diabetic rat given telmisartan (DM+T, n=10). Compared with control: * p<0.05; ** p<0.01; *** p<0.001.

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expression of ddah and prmt isoforms in the diabetic rat ... · 10/1/2007 · methylated by class...

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