Up-regulation of protease-activated receptor-1 in diabetic glomerulosclerosis

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Abstract

Patients with diabetes are under a hypercoagulable state leading to generation of thrombin. It is not known whether thrombin plays a role in the progression of diabetic nephropathy. We analyzed gene expression of two thrombin receptors, protease-activated receptor-1 (PAR-1) and PAR-4 in the kidney of diabetic db/db mice. Mice developed hyperglycemia from 7 to 10 weeks of age and showed renal abnormalities such as mesangial expansion and urinary albumin excretion at 10 weeks of age. PAR-1 mRNA was up-regulated in isolated glomeruli in db/db mice compared with age-matched db/m littermates, but PAR-4 mRNA was not. In situ hybridization studies showed that PAR-1 mRNA was detected mainly at the glomerulus, and that intensive signals were observed in mesangial cells and podocytes. The up-regulation of PAR-1 in glomeruli in diabetic mice may play a role in the progression of glomerulosclerosis and abnormal urinary albumin excretion in diabetic nephropathy.

Introduction

Thrombin is a key enzyme for blood coagulation. Thrombin generates fibrin and is a potent activator of platelets. Several studies have established that thrombin has direct effects on various cell types, including endothelial cells, smooth muscle cells, fibroblasts, and neural cells [1], [2], [3], [4]. To date, four members of the thrombin receptor (protease-activated receptor, PAR) family have been cloned [5]. Thrombin exerts most of its cellular effects via activation of at least three PARs (PAR-1, -3, and -4) by limited proteolytic cleavage of the N-terminal extracellular domain and unmasking of a tethered ligand. Among them, PAR-1 mRNA has been reported to be increased in human crescentic glomerulonephritis. Expression of PAR-1 in the kidney has been observed in glomerular mesangial cells and epithelial cells in human [6]. Results of an experimental model of crescentic glomerulonephritis in PAR-1-deficient mice suggested that PAR-1 contributed to inflammatory cell-mediated renal injury and glomerulosclerosis [7]. Thrombin also regulates renal hemodynamics. Thrombin or PAR-1 agonistic peptide causes contraction of the isolated renal artery in humans and mice [8], [9]. In the rat’s isolated kidney perfusion system, thrombin and PAR-1 agonistic peptide also induce renal vasoconstriction and a marked reduction in glomerular filtration rate [10].

Diabetic patients show enhanced activation of the blood coagulation system. This hypercoagulable state contributes to the high incidence of premature atherosclerosis and increased morbidity and mortality in diabetic patients [11]. In patients with diabetic nephropathy, markers of thrombin activity are elevated [12], [13]. The increases in urinary albumin excretion are associated with levels of plasma fibrinogen and thrombin–antithrombin III complex, a clinical marker of blood coagulability and thrombin activity [13]. From these observations, we hypothesized that a hypercoagulable state in diabetes might contribute to progression of diabetic nephropathy through the activation of thrombin receptors in the kidney.

In this study, we used db/db mice, a genetically diabetic rodent model of type II diabetes. The db/db mice develop hyperglycemia in association with insulin resistance and obesity, and have emerged as a suitable small animal model to investigate diabetic nephropathy [14]. The db/db mice develop abnormalities in renal glomerular morphology and function that parallel in nature and chronology the features of human diabetic nephropathy [15], [16]. We analyzed gene expression profiles of thrombin receptors in the kidney of diabetic db/db mice to investigate whether thrombin receptor expression was changed in diabetic nephropathy.

Section snippets

Materials and methods

Animals. Male diabetic db/db mice and their lean non-diabetic db/m littermates were obtained from Clea Japan (Tokyo, Japan). Mice were housed at the department of Banyu Pharmaceutical Co. Mice were housed in plastic cages in an air-conditioned room at 24 °C, fed on standard laboratory diet (CE-2, containing 0.5% NaCl; Clea Japan), and given water ad libitum. All animal experimental procedures followed the National Institutes of Health guidelines and were approved by the Banyu Animal Care and

Development of diabetes and renal abnormalities in db/db mice

At 6 weeks of age, the body weight of db/db mice was significantly greater than age-matched db/db mice (Table 1). Mean body weights progressively rose in db/db and db/m mice, but remained significantly greater in db/db mice compared with db/m mice at all timepoints. The mean blood glucose level in db/db mice was normal at 6 weeks of age, but was significantly elevated from 7 to 10 weeks of age in db/db mice as body weight increased, whereas glucose levels in db/m mice remained normal. In addition

Discussion

In this study, we found that PAR-1 mRNA was markedly up-regulated in diabetic db/db mice glomeruli at the early stage of progression of diabetes and glomerulosclerosis, whereas another signal transduction receptor of thrombin, PAR-4, was not changed. The distribution of PAR-1 mRNA expression in the kidney was observed mainly in glomeruli and interstitial peritubular cells. These observations suggest that thrombin and PAR-1 pathways are activated in the development of glomerulosclerosis in db/db

References (25)

  • M. Eto et al.

    Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways

    Circ. Res.

    (2001)
  • C.A. McNamara et al.

    Thrombin stimulates proliferation of cultured rat smooth muscle cells by a proteolytically activated receptor

    J. Clin. Invest.

    (1993)
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