Proteoglycans synthesized by human glomerular mesangial cells in culture

Human fetal kidney mesangial cells were cultured for 24 h in the presence of 3H-amino acids and [35S] sulfate and chased for 24 h in nonradioactive medium. Incubation medium and cell layer proteoglycans were purified twice by high performance liquid chromatography-DEAE chromatography followed by gel filtration chromatography. The major medium 35S-macromolecules were chondroitin/dermatan-35SO4 proteoglycans. A small, Sepharose CL-6B Kav 0.14 dermatan-35SO4 proteoglycan was detected in the labeling medium and was released into both the early (time 0-0.5 h) and late (6-24 h) chase media. It contained 38 kDa 4-sulfated 35S-GAGs with a high content of iduronic acid and a 45-kDa protein core. A protein core of similar molecular weight was detected in the culture medium by Western analysis using antibodies to biglycan or proteoglycan-I (Fisher, L. W., Termine, J. D., and Young, M. F. (1989) J. Biol. Chem. 264, 4571-4576). This 35S-proteoglycan was not detected in the cell layer. However, a small dermatan-35SO4 with little or no protein core was present in the intracellular compartment. A large, Sepharose CL-6B excluded chondroitin-35SO4 proteoglycan was released into the culture medium and was detected between 6 and 24 h in chase medium. It eluted near the void volume of both associative and dissociative Sepharose CL-4B columns. It contained 30-kDa 4- and 6-sulfated 35S-GAGs and a 253-kDa protein core. A chondroitin-35SO4 proteoglycan with similar sized 35S-GAGs was detected in both the detergent-soluble and insoluble cell layer compartments. A Sepharose CL-6B Kav 0.11 heparin-35SO4 proteoglycan with a 220-kDa protein core and 38-kDa 35S-GAGs was rapidly released from the cell layer. This proteoglycan was larger than that previously described in isolated rat glomeruli or glomerular basement membranes, but had a core protein similar in size to one previously detected in these tissues. A larger heparan-35SO4 proteoglycan with larger 35S-GAGs was present in the detergent-insoluble cell layer compartment. The proteoglycans released by glomerular mesangial cells in culture resembled those synthesized by aortic smooth muscle cells in culture or extracted from aorta, supporting the notion that these cells are of vascular origin.     

Lipoprotein lipase is expressed by glomerular mesangial cells

Lipoprotein lipase expressed by the vasculature plays a key role in atherogenesis by enhancing the binding and uptake of lipoproteins and, thereby, leading to the formation of lipid-loaded foam cells. Hyperlipidemia also accelerates the progression of glomerular diseases and addition of exogenous lipoprotein lipase to mesangial cells has been shown to lead to an enhanced binding of lipoproteins to these cells. Despite such potential importance, the expression of endogenous lipoprotein lipase by cells of the glomeruli has, as yet, not been investigated. We show here for the first time that mesangial cells, but not epithelial cells, express lipoprotein lipase. The minimal lipoprotein lipase gene promoter was active in mesangial cells and inhibited by interferon-gamma, which is known to suppress its expression.     

Up-regulation of Bcl-2 by redox signals in glomerular mesangial cells

The mediators nitric oxide (NO) and superoxide (O2-) are known to regulate cell death and survival. In mesangial cells (MC), NO induced apoptosis and in higher concentrations necrosis. Intriguingly, cogeneration of NO and O2- in a balanced ratio promoted cell protection. Under these conditions, we noticed the accumulation of the anti-apoptotic protein Bcl-2. Its up-regulation is based on an increase in mRNA and protein level. To investigate whether oxidative stress elicits Bcl-2 expression in general, we further used the chemically unrelated oxidative agents diamide and butyl hydroperoxide. Both stimulated mRNA and protein up-regulation of Bcl-2. But in contrast to diamide, butyl hydroperoxide evoked apoptosis and necrosis despite Bcl-2 accumulation. As diamide was non-toxic, we used diamide as a Bcl-2 activator to protect MC against a subsequent toxic dose of NO. We conclude that redox changes promote Bcl-2 up-regulation that may confer cellular protection towards apoptosis.     

IGF-1 induces foam cell formation in rat glomerular mesangial cells.

When rat glomerular mesangial cells (MCs) are cultured with IGF-1 they accumulate intracellular lipid and take on foam cell morphology. These changes were characterized by electron microscopy and Nile red staining. To define the mechanism responsible for IGF-1-mediated lipid uptake, MCs were evaluated for endocytosis, scavenger receptor activity, and receptor-mediated uptake by the LDL receptor. Lipid accumulation was markedly increased when MCs were cultured with IGF. The primary route of uptake was through enhanced endocytosis. Lipid-laden MCs have decreased phagocytic capacity and disrupted cytoskeletons. These data show that IGF-1 induces MC to take on a foam cell morphology and that lipid-laden MCs have impaired phagocytic function.     

Cystatin C secretion by rat glomerular mesangial cells: autocrine loop for in vitro growth-promoting activity.

Cystatin C, the major inhibitor of the cysteine proteinases found in human and rat body fluids, is particularly abundant in seminal plasma and cerebrospinal fluid. In a precedent report, we have evidenced noteworthy levels of cystatin C in rat kidney cortex. In the present study, we show that rat mesangial glomerular cells produce cystatin C. Immunoprecipitation of extracts of metabolically labeled cells and culture media showed that the synthesized cystatin C is a 15.5 +/- 0.5 kDa protein. The protein was released into the culture supernatant (1.6 +/- 0.26 micrograms/10(6) cells/24 h). Urinary rat cystatin C and PPPR synthetic peptide (5-8 N-terminal sequence of rat cystatin C) increased mesangial cell proliferation. Affinity chromatography on Ultrogel-avidin-biotin-PPPR of extracts of metabolically labeled cells indicate the existence of a PPPR binding protein of 46 kDa. The results described in this work suggest, for glomerular rat mesangial cells in vitro, an autocrine regulation of proliferation by cystatin C.     

Sphingosine kinase-1 pathway mediates high glucose-induced fibronectin expression in glomerular mesangial cells

Diabetic nephropathy is characterized by accumulation of glomerular extracellular matrix proteins, such as fibronectin (FN). Here, we investigated whether sphingosine kinase (SphK)1 pathway is responsible for the elevated FN expression in diabetic nephropathy. The SphK1 pathway and FN expression were examined in streptozotocin-induced diabetic rat kidney and glomerular mesangial cells (GMC) exposed to high glucose (HG). FN up-regulation was concomitant with activation of the SphK1 pathway as reflected in an increase in the expression and activity of SphK1 and sphingosine 1-phosphate (S1P) production in both diabetic kidney and HG-treated GMC. Overexpression of wild-type SphK1 (SphK(WT)) significantly induced FN expression, whereas treatment with a SphK inhibitor, N,N-dimethylsphingosine, or transfection of SphK1 small interference RNA or dominant-negative SphK1 (SphK(G82D)) abolished HG-induced FN expression. Furthermore, addition of exogenous S1P significantly induced FN expression in GMC with an induction of activator protein 1 (AP-1) activity. Inhibition of AP-1 activity by curcumin attenuated the S1P-induced FN expression. Finally, by inhibiting SphK1 activity, both N,N-dimethylsphingosine and SphK(G82D) markedly attenuated the HG-induced AP-1 activity. Taken together, these results demonstrated that the SphK1 pathway plays a critical role in matrix accumulation in GMC under diabetic condition, suggesting that the SphK1 pathway could be a potential therapeutic target for diabetic nephropathy.     

Hyperosmolarity induced by high glucose promotes senescence in human glomerular mesangial cells

Hyperglycemia is involved in the diabetic complication of different organs and can elevate serum osmolarity. Here, we tested whether hyperosmolarity promoted by high glucose levels induces cellular senescence in renal cells. We treated Wistar rats with streptozotocin to induce diabetes or with consecutive daily injections of mannitol to increase serum osmolarity and analyzed p53 and p16 genes in renal cortex by immunohistochemistry. Both diabetic and mannitol treated rats showed a significant increase in serum osmolarity, without significant signs of renal dysfunction, but associated with increased staining for p53 and p16 in the renal cortex. An increase in p53 and p16 expression was also found in renal cortex slices and glomeruli isolated from healthy rats, which were later treated with 30 mM glucose or mannitol. Intracellular mechanisms involved were analyzed in cultured human glomerular mesangial cells treated with 30 mM glucose or mannitol. After treatments, cells showed increased p53, p21 and p16 expression and elevated senescence-associated β-galactosidase activity. Senescence was prevented when myo-inositol was added before treatment. High glucose or mannitol induced constitutive activation of Ras and ERK pathways which, in turn, were activated by oxidative stress. In summary, hyperosmolarity induced renal senescence, particularly in glomerular mesangial cells, increasing oxidative stress, which constitutively activated Ras-ERK 1/2 pathway. Cellular senescence could contribute to the organ dysfunction associated with diabetes.     

Co-culture of glomerular epithelial cells and mesangial cells on collagen-gauze-fiber gel

A novel collagen-gauze-fiber gel was created as a scaffold for the co-culture of renal glomerular epithelial cells and mesangial cells at its opposite sides. This gauze-fiber-gel provides a mimic environment like that of renal glomeruli in vivo. The cell morphology, cell growth and cell viability were investigated and the results showed that this novel scaffold maintains cell growth and cell viability without changing cell morphology for more than 3 weeks. Interestingly, glomerular epithelial cells co-cultured with mesangial cells on the gauze-fiber gel resulted in the polarity formation which usually appears on the normal epithelial cells existing at glomerular basement membrane in vivo, but seldom appears on the epithelial cells when cultured in vitro.     

Bioflavonoid quercetin inhibits mitosis and apoptosis of glomerular cells in vitro and in vivo

Bioflavonoids have been regarded as therapeutic agents for a wide range of disease including inflammation. In this report, we investigated effects of bioflavonoid quercetin on mitosis and apoptosis of glomerular cells in vitro and in vivo. Serum-stimulated rat mesangial cells were treated with or without quercetin, and total cell number, percentages of mitotic cells, and incorporation of [(3)H]-thymidine were evaluated. All three assays showed that mitogenic activity of mesangial cells was markedly attenuated by quercetin. To examine the effect of quercetin on apoptosis, mesangial cells were pretreated with or without quercetin and stimulated by hydrogen peroxide or tumor necrosis factor-alpha. Hoechst staining and DNA ladder assay showed that both apoptotic responses were dramatically inhibited by quercetin. We further investigated effects of quercetin on in vivo mitosis and apoptosis of glomerular cells. Rats were administered with or without quercetin intraperitoneally, and nephrotoxic serum nephritis was induced. Immunohistochemical analyses showed that treatment with quercetin significantly reduced the number of proliferating cell nuclear antigen (+) cells and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (+) cells in the glomerulus. These data suggested that quercetin has the potential for inhibiting mitosis and apoptosis of glomerular cells both in vitro and in vivo.     

Vasoconstrictor hormones depolarize renal glomerular mesangial cells by activating chloride channels

Mesangial cells are smooth muscle-like cells of the renal glomerulus which contract and produce prostaglandins in response to vasopressin and angiotensin. These responses serve to regulate the glomerular capillary filtering surface area. We have used the membrane potential-sensitive fluorescent dye bis-oxonol and the intracellular fluorescent calcium-sensitive probe Indo-1 to study the changes in membrane potential (Em) and intracellular free calcium concentration ([Ca2+]i) in cultured rat mesangial cells in response to vasoconstrictor hormones. Basal [Ca2+]i was 227 +/- 4 nM, and stimulation by maximal concentrations of either vasopressin or angiotensin resulted in a transient 4-6-fold rise. Resting membrane potential was 45.8 +/- 0.9 mV and vasoconstrictor hormones caused a depolarization of 14-18 mV. The following extracellular ion substitutions indicated that chloride efflux was the predominant ion flux responsible for depolarization: 1) depolarization persisted when sodium in the medium was substituted with N-methylglucamine; 2) substitution of medium sodium chloride with sodium gluconate, which enhances the gradient for chloride efflux, augmented vasoconstrictor-stimulated depolarization; 3) suspension of cells in potassium chloride medium resulted in depolarization, following which, stimulation by either vasopressin or angiotensin resulted in hyperpolarization; and 4) this hyperpolarization did not occur when potassium gluconate medium was used to depolarize the cells. The calcium ionophore ionomycin also resulted in membrane depolarization. However, prevention of the rise in [Ca2+]i by prior exposure to ionomycin in calcium-free medium or by loading mesangial cells with the intracellular calcium buffer BAPTA did not abrogate the depolarization response to vasoconstrictor hormones. This indicates that a rise in intracellular calcium is not necessary for depolarization. In contrast, prior depolarization of the cells using varying concentrations of KCl in the external medium, which dissipated the electrochemical gradient for chloride efflux, resulted in a corresponding prolongation of the transient calcium response to vasopressin and angiotensin. These findings indicate that angiotensin and vasopressin depolarize mesangial cells by activating chloride channels and that this activation can occur by both calcium-dependent and -independent mechanisms. In addition, activation of chloride channels with resulting depolarization may serve to modulate the calcium signal.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Cellular and molecular actions of adrenomedullin in glomerular mesangial cells.

Adrenomedullin (AM), a potent vasodilatory and hypotensive peptide produces several biological outcomes in glomerular mesangial cells. Mesangial cells are important in the pathogenesis of glomerulonephritis, and therefore the actions of AM on mesangial cells have important clinical and therapeutic implications. This minireview describes the various actions of AM on mesangial cell function and the signal transduction mechanisms involved. As in other systems, most actions of AM can be explained by increase in cAMP levels in the cell, although a few exceptions remain. The fact that most data obtained to date has been in culture, the physiological significance of the actions of AM in mesangial cells is discussed.