Functional characterization of podocan, a member of a new class in the small leucine-rich repeat protein family.

An important component of the extracellular matrix is the group of non-collagenous proteins belonging to the small leucine-rich repeat (SLR) protein family. A new SLR protein, podocan, with structural characteristics different from the known classes of the SLR protein family has been identified recently from the kidney. In this study, we examined the functional characteristics of this SLR protein expressed in cultured cells. Podocan was clearly observed intracellularly and was also detectable in the supernatant. Treatment of the expressed protein with various glycoenzymes suggested that podocan is a glycoprotein containing N-linked oligosaccharides but not a classical proteoglycan. Moreover, podocan was found to bind type 1 collagen. Cells transfected with podocan showed reductions in cell growth and migration, concomitant with increased p21 expression. Podocan mRNA was detected by reverse transcription polymerase chain reaction not only in the kidney, but also in other tissues including the heart and vascular smooth muscle cells, suggesting that podocan may have a potential role in growth regulation in cardiovascular tissues.

Extracellular matrix glycoprotein biglycan enhances vascular smooth muscle cell proliferation and migration.

Proteoglycans are produced and secreted by vascular smooth muscle cells, but the pathophysiological role of these glycoproteins in the vasculature is an enigma. Because the small leucine-rich proteoglycan (SLRP) biglycan is overexpressed in arteriosclerotic lesions, we produced mice constitutively overexpressing biglycan in the vascular smooth muscle, in order to examine the effects on vascular pathology. In the aorta and renal vasculature, increased vascular proliferation was seen both in the basal state and after infusion of angiotensin II (Ang II) in the transgenic mice compared with wild-type controls. In addition, the combination of biglycan overexpression and Ang II infusion resulted in marked increases in vascular smooth muscle cell proliferation and migration in the coronary arteries, as well as increases in fibrosis surrounding the vessels. In vitro, biglycan caused an increase in thymidine incorporation and migration of vascular smooth muscle cells, whereas these parameters were unchanged or reduced in endothelial cells. Moreover, addition of biglycan resulted in an increase in cdk2 expression and decrease in p27 levels in the vascular smooth muscle cells. These results suggest that this extracellular matrix SLRP may be involved in the regulation of vascular smooth muscle growth and migration through cdk2- and p27-dependent pathways. Furthermore, changes in biglycan expression could be a factor influencing the susceptibility of arteries to vascular injury, and may play a direct role in the pathogenesis of vascular lesions.

Hepatocyte growth factor regulates proteoglycan synthesis in interstitial fibroblasts.

BACKGROUND: Hepatocyte growth factor (HGF) is a clinically important growth factor with therapeutic potential for the treatment of interstitial fibrosis and chronic renal failure. Proteoglycans are components of the renal interstitium, which have multiple actions, including growth regulation. In this study, we examined the effects of HGF on proteoglycan synthesis in interstitial fibroblasts, and the mechanisms of these effects. METHODS AND RESULTS: Expression and agonist-induced activation of the HGF receptor c-Met was detected in rat renal interstitial fibroblasts (NRK-49F) by reverse transcription-polymerase chain reaction (RT-PCR) analysis and immune complex/immunoblot assay. Moreover, stimulation of the cells with HGF resulted in a marked increase (five- to tenfold) in phosphorylation of extracellular signal-related protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK), but not of c-Jun NH2 terminal kinase (JNK). Treatment with HGF resulted in a time- and dose-dependent increase (P < 0.01) in both cell-associated and secreted proteoglycan synthesis to two- to fourfold of control levels. This effect was attenuated by the MAPK/ERK kinase (MEK) inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Ion-exchange chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were up-regulated after HGF treatment. Northern blot, RT-PCR, Western blot, and promoter activity assays revealed that HGF caused a significant increase in decorin mRNA and protein, as well as in biglycan mRNA, protein, and promoter activity, suggesting transcriptional control of gene expression. Since the effects of biglycan on fibroblast proliferation are still unclear, the effects of biglycan were examined by thymidine assay, and biglycan was found to attenuate transforming growth factor-beta (TGF-beta)-induced changes in cell proliferation. CONCLUSION: These results suggest that HGF causes an increase in the small leucine-rich proteoglycans biglycan and decorin by ERK1/2- and p38 MAPK-mediated pathways in fibroblasts. These findings may be relevant for understanding potential mechanisms by which HGF can exert TGF-beta inhibitory actions in the kidney.

Spirits and gastrectomy increase risk for chronic pancreatitis in Japanese male alcoholics.

INTRODUCTION: Most cases of chronic pancreatitis are alcohol-related, but not all alcoholics develop pancreatitis. AIM: To elucidate historical and biologic risk factors for this disease. METHODOLOGY: Alcoholic Japanese men (n = 132) consecutively admitted to the National Alcoholism Center over 24 months, including 54 with chronic pancreatitis (diagnosed by endoscopic retrograde cholangiopancreatography) and 78 without, were surveyed about drinking history, smoking, education, and marital status, and tested for amylase, glycosylated hemoglobin, body mass, alcohol and aldehyde dehydrogenase genotypes, and K-ras gene mutations in pancreatic juice. RESULTS: Higher risk for chronic pancreatitis was associated with drinking spirits rather than lower-alcohol beverages (odds ratio [OR], 2.58; p= 0.01). Daily ethanol consumption by those who drank spirits was greater than that among those who drank lower-alcohol beverages; however, no differences in either daily ethanol consumption or duration of drinking were observed between alcoholics with and without chronic pancreatitis. Postgastrectomy patients were at higher risk for chronic pancreatitis than unoperated comparison subjects (OR, 4.35, P< 0.05). Elevated glycosylated hemoglobin (OR, 4.62, p< 0.01), decreased amylase (OR 4.20, P<0.02), and low body mass (OR 1.89, P<0.1) were more frequent in alcoholics with chronic pancreatitis. K- gene mutations existed in 18.8% of alcoholics with chronic pancreatitis but in only 11.4% of those without the disorder. The frequencies of alcohol and aldehyde dehydrogenase genotypes in alcoholics with and without pancreatitis did not differ significantly. CONCLUSION: Our study strongly suggested that spirits and partial gastrectomy increase the risk for chronic pancreatitis in male alcoholics.

Glucocorticoid regulation of proteoglycan synthesis in mesangial cells.

BACKGROUND: Proteoglycans are integral components of the mesangial matrix and glomerular permeability barrier. Recent studies have shown that changes in glomerular proteoglycan expression may play a major role in the pathogenesis of renal disease. Steroid hormones are used as first-choice therapy for the treatment of glomerular diseases, however, the effects of glucocorticoids on expression of glomerular proteoglycans are unknown. METHODS: This study examined the effects of in vitro and in vivo administration of dexamethasone on proteoglycan synthesis and gene expression of proteoglycan core proteins using rat (RMC) and human (HMC) mesangial cells. RESULTS: Treatment of cultured RMC with dexamethasone resulted in a dose- and time-dependent decrease (P < 0.05) in both cell-associated and secreted proteoglycan synthesis to approximately 50% of control levels. This effect was inhibited by the glucocorticoid antagonist mifepristone, and mimicked by prednisolone or corticosterone treatment. Separation of proteoglycans by ion-exchange and gel permeation chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were down-regulated after steroid treatment. Northern blot analysis, RT-PCR, Western blot, and promoter activity assays revealed that dexamethasone caused a significant decrease in decorin mRNA (to 61 +/- 8% of controls), whereas biglycan expression and promoter activity were increased after steroid treatment. A similar trend was found in glomeruli isolated from rats treated in vivo with dexamethasone. CONCLUSIONS: These results demonstrate that treatment of mesangial cells with steroids results in a decrease in total proteoglycan synthesis, as well as subtype-specific changes in proteoglycan core protein gene expression by transcriptional control, furthering our understanding of the effects of steroid treatment on the renal glomeruli.

Prepubertal treatment with angiotensin receptor blocker causes partial attenuation of hypertension and renal damage in adult Dahl salt-sensitive rats.

Recently, we have shown that treatment of stroke-prone spontaneously hypertensive rats with angiotensin inhibitors for a limited time-window before puberty results in an attenuation of hypertensive nephrosclerosis in later life. The aim of this study was to examine the applicability of this therapeutic paradigm to a low-renin model. Dahl salt-sensitive (Dahl-S) rats were fed a high-salt diet from age 6 weeks. Some rats were treated with the angiotensin receptor blocker (ARB) candesartan cilexetil (2 mg/kg/d) from weaning to puberty (age 3-10 weeks), whereas other rats were treated continuously until overt renal damage was seen (age 3-16 weeks). Dahl-S rats on a high salt diet had increased blood pressure (207 +/- 3 vs. 125 +/- 2 mm Hg), proteinuria, and glomerular/vascular histological changes. The prepubertal treatment with ARB resulted in a continued suppression of blood pressure (153 +/- 2 mm Hg) at 16 weeks. Both proteinuria and renal histological changes were significantly (p < 0.05) attenuated, but not completely prevented by the treatment. No significant differences in plasma renin activity, renin mRNA, or AT1/AT2 mRNA were seen between groups. These results suggest that prepubertal treatment affords sustained renoprotection, even in an animal model with a suppressed renin-angiotensin system, and support the notion that appropriate prepubertal intervention may lead to a partial attenuation in the susceptibility to inherited renal diseases.

Regulation of vascular proteoglycan synthesis by angiotensin II type 1 and type 2 receptors.

Recent studies have shown that proteoglycans play an important role in the development of vascular disease and renal failure. In this study, the effects of angiotensin II (AngII) type 1 (AT1) and type 2 (AT2) receptor stimulation on glycosaminoglycan and proteoglycan core protein synthesis in vascular smooth muscle cells (VSMC) were examined. Treatment of AT1 receptor-expressing VSMC with AngII resulted in a dose-dependent and time-dependent increase (2- to 4-fold) in (3)H-glucosamine/(35)S-sulfate incorporation, which was abolished by pretreatment with the AT1 receptor antagonist, losartan. The effects of AngII were inhibited by the epidermal growth factor receptor inhibitor, AG1478, and the mitagen-activated protein kinase kinase inhibitor, PD98059, but not the protein kinase C inhibitors, chelerythrine and staurosporine. AngII treatment also resulted in significant increases in the mRNA of the core proteins, versican, biglycan, and perlecan. The effects of AT2 receptor stimulation were examined by retroviral transfection of VSMC with the AT2 receptor. Stimulation of the AT2 receptor in these VSMC-AT2 cells resulted in a significant (1.3-fold) increase in proteoglycan synthesis, which was abolished by the AT2 receptor antagonist, PD123319, and attenuated by pretreatment with pertussis toxin. These results implicate both AT1 and AT2 receptors in the regulation of proteoglycan synthesis and suggest the involvement of epidermal growth factor receptor-dependent tyrosine kinase pathways and G alpha i/o-mediated mechanisms in the effects of the two receptors.