Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells

Receptor binding and degradation of urokinase-type plasminogen activator by human mesangial cells. The binding of [125I] labeled urokinase-type plasminogen activator (u-PA) was studied on human mesangial cells (MC) in culture. The binding of active [125I]u-PA at 37°C reached a plateau after 30 minutes of incubation and remained stable for at least four hours. When the supernatant was analyzed with trichloracetic acid (TCA), TCA soluble radioactive material could be detected after a lag phase of 30 minutes, and then increased linearly for four hours. Analysis by electrophoresis on SDS PAGE and autoradiography of the cell associated radioactivity and of the intracellular content showed that active u-PA and u-PA complexed to plasminogen activator inhibitor type-1 (PAI-1) were bound to the cell surface, but only u-PA/PAI-1 complexes were internalized and degraded. Therefore, the Kd and the number of binding sites were determined by competitive inhibition curves at 4°C using diisopropyl-fluorophosphate (DFP) u-PA. Scatchard plots showed a Kd = 400 ± 30 pM, and Bmax = 240,000 ± 25,000 sites/cell. Excess of the amino terminal fragment of u-PA (ATF) completely blocked the specific binding of [125I]u-PA, confirming that the binding of u-PA was independent of the presence of the active site and/or of the formation of complexes with PAI-1. 3H thymidine incorporation by mesangial cells after stimulation with 100nM active u-PA showed that u-PA had a moderate but significant mitogenic effect, in contrast to inactive u-PA and ATF. However, this mitogenic effect was not accompanied by a proliferative effect. Pretreatment of mesangial cell with a phosphoinositol-specific phospholipase C decreased the binding of [125I]u-PA by 60%, indicating that the majority of the u-PA receptor is anchored in the membrane by a phosphatidylinositol group. These results, together with a positive labeling of MC with monoclonal antibodies to the receptor of U937 cells, and the positive RNA hybridization with the cDNA probe for the human receptor cloned from U937 cells, indicate that the u-PA receptor on mesangial cells is identical to the one of U937 cells. In conclusion, human mesangial cells in culture express a specific receptor for u-PA, which could play a major role in the regulation of u-PA activity by degrading u-PA complexed to PAI-1

Estimating Extracellular Fluid Volume in Healthy Individuals: Evaluation of Existing Formulae and Development of a New Equation

peer reviewedIntroduction: Several clinical settings require an accurate estimation of the physiologically expected extracellular fluid volume (ECFV). We aimed to analyze the performances of existing ECFV-estimating equations and to develop a new equation. Methods: The performances of 11 ECFV-estimating equations were analyzed in 228 healthy kidney donor candidates (Bichat Hospital, Paris, France) who underwent ECFV measurement using the distribution volume of 51Cr-labeled EDTA (51Cr-EDTA). An equation was developed using a penalized linear modeling approach (elastic net regression) and externally (Tenon Hospital, Paris, France, N = 142) validated. Results: Participants from Bichat (mean age 45.2 ± 12.0 years, 43.0% men) and Tenon (47.8 ± 10.3 years, 29.6% men) hospitals had a mean measured ECFV of 15.4 ± 2.8 l and 15.1 ± 2.1 l, respectively. Available ECFV-estimating formulae have highly variable precision and accuracy. The new equation incorporating body weight, height, sex, and age had better precision and accuracy than all other equations in the external validation cohort, with a median bias of −0.20 (95% CI: −0.35 to −0.05) l versus −2.63 (−2.87 to −2.42) l to −0.57 (− 0.83 to −0.40) l and 0.21 (0.12 to 0.43) l to 2.89 (2.65 to 3.11) l, for underestimating and overestimating equations, respectively, an interquartile range for the bias of 0.88 (0.70 to 1.08) l versus 0.91 (0.71 to 1.20) l to 1.93 (1.67 to 2.25) l, and an accuracy within 10% of 90.9% (83.8 to 94.4) versus 88.0% (81.0 to 92.3) to 8.5% (4.2 to 13.4). These results were consistent across subgroups defined by sex, body mass index (BMI), body surface area (BSA), age, and ethnicity. Conclusion: We developed and validated a new equation to estimate the individual reference value of ECFV, which is easily usable in clinical practice. Further validation in cohorts including individuals of extreme age and corpulence remains needed

Les atteintes renales de l' hémoglobinurie paroxystique nocturne (une complication sous-estimée)

L hémoglobinurie paroxystique nocturne est une maladie clonale acquise de la cellule souche hématopoïétique caractérisée par une augmentation de la sensibilité des globules rouges à l action du complément activé, conduisant à une hémolyse intra-vasculaire. Des complications rénales sont décrites mais leur prévalence n a jamais été étudiée. Un effet rénal de l Eculizumab a été récemment évoqué. Nous avons mené une étude prospective, longitudinale portant sur 7 patients atteints d HPN afin de décrire les atteintes rénales, avant et après traitement par Eculizumab, par une mesure du débit de filtration glomérulaire et une exploration des fonctions tubulaires.Aucun sujet n était exempt d atteinte rénale. Un sujet était hyperfiltrant et 6 avaient une maladie rénale chronique (1 en stade 1, 2 en stade 2 et 3 en stade 3a) associée à une microalbuminurie dans la moitié des cas. Par analogie avec la néphropathie drépanocytaire, l atteinte glomérulaire pourrait être liée à l hémolyse, débutant par une hyperfiltration d origine hémodynamique et conduisant progressivement à une glomérulosclérose. L Eculizumab pourrait avoir un effet sur la MRC, par la suppression des facteurs causaux que sont l hémolyse et l anémie lorsqu initié précocement. Tous avaient des signes de tubulopathie proximale d intensité variable, d apparition précoce, liés aux dépôts d hémosidérine. L effet de l Eculizumab sur l atteinte tubulaire était limité.Par ailleurs, il est apparu que la créatininémie était un mauvais marqueur d évaluation et de suivi de la fonction rénale dans cette population. La mesure ponctuelle du DFG par un traceur exogène permettrait de ne pas sous-estimer une dysfonction rénale.PARIS6-Bibl.Pitié-Salpêtrie (751132101) / SudocSudocFranceF

Production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide

Production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide. Tumor necrosis factor (TNF) is a cytokine which is produced by mononuclear phagocytes upon activation by bacterial lipopolysaccharide (LPS) and various other stimuli. In immune-mediated glomerulonephritis, infiltration of glomeruli by monocytes-macrophages is associated with production of TNF. The purpose of the present experiments was to determine whether mesangial cells could also contribute to glomerular TNF synthesis. TNF activity has been determined in the culture medium of rat mesangial cells using a L-929 fibroblast lytic assay. This activity was detectable only when the cells were exposed to LPS (0.1 to 10 µg/ml) and for periods longer than one hour. The cytotoxic factor was identified as TNF since: (1) the lytic activity was completely inhibited by an anti-mouse TNF polyclonal antibody and was associated with suppression of lipoprotein lipase activity in adipocytes; (2) its molecular weight (110,000 daltons) corresponded to that observed for murine TNF under non-denaturing conditions; and (3) mRNA encoding TNF was expressed by mesangial cells two hours after addition of LPS. To assess the mechanisms whereby TNF production was regulated, the role of prostaglandin E2 (PGE2) was determined. LPS caused a dose-dependent increase of PGE2 synthesis by mesangial cells. Treatment by indomethacin promoted a suppression of PGE2 production together with an increase of TNF synthesis, indicating that PGE2 acted in a negative feedback manner to regulate the production of TNF. Addition of PGE2 (0.1 to 300nM) or 8-bromo cyclic AMP (0.1 to 100 µM) induced similar dose-dependent reductions of TNF synthesis. Thus the inhibitory effect of PGE2 probably required in part cyclic AMP accumulation. It is concluded that TNF is released by mesangial cells upon stimulation by LPS, and that its synthesis is regulated by both PGE2 and cyclic AMP. Production of TNF could participate in the mechanism of endotoxin-induced glomerular injury

Role of the renin-angiotensin system on the renal functional reserve in renal transplant recipients

Role of the renin-angiotensin system on the renal functional reserve in renal transplant recipients. To determine the renal functional reserve in renal transplant recipients, we measured the glomerular filtration rate by inulin clearance and the renal plasma flow by PAH clearance before and during an amino acid infusion (Totamine, 6 to 8 mg/kg/min for 90 to 120 min) in 18 transplanted patients with stable renal function. To test the role of the renin-angiotensin system on the renal functional reserve, we performed a crossover placebo-controlled randomized trial of acute blockade of the renin-angiotensin system by injection of perindoprilat (2 mg i.v.), an inhibitor of angiotensin converting enzyme before amino acid infusion, each patient being studied twice at seven day intervals. Amino acid infusion induced a time-dependent increase in the glomerular filtration rate (P = 0.04), whether or not the renin-angiotensin system was blocked. Maximal increases were from 49.1 ± 4.1 to 58.9 ± 5.4, mean ± SE (18.5%), in control conditions and from 52.4 ± 5.6 to 62.1 ± 5.5 ml/min/1.73 m2 (19.7%) after perindoprilat. The increase in glomerular filtration rate was less pronounced in patients taking cyclosporin A than in patients treated with steroid and azathioprine. Amino acid infusion also induced a significant and time-dependent increase (15.2 to 20.2%) in the renal plasma flow (P < 0.01) whether or not perindoprilat had been given. Furthermore, perindoprilat alone increased renal plasma flow by 13.6%, and this effect seemed additive with that of amino acids. Perindoprilat injection decreased filtration fraction (from 0.20 ± 0.01 to 0.19 ± 0.01). This parameter returned to basal values after amino acid infusion (0.20 ± 0.01). Amino acid infusion decreased renal vascular resistances as did perindoprilat alone. After perindoprilat administration, renal vascular resistances further decreased during amino acid infusion (P = 0.03). These results demonstrate that renal transplant recipients possess a renal functional reserve of which the mobilization is not inhibited in acute experiments by blockade of the renin-angiotensin system

Thrombin regulates components of the fibrinolytic system in human mesangial cells

Thrombin regulates components of the fibrinolytic system in human mesangial cells. Besides its procoagulant activity, thrombin has been shown to stimulate cell proliferation and to regulate the fibrinolytic pathway. We report here the effect of purified human alpha thrombin on the synthesis of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) by cultured human mesangial cells. Thrombin (0 to 2.5 U/ml) increased in a time- and dose-dependent manner the production of t-PA and PAI-1 (2- to 3-fold increase of secreted t-PA and PAI-1 release during a 24 hour incubation). This effect was associated with a twofold increase in DNA synthesis measured by 3H-thymidine incorporation. Zymographic analysis and reverse fibrin autography showed that thrombin also increased the level of the 110 Kd t-PA-PAI-1 complex, whereas PAI-1 was present as a free 50 Kd form in the culture medium conditioned by unstimulated and thrombin-stimulated cells. Free t-PA was never observed. Both membrane binding and catalytic activity of thrombin were required since the effects of 1 U/ml thrombin were inhibited by addition 2 U/ml hirudin, which inhibits the membrane binding and catalytic activity of thrombin, and since DFP-inactivated thrombin, which has the ability to bind but which has no enzymatic activity, did not induce t-PA or PAI-1. Gamma thrombin, which does not bind to thrombin receptor, did not increase t-PA and PAI-1 releases. The effects of thrombin were probably mediated by protein kinase C activation since H7, an inhibitor of protein kinases, inhibited significantly thrombin effects on t-PA and PAI-1 production, and since addition of an activator of protein kinase A, 8-bromocyclic AMP (100 µM), induced a significant inhibition of the thrombin effect. The effects of thrombin were also suppressed by 1.25 µg/ml alpha amanitin, suggesting a requirement of de novo RNA synthesis. Northern blot analysis indicated that thrombin induced an increase in the mRNA levels of t-PA and of PAI-1. We conclude that thrombin increases DNA synthesis in human mesangial cells and enhances the synthesis of both t-PA and PAI-1. The latter is released in a large excess as compared to t-PA. Hence, thrombin may have a role in provoking a localized hypofibrinolytic state and may contribute to the persistence of glomerular fibrin deposits during proliferative glomerulonephritis

BK virus genotypes and humoral response in kidney transplant recipients with BKV associated nephropathy

Background: Among kidney transplant recipients (KTR) with BK virus associated nephropathy (BKVN), BKV genotypes' evolution and anti-BKV humoral response are not well established. We aim to analyze BKV replication and genetic evolution following transplantation, and characterize concomitant anti-BKV-VP1 humoral response.Methods: We retrospectively analyzed 32 cases of biopsy-proven BKVN. Stored plasma and kidney biopsies were tested for BKV viral load, and VP1 sequencing performed on positive samples. BKV-VP1 genotype-specific neutralizing antibodies (NAbs) titers were determined at transplantation and BKVN.Results: At the time of BKVN diagnosis, BKV viral load was 8.2 log10 IU/106 cells and 5.4 log10 IU/mL in kidney and plasma, respectively. VP1 sequencing identified the same BKV-subtype in both compartments in 31/32 cases. At the time of transplantation, 8/20 (40%) of biopsies tested positive for BKV detection, whereas concomitant BKV viremia was negative. VP1 sequencing identified a different subtype compared to BKVN in 5/6 of these samples. This was confirmed following transplantation: 8 patients had a BKV+ biopsy before BKV viremia, and VP1 sequencing identified a different subtype compared to BKVN in all of them. After the onset of BKV viremia and prior to BKVN diagnosis, the BKV subtype in BKV+ plasma and kidney biopsy was the same as the one isolated at BKVN. BKV-VP1 NAbs titers were significantly higher at the time of BKVN compared to transplantation (p = .0031), with similar titers across genotypes.Conclusion: Altogether, our data suggest that among some KTR with BKVN, the BKV genotype from the donor may not be responsible for BKVN pathogenesis

Long-term survival benefit from dual kidney transplantation using kidneys from donors with very extended criteria-a French cohort between 2002 and 2014

International audienceBACKGROUND: This national multicentre retrospective cohort study aimed to assess the long-term outcomes of dual kidney transplantation (DKT) and compare them with those obtained from single kidney transplantation (SKT). METHODS: Our first analysis concerned all first transplants performed between May 2002 and December 2014, from marginal donors, defined as brain death donors older than 65 years, with an estimated glomerular filtration rate (eGFR) lower than 90 mL/min/1.73 m2. The second analysis was restricted to transplants adequately allocated according to the French DKT program based on donor eGFR: DKT for eGFR between 30 and 60, SKT for eGFR between 60 and 90 mL/min/1.73 m2. Recipients younger than 65 years or with a panel-reactive antibody percentage ≥25% were excluded. RESULTS: The first analysis included 461 DKT and 1131 SKT. DKT donors were significantly older (77.6 versus 74 years), had a more frequent history of hypertension and a lower eGFR (55.1 versus 63.6 mL/min/1.73 m2). While primary nonfunction and delayed graft function did not differ between SKT and DKT, 1-year eGFR was lower in SKT recipients (39 versus 49 mL/min/1.73 m2, P < 0.001). Graft survival was significantly better in DKT, even after adjustment for recipient and donor risk factors. Nevertheless, patient survival did not differ between these groups. The second analysis included 293 DKT and 687 SKT adequately allocated with donor eGFR and displayed similar results but with a smaller benefit in terms of graft survival. CONCLUSIONS: In a context of organ shortage, DKT is a good option for optimizing the use of kidneys from very expanded criteria donors

Presentation and Outcome of Patients with Systemic Amyloidosis Undergoing Dialysis

Background and objectives: Light chain (AL) and secondary (AA) amyloidosis usually present as a systemic disease frequently involving the kidney and leading to ESRD. Data regarding patients with AA or AL amyloidosis undergoing dialysis remain scarce