Cytokines and Fas regulate apoptosis in murine renal interstitial fibroblasts.

Renal fibrosis is characterized by an increased number of fibroblasts and excessive deposition of extracellular matrix. Apoptotic cell death is a physiological mechanism to limit cell numbers, and an insufficient rate of death may contribute to fibroblast accumulation. However, little is known about the regulation of renal fibroblast survival. The authors have studied the interaction of cytokines and the Fas receptor in the regulation of apoptosis of renal fibroblasts and have observed that murine renal fibroblasts express Fas and the Fas ligand. Tumor necrosis factor alpha (TNFalpha) and agonistic anti-Fas antibodies induce apoptosis of renal fibroblasts in a time- and dose-dependent manner. Serum contains survival factors for renal fibroblasts. Both serum deprivation and TNFalpha increase the sensitivity to Fas-induced death and the expression of fas mRNA and Fas receptor. By contrast, insulin-like growth factor-1 decreases apoptosis induced by both serum deprivation and Fas activation and partially prevents the increase in Fas receptor expression induced by serum deprivation. Murine renal fibroblasts express constitutively both fas ligand mRNA and cell-surface Fas ligand, but the authors could not demonstrate a role for Fas ligand in the autocrine regulation of fibroblast survival. These data suggest that Fas and other cytokines cooperate to regulate renal fibroblast apoptosis. Modulation of the Fas death-signaling pathway in renal fibroblasts could represent a new therapeutic target for renal fibrosis.

Agonistic anti-Fas antibodies induce glomerular cell apoptosis in mice in vivo.

Recent studies suggest that apoptotic cell death regulates the cell complement in glomerular diseases. However, little is-known about the factors that promote glomerular cell apoptosis. Activation of the Fas receptor by the Fas ligand or agonistic antibodies triggers apoptosis in some cell types that express Fas. Cultured human mesangial cell are among the cells that undergo apoptosis upon Fas activation, but it is unclear whether mesangial cells are sensitive to death induced by Fas in vivo. We have now explored the role of Fas in experimental glomerular injury. Murine mesangial cells in culture express fas and undergo apoptosis when stimulated with the Jo2 agonistic anti-Fas mAb. A fas mRNA transcript is present in normal murine kidney and freshly isolated glomeruli. Balb-c mice developed hematuria and proteinuria within 24 hours of the intraperitoneal injection of 10 micrograms Jo2 anti-Fas mAb. In addition to liver cell apoptosis, glomerular cell apoptosis and mesangial cell depletion were evident in the kidney at three hours and more pronounced at 24 hours. Glomerular and liver injury were not prevented by decomplementation. These data suggest that Fas activation in vivo by specific antibodies induces glomerular and mesangial cell apoptosis in mice.

Expression of leucocyte chemoattractants by interstitial renal fibroblasts: up-regulation by drugs associated with interstitial fibrosis.

Interstitial inflammation is a strong predictor of long-term renal damage. The potential role of renal interstitial fibroblasts in recruitment of inflammatory leucocytes into the interstitium is unclear. We have thus studied the mRNA expression of several leucocyte chemotactic factors by rat renal interstitial fibroblasts and its modulation by cytokines. In addition, the effects of two unrelated drugs associated with the development of interstitial fibrosis, namely puromycin aminonucleoside (PAN) and cyclosporin A (CsA), were also studied. Rat renal interstitial fibroblasts showed constitutive mRNA expression for the chemokines monocyte chemoattractant protein 1 (MCP-1) and interferon-inducible protein 10 (IP-10). In addition, these cells also exhibited constitutive mRNA expression for cyclophilin B, an immunophilin recently found to have leucocyte chemoattractant properties. The inflammatory cytokine tumour necrosis factor-alpha up-regulated IP-10 and MCP-1 mRNA expression (10- and four-fold, respectively), but had no effect on cyclophilin B mRNA levels. IP-10 and MCP-1 produced about a four-fold increase in MCP-1 and cyclophilin B mRNA expression, but did not affect IP-10 mRNA. PAN caused an augmentation in IP-10, MCP-1 and cyclophilin B mRNA levels (12-, 9.5, and two-fold, respectively), while CsA increased only cyclophilin B mRNA in a dose-dependent manner. In conclusion, rat renal interstitial fibroblasts express mRNA for chemotactic factors and this expression is up-regulated by inflammatory cytokines, PAN and CsA. The present findings suggest that renal interstitial fibroblasts may play an active role in the recruitment of inflammatory leucocytes into the interstitium.

Regulation of Fas and Fas ligand expression in cultured murine renal cells and in the kidney during endotoxemia.

Fas ligand (FasL) and Fas belong to a recently described family of cytokines and receptors with similarities to tumor necrosis factor (TNF) and its receptors. Upon engagement by specific antibodies or by FasL, Fas transduces a signal for apoptosis in permissive cells. Although apoptosis occurs during renal development and following injury to mature cells, the factors responsible for programmed renal cell death are uncertain. We have studied Fas expression by renal cells in vitro and during endotoxemia in mice. Several renal cell types, including glomerular mesangial cells and tubular epithelial cells express a Fas transcript in culture. Lipopolysaccharides (LPS), interleukin-1 beta, interferon-gamma (IFN-gamma), and TNF-alpha increase the levels of Fas mRNA in cultured mesangial and tubular cells. TNF-alpha and LPS raise the level of Fas mRNA in a time- and dose-dependent manner with Fas receptor expression peaking after 72 h of exposure to LPS. Anti-Fas antibodies can induce the death of cultured mesangial cells. This cell death shows the characteristic changes of apoptosis, including DNA fragmentation and pyknotic changes of the nucleus. Increases in Fas by LPS, TNF-alpha, and IFN-gamma enhance the killing induced by the anti-Fas antibody. FasL is also expressed by cultured renal cells, and TNF-alpha treatment of mesangial cells increases its expression. In vivo, Fas mRNA is present at low level in normal kidney. LPS increases the levels of Fas mRNA and protein in kidney and produces evidence of apoptosis along nephrons. These data suggest that transcripts encoding natural FasL and Fas are induced by LPS and may play a role in endotoxemia-induced acute renal failure and organ dysfunction.

Anti-Fas antibodies induce cytolysis and apoptosis in cultured human mesangial cells.

Death of renal cells often occurs during the acute and resolution phases of some forms of glomerulonephritis. The apoptotic Fas protein belongs to a recently described family of cytokine receptors with similarities to tumor necrosis factor (TNF) receptors, and may contribute to the necrobiology of renal cells. Fas transduces a signal for apoptosis in sensitive cells after binding by specific antibodies or following contact with natural Fas ligand. We have studied Fas in cultured human mesangial cells. Cytoflurography demonstrated Fas expression on the surface of human mesangial cells that was increased by stimulation with interferon gamma (IFN gamma). Agonistic anti-human Fas antibodies were cytotoxic to these cells. Cytotoxicity was time- and dose-dependent, and was modulated by pre-stimulation of the mesangial cells with IFN gamma and/or by co-treatment with actinomycin-D. Mesangial cell death following exposure to anti-Fas antibodies has features consistent with apoptosis, such as internucleosomal DNA fragmentation, nuclear shrinkage and condensation, and decreased DNA content. These data suggest that Fas and its ligand could play a mechanistic role in human glomerular cell injury.

Apoptosis in renal diseases.

Abnormalities of cell number are a frequent feature of renal disorders. Cell death is a key factor in the regulation of cell number. Apoptosis is an active form of cell death that is modulated by extracellular lethal and survival signals. Regulation of apoptosis also involves a complex system of sensors of the extracellular signals, triggers of the apoptosis program, effectors of apoptosis as well as intracellular survival factors. This paper first reviews current knowledge on the regulation of apoptosis with particular emphasis on renal cell death. Subsequently, it deals with the role of apoptosis in triggering renal disease and its participation in the progression and resolution of renal disorders. This section includes information on the occurrence of apoptosis and expression of apoptosis-related genes in glomerular injury, acute renal failure, chronic renal atrophy, renal fibrosis, polycystic renal disease and kidney development. The final section presents an overview of possible approaches to the therapeutic manipulation of apoptosis in the kidney.

Urinary excretion of platelet-activating factor in human and experimental nephrosis.

BACKGROUND: Platelet-activating factor (PAF) is a phospholipid that has been implicated in the pathogenesis of glomerulonephritis and can be synthesized by glomerular cells in response to different stimuli. PAF increases glomerular permeability to proteins and urinary PAF has been determined to be of renal origin. In order to assess whether urinary PAF can be found augmented in situations of glomerular damage without glomerular leukocyte infiltration, urinary PAF was quantified in human and experimental nephrosis. METHODS: Urinary PAF was quantified by platelet bioassay and glomerular PAF by incorporation of 3H-acetate into PAF. PAF was characterized by its behaviour on thin-layer chromatography and high performance liquid chromatography and the blockade of its bioactivity by receptor antagonists. RESULTS: Urinary PAF excretion was significantly higher in patients with active idiopathic nephrotic syndrome than in controls (5.8+/-1.5 versus 1.7+/-0.75 mg/24 h; P<0.05) and patients in remission (1.63+/-0.75 ng/24 h; P<0.02). In rats with nephrosis induced by puromycin aminonucleoside there was an early increase in urinary PAF excretion (138+/-19 versus 49+/-22 pg/24 h in controls; P<0.035) that coincided with the augmented glomerular PAF synthesis (67+/-3.4 versus 36+/-1.2 DPM/mg protein in controls; P<0.003). CONCLUSIONS: These results suggest that the synthesis of PAF in the kidney may be involved in the pathogenesis of the proteinuria in idiopathic nephrotic syndrome and that urinary PAF excretion may be a good marker of disease activity.

Interferon-inducible protein-10 is highly expressed in rats with experimental nephrosis.

Interferon-inducible protein (IP)-10 is a small glycoprotein member of a family of chemotactic cytokines structurally related to interleukin-8. We have recently described the induction of IP-10 mRNA in mouse mesangial cells stimulated with lipopolysacharide, interferon-gamma, and tumor necrosis factor-alpha. To further evaluate a possible role for this chemokine in renal injury, we have studied IP-10 in an experimental model of nephrosis induced in rats by adriamycin. High levels of glomerular IP-10 mRNA expression and glomerular and tubulointerstitial IP-10 protein were seen on day 21, coinciding with maximal proteinuria, glomerular tumor necrosis factor mRNA expression, and interstitial cellular infiltrates. Maintenance on a low protein diet not only delayed the appearance of proteinuria and interstitial cellular infiltrate but also decreased glomerular IP-10 mRNA expression. Isolated normal glomeruli and cultured glomerular epithelial and mesangial cells from normal rats expressed IP-10 mRNA upon stimulation with 100 U/ml interferon or 1 microgram/ml lipopolysaccharide for 3 hours. IP-10 mRNA expression was also inducible by lipopolysaccharide and cytokines in NRK 49F renal interstitial fibroblasts and, to a lesser extent, in NRK 52E tubular epithelial cells. Furthermore, IP-10 protein was inducible in murine mesangial cells. We conclude that IP-10 is highly inducible in vitro and in vivo in resident glomerular and tubulointerstitial cells. IP-10 may participate in the modulation of renal damage in experimental nephrosis.

Cyclosporin A (CsA) modulates the glomerular production of inflammatory mediators and proteoglycans in experimental nephrosis.

Nephrosis is characterized by glomerular epithelial cell injury and a decrease in the glomerular basement membrane (GBM) proteoglycan content. Although CsA is a useful treatment for a group of patients with this disease, its mechanism of action is unclear. We have previously shown that in experimental nephrosis there is an increase in the glomerular production of tumour necrosis factor-alpha (TNF-alpha) and platelet-activating factor (PAF). Here we have studied the effect of CsA on kidney generation of TNF-alpha and PAF in puromycin aminonucleoside (PAN) nephrosis as well as on the synthesis of proteoglycans by cultured glomerular epithelial cells. Rats receiving CsA had, on day 8 of PAN injection, a significant reduction in proteinuria, blood cholesterol levels and in interstitial mononuclear cells. A diminution in glomerular production and urinary excretion of TNF-alpha and PAF was also noted. In in vitro studies, at 24 h of incubation PAF and TNF-alpha induced in glomerular epithelial cells a significant decrease in proteoglycan synthesis. Neither PAF nor TNF-alpha had any significant effect on glomerular epithelial cell proliferation. CsA alone induced a dose-response increase in proteoglycan synthesis and a slight decrease in cell proliferation. CsA also reversed the inhibitory effect of PAF and TNF-alpha on proteoglycan synthesis. However, CsA did not alter the pattern of proteoglycan production, remaining around 50% chondroitinase ABC-, 15% heparitinase-sensitive. Our results indicate that PAF and TNF-alpha could be implicated in the pathogenesis of nephrosis through the inhibition of proteoglycan synthesis by glomerular epithelial cells. The beneficial effect of CsA in nephrosis may be due to the recovery of the GBM charge selectivity caused by the normalization of glomerular PAF and TNF-alpha synthesis and the increase in proteoglycan synthesis by glomerular epithelial cells.

[Coxsackie B4 and cytomegalovirus in patients with insulin-dependent diabetes]. / Coxsackie B4 y citomegalovirus en pacientes con diabetes insulín dependiente.

The purpose of this work is to analyze the potential relationship between infection by Coxsackie and cytomegalovirus and the appearance of insulin-dependent diabetes (IDD). We have assessed complement fixing (CF) antibodies against both viruses in 22 samples of serum from other individuals, among which 11 were patients with a recent diagnosis of IDD--less than 3 months of evolution--(GP) and the remainder 11, supposed healthy individuals with similar age, sex and socioeconomic level (GC). In addition, the presence of anti-pancreatic islets antibodies (ICA) was assessed in 22 serum samples using the indirect immunofluorescence reaction. We have not observed any relationship between the presence of Coxsackie B or CMV antibodies and the appearance of IDD, since although the seroprevalence against CMV was higher in GP than in GC, such difference was not statistically significant. With respect to ICAs, they were only present in the GP samples as expected. Given the small size of the sample, this results are not conclusive and larger series need to be studied.

[Prevalence of the Epstein-Barr virus among different populations in the central area]. / Prevalencia del virus de Epstein-Barr en diferentes poblaciones de la zona centro.

In order to estimate the seroprevalence of the Epstein-Barr virus in the centre area, a serological study is carried out using the indirect immunofluorescence technique on 759 supposed healthy people, from both sexes and with ages between 6 months and 50 years, 565 from which were living in urban areas (Madrid and Guadalajara city) and 194 in rural areas (Arganda del Rey, Madrid) and Chiloeches (Guadalajara). Global seroprevalence was of 81% in Madrid city, 90% in Guadalajara city and 85% in both rural populations. Comparing groups of the same age, we have not found any statistically significant differences between the urban (86%) and the rural (85%) areas. Neither we have found any relation between seropositivity and sex (74.7% for women and 81% for men). On the other hand, we observed that in our study group, seroprevalence was related to socioeconomic status--the higher the antibody percentages, the lower the socioeconomic status, both in the rural and in the urban areas (p less than 0.001)-- and age antibody percentages increased with age, with p less than 0.025 for urban areas and p less than 0.05 for rural areas.

[Coxiella burnetii: serologic study in various populations]. / Coxiella burnetii: estudio serológico en distintas poblaciones.

Complement binding antibodies against Coxiella burnetii were measured in 699 seric samples in order to ascertain the possible relationship between seric prevalence and the following factors: age, sex, living in rural or urban areas and socio-economical level. All patients were healthy: 180 were from a rural area 86 from Guijo (Cáceres); 36 from Chiloeches (Guadalajara) and 58 from Bustarviejo (Madrid); 519 were from an urban area (City of Madrid). The samples from Madrid city were classified by socio-economical level: 386 from medium-low level (MLL) and 133 from medium-high socio-economical (MHL) level. The global seroprevalence was 32.3%, being 46.1% from rural areas; --59.3 from Guijo, 33.3% from Chiloeches and 34.5% from Bustarviejo. In urban areas the seroprevalence was 27.5% this being 30.8% MLL and 18% MHL. We found a statistically significant relationship between both socio-economical levels and the area of living.