Bioactive lysophospholipids and mesangial cell intracellular signaling pathways: role in the pathobiology of kidney disease.

Lysophosphatidic acid (LPA), lyso-phosphatidylcholine (LPC), and sphingosine-1-phosphate (S1P) are major biologically active lysophospholipids (LPLs) that are produced by activated platelets, monocyte/macrophages, and many types of mammalian cells. LPLs have been shown to induce a wide array of physiological and pathophysiological properties including cellular differentiation, proliferation, migration, extracellular matrix deposition, change in morphology, and chemotactic responses. The recent cloning and identification of G protein-coupled receptors as specific receptors for LPLs created a great deal of interest in LPLs signaling and diverse biological responses. The pathobiological role of LPLs has been implicated in a number of pathological states and human diseases including atherosclerosis, glomerulosclerosis, post-ischemic renal failure, polycystic kidney disease, and ovarian cancer. Although the research in this area is growing at an enormous rate, this review is specifically focused on the recent understanding of the pathophysiological properties of LPA and LPC with special reference to kidney diseases, and their specific G-protein-coupled receptors and intracellular signaling pathways.

Atherogenic lipoproteins and tyrosine kinase mitogenic signaling in mesangial cells.

BACKGROUND: Mesangial hypercellularity is a critical early histopathological finding seen in human and experimental glomerular diseases. Hyperlipidemia and the glomerular deposition of atherogenic lipoproteins [for example, low-density lipoprotein (LDL) and its oxidized variants, minimally oxidized/modified LDL (mm-LDL)] are commonly associated with mesangial hypercellularity and the development of glomerular disease. This article reviews signal transduction pathways involved in cell proliferation and provides evidence for the participation of atherogenic lipoproteins in intracellular signaling pathways for mesangial cell proliferation. The mitogenic intracellular signaling pathways are regulated by the activation of a series of transmembrane and cytoplasmic protein tyrosine kinases that converge into the activation of Ras and downstream mitogen-activated protein (MAP) kinase. Activated MAP kinase, through translocating into the nucleus and the activation of various transcription factors and proto-oncogenes, regulates cellular proliferation. METHODS: Murine mesangial cells were stimulated with LDL and mm-LDL and were analyzed for the tyrosine kinase activity, phosphorylation of membrane proteins, activation of Ras and MAP kinase, and cell proliferation. RESULTS: The results indicated that the stimulation of mesangial cells with LDL and, with greater activity, mm-LDL induced the phosphorylation of membrane platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors, activated Ras, and resulted in sustained (up to 24 hr) activation of MAP kinase. LDL/mm-LDL-mediated mesangial cell proliferation and MAP kinase activation were dependent on the activation of tyrosine kinases. CONCLUSIONS: We suggest that the accumulation of LDL and more potently its oxidized forms within the glomerulus, through the activation of membrane receptor tyrosine kinases, activate the Ras and MAP kinase signaling cascade leading to DNA synthesis and subsequent cell proliferation.

Atherogenic lipoproteins enhance mesangial cell expression of platelet-derived growth factor: role of protein tyrosine kinase and cyclic AMP-dependent protein kinase A.

Mesangial cell proliferation and extracellular matrix accumulation are fundamental in the pathogenesis of glomerulosclerosis. Platelet-derived growth factor (PDGF) is a major cytokine involved in mesangial cell proliferation, and its increased expression is seen in glomerular injury. Atherogenic lipoproteins stimulate mesangial cell proliferation and induce glomerular injury in experimental animals. We examined the effect of low-density lipoprotein (LDL) and its more atherogenic oxidized forms, minimally modified LDL (mm-LDL) and oxidized LDL (ox-LDL) on mesangial cell PDGF mRNA expression. Incubation with 2.5 to 25 microg/ml LDL or mm-LDL for 1 to 4 hours stimulated mesangial cell PDGF mRNA expression (mm-LDL 2 to 3 times greater than LDL); ox-LDL had no effect. Similarly, both LDL and mm-LDL induced mesangial cell DNA synthesis (mm-LDL 1.5 to 2 times greater). In further studies evaluating key associated intracellular signal transduction mechanisms, the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein markedly decreased basal and lipoprotein-induced PDGF mRNA expression. Both pertussis toxin and isoproterenol, cyclic AMP-generating substances, stimulated PDGF mRNA expression. Preincubation with H-8 or H-89, cyclic AMP-dependent protein kinase A (PKA) inhibitors, blocked the lipoprotein-induced PDGF message, whereas preincubation with calphostin C, a protein kinase C inhibitor, did not alter LDL- or mm-LDL-mediated PDGF mRNA expression. These data suggest that the accumulation of atherogenic lipoproteins and their endogenous oxidized forms within the glomerulus may regulate mesangial cell PDGF expression and related cellular responses. These events appear to be modulated by signal transduction pathways involving PTK and PKA.

Atherogenic lipoproteins enhance murine cortical epithelial cell fibronectin protein synthesis and gene expression.

Tubulointerstitial changes, characterized by the accumulation of extracellular matrix proteins (ECM) and fibrosis, are often associated with primary glomerular injury. Furthermore, these changes may be better prognostic indicators for decline in renal function than the anatomical changes seen within the glomerulus itself. Although hyperlipidemia and the increased renal accumulation of atherogenic lipoproteins are commonly seen in both human and experimental models of renal disease, the possible role that atherogenic lipoproteins may play in the cellular and molecular events associated with the development of tubulointerstitial injury remains unclear. Since atherogenic lipoproteins have been shown to be mediators of renal injury, we examined the effects of native LDL and oxidatively-modified LDL (ox-LDL, a more atherogenic form of LDL) on fibronectin protein synthesis and gene expression in proximal tubular epithelial cells (TEC). Human LDL was freshly isolated and ox-LDL prepared by incubation of LDL with 100 microM CuS04. Incubation of TEC with LDL or ox-LDL (25-50 micrograms/ml) for 24 h increased the steady-state mRNA expression of fibronectin by 16-135% over control as measured by Northern blot analysis and the effect was greater with ox-LDL than native LDL. Additional studies were done to examine whether the increased fibronectin message in response to lipoprotein activation was translated into TEC protein synthesis. The activation of TEC by LDL or ox-LDL stimulated the synthesis and secretion of fibronectin (52-150%, over control) as measured by Western blot analysis. The data show that LDL and ox-LDL stimulate TEC fibronectin gene message and protein synthesis supporting a pathobiological role for these atherogenic lipoproteins in tubulointerstitial fibrosis.

Effect of serum subfractions from peritoneal dialysis patients on Hep-G2 cell apolipoprotein A-I and B metabolism.

We previously showed that uremic serum subfractions isolated from hemodialysis (HD) patients inhibited the production of apolipoprotein (apo) A-I by human hepatoblastoma cells, Hep-G2. Because of the reported differences in atherogenic cardiovascular mortality between HD and peritoneal dialysis (PD) patients, we examined the effect of similar subfractions from PD patients on apo A-I and apo B synthesis. After obtaining informed consent, serum samples from five normal subjects and nine stable PD patients were applied to Sephadex G-25 columns to obtain the serum subfractions used in the various experiments. Sephadex G-25 chromatograms of PD sera showed a broad peak from fractions 30 through 60 (molecular wt 500 to 2000 Da). Control serum showed no peak in this region. PD serum subfractions decreased apo A-I synthesis, secretion, and apo A-I mRNA expression by Hep-G2 cells when compared to subfractions from control subjects. Cholesterol efflux studies showed that conditioned media obtained from Hep-G2 cells incubated with PD serum subfractions inhibited cholesterol efflux from fibroblasts, suggesting a biologically-significant decrease in apo A-I synthesis. PD serum subfractions increased protein synthesis and mRNA expressions of apo B by Hep-G2 cells. Therefore, serum subfractions obtained from PD patients decreased apo A-I and increased apo B synthesis, findings consistent with their serum lipoprotein profiles suggesting that a biologically-active component in these subfractions could contribute to the risk of atherogenic cardiovascular disease in PD.

Uremic serum subfraction inhibits apolipoprotein A-I production by a human hepatoma cell line.

Abnormalities in lipoprotein metabolism are common in uremic patients and may represent an additional risk factor for the development of atherosclerosis. Despite the frequent occurrence of lipoprotein abnormalities, the role of various serum toxins and subfractions that accumulate in uremic patients on lipoprotein metabolism is not clearly understood. This study addressed the role of uremic toxins on lipoprotein metabolism by examining the effect of a 500 to 2,000-d subfraction obtained from the serum of uremic and control subjects on the synthesis of apolipoprotein (apo) A-I in a human hepatoma cell line (Hep-G2). Serum subfractions obtained from uremic patients inhibited apo A-I synthesis and secretion by Hep-G2 cells in a dose-dependent manner as measured by (3H)leucine incorporation into apo A-I, immunoprecipitation, and ELISA. The uremic serum subfraction decreased the mRNA expression for apo A-I in Hep-G2 cells when compared with controls. These observations suggest that a component of uremic serum can have the potential to inhibit hepatic apo A-I synthesis and may adversely influence high-density lipoprotein metabolism, thus increasing the risk for the development of atherosclerotic vascular complications in uremic patients.

Resolution and identification of the "subunits" of cytochrome C oxidase from mouse liver.

Mouse liver cytochrome c oxidase was purified to a specific heme a content of 9.3 nmol/mg protein and the constituent polypeptides separated on SDS-polyacrylamide gel electrophoresis. Using this system, the enzyme was resolved into 12 to 14 polypeptide bands.