Human Mesangial Cells In Culture Research Articles

Glomerular expression of myxovirus resistance protein 1 in human mesangial cells: Possible activation of innate immunity in the pathogenesis of lupus nephritis

Since viral infections activate type I interferon (IFN) pathways and cause subsequent release of IFN-dependent proinflammatory chemokines and cytokines, the innate immune system plays an important role in the pathogenesis of lupus nephritis (LN). It has been reported that human myxovirus resistance protein 1 (Mx1), a type I IFN-dependent transcript, acts against a wide range of RNA viruses. Although the expression of Mx1 in biopsy specimens obtained from patients with dermatomyositis and cutaneous lupus has been described, the expression of Mx1 in human mesangial cells (MCs) has remained largely unknown. We treated normal human MCs in culture with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expression of Mx1 by reverse transcription-polymerase chain reaction and western blotting. To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference against IFN-β. We also conducted an immunofluorescence study to examine mesangial Mx1 expression in biopsy specimens from patients with LN. Poly IC-induced Mx1 expression in MCs are shown both time- and dose-dependently, and RNA interference against IFN-β inhibited poly IC-induced Mx1 expression. Intense glomerular Mx1 expression was observed in biopsy specimens from patients with LN, whereas negative staining occurred in specimens from patients with IgA nephropathy or purpura nephritis. These preliminary observations support, at least in part, the theory of innate immune system activation in the pathogenesis of LN.

TLR4 signaling induces retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5 in mesangial cells

It is known that recognition of bacterial lipopolysaccharide (LPS) and various endogenous ligands by Toll-like receptor 4 (TLR4) induces inflammatory reactions. However, the role of TLR4 activation in mesangial inflammation remains to be elucidated. Retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are putative RNA helicases and are involved in immune and inflammatory reactions. The purpose of the present study was to investigate the implication of RIG-I and MDA5 in TLR4 signaling in mesangial cells. Normal human mesangial cells in culture were treated with LPS. Expression of RIG-I, MDA5, interferon-β (IFN-β), CXCL10 and CXCL8 was examined using real-time RT-PCR, Western blotting and ELISA. The cells were also subjected to RNA interference against TLR4, IFN-β, RIG-I or MDA5. LPS induced the expression of IFN-β, RIG-I, MDA5, CXCL8 and CXCL10 in human mesangial cells. RNA interference against either TLR4 or IFN-β inhibited LPS-induced RIG-I and MDA5 expression. Knockdown of TLR4, IFN-β, RIG-I or MDA5 resulted in decreased induction of CXCL10, while only TLR4 knockdown inhibited CXCL8 induction. TLR4 signaling induces the expression of RIG-I and MDA5 in mesangial cells. RIG-I and MDA5 may be involved in inflammatory reactions by regulating CXCL10 expression in the downstream of TLR4 signaling in human mesangial cells.

TNFα enhances TLR3‐dependent effects on MMP‐9 expression in human mesangial cells

The role of MMPs (matrix metalloproteinases) in kidney diseases has been widely accepted, where they can regulate inflammatory response because of their effects on both recruitment and survival of inflammatory cells. TNFα (tumour necrosis factor α) has also been implicated in the pathogenesis of inflammatory kidney diseases, including forms of glomerulonephritis associated with viral diseases. Previously, we established the functional linkage between viral receptors of the innate immune system, the TLRs (Toll-like receptors) and control of MMP activity in human MC (mesangial cells). Expression levels of MMP-2, MMP-7, MMP-9, TIMP-1 (tissue inhibitor of metalloproteinase 1) and TIMP-2 in human MC in culture were analysed by RT-PCR (reverse transcription-PCR). TNFα significantly enhanced the TLR3-dependent induction of MMP-9 in human MC. Expression levels of MMP-2, TIMP-1 and TIMP-2 were not significantly affected by the activation of TLR3 or TNFα stimulation. No significant MMP-7 expression was found. We conclude that the role of MMP-9 in chemotaxis, activation and proliferation of inflammatory cells is amplified by TNFα originating from infiltrating cells, especially monocytes, producing a regulatory loop that potentially leads to a self-propagating inflammation.

Quercetin suppresses NF-κB and MCP-1 expression in a high glucose-induced human mesangial cell proliferation model

Diabetic nephropathy (DN), which is characterized by mesangial cell proliferation, is a common complication observed in diabetic patients. The protective effects of quercetin for DN have been reported; however, the mechanism has yet to be determined. We aimed to identify the underlying mechanism for quercetin protection against DN. High glucose (HG)-induced human mesangial cell (HMC) proliferation, a feature of the early stages of diabetic nephropathy, was employed as an in vitro model. Cells were grown in normal glucose (5.6 mM), high glucose (30 mM) or high glucose with various concentrations of quercetin. Cell proliferation, cell cycle progression, and expression of NF-κB and MCP-1 were examined by MTT assay, DNA staining, immunocytochemistry and western blot analysis, respectively. HMCs cultured in high glucose had signficantly greater proliferation, accumulation in the G1 phase, upregulated NF-κB and MCP-1 expression. Quercetin treatment reversed the effects of high glucose in a dose-dependent manner. Cotreatment of quercetin with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB activation, suggest that the effects of quercetin are partially mediated by NF-κB signaling. Quercetin partially suppresses the effects of high glucose in HMC cultures, which are mediated at least in part through the suppression of NF-κB.

Retinoic acid-inducible gene-I (RIG-I) is induced by IFN-γ in human mesangial cells in culture: possible involvement of RIG-I in the inflammation in lupus nephritis

Interferon-gamma is a potent Th1-type cytokine and a key molecule in the pathogenesis of autoimmune diseases including lupus nephritis. Retinoic acid-inducible gene-I is a putative RNA helicase that plays an important role in immune and inflammatory reactions. We previously demonstrated the increased expression of the retinoic acid-inducible gene-I protein in the kidney tissue of patients with lupus nephritis, and the presence of a significant amount of retinoic acid-inducible gene-I mRNA in the urinary sediment of patients with this inflammatory renal disease. In the present study, interferon-gamma was found to induce the expression of retinoic acid-inducible gene-I in human mesangial cells in culture. Knockdown of retinoic acid-inducible gene-I inhibited the interferon-gamma-induced upregulation of interferon regulatory factor 7, a transcriptional factor involved in immune and inflammatory reactions. These findings suggest that retinoic acid-inducible gene-I produced by mesangial cells may be involved in the pathogenesis of lupus nephritis.

Retinoic acid-inducible gene-I is induced by double-stranded RNA and regulates the expression of CC chemokine ligand (CCL) 5 in human mesangial cells

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase involved in immune reactions against RNA viruses and various inflammatory and autoimmune diseases. The purpose of the present study was to investigate the role of RIG-I in glomerular diseases. We treated human mesangial cells in culture with polyinosinic-polycytidylic acid (poly IC), which is an authentic double-stranded RNA, and analysed the expression of RIG-I, CC chemokine ligand 5 (CCL5) and interferon (IFN)-β by western blotting, reverse transcriptase-polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA). To elucidate the poly IC-signalling pathway, we subjected the cells to RNA interference (RNAi) against RIG-I, IFN-β or Toll-like receptor (TLR) 3. Furthermore, we studied the effects of IFN-β receptor blocking and IFN-β overexpression. Poly IC induced the expression of RIG-I and CCL5 in human mesangial cells, and RNAi against RIG-I inhibited this poly IC-induced CCL5 expression. Poly IC-induced RIG-I expression was also inhibited by RNAi against IFN-β and by an antibody against the IFN-β receptor. IFN-β overexpression induced the expression of both RIG-I and CCL5. The knockdown of TLR3 abolished poly IC-induced RIG-I expression. The TLR3/IFN-β/RIG-I/CCL5 signalling pathway may mediate immune and inflammatory responses against viral infection in mesangial cells, suggesting the role of this pathway in the aggravation of glomerulonephritis due to viral infection.

Role of aldose reductase in the high glucose induced expression of fibronectin in human mesangial cells

Aldose reductase (AR) has emerged as a key contributor to the diabetic nephropathy (DN), however, the mechanisms by which AR increases DN remain poorly understood. Here, we report that exposure to high glucose (HG) stimulates fibronectin (FN) from human mesangial cells in culture. Our results show that exposure to HG and overexpression AR increase the expression of FN. This increase was prevented by the AR inhibitors sorbinil and zopolrestat. Treatment with HG and transfected with plasmid PcDNA3.0-AR, resulted in phosphorylation and activation of ERK, JNK and AKT signaling pathway, and increase the expression of FN. Treatment with inhibitor of JNK and AKT signaling pathway decreased the expression of FN. These results show that inhibition of AR may be useful to prevented extracellular matrix (ECM) deposition in diabetic nephropathy, which is regulated by JNK and AKT.

Macromolecular IgA1 taken from patients with familial IgA Nephropathy or their asymptomatic relatives have higher reactivity to mesangial cells in vitro

Multiple cases of IgA nephropathy (IgAN) may occur in families; we compared their prognosis to sporadic cases of this disease. We isolated macromolecular IgA1 from 60 patients with familial IgAN, 91 of their asymptomatic relatives, 43 patients with sporadic IgAN (SpIgAN), 90 of their asymptomatic relatives, and 43 healthy subjects. Compared with SpIgAN patients, those with multiplex familial IgAN (MpIgAN) had more advanced renal histopathology and more galactose-deficient macromolecular IgA1 in their serum. Further, when we tested the effects of the macromolecular IgA1 on human mesangial cells in culture, we found that the macromolecular IgA1 taken from familial clusters had enhanced binding to mesangial cells and caused increased expression of interleukin-6, tumor necrosis factor-alpha, and monocyte chemotactic peptide-1. The macromolecular IgA1 isolated from asymptomatic relatives caused increased cytokine expression in the mesangial cells when derived from MpIgAN compared with SpIgAN or healthy controls. While these studies suggest that macromolecular IgA1 isolated from patients with MpIgAN is more pathogenic than that from patients with SpIgAN, long term follow-up will be needed to clarify the risk in asymptomatic relatives of the patients with multiplex familial disease.

Role of matrix metalloproteinases in viral-associated glomerulonephritis

Viral infections are a major problem worldwide. Many of these infections are frequently complicated by a virus-associated glomerulonephritis. In glomerulonephritis, progression of renal failure is mainly attributed to the development of extensive glomerular and interstitial fibrosis. Advanced glomerular disease is characterized by the accumulation of extracellular matrix components in the mesangial matrix and glomerular basement membrane. These matrix components are metabolized by matrix metalloproteinases (MMPs) as well as tissue inhibitors of metalloproteinase (TIMPs). The expression of MMP2, MMP9 and TIMP-1 in human mesangial cells in culture was analysed by RT-PCR. Mesangial cells express the viral receptors toll-like receptor 3 and RIG-I. Activation of these viral receptors by viral RNA exemplified by poly (I:C) RNA leads to a time- and dose-dependent expression of MMP9 without affecting the expression of MMP2 and TIMP-1. To show the specific effect of viral receptors, knockdown experiments with siRNA specific for TLR3 and RIG-I were performed. This novel finding of the functional expression of these viral sensors on glomerular fibrosis may indicate a novel link between viral infections and glomerular inflammation and indicates a pathophysiologic role of viral receptors in these processes.

Critical Review of Prorenin and (Pro)renin Receptor Research

The renin-angiotensin system (RAS) plays an important role in cardiovascular and renal physiology and disease, and the benefits of angiotensin-converting enzyme inhibitor, angiotensin type 1 receptor blocker, and renin inhibitor therapies are mediated in part by their modification of the levels and actions of angiotensin peptides. Despite its long history, the RAS remains an active area of research. Contrary to the classical view of the RAS as an endocrine system whereby kidney-secreted renin acts on circulating angiotensinogen to produce angiotensin peptides in the circulation, it is now recognized that tissues are the main sites of angiotensin peptide formation by the action of plasma- (kidney-) derived renin on plasma-derived and locally synthesized angiotensinogen.1–3 A receptor for prorenin and renin was recently identified4,5 that is hereby referred to as the (pro)renin receptor. Prorenin and (pro)renin receptor research offers the exciting possibility of a new paradigm for the RAS whereby renin and prorenin binding to the (pro)renin receptor not only target and facilitate angiotensin generation but also lead to activation of (pro)renin receptor signal transduction pathways distinct from angiotensin II receptor signals.6,7 By revealing novel potential mechanisms of disease pathogenesis, this new paradigm offers the possibility of new therapies that may be more effective than those currently available. It also suggests that stimulation of the (pro)renin receptor by the increased renin and prorenin levels that accompany angiotensin-converting enzyme inhibitor, angiotensin type 1 receptor blocker, and renin inhibitor therapies may attenuate the benefits these therapies.7,8 Recent reviews and editorials have discussed this research.6,9,10 The purpose of this brief review is to critically assess the evidence for this new paradigm from the perspective of disease pathogenesis and to advocate caution in its interpretation. The available evidence presents contradictions and difficulties of interpretation that preclude any conclusion about the reality of …

Human glomerular mesangial IP15 cell line as a suitable model for in vitro cadmium cytotoxicity studies

Cadmium represents a major environmental pollutant that may induce severe damage, especially in the kidney where cadmium accumulates. While cadmium is known to severely impair renal tubular functions, glomerular structures are also potential targets. Owing to their contractile properties, glomerular mesangial cells play a major role in the control of glomerular hemodynamics and influence the ultrafiltration coefficient. Cell cultures provide alternative and fruitful models for study of in vitro toxicology. However, the use of primary human mesangial cell cultures is hampered by their limited survival span and their rapid dedifferentiation during passages. This study presents a human stable immortalized mesangial cell line, designated IP15. Cell characteristics were investigated by the detection of known mesangial markers, as well as their ability to contract in response to angiotensin II. IP15 cells were used to investigate cadmium uptake and morphological changes such as cell contraction and cytoskeleton protein expression. The IC(50) cytotoxicity index was obtained with 3.55 micromol/L using neutral red assay for 24 h. After cadmium exposure (1 micromol/L, determined as nonlethal concentration), 0.38 microg Cd/mg protein was internalized by the cells as evaluated by inductively coupled plasma optical emission spectrometry (ICP/OES). Cadmium induced a significant cell surface reduction that correlated with smooth-muscle alpha-actin disorganization. Thus, the IP15 cell line is a suitable model for study of in vitro cadmium cytotoxicity in mesangial cells and allows sufficient material to be obtained for future studies of the intracellular effects of cadmium exposure.

TGF-β1 induces COX-2 expression and PGE2 synthesis through MAPK and PI3K pathways in human mesangial cells

Transforming growth factor-beta1 (TGF-beta1) plays a fundamental role in the progression of renal diseases. Accumulating evidence has suggested that eicosanoids derived from cyclooxygenase-2 (COX-2) participate in a number of pathological processes in immune-mediated renal diseases. Mesangial cells (MC) play a major role in physiological and pathophysiological renal processes. MC express receptors for TGF-beta1, and COX-2 expression can be induced in MC. However, to date, there are no published data on the possible role of TGF-beta1 in COX-2 expression in human mesangial cells (HMC). We designed studies to determine (1) whether TGF-beta1 stimulates COX-2 expression in primary HMC, (2) whether mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades are involved in TGF-beta1-induced COX-2 expression, and (3) whether prostaglandin (PG)E2 synthesis is affected by TGF-beta1 and MAP kinases and PI3K activation. Studies were performed in primary cultures of HMC and in an immortalized line of HMC. TGF-beta1 induces COX-2 promoter activity and COX-2 mRNA and protein expression in HMC. COX-2 induction is accompanied by increased PGE2 synthesis. Extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, and PI3K pathway inhibition blunted TGF-beta1-induced COX-2 overexpression. We demonstrate that TGF-beta1 regulates COX-2 expression in HMC through the activation of ERK1/2, p38 MAPK, and PI3K. These results can help to elucidate the molecular mechanisms underlying the regulation of COX-2 and open up specific strategies for the treatment of glomerular disease.

Prorenin and Its Ancient Receptor

Prorenin, the precursor of renin, has for a long time been considered to be functionally inert, despite high circulating concentrations. Plasma levels of prorenin usually exceed renin levels and are even further increased under pathological conditions, such as in diabetes. First support of a functional significance of prorenin came from studies in transgenic rats, which overexpress a renin gene in the liver resulting in high circulating prorenin levels.1 Without systemic renin–angiotensin system activation and with normal blood pressure, these animals developed a vascular phenotype reminiscent of malignant hypertension. Moreover, in a series of elegant experiments, Ichihara et al2–4 supported the concept of prorenin as a relevant factor in the pathophysiology of diseases, such as hypertension and diabetes, by using a peptide that binds and inhibits the activation of the precursor. Thus, this peptide attenuated nephropathy in streptozotocin-induced diabetes2 and cardiac fibrosis in stroke-prone spontaneously hypertensive rats.3 By successfully repeating the streptozotocin experiment in mice deficient for the angiotensin II type 1a (AT1a) receptor, they reported recently that these effects are independent of renin activation or angiotensin II generation and concluded that the peptide, in fact, inhibits the interaction of prorenin with a receptor.4 In this issue of Hypertension , Saris et al5 contribute further evidence for an angiotensin-independent action of prorenin on cells by showing that neonatal cardiomyocytes respond to purified prorenin by p38 mitogen-activated protein (MAP) kinase phosphorylation and broad changes in gene expression partially also caused by the MAP kinase activation. Because the authors had shown before that these cells are not able to synthesize angiotensin II after the addition of prorenin, they consider the observed effects as being …

C-peptide increases the expression of vasopressin-activated calcium-mobilizing receptor gene through a G protein-dependent pathway

Although an increasing number of reports suggest that physiological concentrations of C-peptide protect against the development of diabetic nephropathy, possibly through the modulation of Na-K pump activity, the intracellular pathways controlled by C-peptide are still unrecognized. C-peptide and vasopressin share similar intracellular effects including the activation of calcium influx and endothelial nitric oxide synthase. Both hormones stimulate also the activity of Na-K pump activity. Whether the activity of C-peptide is mediated by the recently identified vasopressin-activated calcium-mobilizing receptor (VACM-1) has never been previously investigated. To clarify this issue, we evaluated the effect of C-peptide on VACM-1 RNA (measured by semiquantitative RT-PCR) and protein expression (measured by immunoblotting) in human skin fibroblasts (where a specific binding of C-peptide was demonstrated) and in human mesangial cells, the cellular target of diabetic nephropathy. C-peptide-induced activation of VACM-1 was demonstrated in fibroblasts from six healthy individuals (0.51+/-0.1 vs 1.48+/-0.4, arbitrary units+/-s.e., P = 0.025). This finding was paralleled by an increased VACM-1 protein expression (5.64+/-1.0 vs 8.47+/-1.2, arbitrary units+/-s.e., P= 0.043). Similar results were confirmed in three independent cultures of human mesangial cells. VACM-1 activation in fibroblasts was insensitive to phosphatidylinositol-3-kinase inhibitor LY294002, but was inhibited by pertussis toxin, suggesting that activation of VACM-1 could be mediated by a G protein-coupled receptor. This study demonstrates for the first time that C-peptide activates VACM-1, possibly through a G protein-coupled receptor. Further studies are needed to clarify whether VACM-1 is involved in the protective effect of C-peptide against the development of diabetic nephropathy.

P2Y-receptors stimulating the proliferation of human mesangial cells through the MAPK42/44 pathway.

1. Mesangial cell proliferation is observed in a number of kidney diseases. The sympathetic cotransmitter ATP is suspected to play a major role in proliferative processes. Therefore, the effects of exogenous ATP on human mesangial cells in culture were studied. 2. Fresh human kidney cortex was processed to obtain mesangial cells in culture. Effects of nucleotides on [(3)H]thymidine incorporation, the activation of mitogen-activated protein kinase and the cell number were studied. The involved P2-receptors were characterized pharmacologically. In addition, we searched for mRNA for P2Y- and P2X-receptors by RT-PCR. 3. ATP (0.1-300 micro M) and related nucleotides induced a significant increase in [(3)H]thymidine incorporation up to 220% of control. The adenine nucleotides ATP and ADP were about equally effective. Also ATP-gamma-S, UTP, ADP-beta-S and 2-m-thio-ADP induced a weaker response. UDP and alpha-beta-methylene-ATP failed to induce an effect on [(3)H]thymidine uptake. 4. ATP (100 micro M) induced a fast activation of the MAPK(42/44) pathway. The effects of ATP on MAPK(42/44) activation and [(3)H]thymidine incorporation were reduced by the MAPK inhibitor PD 98059. Platelet-derived growth factor (PDGF 5 ng ml(-1)) increased the cell number to more than 122% of control. ATP (10 micro M) on top of PDGF amplified PDGF induced cell proliferation to 136% of control. 5. RT-PCR products for P2Y(1,2,4,6,11,12)- and P2X(1,2,4,5,6,7)-receptor subtypes were detected in human mesangial cells. 6. ATP has mitogenic effects on human mesangial cells. DNA synthesis is increased by the activation of the MAPK(42/44) pathway. ATP amplifies PDGF-induced cell hyperplasia.

CTGF mediates TGF-beta-induced fibronectin matrix deposition by upregulating active alpha5beta1 integrin in human mesangial cells.

Excessive deposition of fibronectin in the glomerular mesangium in diabetic nephropathy (DN) is partly due to the induction of transforming growth factor-beta (TGF-beta) by high glucose. TGF-beta induces its downstream mediator connective tissue growth factor (CTGF), which stimulates fibronectin matrix synthesis, a process that requires the presence of alpha5beta1 integrin. Although TGF-beta has been shown to upregulate alpha5beta1 integrin expression in human mesangial cells (HMC), little is known about the effect of CTGF on levels of this receptor. This study tested whether CTGF modulates alpha5beta1 expression by HMC in culture and whether changes induced by TGF-beta are mediated through the induction of CTGF. FACS analysis showed that both TGF-beta and CTGF significantly increased cell-surface alpha5beta1 levels compared with basal conditions. RT-PCR indicated that the changes were at the level of transcription. Treatment of cells with TGF-beta and antisense CTGF oligonucleotides significantly reduced the TGF-beta-induced increases in alpha5beta1 levels. CTGF and TGF-beta also significantly increased levels of ligand-occupied cell-surface beta1 integrins and cell adhesion to fibronectin, the main alpha5beta1 substrate. Antisense CTGF significantly reduced the number of adherent cells from TGF-beta-stimulated cultures. Finally, alpha5beta1 blocking antibodies inhibited HMC fibronectin matrix deposition, confirming the importance of this receptor for this process. Taken together, these data provide evidence that CTGF controls alpha5beta1 expression by HMC in vitro. Alterations in alpha5beta1 levels induced by TGF-beta are mediated at least in part through the induction of CTGF, and specific targeting of either alpha5beta1 or CTGF could be useful in controlling excessive fibronectin matrix production in DN.

Surface antigens of human mesangial cells: impact of growth surface or IL-1alpha.

The interactions of mesangial cells (MC) with their environment are important events in glomerular physiology and pathology, yet a detailed characterization of the MC-surface antigens mediating these interactions is still lacking. In this study, a comparative phenotype analysis of primary human MC in culture using 191 monoclonal antibodies directed against 108 antigens was performed by flow-cytometry. The MC were grown on three different surfaces (human matrix, fibronectin, polystyrene) and cultured in the presence or absence of IL-1alpha. Seventy-one antibodies recognizing 35 different antigens (integrins: CD29, 49b, 49c, 49e, 51, 61; immunoglobulin gene family: CD54, 58, 90, 106, 146, 147, 166; growth factor receptors: CD105, 140b; apoptosis related: CD95; hemostatis related: CD141, 142; miscellaneous: CD44, 109, 138, 151, 157, 165, and 11 nonclustered antigens) reacted with mesangial cells. CD58, 109, 146, 147, 151, 157, 165, and 166 are reported for the first time to be present on human mesangial cells. In comparison to growth on polystyrene, CD44, 54, 95, 105, 109, 140b, 146, 147, 157, 165 and 166, were up-regulated on fibronectin, and CD44, 54, 90, 95, 105, 106, 109, 138, 140b, 141, 142, 146, 147, 151, 157, 165 and 166 were up-regulated on human matrix. The stimulation by IL-1alpha up-regulated CD44, 49e, 51, 54, 61, 106 on MC on polystyrene; CD49e, 51, 61, 106, 146, 165 on MC on fibronectin, and CD49e, 51, 54 on MC grown on human matrix. This analysis of surface antigen expression provides new information to enable a better understanding of the role of mesangial cells in glomerular pathophysiology.

Mycophenolic acid antagonizes the activation of cultured human mesangial cells

Activation of mesangial cells is observed in several forms of chronic renal disease, and in culture conditions upon stimulation by fetal calf serum (FCS), or agonists such as transforming growth factor beta (TGF-beta). Mycophenolate mofetil (MMF), the precursor of mycophenolic acid (MPA), is currently used in organ transplantation and has been shown to be protective in clinical and experimental glomerulonephritis. This study assessed the effects of MPA on markers of human mesangial cells (HMC) activation. Primary cultures of HMC and of an immortalized HMC clone (IP15 cells characterized in this report) were stimulated either by FCS or by TGF-beta, and treated by MPA at clinically relevant concentrations (1 to 10 micromol/L) for 24 hours to 14 days. HMC proliferation, smooth muscle alpha-actin (SMA), collagen type I alpha-1 chain (coll I) and fibronectin synthesis were used as markers of HMC phenotypic activation. Exposure of HMC to MPA inhibited proliferation induced by FCS without cytotoxicity. MPA counteracted the stimulatory effects of FCS and TGF-beta on coll I mRNA and protein and fibronectin protein. SMA expression was increased upon exposure to MPA, without cell hypertrophy. Treatment of cultured HMC with MPA inhibited mesangial cell proliferation and matrix production induced by stimulation with either FCS or TGF-beta. Such mechanisms may contribute to the favorable effects of treatment using mycophenolate mofetil in chronic fibrotic kidney diseases, including chronic allograft rejection.

IP-10 and Mig production by glomerular cells in human proliferative glomerulonephritis and regulation by nitric oxide.

High levels of expression of mRNA and protein for the chemokines interferon-gamma (IFN-gamma)-inducible protein of 10 kD (IP-10) (CXCL10) and the monokine induced by IFN-gamma (Mig) (CXCL9) were observed, by using in situ hybridization and immunohistochemical analyses, in kidney biopsy specimens from patients with glomerulonephritis (GN), particularly those with membranoproliferative or crescentic GN, but not in normal kidneys. Double-immunostaining or combined in situ hybridization and immunohistochemical analyses for IP-10, Mig, and proliferating cell nuclear antigen (PCNA) or alpha-smooth muscle actin (alpha-SMA) revealed that IP-10 and Mig production by resident glomerular cells was a selective property of glomeruli in which mesangial cells demonstrated active proliferation. IP-10 and Mig mRNA and protein were also expressed by primary cultures of human mesangial cells and human visceral epithelial cells after stimulation with IFN- gamma or with IFN-gamma plus tumor necrosis factor-alpha (TNF-alpha) (which produced greater stimulation). The induction of IP-10 and Mig mRNA and protein expression by IFN-gamma plus TNF-alpha was strongly inhibited by nitric oxide (NO) donors, such as sodium nitroprusside or S-nitroso-N-acetylpenicillamine, but not by cGMP analogues. Electrophoretic mobility shift assays demonstrated that NO donors repressed IP-10 gene transcription induced by IFN-gamma plus TNF-alpha through the inhibition of NF-kappaB activation. These data demonstrate that resident glomerular cells in kidneys of patients with proliferative GN produce large amounts of IP-10 and Mig, which may play important pathogenic roles in this disease. These data also indicate that the production of IP-10 and Mig by human mesangial cells can be downregulated by NO donors through cGMP-independent inhibition of NF-kappaB activation.

Transmembrane signalling in human monocyte/mesangial cell co-cultures: role of cytosolic Ca(2+).

Adhesion of monocytes triggers apoptosis, cytotoxicity, cytokine release, and later proliferation of cultured human mesangial cells (HMC). In the search for transmembrane signals transducing the interaction of HMC adhesion molecules with leukocyte counterreceptors, we measured variations of cytosolic Ca(2+) ([Ca(2+)](i)) in HMC and monocytes of the U937 cell line during 6-h co-cultures. Monolayer cultures of HMC and suspensions of U937 cells were loaded with the fluoroprobe fura 2-AM and subsequently co-cultured for 6 h while separately monitoring by microfluorometry the Ca(2+)-dependent 500 nm fluorescent emission of each cell line at fixed intervals upon excitation at 340/380 nm. U937 and peripheral blood monocyte adhesion was followed in HMC by a slow, progressive rise of [Ca(2+)](i) from basal levels of 96+/-9 nM to 339+/-54 at 60 min and 439+/-44 nM at 3 h. The [Ca(2+)](i) elevation reached a steady state thereafter, while parallel monolayers incubated with control media maintained resting levels throughout the co-culture with stable fluoroprobe retention. Receptor sensitivity to vasoconstrictor agents, including compounds not released by monocytes, such as angiotensin II, was rapidly downregulated in HMC co-cultured with U937 cells. No [Ca(2+)](i) changes could be elicited by the octapeptide or by the TxA(2) analogue, U-46619, as early as 30 min after exposure to U937 cells. No [Ca(2+)](i) changes were observed in U937 cells throughout the co-culture. Conditioned media from monocytes and from co-cultured HMC+U937 cells had no effect on [Ca(2+)](i) of HMC. Ca(2+) entry leading to fura 2 saturation was still inducible by Ca(2+) ionophores, such as ionomycin and 4-Br-A23187, which also inhibited the responses to vasoconstrictors. Ca(2+)-free solutions prevented the [Ca(2+)](i) rise as well as subsequent receptor inactivation, implicating Ca(2+) influx through store-operated Ca(2+) channels (SOC), a major pathway for Ca(2+) entry in these cultured cells. Ca(2+) influx was confirmed by Mn(2+)-quenching of fura 2. In HMC, early changes in [Ca(2+)](i) signal for monocyte adhesion in a co-culture model of glomerular inflammation. This signalling mechanism may mediate the functional responses elicited in glomerular cells by leukocytes, including downregulation of receptors for vasoactive agents.