Background-Exposure to risk factors such as hypertension or hypercholesterolemia decreases the bioavailability of endothelium-derived nitric oxide (NO) and impairs endothelium-dependent vasodilation. Recently, a circulating endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), has been detected in human plasma. The purpose of this study was to examine the relationship between plasma ADMA and atherosclerosis in humans. Methods and Results-Subjects (nϭ116; age, 52Ϯ1 years; male:female ratio, 100:16) underwent a complete history and physical examination, determination of serum chemistries and ADMA levels, and duplex scanning of the carotid arteries. These individuals had no symptoms of coronary or peripheral artery disease and were taking no medications. Univariate and multivariate analyses revealed that plasma levels of ADMA were positively correlated with age (PϽ0.0001), mean arterial pressure (PϽ0.0001), and ⌺ glucose (an index of glucose tolerance) (Pϭ0.0006). Most intriguingly, stepwise regression analysis revealed that plasma ADMA levels were significantly correlated to the intima-media thickness of the carotid artery (as measured by high-resolution ultrasonography). Conclusions-This study reveals that plasma ADMA levels are positively correlated with risk factors for atherosclerosis.Furthermore, plasma ADMA level is significantly correlated with carotid intima-media thickness. Our results suggest that this endogenous antagonist of NO synthase may be a marker of atherosclerosis.

Glomerulonephritis is an inflammation of the kidney characterized by the accumulation of extracellular matrix within the damaged glomeruli, impaired filtration and proteinuria. In its progressive form, the disease destroys kidney function leading to uraemia and death, unless dialysis therapy or kidney transplantation is available. The pathogenesis of glomerulonephritis is incompletely understood, but the eliciting factor is thought often to be an immunological injury to mesangial and/or other resident cells in the glomeruli. We have used an animal model of acute mesangial proliferative glomerulonephritis to show that this disease is associated with increased production and activity of transforming growth factor beta 1 (TGF-beta 1), an inducer of extracellular matrix production. Here we report that administration of anti-TGF-beta 1 at the time of induction of the glomerular disease suppresses the increased production of extracellular matrix and dramatically attenuates histological manifestations of the disease. These results provide direct evidence for a causal role of TGF-beta 1 in the pathogenesis of the experimental disease and suggest a new approach to the therapy of glomerulonephritis.

IntroductionGlomerular accumulation of extracellular matrix is a prominent feature of progressive glomerulonephritis. Previously, we have shown that transforming growth factor-,8 (TGF-ft) is unique among growth factors in regulating the production of the proteoglycans biglycan and decorin by glomerular mesangial cells in vitro. We now provide evidence of an elevated expression of TGF-,8, proteoglycans, and fibronectin in glomerulonephritis induced in rats by injection of anti-thymocyte serum (ATS). Glomeruli were cultured from rat kidneys at 1, 4, 7, 14, and 28 d after ATS administration. Increased proteoglycan synthesis was detected beginning on day 4, which peaked at a 4,900% increase compared with control on day 7, and returned toward control levels by day 28. The increased proteoglycan synthesis by cultured nephritic glomeruli, as well as that of fibronectin, were greatly reduced by addition of antiserum raised against a synthetic peptide from TGF-ft. Conditioned media from ATS glomerular cultures, when added to normal cultured mesangial cells, induced elevated proteoglycan synthesis that also peaked on day 7 and that mimicked the response to added exogenous TGF-ft. The stimulatory activity of the conditioned media was blocked by addition of TGF-,iantiserum. Prior addition of the immunizing peptide to the antiserum abolished the blocking effect. The main induced proteoglycans were identified as biglycan and decorin by immunoprecipitation with antiserum made against synthetic peptides from the proteoglycan core proteins. Glomerular histology showed mesangial matrix expansion in a time course that roughly paralleled both the elevated proteoglycan synthesis by the ATS glomeruli and the ability of the conditioned media from these glomeruli to induce proteoglycan synthesis.

Serial changes in urine protein, blood chemistry, and histology of the kidney were investigated in rats for 28 weeks after injections of adriamycin (ADR). Massive proteinuria, hypoalbuminemia, and hyperlipidemia were observed at week 4 and throughout the experiment. Both BUN and serum creatinine began to increase at week 16 and reached the uremic level at week 28. Light microscopic study of the kidney demonstrated a normal appearance at week 4, vacuole formation in glomerular tuft at weeks 8 and 12, focal and segmental glomerular sclerosis at weeks 16 and 20, and extensive glomerular sclerosis with tubulointerstitial degenerations at weeks 24 and 28. Immunohistologically, IgM with a small amount of IgG and C3 appeared in the sclerosing glomeruli from week 16. Aggregated human IgG, injected intravenously at week 24, had accumulated mainly in the glomeruli. Electron microscopy revealed degenerative changes of glomerular epithelial cells with small vacuoles in the cytoplasm at week 4. Size of vacuoles increased at the later stage. In conclusion, ADR produced chronic, progressive glomerular changes in rats, which led to terminal renal failure. The segmental glomerular sclerosis and IgM-dominant glomerular deposition in these animals are similar to pathological characteristics of focal and segmental glomerular sclerosis seen clinically.

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The present study suggests that AGE-RAGE-mediated ROS generation activates TGF-beta-Smad signaling and subsequently induces mesangial cell hypertrophy and fibronectin synthesis by autocrine production of Ang II. This pathway may provide an important link between metabolic and haemodynamic factors in promoting the development and progression of diabetic nephropathy.

Accumulation of glomerular extracellular matrix is a prominent feature of most forms of progressive glomerular disease. Since some growth factors may play a role in extracellular matrix production, we examined the effects of transforming growth factor-beta (TGF-beta), interleukin 1, platelet derived growth factor, and tumor necrosis factor on the production of extracellular matrix components by cultured rat mesangial cells. In control experiments we found that mesangial cells produced two distinct proteoglycans identified as the small chondroitin/dermatan sulfate proteoglycans biglycan (PG I) and decorin (PG II) by showing that their mobility on SDS-PAGE changed upon digestion by chondroitinase ABC, and that they reacted with antibodies raised against synthetic peptides from the core protein sequence of human biglycan and decorin. Exposure to TGF-beta for 48 hours stimulated an 8- to 10-fold increase in the biglycan and decorin bands, and induced a structural change detected as a shift in electrophoretic mobility. TGF-beta did not demonstrably affect the production of other matrix proteins by the mesangial cells. The other growth factors tested had no comparable effect on the production of proteoglycans or other extracellular matrix components by these cells. Our results show that TGF-beta is unique among growth factors in its regulatory effects on mesangial cell proteoglycan production. The release or activation of TGF-beta during glomerular injury could mediate the accumulation of proteoglycans in the extracellular matrix and predispose the kidney to development of glomerulosclerosis.

Nc,NG-dimethyl-L-argmme (ADMA) IS an endogenously synthesized mtnc oxide (NO) synthase mhibltor which has potent pressor/vasoconstnctor effects Dimethylargmmase metabolizes ADMA to L-citrullme and plays a key role m deterrmmng the m VIVO levels of ADMA To mvestlgate the role of ADMA m the pathogenesls of hypertension, we measured 24-hour urinary excretion of ADMA (UADMA) and nitrate/nitrite (NOx) m Dahl salt-sensitive hypertensive rats and spontaneously hypertensive rats (SHR) In Dahl salt-resistant rats, high-salt &et (8% NaCI) did not increase blood pressure and increased urinary NOx (P< 01) wlthout changes in UADMA cornpaled with lowsalt diet (0 3% NaCI) In contrast, m Dahl salt-sensitive rats, hlghsalt diet Increased blood pressure (P< Ol), did not change urinary NOx excretion, and Increased UADMA (P< 01) There was a slgmficant (Y= 65, P< 01) correlation between UADMA and the level of blood pressure m Dahl salt-sensltlve rats Plasma levels of NOx and ADMA and renal chmethylargmmase content were comparable among them These results may suggest that m Dahl salt-resistant rats, blood pressure 1s kept constant during high-salt intake, possibly due to the compensatory increased production of NO, and that m Dahl salt-sensitive rats, high-salt intake increases the production of ADMA, attenuates the compensatory Increase\ m NO, and increases blood pressure These results also suggest that the systemic production of ADMA 19 not dependent on renal dlmethylargmmase SHR had significantly greater urinary NOx excretion (P< 05) and smaller UADMA than Wlstar-Kyoto rats nously have suggested that the NO production 1s preserved m SHR * Furthermore, recent evidence demonstrated the increased but not decreased production of NO m the heart9.10 and aorta" of SHR The synthesis of NO can be inhibited experimentally by some analogues of argmme mcludmg L-NMMA and ADMA), both of which have equally potent vasoconstnctor and pressor actions 12 Acute admmlstratlon of ADMA into guinea pigsI and rats'4 causes blood pressure elevation partly via elevation of total peripheral resistance I4 In humans, mtra-arterial admmlstratlon of ADMA lowers forearm blood flow 1~15 Although both L-NMMA and ADMA are synthesized I6 and metabolized I7 endogenously, the plasma concentration of dlmethylargmme 1s ten times greater than that of L-NMMA 13 Since studies m ammals suggest that the kidney may be mvolved m the excretion lx and metabolism 1Y.*O of ADMA, abnormalities of the ADMA production or ehmmatlon have been reported m human kidney diseases 13 21 Although recent evidence demonstrated high accumulation of ADMA m plasma from hypercholesterolexmc ammals**J3 and m balloon-injured vessels,*4 the role of this endogenous NO synthase mhlbltor m the pathogenesls of hypertension has not been elucidated at all Accordmgly, we hypothesized that endogenous ADMA may play a role m salt-sensitive hypertension by competltlve inhibition of NO synthesis. To test this hypothesis, urinary and plasma ADMA and NOx were evaluated m Dahl rats on different salt intakes....