Regulation of filtration rate by glomerular mesangial cells in health and diabetic renal disease

The rate of renal filtration is in large part responsible for volume and electrolyte balance in an organism. Integral components of the renal glomerulus are the mesangial cells (MCs), excitable renal pericytes that regulate the glomerular filtration rate by modulating the surface area of the capillaries. Similar to vascular smooth muscle, the signal transduction pathways and ion selective channels regulating isotonic and isometric contraction of MCs are dependent on the voltage-gated Ca influx. During the response to contractile agonists, both Cl and nonselective cation channels play critical roles to depolarize the membrane potential and activate Ca channels. The relaxation pathways involve a negative-feedback mechanism that counteracts mesangial contraction by regulating voltage-dependent Ca signaling. Part of the feedback response involves the activation of plasmalemmal K channels, which hyperpolarize the membrane potential and inhibit voltage-gated Ca entry. This calcium- and voltage-activated feedback K (BKCa) channel shares biophysical, pharmacologic, and molecular properties with the BKCa channels identified in brain and muscle, and with the sio gene product as expressed in Xenopus laevis oocytes. Systemic hormones, such as atrial natriuretic peptide, and paracrine factors, such as nitric oxide (NO), use guanosine 3',5'-cyclic monophosphate (GMP) as a second messenger and enhance the gain in this feedback system by decreasing the voltage and Ca activation thresholds for BKCa. Diabetes mellitus is often associated with high rates of glomerular filtration, mesangial expansion, and secretory abnormalities of the basement membrane. NO-mediated increases in negative-feedback regulation of mesangial tone may attribute, in part, to the pathology of hyperfiltration. Stimulation of inducible nitric oxide synthetase in glomerular MCs by inflammatory cytokines is a possible positive-feedback pathway that contributes to further glomerular destruction. In addition, high ambient glucose, through modulation of BKCa activity, facilitates MC relaxation and thus propagates hyperfiltration. Since cellular arachidonic acid is metabolically linked to extracellular glucose, this fatty acid is a possible mediator of the pathologic actions of hyperglycemia. Clarification of the signal transduction pathways and ionic mechanisms regulating the normal and dysfunctional tones of MCs is essential for rational clinical management of glomerular disease and critical to understanding fluid and electrolyte homeostasis.     

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.     

Natriuretic peptides increase a K+ conductance in rat mesangial cells

Mesangial cells (MC) are a main target of natriuretic peptides in the kidney and are thought to play a role in regulating glomerular filtration rate. We examined the influence of cGMP-generating (i.e. guanosine 3',5'-cyclicmonophosphate) peptides on membrane voltages (Vm) of rat MC by using the fast whole-cell patch-clamp technique. The cGMP-generating peptides were tested at maximal concentrations ranging from 140 to 300 nmol/l. Whereas human CNP (C natriuretic peptide), rat guanylin and human uroguanylin had no significant effect on Vm these cells, human BNP (brain natriuretic peptide), rat CDD/ANP-99-126 (cardiodilatin/atrial natriuretic peptide) and rat CDD/ANP-95-126 (urodalatin) hyperpolarized Vm significantly by 1.6 +/- 0.4 mV (BNP, n=8), 3.7 +/- 0.3 mV (CDD/ANP-99-126, n=25) and 2.8 +/- 0.4 mV (urodilatin, n=9), respectively. The half-maximally effective concentration (EC50) for the latter two was around 400 pmol/l each. This hyperpolarization could be mimicked with 0.5 mmol/l 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br-cGMP) and was blocked by 5 mmol/l Ba2+. The K+ channel blocker 293 B (100 micromol/l) depolarized basal Vm by 4.3 +/- 0.4 mV (n=8), but failed to inhibit the hyperpolarization induced by CDD/ANP-99-126 (160 nmol/l) (n=8). The K+ channel opener cromakalim (10 micromol/l) neither influenced basal Vm nor altered the hyperpolarization induced by 160 nmol/l CDD/ANP-99-126 (n=8). Adenosine (100 micromol/l) hyperpolarized Vm by 13.4 +/- 1.3 mV (n=16). At 100 micromol/l, 293 B did not inhibit the adenosine-induced hyperpolarization (n=6). At 160 nmol/l, CDD/ANP-99-126 enhanced the adenosine-induced hyperpolarization significantly by 1.5 +/- 0.6 mV (n=10). CDD/ANP-99-126 (160 nmol/l) failed to modulate the value to which Vm depolarized in the presence of 1 nmol/l angiotensin II (n=10), but accelerated the repolarization to basal Vm by 49 +/- 20% (n=8). These results indicate that the natriuretic peptides CDD/ANP-99-126, CDD/ANP-95-126 and BNP hyperpolarize rat MC probably due to an increase of a K+ conductance. This effect modulates the voltage response induced by angiotensin II. The natriuretic-peptide-activated conductance can be blocked by Ba2+, but not by 293 B and cannot be activated by cromakalim. This increase in the K+ conductance seems to be additive to that inducable by adenosine, indicating that different K+ channels are activated by these hormones.     

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.     

Differential response of glomerular epithelial and mesangial cells after subtotal nephrectomy

Recent studies in both human and experimental chronic renal disease suggest that there is a linkage between glomerular hypertrophy and glomerulosclerosis. To further define these relationships, we studied the changes in glomerular hypertrophy, procollagen alpha 1(IV) mRNA levels and glomerulosclerosis in rats undergoing 1 2/3 nephrectomy (Nx) or sham nephrectomy (SNx). Glomerular hypertrophy, measured biochemically by RNA/DNA and protein/DNA ratios, was significantly increased in Nx compared to SNx two days after subtotal renal ablation (RNA/DNA: Nx = 133 +/- 8%, SNx = 100 +/- 3% of the mean control value, P < 0.01; protein/DNA: Nx = 164 +/- 22%, SNx = 100 +/- 10%, P < 0.05) and remained elevated after 7 and 15 days (RNA/DNA: seven days Nx = 155 +/- 3%, SNx = 100 +/- 13%, P < 0.01; 15 days Nx = 303 +/- 21%, SNx = 100 +/- 24%, P < 0.001; protein/DNA: seven days Nx = 228 +/- 57%, SNx = 100 +/- 18%, P < 0.05; 15 days Nx = 341 +/- 23%, SNx = 100 +/- 18%, P < 0.01). Light microscopic measures of glomerular tuft volume (GTV) were too insensitive to detect glomerular enlargement until 15 days postoperatively, but GTV measured ultrastructurally demonstrated a 20% increment in Nx compared to SNx as early as two days postoperatively (P < 0.01). The latter increment in GTV was due exclusively to glomerular visceral epithelial cell (GVEC) expansion. Glomerular procollagen alpha 1(IV) mRNA levels were significantly elevated only 15 days after nephrectomy (Nx = 265 +/- 58% of the mean control value, SNx = 100 +/- 12%, P < 0.05; corrected for beta-actin mRNA levels). As this time, exuberant mesangial expansion measured ultrastructurally contributed to a 1.6 +/- 0.1-fold increase in GTV (P < 10(-5)), and to a relative decrement in the GVEC contribution to glomerular cells plus matrix (P < 0.01). Segmental sclerosis was observed only 15 days postoperatively in Nx (Nx = 1.3 +/- 0.4% of glomeruli evaluated, SNx = 0.0%, P < 0.05), and there was a strong correlation between the prevalence of segmental sclerosis and the procollagen alpha 1(IV) mRNA levels in Nx at 15 days (r = 0.93, P < 0.01). There was no significant correlation between the RNA/DNA and protein/DNA ratios and procollagen alpha 1(IV) mRNA levels. Thus, glomerular regions responded differentially to subtotal nephrectomy. Early epithelial cell expansion was followed by later mesangial expansion. Glomerular procollagen alpha 1(IV) mRNA levels were elevated only during the second (mesangial) phase of glomerular hypertrophy, when it correlated with glomerulosclerosis, but not during the initial (epithelial) phase, a pattern consistent with a mesangial origin of the procollagen alpha 1(IV) mRNA.     

Granulosa cells have calcium-dependent action potentials and a calcium-dependent chloride conductance

We have found chicken granulosa cells to be excitable. Experiments using the whole-cell patch-clamp technique showed that they had membrane resting potentials of -62 +/- 3 mV (n = 8) and generated action potentials, either in response to 10-ms depolarizing current pulses or, on occasion, spontaneously. The action potentials persisted in a Na(+)-free bath and were reversibly blocked by 4 mM Co2+. They lasted 0.9-3.0s with 64 mM Cl- in the pipette, were shortened 67 +/- 8% by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 20 microM), and lengthened to 8.7 +/- 2.2 when the Cl- equilibrium potential (Vcl) was changed from -20 mV to -2 mV by using 134 mM Cl- in the pipette. With conventional whole-cell voltage-clamp, slowly activating and inactivating currents, which reached maximum amplitude after 0.35-1.40 s, were evoked by depolarizing voltage steps. These slow currents activated between voltage steps of -60 mV and -50 mV and reached a maximum inward amplitude at about -40 mV. Changing the Cl- concentration in the pipette (VCl of -2MV or -20 mV) or bath (VCl of -2 mV or + 18 mV) shifted their reversal potential in a direction consistent with a Cl- electrode. They were inhibited by the Cl- channel antagonists 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.5 mM), NPPB (20 microM), and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS; 0.5 mM). The slow currents were blocked by Ca2+ deprivation, or by CO2+ (4 mM), or by replacing external Ca2+ with Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

P2U-purinergic receptor activation mediates inhibition of cAMP accumulation in cultured renal mesangial cells

Extracellular ATP has been reported to exert mitogenic and contractile effects on cultured renal mesangial cells (MCs). Since it is possible that these actions involve changes in the cAMP second messenger system, we examined the effect of extracellular nucleotides on the accumulation of cAMP in rat MCs. ATP, UTP and adenosine 5'-0-(3-thio)triphosphate (ATP gamma S) (100 microM) had no significant effects on baseline cAMP levels, but inhibited forskolin-stimulated accumulation of cAMP by 21-75% in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Maximal inhibitory effects were observed at 100 microM of ATP gamma S with a threshold dose of 1 microM. ATP gamma S, ATP and UTP were the most potent inhibitors indicating stimulation of the P2u receptor. The P2x agonists adenosine 5'-(alpha, beta-methylene) triphosphate and adenosine 5'-(beta, gamma-methylene) triphosphate, and the P2y agonist 2-methylthio-ATP did not affect cAMP accumulation. Treatment with the P2 receptor antagonist suramin (200 microM) reduced the inhibition by 58%. The inhibitory effects of the nucleotides were significantly attenuated by preincubation with pertussis toxin (10-100 ng/ml). Inhibition of phospholipase C and protein kinase C did not prevent the inhibitory effect of the nucleotides. Inhibitors of forskolin-stimulated cAMP accumulation had different effects on DNA synthesis in cultured MCs as measured by 3H-thymidine uptake at 48 h: ATP, ATP gamma S and the inhibitor of adenylyl cyclase, SQ 22536, stimulated DNA synthesis in MCs, while UTP showed no significant mitogenic effect. Agents which increased baseline levels of intracellular cAMP (forskolin, IBMX, dibutyryl-cAMP) significantly diminished DNA synthesis in MCs. The results indicate that the P2u-purinergic receptor mediates inhibition of forskolin-induced cAMP accumulation which is likely due to inhibition of adenylyl cyclase. This effect appears to be partially mediated by PTX-sensitive G proteins. While the increase in cAMP accumulation is anti-mitogenic, inhibition of cAMP accumulation by P2u receptors is not correlated with MC growth control. Thus, additional mechanisms other than inhibition of cAMP accumulation by P2u receptors are likely to be involved in the mitogenesis of extracellular ATP.     

Endothelin-1 mediates erythropoietin-stimulated glomerular endothelial cell-dependent proliferation of mesangial cells

These experiments were performed in an attempt to determine whether chronic stimulation of glomerular endothelial cells with recombinant human erythropoietin would alter mesangial cell proliferation. Glomerular endothelial cells in culture incubated with various concentrations of erythropoietin for up to 4 days exhibited dose-dependent endothelin-1 production. Moreover, the conditioned medium from erythropoietin-stimulated glomerular endothelial cells enhanced [3H]thymidine incorporation into mesangial cells. This enhancement was significantly attenuated in the presence of a endothelin A receptor antagonist, BQ-123. These results suggest that endothelin-1 mediates erythropoietin-stimulated glomerular endothelial cell-dependent mesangial cell proliferation, resulting in the progression of glomerulonephritis.     

Apoptosis is triggered by the cyclic AMP signalling pathway in renal mesangial cells

Glomerular mesangial cells are regarded as specialized smooth muscle cells located within the renal glomeruli and fulfilling important functions in glomerular physiology and pathophysiology. Here, we report that activation of the cyclic AMP signalling pathway by dibutyryl cyclic AMP, forskolin, or the beta 2-adrenergic receptor agonist salbutamol results in induction of apoptosis in mesangial cells. Activation of the apoptotic programme results in DNA fragmentation which is visible for most forms of apoptosis and is paralleled by enrichment of cytosolic DNA/histone complexes, an increasing number of cellular 3'-OH-fragmented DNA ends and typical nuclear chromatin condensation. Induction of apoptosis was found to be dependent on translation and independent of nitric oxide synthase activity.     

Atrial natriuretic peptide inhibits endothelin-1-induced activation of JNK in glomerular mesangial cells

Atrial natriuretic peptide (ANP) has been shown to counteract various actions of endothelin-1 (ET-1) in mesangial cells. We have reported that both extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) are activated by ET-1 and ET-1-induced activation of ERK is inhibited by ANP. To further clarify the action of ANP, we examined the effect of ANP on ET-1-induced activation of JNK. ANP inhibited ET-1-induced activation of JNK in a dose-dependent manner. This inhibitory effect of ANP was reversed by HS-142-1, an antagonist for biological receptors of ANP, while C-ANP, an analog specific to clearance receptors of ANP, failed to inhibit ET-1-induced activation of JNK. 8-Bromo-cGMP and sodium nitroprusside were also able to inhibit ET-1-induced activation of JNK, suggesting cGMP-dependent action of ANP. In contrast, ANP failed to inhibit interleukin-1 beta (IL-1 beta)-induced activation of JNK. Since an increase in intracellular calcium ([Ca2+]i) was shown to be necessary for ET-1-induced activation of JNK in mesangial cells, we measured [Ca2+]i using fura-2. ANP attenuated the ET-1-induced increase in [Ca2+]i in concentrations enough to inhibit ET-1-induced activation of JNK. Finally, ANP was able to inhibit ET-1-, but not IL-1 beta-induced increase in DNA-binding activity of AP-1 by gel shift assay. These results indicate that ANP is able to inhibit ET-1-induced activation of AP-1 by inhibiting both ERK and JNK, suggesting that ANP might be able to counteract the expression of AP-1-dependent genes induced by ET-1.     

Ca2+ signaling induced by sphingosylphosphorylcholine and sphingosine 1-phosphate via distinct mechanisms in rat glomerular mesangial cells

BACKGROUND: To elucidate the molecular mechanism underlying sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) mediated signaling, we compared their effects with those of adenosine triphosphate (ATP) and angiotensin II (Ang II) on the cytosolic free Ca2+ concentration ([Ca2+]i), inositol 1,4, 5-trisphosphate (IP3) generation and arachidonic acid release in rat glomerular mesangial cells. METHODS: The fluorescent Ca2+ indicator, Fura-2, was used to measure the [Ca2+]i changes in cultured rat glomerular mesangial cells either in suspension or attached to the coverslips. RESULTS: SPC 5 microM, S1P 5 microM, ATP 100 microM and Ang II 90 nM all induced increases in the [Ca2+]i, and the effect showed marked homologous desensitization, while heterologous desensitization was less. After the initial exposure of the cells to SPC, the increase in [Ca2+]i induced by subsequent addition of ATP or Ang II was only reduced by about 14.3% and 4.8%, respectively. After the initial exposure to S1P, a greater reduction was seen (42. 1% and 47.7%, respectively). Both arachidonic acid release and IP3 generation were activated by all four agonists with an identical rank order of effectiveness of SPC > S1P > ATP = Ang II; both were pertussis toxin-sensitive and cholera toxin-resistant. The arachidonic acid release induced by all four agonists showed identical susceptibility to removal of extracellular Ca2+, whereas IP3 generation displayed differential extracellular Ca2+ dependence. Only SPC-induced IP3 generation was highly sensitive to extracellular Ca2+ level, and this Ca2+ dependence was abolished after pretreatment of cells with arachidonyl trifluoromethyl ketone (AACOCF3), a phospholipase A2 inhibitor. Furthermore, the Mn2+ influx was markedly greater in SPC-stimulated cells than in either control or other agonist-stimulated cells, and was decreased by prior exposure of cells to AACOCF3. After phospholipase A2 was inhibited or in the absence of extracellular Ca2+, SPC displayed identical effectiveness as S1P on desensitizing the action of ATP or Ang II on the increase in [Ca2+]i. Conclusions. Our results indicate that all four agents primarily activate phospholipase C through their receptor occupancies, but that SPC alone also induces further significant Mn2+ influx and IP3 generation attributable to its primary stimulatory effect on arachidonic acid release. Thus, the heterologous desensitization to ATP or Ang II induced by SPC was less profound than that induced by S1P, since SPC induced a Ca2+ influx.     

Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.     

Inhibition by protein kinase C of the 86Rb+ efflux and vasorelaxation induced by P1075, a K(ATP) channel opener, in rat isolated aorta

The expression of GABA in the human fetal (12-25 weeks of gestation), postnatal (five-month-old), and adult (35-year-old) retinas was investigated by immunohistochemistry. GABA expression was seen as early as 12 weeks in the undifferentiated cells of the inner neuroblast zone; a few optic nerve fiber layer axons were clearly labeled, suggesting that some of the stained cell bodies were prospective ganglion cells, others could be displaced amacrine cells. From 16-17 to 24-25 weeks, intense labeling was found in the amacrine, displaced amacrine, and some ganglion cells. During this time period, horizontal cells (identified by calbindin immunohistochemistry), undergoing migration (periphery) and differentiation (center), expressed GABA prominently. In the postnatal retina, some horizontal cells were moderately labeled, but very weakly in a few cells, in the adult. The Müller cells developed immunoreactivity first weakly at 12 weeks and then moderately from 16-17 weeks onward. The staining was also evident in the postnatal and adult retinas, showing labeled processes of these glial cells. Virtually no axons in the adult optic nerve and nerve fiber layer were stained; the staining was restricted to a few, large ganglion cells and displaced amacrine cells: Some amacrines were also labeled. The possibility that GABA might play a role in horizontal cell differentiation and maturation is highlighted. Other evidences suggest that GABA might play a role in metabolism during retinal development.     

Soluble aggregates of IgG and immune complexes enhance IL-6 production by renal mesangial cells

Primary rat mesangial cells (MC) were cultured in RPMI-1640 containing 5% fetal calf serum (FCS). The cells produced interleukin-6 (IL-6) in vitro depending on the concentration of FCS in the medium. Binding of soluble aggregates of IgG (AIgG) to MC was visualized with AIgG coupled with aminomethyl coumarin acetic acid (AMCA). There was a dose-dependent binding of 125I-AIgG to MC at 4 degrees C. Scatchard analysis revealed binding of AIgG containing 20 to 24 molecules per aggregate, with an affinity of 2.2 x 10(8) M-1 and a total average number of 2.7 x 10(5) sites per cell. The binding of AIgG or immune complexes to MC resulted in enhanced production of IL-6 by MC in culture. This enhanced production of IL-6 was dependent on the concentration of AIgG. To our surprise, preparations of monomeric IgG (mIgG) also enhanced the production of IL-6 by MC, but to a lower extent when compared to levels induced by AIgG. Very little amounts of aggregated F(ab')2 fragments [AF(ab')2] bound to MC and consequently no significant enhancement of IL-6 release by AF(ab')2 was seen, suggesting that IL-6 production is an Fc receptor-mediated phenomenon. The production of IL-6 by MC is inhibitable by cycloheximide, thus indicating de novo synthesis. Northern blot analysis revealed a threefold increase in mRNA for IL-6 by AIgG in vitro. The increase in mRNA expression was directly related to the quantity of IL-6 released in the supernatant by MC. These results suggest that binding of AIgG or immune complexes to MC in vivo may induce IL-6 production by MC, thus leading to proliferation of MC.     

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.     

Signaling role of PDE isozymes in pathobiology of glomerular mesangial cells. Studies in vitro and in vivo

Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazinone, inhibited (> 50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with cilostamide or with rolipram increased in situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated protein kinase. The efficacy of PDE isozyme inhibitors (IC50) to suppress mitogenesis or ROM generation paralleled IC50 for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazinone in combination with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should be investigated for potential use for "signal transduction pharmacotherapy" of glomerulonephritis.     

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.     

High glucose-induced transforming growth factor beta1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.