SPARC is expressed by mesangial cells in experimental mesangial proliferative nephritis and inhibits platelet-derived-growth-factor-medicated mesangial cell proliferation in vitro

Mesangial cell proliferation is a characteristic feature of many glomerular diseases and often precedes extracellular matrix expansion and glomerulosclerosis. This study provides the first evidence that SPARC (secreted protein acidic and rich in cysteine) could be an endogenous factor mediating resolution of experimental mesangial proliferative nephritis in the rat. SPARC is a platelet-derived-growth-factor-binding glycoprotein that inhibits proliferation of endothelial cells and fibroblasts. We now show that SPARC is synthesized by mesangial cells in culture and that SPARC mRNA levels are increased by platelet-derived growth factor and basic fibroblast growth factor. Recombinant SPARC or the synthetic SPARC peptide 2.1 inhibited platelet-derived-growth-factor-induced mesangial cell DNA synthesis in vitro. In a model of experimental mesangioproliferative glomerulonephritis, SPARC mRNA was increased 5-fold by day 7 and was identified in the mesangium by in situ hybridization. Similarly, SPARC was increased in glomerular mesangial cells and visceral epithelial cells by day 5 and reached maximal expression levels by day 7. Mesangial cell proliferation increased by 36-fold on day 5 and decreased abruptly on day 7. Maximal expression of SPARC was correlated with the resolution of mesangial cell proliferation. We propose that SPARC functions in part as an endogenous inhibitor of platelet-derived-growth-factor-mediated mesangial cell proliferation in glomerulonephritis and that it could account for the resolution of cellular proliferation in this disease.     

Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis

Increases in mesangial cell number may herald glomerular scarring, but they are not irreversible. This study sought mechanisms by which surplus glomerular mesangial cells can be cleared. A small proportion of cultured mesangial cells exhibited typical morphological features of apoptosis (programmed cell death), which was increased by growth factor deprivation or exposure to cycloheximide, stimuli known to increase apoptosis in other cell types. Apoptosis was confirmed by typical internucleosomal chromatin cleavage. In vivo, clear morphological evidence of mesangial apoptosis leading to phagocytosis by neighboring mesangial cells was obtained in self-limited mesangial proliferation induced in rats by Thy1.1 antibody, apoptosis occurring approximately 10-fold more frequently than in the healthy rat glomerulus. Indeed, changes in glomerular cell number in Thy1.1 nephritis strongly suggested that apoptosis is the major cell clearance mechanism counterbalancing cell division, thereby mediating resolution of glomerular hypercellularity in experimental mesangial proliferation.     

Inhibition of glomerular mesangial cell proliferation and extracellular matrix deposition by halofuginone

Mesangial cell proliferation, increased deposition of collagen, and expansion of the mesangial extracellular matrix (ECM) are key features in the development of mesangioproliferative diseases. Halofuginone, a low molecular weight anti-coccidial quinoazolinone derivative, inhibits collagen type alpha 1(I) gene expression and synthesis. We investigated the effect of halofuginone on both normal and SV40 transformed mesangial cell proliferation, collagen synthesis, and ECM deposition. Proliferation of both cell types was almost completely inhibited in the presence of 50 ng/ml halofuginone. The cells were arrested in the late G1 phase of the cell cycle and resumed their normal growth rate following removal of the compound from the culture medium. The antiproliferative effect of halofuginone was associated with inhibition of tyrosine phosphorylation of cellular proteins. Similar results were obtained whether the mesangial cells were seeded on regular tissue culture plastic or in close contact with a naturally produced ECM resembling their local environment in vivo. Halofuginone also inhibited synthesis and deposition of ECM by mesangial cells as indicated by a substantial reduction in 14C-glycine and Na2(35)SO4 incorporation into the ECM, and by the inhibition of collagen type I synthesis and gene expression. It is proposed that by inhibiting collagen type I synthesis and matrix deposition, halofuginone exerts a potent antiproliferative effect that may be applied to inhibit mesangial cell proliferation and matrix expansion in a variety of chronic progressive glomerular diseases.     

Effects of human immunodeficiency virus sera and macrophage supernatants on mesangial cell proliferation and matrix synthesis

Patients with human immunodeficiency virus (HIV) infection are prone to the development of focal segmental glomerulosclerosis, a lesion in which increased mesangial cell proliferation and matrix synthesis may play a role. We undertook the present study to determine whether HIV sera may affect mesangial cell proliferation and matrix synthesis either directly or indirectly via effects on macrophage supernatants. Pooled HIV sera was found to significantly enhance (P < 0.01) mesangial cell proliferation in a concentration-related manner. Mesangial cell proliferation was significantly suppressed by two medications commonly utilized in HIV-infected patients, azidothymidine and trimethoprim/sulfamethoxazole, and was not significantly altered by lipopolysaccharide, suggesting that these medications as well as recurrent infection are unlikely to account for the proliferative effect of HIV sera. Supernatants from HIV sera-treated macrophages were found to significantly enhance (P < 0.01) mesangial cell incorporation of [3H]proline, a marker for synthesis of the matrix component collagen, compared to supernatants from control sera-treated macrophages. These results suggest that HIV sera may directly enhance mesangial cell proliferation and may indirectly increase mesangial cell matrix synthesis by altering macrophage secretory products. These effects may play a role in the development of glomerulosclerosis in patients with HIV infection.     

Cationic dextran induces mesangioproliferative nephritis in rats independent of glomerular IgA deposition

BACKGROUND: Dextran-induced mesangioproliferative glomerulonephritis in mice and, as recently reported, in rats is used as a model of IgA nephropathy. The pathogenetic role of the glomerular IgA deposits in this model, however, is unclear since IgG is often deposited in parallel. METHODS: Lewis rats were immunized with cationic DEAE-dextran. Following this, rats received 5 x/week i.v. injections of 3 mg DEAE-dextran each, from days 20 to 80 and were then followed until day 120. RESULTS: Rats developed transient proteinuria (range 63-152 mg/24 h) and haematuria on day 80. Renal biopsies obtained at days 60, 80, 100 and 120 showed mild to severe mesangioproliferative changes at days 80 and 100 which did not persist at day 120. Electron-microscopy revealed mesangial immune deposits, signs of endothelial activation and vacuoles in mesangial cells and podocytes. Compared to normal, age-matched controls, in the nephritic rats significant (P < 0.05) increases were noted for glomerular total cellularity, alpha-smooth-muscle actin expression (a marker of activated mesangial cells), monocyte/macrophage counts, and matrix proteins. Using three different antibodies, no evidence of glomerular IgA deposition was detected at any time point. In contrast, glomerular IgG, IgM, C3, and occasional small C5b-9 deposits were present in nephritic rats. Circulating IgG but not IgA anti-dextran antibodies could be demonstrated in nephritic rats. CONCLUSIONS: The data confirm that mesangioproliferative glomerulonephritis can be induced in rats by immunization and chronic challenge with cationic dextran. Our data also show that in rats glomerular IgG deposition rather than IgA, appears to play an important pathogenetic role in this mesangioproliferative glomerulonephritis model.     

Cytokines promote glomerular mesangial cell survival in vitro by stimulus-dependent inhibition of apoptosis

Resolution of glomerular inflammation requires the removal of proliferating resident glomerular mesangial cells, but excessive loss of glomerular cells is a feature of postinflammatory scarring. Because apoptosis regulates mesangial cell number in glomerular inflammation, we have studied the exogenous control of apoptosis triggered in cultured mesangial cells by stimuli likely to be important in vivo. Apoptosis could be induced by serum deprivation to model decreased availability of survival factors, by etoposide as an example of DNA-damaging agents, by ligation of mesangial cell Fas, and by protein synthesis inhibition by cycloheximide. Insulin-like growth factor I (IGF-I), IGF-II, and basic fibroblast growth factor were each able to suppress apoptosis induced by serum deprivation, whereas TGF-beta 1, epidermal growth factor, and platelet-derived growth factor had no effect. IGF-I and IGF-II (but not basic fibroblast growth factor) were also able to protect cells from apoptosis induced by etoposide or cycloheximide. However, Fas-mediated apoptosis was resistant to suppression by all three cytokines. None of the cytokines tested caused a change in the levels of expression of Bcl-2, Bax, Bcl-x, or Bak proteins. The survival-promoting properties of serum-free medium conditioned by mesangial cells was abrogated by neutralizing IGF-I Ab. These experiments are the first to define cytokines that inhibit apoptosis and thereby promote survival of mesangial cells, and the data indicate a paracrine survival signaling role for IGF-I. Finally, the data show that Fas ligation can override cytokine survival signaling, emphasizing a candidate role for this molecule in the undesirable apoptotic loss of mesangial cells during the progression of glomerular scarring.     

Cell proliferation and apoptosis of the glomerular epithelial cells in rats with puromycin aminonucleoside nephrosis

Injury and repair of the glomerular epithelial cells (GECs) play an important role in the pathogenesis of focal segmental glomerulosclerosis (FSGS). To obtain a better understanding of proliferation and apoptosis of GECs, we examined immunohistochemical and in situ hybridization findings in puromycin aminonucleoside nephrosis (PAN) of rats. The minimal-change nephrotic syndrome model (PAN-MCNS) was induced by administering 5 subcutaneous injections of puromycin aminonucleoside (PA; each 1.5 mg/100 g B/W to one group of rats), whereas the FSGS model (PAN-FSGS) was induced by administering an additional 5 injections of PA to another group of rats. The cell kinetics of the GECs were assessed with labeling 5-bromo 2'-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). To investigate regulation of apoptosis in rats with PAN, we evaluated the expression of p53, Fas antigen, Fas ligand and Bc1-2. Rats with PAN-MCNS exhibited a significantly greater number of BrdU- and PCNA-labeled GECs as compared with control rats. In rats with PAN-FSGS, the number of PCNA-labeled GECs was greater than in rats with PAN-MCNS, but the number of BrdU-labeled GECs was lower. Apoptotic cells were occasionally observed in the sclerotic lesions, with the number being significantly higher in rats with PAN-FSGS than in rats with PAN-MCNS and control. Apoptotic cells were observed in the GECs of PAN-FSGS rats. However, they were negative for p53, Fas antigen, and Fas ligand. Immunohistochemical and in situ hybridization studies revealed a greater intraglomerular overexpression of Bc1-2 protein and bc1-2 mRNA in the PAN-FSGS rats as compared with control rats. These results suggest that insufficient proliferation and apoptosis in GECs may be involved in the progression of FSGS.     

Diffuse proliferative lupus nephritis in a patient with ulcerative colitis

PURPOSE: To compare activation of the ipsilateral cerebral hemisphere during tactile sensory and motor tasks involving the right and left hands. METHODS: Eight volunteers had functional MR imaging to measure the extent of cerebral hemisphere activation during a motor task and sensory task involving each hand. Hemispheric indexes (left hemisphere activation minus right hemisphere activation)/(left hemisphere activation plus right hemisphere activation) were computed for each hand and each task. The indexes for two tasks and the two hands were compared. RESULTS: The left-hand motor tasks activated the ipsilateral hemisphere in right handers significantly more than did the right-hand tasks. Motor tasks produced a greater activation of the ipsilateral hemisphere than did the sensory tasks. No significant differences were found between the hemispheric indexes for the right-hand and left-hand sensory tasks. CONCLUSION: This study confirms findings of a previous study, showing that the left hemisphere is active in left-hand motor tasks. Activation of the ipsilateral hemisphere is significantly less pronounced during sensory tasks than during motor tasks.     

Thrombin increases clusterin mRNA in glomerular epithelial and mesangial cells

Clusterin, a multifunctional protein with complement blocking activity, and fibrin, a product of thrombin's enzymatic activity, are present in the kidney during acute and chronic renal failure. The role of thrombin in regulating clusterin mRNA in the kidney is not known. The effect of thrombin on clusterin mRNA expression was examined in rat glomerular mesangial and glomerular epithelial cells, and cultured human renal proximal tubular epithelial cells by northern blot. Thrombin (10(-8) M) increased clusterin mRNA levels two- to fourfold in glomerular mesangial, glomerular epithelial, and proximal tubule epithelial cells. This was a specific effect of thrombin receptor activation because peptides corresponding to the tethered ligand of the thrombin receptor were also able to increase clusterin mRNA levels. Epidermal growth factor, insulin-like growth factor-1, and transforming growth factor-beta 1 had little or no effect on clusterin mRNA levels. The protein kinase C inhibitor RO-32-0432 (1 microM) inhibited the thrombin-induced increase in clusterin mRNA, suggesting that thrombin receptor activation may regulate renal clusterin mRNA levels through protein kinase C.     

Endothelin-induced activation of mitogen-activated protein kinases in glomerular mesangial cells from normotensive and stroke-prone spontaneously hypertensive rats

1. Kinase assay in myelin basic protein (MBP) containing polyacrylamide gels revealed that endothelin-1 (ET-1) and ET-3 increased MBP kinase activities in glomerular mesangial cells (MC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rat (SHRSP). ET-1 stimulated MBP kinase activities more potently than ET-3. 2. Immunoprecipitation with anti-41-kDa MAPK antiserum showed that the MBP kinases activated by ET-1 correspond to 43- and 41-kDa MAPK. 3. Since Phorbol 12-myristate 13-acetate, a direct activator of protein kinase C, also activated MAPK, protein kinase C was suggested to mediate ET-induced activation of MAPK. 4. These results suggest that MAPK may mediate the ET actions in glomerular mesangial cells from normotensive rats as well as spontaneously hypertensive rats. Since ET is produced by vascular endothelial cells of the kidney and glomerular mesangial cells, the ET signalling pathway may have some physiological and pathophysiological significance in vivo glomerulus.     

Suppression of mesangial proliferative glomerulonephritis development in rats by inhibitors of cAMP phosphodiesterase isozymes types III and IV

Excessive mesangial cell (MC) proliferation is a hallmark of many glomerulopathies. In our recent study on cultured rat MC (Matousovic, K., J.P. Grande, C.C.S. Chini, E.N. Chini, and T.P. Dousa. 1995. J. Clin. Invest. 96:401-410) we found that inhibition of isozyme cyclic-3',5'-nucleotide phosphodiesterase (PDE) type III (PDE-III) suppressed MC mitogenesis by activating cAMP-dependent protein kinase (PKA) and by decreasing activity of mitogen-activated protein kinase (MAPK). We also found that inhibition of another PDE isozyme, PDE-IV, suppresses superoxide generation in glomeruli (Chini, C.C.S., E.N. Chini, J.M. Williams, K. Matousovic, and T.P. Dousa. 1994. Kidney Int. 46:28-36). We thus explored whether administration in vivo of the selective PDE-III antagonist, lixazinone (LX), together with the specific PDE-IV antagonist, rolipram (RP), can attenuate development of mesangioproliferative glomerulonephritis (MSGN) induced in rats by anti-rat thymocyte serum (ATS). Unlike the vehicle-treated MSGN rats, rats with MSGN treated with LX and RP did not develop proteinuria and maintained normal renal function when examined 5 d after injection of ATS. In PAS-stained kidneys from PDE-antagonists-treated MSGN-rats the morphology of glomeruli showed a reduction in cellularity compared with control rats with ATS. Compared with MSGN rats receiving vehicle, the MSGN rats receiving PDE-antagonists had less glomerular cell proliferation (PCNA delta -65%), a significantly lesser macrophage infiltration (delta -36% ED-1) and a significant reduction of alpha-smooth muscle actin expression by activated MC; in contrast, immunostaining for platelet antigens and laminin were not different. The beneficial effect of PDE inhibitors was not due to a moderate decrease (approximately -20%) in systolic blood pressure (SBP); as a similar decrease in SBP due to administration of hydralazine, a drug devoid of PDE inhibitory effect, did not reduce severity of MSGN in ATS-injected rats. We conclude that antagonists of PDE-III and PDE-IV administered in submicromolar concentrations in vivo to ATS-injected rats can decrease the activation and proliferation of MC, inhibit the macrophage accumulation, and prevent proteinuria in the acute phase of MSGN. We propose that PDE isozyme inhibitors act to block (negative "crosstalk") the mitogen-stimulated intracellular signaling pathway which controls MC proliferation due to activating of the cAMP-PKA pathway. These results suggest that antagonists of PDE-111 and IV may have a suppressive effect in acute phases or relapses of glomerulopathies associated with MC proliferations.     

Inhibitory effects of antihypertensive drugs on mesangial cell proliferation after anti-thymocyte serum (ATS)-induced mesangiolysis in spontaneously hypertensive rats

The effects of antihypertensive drugs on mesangial cell proliferation were studied in spontaneously hypertensive rats (SHR) with anti-thymocyte serum (ATS)-induced glomerulo-nephritis. Rats were treated with either enalapril (Group 1), nifedipine (Group 2), or reserpine + hydrochlorothiazide + hydralazine (Group 3), or were untreated (Group 4). The animals were sacrificed 2, 4 and 7 days after ATS injection and the glomerular cell number and degree of mesangial area expansion were examined. A marked, similar decrease in glomerular nuclear cell number (NC) due to severe mesangiolysis was observed in all of the groups on day 2. Thereafter, an increase in NC reflecting mesangial cell proliferation after mesangiolysis occurred in Group 4 on days 4 and 7. In Group 1 and 2, the NC was significantly smaller than that in Group 4 on days 4 and 7, indicating suppression of mesangial cell proliferation. In Group 3, however, the number of NCs did not differ from that in Group 4 on days 4 and 7, indicating a lack of such suppression by conventional antihypertensive drugs. The degree of mesangial area expansion (MS) showed the same pattern as mesangial cell proliferation. That is, the rapid increases in MS seen in Group 4 on days 4 and 7 were apparently suppressed in Groups 1 and 2, but not in Group 3. Our in vivo observations that both an angiotensin converting enzyme (ACE) inhibitor and a calcium channel blocker suppress mesangial cell proliferation and mesangial area expansion suggest that these agents have practical implications in the treatment of mesangial proliferative glomerular diseases through the suppression of excess mesangial cell proliferation.     

Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration

We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.     

Inflammation measurement and immunocharacterization of cell proliferation in an experimental model of proliferative vitreoretinopathy

An experimental model of proliferative vitreoretinopathy was developed in the rabbit eye by injecting a solution of human platelet-rich plasma. In this model we evaluated the progression with time of intraocular inflammation and the rate and origin of cell proliferation. A sterile solution adjusted to 107 platelets was injected into the right eye of a total of 46 pigmented and 14 albino rabbits. Animals were sequentially sacrificed at days 7, 14, 21 and 1 month after injection. Clinical evaluation of vitreoretinal proliferation, using a classification in six grades, and of anterior segment inflammation assessed by a Laser Flare Meter, were done for 1 month after injection, before histopathological analysis. Eighty percent of eyes developed tractional retinal detachment in 1 month. Histopathology showed intense cell migration and proliferation in the area of the ciliary body, as early as the seventh day, then further increasing rapidly. Infiltrates were composed of cytokeratin- and vimentin-expressing cells. Abnormal expression of vimentin was also found in ciliary and retinal epithelia and in M?ller cells. Inflammation measured by the Laser Flare Meter was maximal at day 11 and then reached a plateau at significantly higher levels than controls. Albino rabbits showed significantly lower grades of proliferation, as compared to pigmented rabbits. This study thus clarified some characteristics of experimental vitreoretinal proliferations that that proved similar to those in human diseases, such as the involvement of ciliary body and retinal pigment epithelium, the existence of inflammatory reactions preceding cell proliferation and strong changes in intermediate filaments. This may provide a simple and valuable model for antiproliferative assays and shed some light on the pathogenesis of intraocular proliferative disorders.     

Local macrophage proliferation in the pathogenesis of glomerular crescent formation in rat anti-glomerular basement membrane (GBM) glomerulonephritis

1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity. 2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity. 3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells. 4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.