T gamma/delta lymphocytes in renal transplant recipients.

T gamma/delta lymphocytes are able to perform allospecific cytotoxicity and natural killer cytotoxicity in vitro. However, very little is known about their function in vivo. To investigate the possible involvement of T gamma/delta lymphocytes in the immune response to renal allografts, fine-needle aspiration biopsies and peripheral blood of 15 renal transplant recipients were studied during the first 4 weeks after transplantation. In addition peripheral blood of patients before transplantation, half a year and one year after transplantation was studied. No increase in the percentage of T gamma/delta lymphocytes in the fine-needle aspiration biopsies, including those taken during acute rejection episodes, was found. A significant decrease in the percentage of T gamma/delta lymphocytes was observed in peripheral blood after transplantation. We conclude that T gamma/delta lymphocytes seem to play no major role in the immune response to renal allografts.     

Endotoxins modulate chronically tumour necrosis factor {alpha} and interleukin 6 release by uraemic monocytes

We examined in vivo the release of tumour necrosis factor alpha(TNF) and interleukin 6 (IL-6) by uraemic monocytes upon stimulationwith endotoxin-contaminated bicarbonate concentrate. Twelveuraemic patients underwent 1-month-subsequent periods of standardhaemodialysis (SHD) with cuprophane (CU), a high-complement-activatingmembrane (6 patients), or haemodiafiltration (HDF) with polyacrylonitrile(PAN), a low-complement-activating membrane (6 patients), byusing a dialysate prepared with either non-sterile bicarbonateconcentrate tanks (phase 1) or sterile bicarbonate concentratebags (phase 2). TNF and IL-6 concentrations were determinedin monocyte supernatants by ELISA; endotoxin levels in bicarbonateconcentrates were measured by a chromogenic limulus amoebocytelysate (LAL) assay. A significant increase in LAL reactivity was found in bicarbonateconcentrate tanks compared to sterile bags (P<0.001). Non-steriledialysate caused a significant (P<0.001) predialytic increasein monocyte TNF release as compared to controls and nondialyseduraemic patients. One month treatment with sterile bicarbonatesignificantly decreased TNF predialytic activity in monocytesupernatants (P<0.001) to levels closer to those of non-dialyseduraemic patients. A similar decrease was observed for IL-6 production.Dialytic treatment induced a further increase in both TNF andIL-6 production, particularly in phase 1. When uraemic patientswere examined separately according to the different dialyticprocedures (SHD-CU or HDF-PAN), the use of sterile dialysate(phase 2) caused a significant decrease of predialysis TNF releasein both SHD CU patients (24.1±8.4 pg/ml versus 55.3±5.7pg/ml, P<0.001) and HDF PAN-treated patients (16.6±5.3pg/ml versus 29.1±5.4pg/ml, P<0.005), so that thedifferences between the dialytic procedures were completelyabolished. In conclusion, TNF and IL-6 release may be induced by endotoxin-contaminateddialysate during haemodialysis. The use of sterile bicarbonatecan ameliorate the bioincompatibility of CU membranes and probablyinfluences the biocompatibility of PAN membranes. Therefore,regardless of the type of dialyser used, all attempts to obtainan ultrapure dialysate are important to optimize dialytic treatment,in order to attenuate the chronic monocyte activation whichoccurs during haemodialysis.     

Non-endotoxinic tumour necrosis factor-{alpha}-inducing factors in haemodialysis

A transmembrane passage of endotoxins may account for the dysfunctionof cytokine production which has been often reported in haemodialysis.We developed an assay based on the ability of patient serumto stimulate tumour necrosis factor (TNF) secretion in normalperipheral blood mononuclear cells. Three groups of subjectswere investigated: normal controls (n=14), patients with chronicrenal failure, CRF (n=15), and patients dialysed with poly-acrylonitrile(n=7), polysulphone (n=8), and cellulose acetate (n=7). Serafrom dialysed patients displayed a significantly higher TNF-inducingactivity than those of controls and CRF patients. The abilityof serum to elicit TNF secretion was neither modified duringthe dialysis session nor influenced by the type of haemodialysismembrane. TNF-inducing activity in serum was not inhibited bypolymyxin B, known to impair endotoxin-dependent cell responses,thus suggesting that it was not related to circulating endotoxins.We conclude that non-endotoxinic factors are present in serumfrom dialysed patients and are able to induce cytokine secretion.     

Interleukin 6 production by human proximal tubular epithelial cells in vitro: analysis of the effects of interleukin-1{alpha} (IL-1{alpha}) and other cytokines

Proximal tubular epithelial cells (PTEC) from human renal tissueobtained from biopsy or nephrectomy were grown in monocultureand evaluated in vitro at passage 2–4 for interleukin6 (IL-6) production in response to medium alone or to interleukin1 alpha (IL-1), tumour necrosis factor alpha (TNF), interleukin2 (IL-2), interferon gamma (INF) or lipopolysaccharide (LPS).IL-6 bioactivity was quantitated using the IL-6-dependent murinehybridoma cell line (B9) and expressed as IL-6 units/ml/10⁵PTEC. PTEC cell lines exposed to medium alone produced intermediateamounts of IL-6 with substantial variability between cell lines.Introduction of IL-1 resulted in a dose- and time-dependentincrease in IL-6 production by PTEC that was maximal at 1 ng/mlIL-1 at 24 h. All PTEC cell lines showed an increased IL-6 productionon exposure to IL-1 varying from 1.3- to 24-fold increase overbaseline production. This response was completely blocked byanti-rIL-1. No significant IL-6 production by PTEC could beinduced by TNF, IL-2, IFN, or LPS over a broad dosage range.Cycloheximide inhibited IL-6 production without irreversiblecell toxicity, indicating de-novo synthesis. IL-6 produced byPTEC had a molecular weight of 26-29 kDa as demonstrated byWestern blot analysis. Using PCR analysis we could demonstrateupregulation by IL-1 of IL-6 mRNA in a dose-response fashion,indicating that IL-1 regulates IL-6 production at a pretranslationalvalue of protein synthesis. These results show that human culturedPTEC produce IL-6 under both normal and IL-1-stimulated conditions,and suggest that they may have a regulatory function in responseto cytokines in the setting of inflammation in the renal cortex.     

Rosiglitazone ameliorates cisplatin-induced renal injury in mice.

BACKGROUND: Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Agonists of the peroxisome proliferator-activated receptor-gamma (PPARgamma), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to examine the protective effects of rosiglitazone on cisplatin nephrotoxicity and to explore the mechanism of its renoprotection. METHODS: Mice were treated with cisplatin with or without pre-treatment with rosiglitazone. Renal functions, histological findings, aquaporin 2 (AQP2) and adhesion molecule expression, macrophage infiltration and tumour necrosis factor-alpha (TNF-alpha) levels were investigated. The effect of rosiglitazone on nuclear factor (NF)-kappaB activity and on viability was examined using cultured human kidney (HK-2) cells. RESULTS: Rosiglitazone significantly decreased both the damage to renal function and histological pathology after cisplatin injection. Pre-treatment with rosiglitazone reduced the systemic levels of TNF-alpha and down-regulated adhesion molecule expression in addition to the infiltration of inflammatory cells after cisplatin administration. Rosiglitazone restored the decreased AQP2 expression after cisplatin treatment. Pre-treatment with rosiglitazone blocked the phosphorylation of the p65 subunit of NF-kappaB in cultured HK-2 cells. Rosiglitazone had a protective effect via a PPARgamma-dependent pathway in cisplatin-treated HK-2 cells. CONCLUSION: These results showed that pre-treatment with rosiglitazone attenuates cisplatin-induced renal damage through the suppression of TNF-alpha overproduction and NF-kappaB activation.     

Polymorphisms of the tumour necrosis factor alpha gene at position -308 and TNFd microsatellite in primary IgA nephropathy.

BACKGROUND: The development of glomerular inflammation in immunoglobulin A nephropathy (IgAN) has been associated with various cytokines, including tumour necrosis factor alpha (TNFalpha). A biallelic polymorphism in the promoter region of the TNFalpha gene (TNFA), at position -308, has been described (TNFA-1 and TNFA-2) and is associated with increased TNFalpha production for the TNFA-2 allele. Another microsatellite polymorphism has been described for TNFd, which is functional and associated with increased production of TNFalpha for the d3 allele. METHODS: We have studied these two polymorphisms in 242 Caucasian patients with biopsy-proven IgAN (169 male, 73 female), who were followed from 1990 to 1999, and in 210 appropriate local Caucasian controls (133 male, 77 female) for comparison of genotypes and allelic distribution. RESULTS: The respective frequencies of A1/A1, A1/A2 and A2/A2 TNFA genotypes were 76.4, 22.3 and 1.3% in IgAN vs 78.1, 19.5 and 2.4% in controls (P=NS). For TNFd, the frequencies of the respective genotypes d3/d3, d3/non-d3 and non-d3/non-d3 were significantly different (chi(2)=12.30, P=0.002, Pc=0.013) with an increased frequency of the low-producer genotype non-d3/non-d3 in IgAN patients (24 vs 12%). The combination of TNFA and TNFd polymorphisms demonstrated that compared with controls, patients with non-A2 and non-d3 alleles (low producers) were more common (18 vs 9%; P=0.006). In the genotype/clinical phenotype correlations, we could not demonstrate significant differences between the different subgroups of patients. However, high-producer TNFalpha patients (A2 and d3 alleles) had more chronic renal failure than others (36.6 vs 22.9%) at last follow-up and their survival without chronic renal failure (Kaplan-Meier) was lower. Nevertheless, TNFalpha polymorphisms were not an independent risk factor for the progression of the disease. CONCLUSIONS: TNFA and TNFd polymorphisms seem to influence the occurrence or initiation of the disease, but do not play a significant role, if any, in the progression of IgA nephritis.     

Alphav integrin regulates TNF-alpha-induced nitric oxide synthesis in rat mesangial cells--possible role of osteopontin.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) induces nitric oxide (NO) synthesis in rat mesangial cells (MCs). We previously demonstrated that osteopontin (OP), a matrix protein that mainly interacts with the alphav integrin family, increased time-dependently by TNF-alpha stimulation at gene and protein levels. The regulation of NO synthesis by integrins or matrix proteins is unclear. METHODS: We examined whether integrin, especially alphav integrin, regulates NO synthesis in rat MCs and whether OP, an alphav integrin ligand, has an effect on TNF-alpha-induced NO synthesis. Furthermore, OP and inducible NO synthase (iNOS) gene expression was examined by Northern blotting. RESULTS: TNF-alpha increased NO synthesis in MCs in a time-dependent manner. Synthetic GRGDSP peptide, which is known to inhibit various integrins that interact with RGD-containing extracellular matrices, increased TNF-alpha-induced NO levels in a dose-dependent manner. Cyclical RGD peptide, the specific inhibitor of alphav integrin, also exhibited a dose-dependent effect of increasing NO levels, while GRGESP peptide, which has very low affinity to integrins, had no effect. In addition, NO synthesis was found to be significantly reduced when MCs were plated on OP-coated dishes compared to type I or IV collagen-coated dishes. Furthermore, anti-OP antibody increased NO synthesis in MCs. iNOS mRNA levels were increased by TNF-alpha, and were abruptly diminished after OP mRNA was significantly induced. CONCLUSIONS: The present study demonstrated the involvement of alphav integrin in TNF-alpha-induced NO synthesis in rat MCs, and the possible role of OP was suggested in the mechanism. TNF-alpha and extracellular matrices can co-operate to regulate the behaviour of MCs at least partly through NO synthesis, which may participate in the course of glomerular diseases.     

Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.

BACKGROUND: Clinical and/or laboratory signs of systemic inflammation occur frequently in patients undergoing long-term haemodialysis. It is likely, therefore, that a compensatory release of endogenous anti-inflammatory molecules occurs to limit host reactions. The aim of the present research was to determine if the potent anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH), a pro-opiomelanocortin derivative, is increased in plasma of haemodialysis patients. Because endotoxin and cytokines induce alpha-MSH in vivo and in vitro, we also measured plasma concentrations of endotoxin, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha), and the two circulating products of activated monocytes, nitric oxide (NO) and neopterin. METHODS: Thirty-five chronic haemodialysis patients, 20 patients with chronic renal failure not yet on dialysis, and 35 normal controls were included in the study. In the haemodialysis group, blood samples were obtained before and at the end of a dialysis session. Plasma alpha-MSH was measured using a double antibody radioimmunoassay, and IL-6, TNF-alpha, and neopterin using specific enzyme-linked immunosorbent assays. Plasma nitrites were determined by a colorimetric method, and endotoxin with the quantitative chromogenic LAL (limulus amoebocyte lysate) method. RESULTS: Mean plasma alpha-MSH was higher in haemodialysis patients than in control subjects, with the peptide concentrations being particularly elevated in dialysed patients with detectable endotoxin. High alpha-MSH concentrations were observed in the pre-dialysis samples, with no substantial change at the end of the dialysis session. Plasma concentrations of IL-6, TNF-alpha, neopterin, and NO were generally elevated in chronic haemodialysis patients and there was a negative correlation between circulating alpha-MSH and IL-6. In patients with renal failure not yet on dialysis, mean plasma alpha-MSH was similar to that of normal subjects. CONCLUSIONS: alpha-MSH is increased in the circulation of chronic haemodialysis patients and particularly so in case of detectable endotoxaemia. Reduction of renal clearance is unlikely to contribute to the observed rise of the peptide because alpha-MSH concentration is not increased in patients with chronic renal failure who are not yet on dialysis. It is likely that dialysis-associated endotoxaemia, directly and/or through cytokine release, enhances the production of the anti-inflammatory mediator alpha-MSH that limits host reactions.     

Pentoxifylline suppresses renal tumour necrosis factor-alpha and ameliorates experimental crescentic glomerulonephritis in rats.

BACKGROUND: Crescentic glomerulonephritis is a rapidly progressive form of glomerulonephritis, but treatment remains non-specific. The methylxanthine derivative pentoxifylline (PTX) is a clinically available phosphodiesterase inhibitor with anti-inflammatory and immunoregulatory activities. This study examined whether PTX has beneficial effects in a rat model of anti-glomerular basement membrane (GBM) crescentic glomerulonephritis. METHODS: Experimental crescentic glomerulonephritis was induced in Wistar rats by intravenous injection of rabbit anti-rat GBM serum and treated with either vehicle (phosphate-buffered saline) or PTX (0.1 g/kg/day) intravenously on a daily basis. Groups of six animals were euthanized at days 3, 7, 14 or 28 after induction of disease. Effects of PTX on renal function, histology and expression of cytokines, chemokines and adhesion molecules were determined. RESULTS: Compared with the vehicle-treated nephritic rats, PTX treatment beginning at the start of the nephritis significantly suppressed mRNA expression of tumour necrosis factor (TNF)-alpha, but not interleukin-1 beta, throughout the course of nephritis. Moreover, PTX decreased renal mRNAs for intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T-cell expressed and secreted (RANTES) and osteopontin (OPN) at all time points examined. These effects were associated with a significant inhibition of macrophage and T-cell infiltration, a reduction of 24-h urinary protein excretion (50-75%, P<0.05), an improvement of histological damage including glomerular crescent formation (60-70%, P<0.01) and a decrease of cortical mRNAs for type I (alpha 1) collagen and fibronectin. The efficacy of PTX could also be seen, though to a lesser extent, in rats with established nephritis. CONCLUSIONS: PTX is an effective anti-inflammatory and immunomodulatory agent capable of suppressing rat crescentic glomerulonephritis. Inhibition of renal TNF-alpha, ICAM-1, RANTES, MCP-1 and OPN expression may be a mechanism whereby PTX suppresses progressive renal injury in rat crescentic glomerulonephritis.     

Urinary tumour necrosis factor-alpha excretion independently correlates with clinical markers of glomerular and tubulointerstitial injury in type 2 diabetic patients.

BACKGROUND: Inflammation is a potential factor in the development and progression of diabetic nephropathy. The aim of this study was to analyse the relationship between the pro-inflammatory cytokine tumour necrosis factor-alpha (TNFalpha) and clinical markers of glomerular and tubulointerstitial damage [urinary albumin excretion (UAE) and urinary N-acetyl-beta-glucosaminidase (UNAG), respectively] in a large group of type 2 diabetic patients. METHODS: A total of 160 diabetic patients and 32 healthy controls were included in the study. High-sensitive C-reactive protein (hs-CRP) as well as serum and urinary levels of TNFalpha were measured. UAE and UNAG were determined by 24-h urine collection. RESULTS: Serum hs-CRP and TNFalpha were significantly higher in diabetic than in control subjects, as well as UAE and UNAG. Diabetic patients had increased urinary TNFalpha compared to non-diabetics [14.5 (2-29) vs 4 (0.8-12), P < 0.001]. Serum hs-CRP and TNFalpha in diabetics with increased UAE were elevated compared to diabetics having normoalbuminuria. Urinary TNFalpha was also higher in diabetic subjects with micro- or macroalbuminuria than in patients with normal UAE [10.5 (4-20) and 18 (9-29) vs 7 (2-18) pg/mg, P < 0.0001, respectively]. Multiple regression analysis showed that urinary TNFalpha (P < 0.0001), hs-CRP (P < 0.0001), serum TNFalpha (P < 0.01) and HbA1c (P < 0.05) were independent of and significantly associated with UAE, whereas duration of diabetes (P < 0.001), urinary TNFalpha (P < 0.01), HbA1c (P = 0.01), hs-CRP (P < 0.05) and serum creatinine (P < 0.05) were associated with UNAG. CONCLUSIONS: In patients with type 2 diabetes, urinary TNFalpha excretion is elevated and correlates with severity of renal disease in terms of both glomerular and tubulointerstitial damage, suggesting a significant role for TNFalpha in the pathogenesis and progression of renal injury in diabetes mellitus.     

Interferon-{alpha}2b treatment of chronic hepatitis C in haemodialysis patients

Nineteen haemodialysis (HD) patients with chronic hepatitisC were treated with interferonalpha2b (IFN-) at a dose of 3or 1 MU thrice weekly for 6 months and were followed-up foranother 14 months without treatment. Six patients discontinuedtreatment because they either presented severe side-effectsto IFN- or had complications of their primary disease. Levelsof AST and ALT were within normal limits on the 2nd month oftreatment and remained so throughout the treatment and the follow-upperiod in all patients except one who showed an elevation oftransaminase levels 2 months after the end of treatment. SerumHCVRNA became negative in 10/13 patients at the end of treatmentand was negative in all patients on the 6th month and in 12/13patients on the 14th month during the follow-up period. Levelsof 2'5' oligosynthetase were increased significantly on the2nd and 4th month of treatment and returned to pretreatmentvalues the 2nd month after treatment. These findings demonstratethat haemodialysis patients with chronic hepatitis C respondwell to interferon treatment and that a long-term response isachieved in a high proportion of patients.     

Association between oestrogen receptor alpha gene polymorphism and mortality in female end-stage renal disease patients.

BACKGROUND: In the general population, genetic variations in the oestrogen receptor alpha (ERalpha) gene may influence lipid abnormalities, cardiovascular disease (CVD), and mortality, but this has not previously been studied in end-stage renal disease (ESRD) patients. METHODS: A total of 227 ESRD (141 men and 86 women) patients starting renal replacement therapy (RRT) were genotyped for three ERalpha gene polymorphisms (Ser10Ser, PvuII and XbaI) and the associations between these polymorphisms and clinical and laboratory parameters and survival were analysed. Patients were followed for a median period of 55 months (range 1-126 months). RESULTS: The PvuII and XbaI polymorphisms were not associated with any of the clinical parameters. The ERalpha Ser10Ser CC genotype was present in 24 (28%) of the female and in 37 (26%) of the male patients. When comparing the CC genotype with the CT and TT genotypes, there were significant differences in lipid levels and inflammatory marker levels, especially in female patients. In female patients, the CC genotype was associated with lower prevalence of protein energy wasting (PEW) (17.4% vs 43.1%; P=0.03), lower median serum triglyceride (1.7 vs 2.1 mmol/l; P=0.001), higher median serum albumin (34.0 vs 32.5 g/l; P=0.03) and lower median high sensitivity-CRP (hsCRP) (2.2 vs 5.5 mg/l; P=0.03) levels compared with the CT plus TT genotypes. In male patients only HDL-cholesterol and ApoA levels were associated with this polymorphism. Whereas this polymorphism did not influence survival in males, the mortality was lower in female patients with the CC genotype (Kaplan-Meier; Log-rank 2.2, P=0.02). Moreover, female patients with the CT plus TT genotypes had a borderline significant increased relative risk (Cox hazard model; 6.6, 95% CI: 0.87-49.9 P=0.06) of death as compared with those with the CC genotype, even after adjustment for age and prevalence of CVD. CONCLUSIONS: Female, but not male ESRD patients with the ERalpha Ser10Ser CC genotype had lower prevalence of PEW, lower serum triglyceride, higher serum albumin and lower hsCRP levels. As this genotype was associated with a significantly decreased risk of all-cause death during the initial years of RRT, its protective properties need further study.     

Hypoxia reduces the expression and anti-inflammatory effects of peroxisome proliferator-activated receptor-gamma in human proximal renal tubular cells.

BACKGROUND: Peroxisome proliferator-activated receptor (PPAR)-gamma may counteract tissue fibrosis via its anti-inflammatory actions, while hypoxia, a new pro-fibrotic factor, reportedly modifies PPAR-gamma expression. However, the effects of hypoxia on the expression and anti-inflammatory actions of PPAR-gamma have yet remained to be clarified in renal tubular cells. METHODS: Confluent human proximal renal tubular epithelial cells (HPTECs) were exposed to hypoxia (1% O2) and/or TNF-alpha at 10 ng/ml for up to 48 h. The cells were incubated with PPAR-gamma agonists, 15d-PGJ2 or pioglitazone, for 30 min before stimulation. Precise amounts of PPAR-gamma and MCP-1 mRNA and protein were measured by TaqMan quantitative PCR and immunoblot or ELISA, respectively. RESULTS: A cDNA array analysis identified PPAR-gamma as one of the hypoxia-affected genes in HPTECs. Hypoxia reduced mRNA levels of PPAR-gamma at 24 and 48 h and protein levels at 6 and 48 h. Knockout of hypoxia-inducible factor-1alpha (HIF-1alpha) with its dominant negative form did not block the hypoxia-induced reduction in PPAR-gamma expression. PPAR-gamma's activation with 15d-PGJ2 or pioglitazone reduced basal and TNF-alpha-stimulated MCP-1 expression at mRNA and protein levels at 24 h under normoxia. MCP-1 reduction rates at basal mRNA and protein levels were slightly but significantly lower during hypoxia than normoxia (9 vs 69% and 36 vs 42%, respectively, for 15d-PGJ2, and 0 vs 34% and 12 vs 21%, respectively, for pioglitazone). Finally, a specific inhibitor for PPAR-gamma, GW9662, weakened the MCP-1-decreasing effect of 15d-PGJ2 by about 30%, under basal conditions, while it abolished the effect of pioglitazone almost completely. CONCLUSIONS: Hypoxia-induced loss of function of PPAR-gamma reduces anti-inflammatory effects of PPAR-gamma activation, possibly modulating inflammatory responses in the diseased kidney.     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

Association between tumour necrosis factor-alpha G-308A polymorphism and risk of nephropathy in obese Chinese type 2 diabetic patients.

BACKGROUND: The G-308A polymorphism in the promoter region of the tumor necrosis factor alpha (TNF-alpha) gene has been reported to be associated with insulin resistance and obesity, both of which may increase the risk of diabetic nephropathy. We hypothesized that this polymorphism might interact with obesity to affect development of diabetic nephropathy. METHODS: A consecutive cohort of 1281 Chinese type 2 diabetic patients was enrolled for analysis. Genotyping of TNF-alpha G-308A polymorphism was performed using a PCR-based RFLP method with NcoI digestion. The mean value of the albumin creatinine ratio (ACR) of a random spot urine sample and a timed urinary collection was used to determine albuminuric status. Diabetic nephropathy was defined as serum creatinine > or =150 micromol/L and/or mean ACR > or =25 mg/mmol. Obesity was defined as body mass index > or =25 kg/m2 using Asian criteria. RESULTS: The G-308A polymorphism was not associated with either obesity or nephropathy. Clinical characteristics were similar between GG and GA/AA genotype carriers. Amongst the obese patients, GG genotype carriers had a higher median (interquartile range) urinary ACR [3.16 (0.70, 59.10) vs 1.28 (0.48, 12.28) mg/mmol; p = 0.01] and albumin excretion rate [38.7 (12.1, 620.3) vs 21.4 (8.9, 224.0) microg/min, p = 0.03] than GA/AA carriers. On multiple logistic regression analysis, compared with non-obese GA/AA carriers, obese subjects with the GG genotype had a 2.5-fold increased risk (95% CI: 1.04-6.03; P = 0.04) of nephropathy after adjustment for confounding factors. Other independent factors for diabetic nephropathy included male sex, systolic blood pressure, triglycerides (logarithmically transformed value), and the presence of cardiovascular and microvascular complications. CONCLUSION: Our findings suggest that the GG genotype of TNF-alpha G-308A polymorphism or a genetic variant in close linkage disequilibrium may interact with obesity to increase the risk of nephropathy in Chinese Type 2 diabetic patients. Apart from the need for replication of these results, functional studies are required to clarify its significance.