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.     

Complement regulatory proteins in glomerular diseases

Complement activation plays a critical role in the pathogenesis of many forms of glomerulonephritis. Complement activation leads to tissue injury through various mechanisms including the generation of chemotactic factors and activation of the resident glomerular cells following C5b-9 insertion. Recent advances have disclosed the mechanisms of regulation of complement activation by discovery of a number of complement regulatory proteins. Decay accelerating factor (DAF), membrane cofactor protein (MCP), and complement receptor type 1 (CR1) act by inactivating C3/C5 convertase. They belong to the gene superfamily known as the regulators of complement activation (RCA), and share a common structural motif called a short consensus repeat (SCR). In contrast, CD59 works by inhibiting formation of C5b-9. The glomerulus is particularly well endowed with these membrane-bound complement regulatory proteins. DAF, MCP, and CD59 are ubiquitously expressed by all three resident glomerular cells, while CR1 is localized exclusively in podocytes. Expression of complement regulatory proteins can be changed by many factors including complement attack itself, and their expression levels are affected in various glomerular disorders. Studies utilizing cultured glomerular cells and animal models of glomerular diseases suggest important protective roles of complement regulatory proteins against immune-mediated renal injury. Recent progress in molecular biological techniques has made new therapeutic strategy feasible. Systemic administration of soluble recombinant complement regulatory proteins and local overexpression of complement regulatory proteins are promising therapeutic approaches.     

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.     

Purification of lipases, phospholipases and kinases by heparin-Sepharose chromatography

Heparin interacts with lipases, phospholipases and kinases. Immobilized heparin can be used for the purification of diacylglycerol and triacylglycerol lipases, phospholipases A2 and C and protein and lipid kinases. The use of heparin-Sepharose is an important development in analytical and preparative techniques for the separation and isolation of lipases, phospholipases and kinases.     

Complement activation accelerates glomerular injury in diabetic rats

A case of hemispheric infarction involving the territory of the right middle cerebral artery and the thalamus showed conspicuous asymmetric degeneration in the substantia nigra, red nuclei, inferior olivary nuclei and dentate nuclei with concomitant changes of progressive supranuclear palsy (PSP). The right substantia nigra and red nucleus showed loss of neurons and proliferation of astrocytes. The right olivary nucleus was hypertrophic, while the neuronal loss and astrocytosis in the dentate nucleus were predominant on the contralateral side. Modified Gallyas-Braak staining revealed the extensive distribution of neurofibrillary tangles (NFTs), threads and intraglial argyrophilic structures in the globus pallidus, subthalamic nuclei, cerebral cortex and dentate nuclei, as well as in the affected brain stem nuclei, with a distinct predominance on the affected side. In this case, the one-sided predominance of the extended degeneration in these brain stem and cerebellar areas is considered, in addition to the PSP changes, to be due to secondary retrograde degeneration via the nigrostriatal and dentato-rubro-thalamic pathways following the hemispheric infarction, and to also be the result of disruption of the dentato-olivary fiber connections. In addition, because of the predominant distribution of NFTs on the more degenerated side, it is surmised that the formation of NFTs may be accelerated by secondary degeneration.     

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.     

Unique phosphorylation of vimentin filaments in glomerular epithelial cells in culture and in disease

Three cDNA clones for the Machado-Joseph disease gene (MJD1) were isolated, two of which have a new exon sequence and a distinct 3' terminal nucleotide sequence resulting in a new carboxyl terminal domain in the translated product. The nucleotide sequence of the other one is similar to the previously published one except for five polymorphisms, one of which is a single nucleotide substitution resulting in a change from the stop codon (TAA; allele A) to a tyrosine residue (TAC; allele C). Genetic analysis results suggest that Japanese MJD mutations are associated with allele A.     

Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes

Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 micromol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 micromol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 micromol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 micromol/L, 45 minutes) or herbimycin A (3 micromol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 micromol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.     

Effect of glycated proteins on the matrix of glomerular epithelial cells

The bactericidal activity of gaseous ozone was investigated using a commercial ozone generator. Five species of fish bacteria, Pseudomonas putida, Shewanella putrefaciens, Brochothrix thermosphacta, Enterobacter sp. and Lactobacillus plantarum, were inoculated on agar surfaces and exposed to different ozonation times in a gas chamber. Results showed ozone in relatively low concentrations (< 0.27 x 10(-3) g l-1) was an effective bactericide of vegetative cells of the five fish bacteria. The age of the cell culture was shown to influence the cell response following exposure. Survival rate was not linearly related to ozonation time, but exhibited biphasic death over an extended period. Similar bactericidal effects were observed on fish skin treated with ozone daily in the laboratory, with decreases of 1.0 log cfu cm-2 for the micro-organisms studied. Whole fish treated daily in the laboratory using a commercial ozone generator showed improved scores for sensory analyses compared with the controls. The results were statistically significant. Fish treated on board ships were also analysed for microbiological and sensory changes. Controls were obtained from a similar vessel without the ozone facility in the hold. Similar trends to those recorded in the laboratory for the microbiological and sensory results on ozonated fish were observed.     

Visceral glomerular epithelial cells can proliferate in vivo and synthesize platelet-derived growth factor B-chain

In glomerular diseases associated with antibody- and complement-mediated injury to endothelial and mesangial cells, cell proliferation is an important early response that precedes matrix accumulation and glomerulosclerosis. In contrast, in diseases in which the visceral glomerular epithelial cell (vGEC) is the principal target of injury, cell proliferation is not a recognized consequence, although vGECs respond in vitro to a variety of growth factors and inflammatory mediators. To explore the possibility that low levels of vGEC proliferation may occur and participate in such diseases, serial studies were done in the passive Heymann nephritis model of membranous nephropathy, in which the vGEC is the primary target of antibody- and C5b-9-mediated injury. The results showed mitotic figures and positive staining for the proliferating cell nuclear antigen in cells whose location defined them as vGECs. The proliferating cell nuclear antigen-positive cells at the edge of the capillary wall were confirmed to be vGECs by double-immunostaining with antibodies to SPARC/osteonectin, which preferentially label vGECs in passive Heymann nephritis. Proliferation of vGECs in vivo was preceded by increased glomerular expression of platelet-derived growth factor (PDGF) B-chain protein and messenger RNA, both of which localized to vGECs. PDGF B-chain protein and messenger RNA were also detected in cultured vGECs. PDGF receptor beta-subunit protein or messenger RNA could not be demonstrated in vGECs in vivo or in vitro, and no growth response of cultured vGECs to PDGF was noted. These results suggest that proliferation of vGECs does occur in a model of glomerular injury induced by antibody and C5b-9 on vGECs. VGEC proliferation and production of PDGF may be involved in the restoration of the capillary wall but could also contribute to local capillary wall injury and proliferation of other cells in Bowman's capsule, interstitium, and tubules.     

Glomerular epithelial protein 1 and podocalyxin-like protein 1 in inflammatory glomerular disease (crescentic nephritis) in rabbit and man

The podocyte is the cell responsible in large part for maintaining the glomerular filtration barrier. Glomerular epithelial protein 1 (GLEPP1) is a novel receptor-like transmembrane protein tyrosine phosphatase present on the apical surface of podocyte foot processes. Podocalyxin-like protein 1 (PCLP1) is a transmembrane sialoglycoprotein which is also present on the foot process apical surface as well as on the surface of endothelial cells. GLEPP1 and PCLP1 are thought to play a role in regulating the structure and function of podocyte foot processes. Glomerular injury affecting the podocyte is likely to be reflected by changes in these proteins. GLEPP1 distribution in human renal biopsy with inflammatory glomerular disease and crescent formation was examined by immunocytochemistry. A model of inflammatory glomerular injury induced by guinea pig anti-rabbit basement membrane (anti-GBM) antibody was used to examine the distribution and amount of GLEPP1 and PCLP1 mRNA and protein. A biopsy study was done to determine whether the extent of GLEPP1 depletion from glomeruli at early time points (Day 7) would predict the severity of crescent formation at Day 30. Glomeruli from human renal biopsies with crescentic nephritis showed focal to diffuse disappearance of GLEPP1 protein. No GLEPP1 was present within the cellular crescent. By Day 4 of the rabbit anti-GBM model, before cellular crescents had formed, GLEPP1 protein was reduced from 127 +/- 28 X 10(7) to 30 +/- 5 X 10(7) molecules per glomerulus (p < 0.001), and GLEPP1 mRNA was reduced by 62% (p < 0.05). In contrast, at this time there was no significant reduction of PCLP1 protein from the normal number of 309 X 10(9) molecules per glomerulus and the PCLP1 mRNA level had not decreased. At Day 4, podocyte foot processes were effaced and proteinuria was present. Glomerular culture supernatants from Day 4 rabbits caused a reduction in GLEPP1 but not PCLP1 protein expression by cultured normal glomeruli, showing that a soluble factor was produced at Day 4 which reduced the number of GLEPP1 molecules in glomeruli. There was no detectable proteolysis of GLEPP1 or PCLP1 in glomeruli and no increase in GLEPP1 or PCLP1 excretion in urine. Therefore, the reduction in glomerular GLEPP1 was associated with reduced synthetic capacity. The proportion of glomeruli with reduced GLEPP1 at Day 7 of the model was significantly associated with the percent of glomeruli which had formed crescents at Day 30 (r = 0.86, p < 0.0001). GLEPP1 appears to be a sensitive indicator of glomerular injury during inflammation in man and in the rabbit model. A reduction in amount of GLEPP1 is associated with worse outcome for the glomerulus.     

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.     

Significance of membrane attack complex inhibitory factor expression in cultured human glomerular epithelial cells

We report five cases of pancreatitis in systemic lupus erythematosus. Three patients died. The cause of death was directly related to pancreatitis in two cases. Several causes of pancreatitis may be suspected: vasculitis, thrombosis when associated to antiphospholipid syndrome, biliary calculi, infection, metabolic abnormalities or adverse effects of therapy. About seventy cases of pancreatitis have been reported in systemic lupus erythematosus in the literature Pancreatitis was the inaugural sign of lupus in six cases. In 12 cases, besides lupus, no other cause of pancreatitis was found. However, pancreatic vasculitis or thrombosis was rarely demonstrated even in post-mortem examinations. The role of corticosteroids in pancreatitis in lupus is controversial since the evolution of pancreatitis in lupus is generally good with corticosteroids. We think that diagnosis of pancreatitis in lupus should not implicate discontinuation or decrease of corticosteroids dosage, unless their responsibility is absolutely demonstrated.