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Regulation of growth-hormone-receptor gene expression by growth hormone and pegvisomant in human mesangial cells.BackgroundMice transgenic for growth hormone develop mesangial proliferation, glomerular hypertrophy, and progressive glomerular sclerosis suggesting that the growth hormone–insulin-like growth factor I (IGF-I) pathway plays an important role. Therefore, we studied the impact of variable concentrations of 22 kD, 20 kD growth hormone, as well as of the growth hormone receptor antagonist pegvisomant (B2036-PEG), on both the growth hormone receptor (GHR/GHBP) gene expression and growth hormone binding protein (GHBP) formation in a human glomerular mesangial cell line. Further, the impact on collagen, IGF-I and IGF binding protein-1 (IGFBP-1) formation was studied.MethodsIn order to assess transcription, quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used.ResultsPhysiologic doses of 22 kD or 20 kD growth hormone caused a dose-dependent and significant (P < 0.01) up-regulation of GHR/GHBP gene transcription, whereas supraphysiologic doses (50 and 500ng/mL) resulted in down-regulation (P < 0.001). Whenever pegvisomant was used, there was no increase in GHR/GHBP expression. These data were confirmed using run-on experiments. Further, the assessment of GHBP presented a constant, dose-dependent increase, which was completely abolished in the experiments where pegvisomant was used.ConclusionWe present data showing that growth hormone has a direct impact on GHR/GHPB gene transcription and that pegvisomant is a potent growth hormone receptor antagonist in human mesangial cells. In addition, although the GHR/GHBP gene transcription is down-regulated by supraphysiologic growth hormone concentrations, this effect was not found when GHBP levels were measured. This finding may reflect a self-inhibitory effect of growth hormone on the level of GHR/GHBP gene transcription, which does not involve the regulation of the shedding of GHBP and may, therefore, be of physiologic interest.

The renin-angiotensin system plays a major role in the control of blood pressure and of salt balance, but it is also involved in physiological and pathological processes, development, inflammation and cardiac hypertrophy. A concept has emerged suggesting that these effects are due to a local activation of the renin-angiotensin system. The search for a receptor of renin was based on the idea that tissue (pro)renin is taken up from the circulation and on data suggesting that renin has cellular effects independent of angiotensin II.Endothelial cells and cardiac myocytes bind (pro)renin via the mannose-6-phosphate receptor, mainly a clearance receptor as no cellular effect has been specifically attributed to prorenin binding. A functional receptor was cloned recently. It mediates intracellular signalling by activating the mitogen activated protein kinases, extracellular signal regulated kinases 1 and 2, and acts as a co-factor by increasing the efficiency of angiotensinogen cleavage by receptor-bound (pro)renin. The receptor is abundantly expressed in heart, brain, placenta and eye, compared with a lower expression in liver and kidney. In normal human kidney and heart, it is localized in the mesangium and in the coronary and kidney artery, associated with smooth-muscle cells and co-localized with renin.This receptor provides a functional role for prorenin and may help to understand the physiological and pathological role of elevated levels of prorenin and of local activation of the renin-angiotensin system. From a practical point of view, it questions the need for a pharmacological compound blocking (pro)renin binding and activity as an alternative to the classical inhibitors of the renin-angiotensin system.

Background. Haemolytic uraemic syndrome (HUS) is a rare and severe disease of various aetiologies in adults. The effect of fresh frozen plasma (FFP) infusion in adults suffering from HUS is not well defined. The aim of this retrospective study was to analyse the causes of HUS in adults admitted in a single renal intensive care unit (ICU) and to determine the life and renal prognosis factors, while most patients (78%) received FFP infusion. Methods. We recorded clinical, biological, and histological data of 55 adults admitted in our renal ICU for HUS between 1990 and 1998. 49 of them having had a renal biopsy. By stepwise logistic regression analysis, we examined the parameters that were associated with the in-hospital mortality and renal function at discharge. Results. HUS complicated different diseases in 40 patients (HIV infection n = 18, nephropathies n = 10, allotransplantation n = 7, malignant diseases n = 5) and appeared as a primary in 15 patients. Factors influencing the in-hospital mortality were positive HIV serology (odds ratio (OR) >20, P=0.0002) and requirement for haemodialysis (OR >35, P = 0.004). A pre-existing nephropathy was a bad prognosis factor for renal function (OR >99, P = 0.02), while fever was associated with better renal prognosis (OR = 1 10, P = 0.033). Conclusions. HUS in adults remains a severe disease, with a high mortality rate in HIV patients and in those who required haemodialysis. However. as compared with previous studies, we observed an improvement in renal outcome, particularly in patients with primary HUS, suggesting a beneficial effect of FFP infusion, at least in these forms.

BACKGROUND Plasminogen activator inhibitor type 1 (PAI-1) exerts antifibrinolytic and profibrotic activities. Inside the glomerulus, PAI-1 is mainly synthesized by mesangial cells. We hypothesized that thrombin, via its receptor protease activated receptor type 1 (PAR-1), present on the membrane of glomerular cells, is an important mediator of PAI-1 synthesis. METHODS Using the technique of Peten et al., we microdissected the glomeruli of 23 kidney transplanted patients admitted in our department from 1993 to 1997, and we followed-up these patients for up to 5 years, with sometimes iterative renal biopsies. With this technique, we also microdissected the glomeruli of three patients who have had a nephrectomy for cancer (control patients). We investigated mRNA expression of the PAI-1, the thrombin receptor PAR-1, the alpha2 chain of type IV (alpha2 IV) collagen, and of a housekeeping gene (cyclophilin) by reverse transcription-polymerase chain reaction. The results were correlated with the renal function and the histological findings classified into acute rejection (9 biopsies), chronic rejection (22 biopsies), or normal (8 biopsies). RESULTS A significant up-regulation of PAI-1 and alpha2 IV collagen mRNA was observed in acute rejection (P

Background. Hemolytic uremic syndrome (HUS) is a rare complication in solid organ transplantation. It can be associated with severe hypertension. Several risk factors have been identified including immunosuppressive drugs such as cyclosporin A and, more recently, tacrolimus. Methods. Here we report a case of tacrolimus-induced HUS in a 61-yr-old woman after liver transplantation. Hypertension, microangiopathic anemia and end-stage renal failure occurred 2 yr after liver transplantation. Results. At admission, she had malignant hypertension with a severe hypertensive retinopathy, renal failure (creatininemia: 800 μmol/L) and microangiopathic anemia (Hb: 7.3 g/dL, a low platelet count and elevated lactate dehydrogenase). At renal biopsy, histologic findings were ischemic and sclerotic glomeruli with hyaline thrombi, severe mesangiolysis and interstitial fibrosis. Conclusion. Despite steroid treatment, antihypertensive agents and fresh frozen plasma therapy, end-stage renal failure was observed and chronic hemodialysis treatment was required.

Objective: To determine the epidemiological trends, spectrum of etiologies, morbidity and mortality of acute renal failure (ARF) in patients over 80 years old.¶Design: Historical cohort analysis.¶Setting: Intensive care unit (ICU) of nephrology, Tenon Hospital, Paris.¶Patients and participants: The criteria of inclusion was ARF, defined on the basis of a creatinine value over 120 μmol/l, in patients over 80 years of age admitted between October 1971 and September 1996. When moderate chronic nephropathy was pre-existing, ARF was defined by the increase of at least 50 % over the basal creatininemia.¶Measurements and results: Three hundred and eighty-one patients over 80 years of age were included. The etiology and mechanism of ARF are detailed. 29 % of the patients received dialysis. Global mortality at the hospital was 40 %. Factors significantly associated with a poor prognosis are identified. Mean survival after hospitalization was 19 months.¶Conclusion: The frequency of admission to ICUs for ARF in patients older than 80 years seems to be on the increase. Mortality is less severe than expected. These patients could benefit from the renal replacement therapy of modern intensive care medicine.

Hyperfiltration, associated with kidney hypertrophy and hyperplasia, are early markers of diabetic nephropathy [1, 2]. Procedures for the identification of renal molecules potentially involved in this early process are of particular interest. In order to detect hyperexpression of major renal proteins in early diabetic nephropathy, we used the well-known model of streptozotocin (STZ)induced diabetes in rats [3] and a robust technique of subtracted hybridization between normal and diabetic kidneys [4 ‐ 6]. Two subtracted probes were elaborated after hydridization of diabetic rat kidney cDNA with control rat kidney mRNA. These probes were used to screen a diabetic rat kidney cDNA library. Two clones were overexpressed in diabetic rat kidneys and corresponded to the multi-drug resistance 1 (mdr 1) cDNA and to the b-amyloid protein precursor (bPP), respectively. This study describes the results of subtractive hybridization cloning in rats, as well as some data obtained in human kidney sections originating from diabetic patients.

Specific receptor binding of renin on human mesangial cells in culture increases plasminogen activator inhibitor-1 antigen. Some proteases possess a membrane receptor that focalizes their proteolytic activity on the cell surface and may mediate a proliferative effect, such as urokinase on glomerular epithelial cells. Since some hypertensive states are associated with high concentrations of renin and proliferation of arteriolar smooth muscle cells, we asked whether renin, an aspartyl-protease, would bind to mesangial cells that are smooth-muscle derived cells, which would induce their proliferation. The binding of 125 I labeled recombinant human renin ( 125 I-R) was studied on human primary mesangial cells and mesangial cells immortalized by transfection with SV40-T antigen. At 37°C, the binding of 125 I-R was time dependent and reached a plateau after two hours. 125 I-R was found to bind in a saturable and specific manner with a K d = 0.4nM and 1nM and 8,000 and 2,000 binding sites/cell, for primary and immortalized cells, respectively. When binding experiments were performed in the presence RO 42-5892, a synthetic inhibitor of renin, RO 42-5892 could inhibit the specific binding of labeled renin only at concentrations 1,000 times superior to the IC 50, indicating that the renin-mesangial receptor interaction did not depend on the active site of renin. Analysis by SDS-PAGE and autoradiography of cross-linking experiments of 125 I-R bound to a membrane preparation showed a band of approximately 110 to 120 kDa, suggesting a Mr of 70 to 80kDa for the renin receptor. Incubation of mesangial cells with 100nM renin for 24 hours provoked a 100% increase of 3 H thymidine incorporation that was not accompanied by an increase of the cell number, even after a seven day period of incubation. However, the incubation of mesangial cells with renin for 24 hours induced a significant increase (170% of control, P = 0.04) of plasminogen activator inhibitor-1 (PAI1) antigen in the conditioned medium. In conclusion, we have shown that human mesangial cells in culture express a specific receptor for renin, and that the binding of renin increases 3 H thymidine incorporation independently of renin enzymatic activity. The absence of cell proliferation, the increase of 3 H thymidine incorporation and the increase of PAI1 antigen suggest that the binding of renin can induce mesangial cell activation, which is reflected by a change in the fibrinolytic capacity of the cells. The role of this receptor remains to be determined in nephropathies and hypertensive states associated with high plasma/tissue renin concentrations, hypertrophy of mesangial or smooth muscle cells and extracellular matrix remodeling.

Numerous glomerular and vascular nephritides are associated with fibrin formation and deposition within the kidney. Thrombin, which induces fibrin formation, also exerts numerous cellular effects on both circulating cells and intrinsic glomerular cells through the activation of the functional thrombin receptor. By immunohistochemistry and in situ hybridization, we demonstrated the constitutive expression of the functional thrombin receptor in the normal human kidney. Both glomerular epithelial and mesangial cells in culture also express the functional thrombin receptor, as shown by the binding of a specific monoclonal antibody against this receptor and Northern blot analysis. Thrombin has a potent mitogenic effect on cultured glomerular cells, suggesting that it could be at least, in part, one of the mediators that induces glomerular cell proliferation in glomerular diseases. Furthermore, we demonstrated that thrombin upregulates the synthesis of plasminogen activators and their type 1 inhibitor in these cells. Thrombin induces protein kinase C activation and an increase in intracellular calcium in these cells. Finally, we demonstrated that the functional thrombin receptor is internalized after addition of thrombin and thrombin receptor-activating peptide (homologous internalization) and also after phorbol myristate acetate addition (heterologous internalization).

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.

The mechanisms of glomerular injury can be separated into nonimmunologically mediated glomerulonephritis (GN) such as diabetes, leading to glomerular hypertension and into immunologically mediated GN. The immunologically mediated GN may induce chronic glomerulopathy such as membranous GN or proliferative GN. The final pathway of these two types of GN is proteinuria and renal failure linked to glomerulosclerosis. In inflammatory GN, most of the mediators could be synthesized either by infiltrating cells or by resident glomerular cells. They include cytokines, lymphokines, complement activation, generation of superoxyde anions, arachidonic acid metabolites, and fibrin deposition. (a) We have investigated the interaction between isolated glomeruli and platelets and have demonstrated that lipidic and proteic extracts of glomeruli enhance thromboxane B2 platelet synthesis. This fact is related to the generation by isolated glomeruli of saturated fatty acids and tissue factor. (b) We investigated the interaction between rat isolated glomeruli and peritoneal macrophages. We have demonstrated that 12-HETE synthesized by isolated glomeruli induce macrophage prostaglandin synthesis which, in turn, inhibits the 12-HETE synthesis. (c) We have demonstrated, using human glomerular epithelial cells, that alpha-thrombin, the active form of thrombin, generated before fibrin formation, is able to induce cell proliferation and abolishes the profibrinolytic activity of these cells. In summary, the mechanisms of glomerular injury are complex, certainly acting by multiple pathways. So far, the mediators leading to proteinuria and renal failure after glomerular injury remain under investigation.

Using an immortalized human glomerular epithelial cell line (E71 A1), we studied the effect of endothelin-1 (ET-1), a potent vasoconstrictor peptide, on the synthesis of urokinase type plasminogen activator (u-PA) and its receptor (u-PAR). The results show that ET-1 had no effect on u-PA synthesis but induced an increase in u-PAR number (2.8 +/- 0.6 x 10(4) vs 1.2 +/- 0.5 x 10(4) sites per cell, p less than 0.001) without change in receptor affinity (280 +/- 80 pM vs 250 +/- 50 pM, NS), maximal effect being observed at 10(-7) M. Time course shows that a plateau was reached after a 24 hour incubation. ET-1 induced-increase in binding capacity was abolished by cycloheximide. ET-1 also induced an increase in u-PAR mRNA level, which was completely blocked by alpha-amanitin (5 micrograms/ml). Cycloheximide (1 microgram/ml) alone induced an increase in u-PAR mRNA level and this effect was enhanced when cycloheximide was combined with ET-1. Our data show that ET-1 can induce an increase in membrane expression of u-PAR through activation of the transcription of the u-PAR gene and de novo protein synthesis.

Tumor necrosis factor α increases antifibrinolytic activity of cultured human mesangial cells. Tumor necrosis factorα (TNFα) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties, TNFα interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNFα on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNFα binds specifically to a single class of high affinity receptors (K d 5.10 -11 M; 1500 receptors/cell). TNFα has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3 H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51 Cr was not increased by 100 ng/ml TNFα as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNFα (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNFα increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours. Finally, in contrast to what was reported for rat mesangial cells, we failed to demonstrate a production of TNFα in human mesangial cells themselves by immunoradiometric assay, immunocytochemistry and Northern blot analysis using a specific TNFα cDNA probe. We conclude that TNFα has an antimitogenic activity on human mesangial cells in culture and enhances the synthesis of PAI-1. Whether TNFα plays a role in glomerular PAI-1 deposition and persistency of fibrin in vivo remains to be determined.

A 24-year-old white woman with a past history of recurrent venous thromboses of the lower extremities was admitted for hypertension and renal failure. She had a chronic cutaneous ulcer on the anterior side of the left leg and oral ulcers of the palatum. Laboratory tests demonstrated rapidly progressive renal failure and the presence of an anticardiolipin antibody (ELISA). Thrombosis of the inferior vena cava was shown by phlebocavography. Renal biopsy revealed typical thrombotic microangiopathy. Tissue-type plasminogen activator (tPA) was visualized by immunofluorescence in endothelial cells of renal arterioles and glomeruli. Normal plasma levels of tPA, urokinase and plasminogen activator inhibitor 1 were found by ELISA, and tPA antigen levels rose after desmopressin acetate infusion. Thus, in this case, the diffuse thrombotic process was not related to defective circulating or renal fibrinolytic systems and could be promoted by the procoagulant effect of antiphospholipid antibodies.

A role for cytomegalovirus (CMV) infection in the etiologies of acute and chronic rejection in renal allograft recipients has been suggested. We previously reported that preemptive treatment of CMV infection with ganciclovir in kidney transplant patients was safe and effective. We now present a retrospective analysis of 169 consecutive renal transplant patients, of whom 87 (51.5%) received preemptive treatment with ganciclovir (CMV(+) group). No patient died of CMV infection. Actuarial graft and patient survival rates were not different between the CMV(+) and the CMV(-) groups (graft survival: 68% and 69%; patient survival: 89% and 88%, respectively). At the end of the study, the mean plasma creatinine levels were not statistically different between the two groups (185+/-13 and 166+/-12 micromol/L for the CMV(+) group and the CMV(-) group, respectively). These results suggest that preemptive treatment of CMV infection with ganciclovir may prevent the CMV-induced renal injury and graft loss.

The renin-angiotensin system (RAS) has become increasingly complex. New components have been identified, and additional roles for angiotensin peptides and their receptors are being uncovered. A functional (pro)renin receptor has been cloned that acts as (pro)renin cofactor on cell surface, enhancing the efficiency of angiotensinogen cleavage by (pro)renin and unmasking prorenin catalytic activity. Binding of (pro)renin to the receptor mediates (pro)renin cellular effects by activating mitogen-activating protein (MAP) kinases, extracellular signal-regulated kinases (ERK)1/2. Immunofluorescence studies have localized the receptor on mesangial and vascular smooth muscle cells in human heart and kidney. This suggests that the renin receptor might represent a means to capture (pro)renin from the circulation and to concentrate (pro)renin at the interface between smooth muscle and endothelial cells. In this article, we review the biochemical characteristics of this receptor and of other renin-binding proteins, and discuss their physiologic significance.

Plasminogen activator inhibitor type 1 is a potential target in renal fibrogenesis. The progression of renal lesions to fibrosis involves several mechanisms, among which the inhibition of extracellular matrix (ECM) degradation appears to play an important role. Two interrelated proteolytic systems are involved in matrix degradation: the plasminogen activation system and the matrix metalloproteinase system. The plasminogen activator inhibitor type 1 (PAI-1), as the main inhibitor of plasminogen activation, regulates fibrinolysis and the plasmin-mediated matrix metalloproteinase activation. PAI-1 is also a component of the ECM, where it binds to vitronectin. PAI-1 is not expressed in the normal human kidney but is strongly induced in various forms of kidney diseases, leading to renal fibrosis and terminal renal failure. Thrombin, angiotensin II, and transforming growth factor-beta are potent in vitro and in vivo agonists in increasing PAI-1 synthesis. Several experimental and clinical studies support a role for PAI-1 in the renal fibrogenic process occurring in chronic glomerulonephritis, diabetic nephropathy, focal segmental glomerulosclerosis, and other fibrotic renal diseases. Experimental models of renal diseases in PAI-1-deficient animals are in progress, and preliminary results indicate a role for PAI-1 in renal fibrogenesis. Inhibition of PAI-1 activity or of PAI-1 synthesis by specific antibodies, peptidic antagonists, antisense oligonucleotides, or decoy oligonucleotides has been obtained in vitro, but needs to be evaluated in vivo for the prevention or the treatment of renal fibrosis.

The binding of Fc fragments of Ig on glomerular epithelial cells (GEC) was observed previously, but the receptor could not be identified. In immunofluorescence and immunohistochemical studies using normal adult human kidney sections, the presence of the so-called neonatal Fc receptor (FcRn) was demonstrated on GEC as well as in the brush border of proximal tubular cells. FcRn transcripts were also detected on isolated glomeruli by reverse transcription-PCR. Using an immortalized GEC line, the presence of the FcRn was confirmed by flow cytometry, reverse transcription-PCR, Western blotting, and by the pH dependence of the binding of heat-aggregated IgG. Because it is well established that the FcRn is involved in IgG transcytosis, it is hypothesized that the FcRn in the kidney may play a role in the reabsorption of IgG. Ongoing studies should clarify the role of the FcRn as a potential target for immune complexes on GEC and should assess its relevance in physiology and pathology.

Abstract —3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (HRIs) have been recently shown to prevent atherosclerosis progression. Clinical benefit results from combined actions on various components of the atherosclerotic lesion. This study was designed to identify the effects of HRI on one of these components, the endothelial fibrinolytic system. Aortas isolated from rats treated for 2 days with lovastatin (4 mg/kg body wt per day) showed a 3-fold increase in tissue plasminogen activator (tPA) activity. In a rat aortic endothelial cell line (SVARECs) and in human nontransformed endothelial cells (HUVECs), HRI induced an increase in tPA activity and antigen in a time- and concentration-dependent manner. In SVARECs, the maximal response was observed when cells were incubated for 48 hours with 50 μmol/L HRI. An increase of tPA mRNA was also in evidence. In contrast, HRI inhibited plasminogen activator inhibitor-1 activity and mRNA. The effects of HRI were reversed by mevalonate and geranylgeranyl pyrophosphate, but not by LDL cholesterol and farnesyl pyrophosphate, and were not induced by α-hydroxyfarnesyl phosphonic acid, an inhibitor of protein farnesyl transferase. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and plasminogen activator inhibitor-1 activity and blocked its reversal by geranylgeranyl pyrophosphate. The effect of HRI was associated with a disruption of cellular actin filaments without modification of microtubules. A disrupter of actin filaments, cytochalasin D, induced the same effect as lovastatin on tPA, whereas a disrupter of microtubules, nocodazole, did not. In conclusion, HRI can modify the fibrinolytic potential of endothelial cells, likely via inhibition of geranylgeranylated Rho protein and disruption of the actin filaments. The resulting increase of fibrinolytic activity of endothelial cells may contribute to the beneficial effects of HRI in the progression of atherosclerosis.

Background Endocarditis-induced crescentic glomerulonephritis is a well-described complication in nontransplant patients. Its occurrence in transplant patients has not been reported to date. Methods A 50-year-old man who had received a renal allograft 13 years before and been treated with prednisone, 10 mg/day, was admitted for progressive renal failure, purpura, edema of the lower limbs, and fever. Results Blood cultures isolated Streptococcus bovis and cardiac ultrasound examination revealed a 23-mm-large vegetation on the mitral valve. His plasma creatinine level was 478 micromol/L and his proteinuria was 5.5 g/day. A renal biopsy showed diffuse crescentic glomerulonephritis. Long-term antibiotic treatment and three methylprednisolone pulses were effective in treating the endocarditis and glomerulonephritis. Conclusion Endocarditis-induced glomerulonephritis is an immune-mediated disease that can also occur on a renal allograft. It is likely that a low daily dose of immunosuppressive treatment may have been a facilitating factor.

Interstitial fibrosis is one of the most deleterious events during the progression of renal deterioration after renal mass reduction. In vivo, hydroxymethylglutaryl CoA reductase inhibitors (HRI) were shown to reduce progression of glomerulosclerosis, but the mechanisms are still unclear. The present study investigates, in vivo, whether lovastatin, a potent HRI, was able to modulate the plasminogen-plasmin pathway, one of the most efficient systems involved in extracellular matrix remodeling, and characterizes in vitro the cellular mechanisms of these effects. Proximal tubules freshly isolated from rats treated for 2 d with lovastatin (4 mg/kg per d) showed increased tissue-type plasminogen activator (tPA) and urokinase (uPA) activities and antigens. Incubation with lovastatin (5 microM) of proximal tubules isolated from untreated rats induced an increase in tPA and uPA and a decrease in plasminogen activator inhibitor-1 (PAI-1) activities. In vitro, supernatants, cytosols, and membranes of renal proximal tubular cells in primary cultures had no detectable uPA activity, and lovastatin (0.1 to 10 microM) induced an increase in tPA and a decrease in PAI-1 activities and antigens. These effects were reversed by mevalonate and geranylgeranyl-pyrophosphate (GGPP) but not by farnesyl-pyrophosphate or LDL cholesterol. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and PAI- activity and blocked its reversion by GGPP. The effect of lovastatin was associated with a disruption of cellular actin stress fibers, which was reversed by GGPP and reproduced by C3 exoenzyme. In conclusion, HRI can modify the fibrinolytic potential of proximal tubules, most likely via inhibition of geranylgeranylated Rho protein and disruption of the cytoskeleton. The resulting increase of proteolytic activity of tubular cells may serve to prevent extracellular matrix deposition and renal interstitial fibrosis.

We evaluated the prevalence of renal cell carcinoma of the native kidneys in renal transplant recipients as well as possible risk factors.A total of 129 consecutive renal transplant recipients underwent ultrasound examination of the native kidneys as part of a routine evaluation. A record was made of acquired cystic kidney disease, defined as 3 cysts or more, and of suspicious masses. When a malignancy was suspected radical nephrectomy was performed.The overall prevalence of renal cell carcinoma of the native kidney was 5 in 129 recipients (3.9%). All cancers were limited to the kidney. No significant relationship was detected between renal cell carcinoma occurrence and patient age, dialysis (when initiated, type and duration), transplantation, drug regimen or incidence of acquired cystic kidney disease.The risk of renal cell carcinoma, a clinically significant cancer, was approximately 100 times greater in our renal transplant patients than in the general population but no significant risk factor could be identified. Routine ultrasonography for early diagnosis in asymptomatic patients on immunosuppressive therapy is strongly recommended to improve prognosis.

The binding of urokinase-type plasminogen activator (u-PA) to a specific cell surface receptor (uPA-R) has been shown to enhance plasminogen activation, a process involved in extracellular matrix degradation and cell migration during angiogenesis and tumor growth. We investigated the expression of u-PA and uPA-R in renal cell carcinomas (n = 11). By immunohistochemistry using monoclonal and polyclonal anti-uPA-R antibodies, we found that tumoral capillary endothelial cells (von Willebrand factor and CD31 positive cells) overexpressed uPA-R, whereas vascular endothelial cells of the normal human kidney do not. In addition, tumor-associated macrophages (CD68-positive cells) strongly expressed uPA-R. In contrast, few tumoral cells and stromal fibroblasts expressed uPA-R. By in situ hybridization using a cDNA S 35 -labeled probe specific for uPA-R, we confirmed the local expression of uPA-R messenger RNA. We also detected the induction of u-PA in tumoral capillary endothelial cells and in tumor-associated macrophages. In two cases, tumoral cells themselves were also stained by anti—u-PA antibodies in focal areas. Finally tissue-type plasminogen activator (t-PA) was also overexpressed by tumoral capillary endothelial cells as compared with endothelial cells of normal human kidney vessels. These findings indicate an active invasive phenotype of endothelial cells in renal cell carcinoma and suggest a role for the plasminogen activation system in tumoral angiogenesis and invasion.

Renal retransplantation can be hampered by the presence of anti-HLA alloantibodies. Previous studies have documented in vitro and in vivo suppression of these antibodies by intravenous immunoglobulins (IVIg). We conducted a randomized study in 41 patients, who have received a second cadaveric transplant between 1989 and 1994. They all were treated with a quadruple-immunosuppressive protocol. In addition, 21 patients received 0.4 g/kg/day of IVIg, on the first 5 days after transplantation. The two groups of patients were identical for age, sex, duration of the first graft, duration of cold ischemia, anti-HLA sensitization, HLA matching, the number of acute rejection episodes, and the incidence of cytomegalovirus infection. The 5-year survival rate was significantly higher in the group of patients treated with IVIg: 68% versus 50% in the control group. The only significant factor associated with IVIg infusion and better survival was a shorter delay of graft function (3.4 +/- 1.0 days versus 9.9 +/- 1.6 days). In conclusion, this randomized study demonstrates that IVIg treatment is associated with better long-term graft survival in retransplanted patients. This beneficial effect may be related to a long-lasting immunosuppressive effect of IVIg and/or to an early protective effect of the graft against ischemia.

Evidence for renal glomerular receptors for angiotensin II. Monoiodinated angiotensin II (2000 mCi/μmole) was found to bind specifically to isolated rat glomeruli. Equilibrium was reached after 12 min and specific binding represented more than 95% of total binding. Dissociation after addition of an excess of unlabelled molecules was rapid. The k 1 and k -1 association and dissociation constants determined from time-course studies were 0.254±0.078 × 10 10 M -1 min -1 and 0.102±0.018 min -1 , respectively and the ratio k -1 /k 1 (K D ) was 4.51±0.55 × 10 -11 M. Angiotensin II, liberated from glomerular binding sites at low pH, retained the ability to bind to fresh membranes. Angiotensin II, angiotensin I, ileu 8 -angiotensin II and sarc 1 -ileu 8 -angiotensin II were all equally effective as competitive inhibitors of 125 I-angiotensin binding. In a preparation of isolated rat glomeruli, mean glomerular diameter decreased as a function of 125 I-angiotensin II concentration according to a sigmoidal effect vs. log dose curve and the calculated K D (6 × 10 -11 M) was similar to that obtained from binding studies. Specific binding of angiotensin II at physiological plasma concentration to rat glomeruli and correlation of this binding with glomerular vasoreactivity suggest a physiological role for this hormone in regulation of glomerular filtration. Recepteurs renaux glomerulaires de l'angiotensine II. L'Angiotensine II monoiodee (2000 mCi/μmole) se lie specifiquement aux glomerules de rat isoles. L'equilibre est atteint au bout de 12 mn et la fixation specifique represente plus de 95% de la fixation totale. L'addition d'un exces d'angiotensine non marquee determine une dissociation rapide de l'hormone fixee. Les constantes d'association (k 1 ) et de dissociation (k -1 ) calculees a partir des experiences de fixation en fonction du temps sont egales a 0,254±0,078 × 10 10 M -1 mn -1 et 0,102±0,018 mn -1 , respectivement. Le rapport k -1 / k1 (K D ) est egal a 4,51 ±0,55 × 10 -11 M. L'angiotensine II, eluee a pH acide de ses sites glomerulaires de liaison, garde la capacite de se lier de nouveau a des membranes fraiches. L'angiotensine II, l'angiotensine I, la ileu 8 -angiotensine II et la sarc 1 -ileu 8 -angiotensine II sont des inhibiteurs competitifs de la 125 I-angiotensine II, tous de meme efficacite. Le diametre glomerulaire moyen mesure dans une preparation de glomerules isoles decroit en fonction de la concentration de la 125 I-angiotensine II selon une courbe sigmoide (effet contre le log de la dose) et le K D calcule (6 × 10 -11 M) est semblable a celui obtenu a partir des experiences de liaison. La fixation specifique aux glomerules de rat de l'angiotensine II a des concentrations identiques a celles presentes physiologiquement dans le plasma, ainsi que la correlation entre la liaison de l'hormone aux glomerules et la vasomotricite glomerulaire suggerent un role physiologique de l'angiotensine II dans la filtration glomerulaire.

Angiotensin II (Ang II) controls renal plasma flow, which depends on perfusion pressure and on renal vascular resistances; the glomerular filtration rate of single nephrons (SNGFR) also depends on these two parameters. The glomerulus can synthesize all components of the renin-angiotensin system. Renin is produced by mesangial cells and cells of afferent arterioles, angiotensinogen by mesangial cells, and angiotensin I-converting enzyme (ACE) by endothelial and mesangial cells. Ang II binds to afferent and efferent arterioles and to mesangial cells, which subsequently contract and enhance their production of vasodilator prostaglandins. Ang II participates in the regulation of the SNGFR by acting on arteriolar resistance, on the glomerular capillary surface area, and finally on the ultrafiltration coefficient. The synthesis of renin is initiated when the cAMP concentration increases under the influence of hormones (parathyroid hormone, catecholamines), of mediators (e.g., prostanoids), or of changes in composition of tubular fluid (in sodium, chlorine, or calcium) detected by the macula densa. Moreover, renin synthesis is stimulated by decreases in intracellular calcium concentration. The renin-angiotensin system plays an important role in the regulation of glomerular filtration. A decrease in renal plasma flow is followed by an increase in glomerular efferent arteriole resistance, so that perfusion pressure and glomerular filtration remain constant. Furthermore, if the decrease in renal plasma flow is more marked, vasodilator prostaglandins, whose synthesis is stimulated by Ang II, dilate afferent arterioles, which sustains glomerular filtration. Prostaglandins are also apt to bind to the juxtaglomerular apparatus and to increase the intracellular cAMP level.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyunsaturated fatty acids increase fibrinolytic activity of human isolated glomeruli. The release of plasminogen activators (PA) from human isolated glomeruli has been studied by a sensitive radioenzymatic assay using 125I-fibrin coated tubes and plasminogen. The glomerular fibrinolytic activity (GFA) was detectable after 15 minutes of incubation. Then it increased with time, the glomerular protein concentration, and with the plasminogen concentration (P < 0.001 for all). CaCl2 (1mM) increased the GFA (9.7 ± 0.9 versus 4.9 ± 0.4 µg fibrin/mg/30 min, P < 0.05). The GFA was also enhanced when pH increased. Arachidonic acid (AA, 1 to 20 µg/ml) increased the GFA in a saturable manner. Inhibitors of cyclooxygenase (aspirin) or of lipoxygenase (nordihydroguaiaretic acid) did not modify the basal and AA-stimulated GFA. Other polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), eicosatrienoic acid (ETA), eicosatetraynoic acid (ETYA), or dihomo-γ-linoleic acid (DHL), also stimulated the GFA whereas linoleic acid and oleic acid did not. Polyunsaturated fatty acids also stimulated the fibrinolytic activity of glomerular superna-tants. Specific antibodies to t-PA, and to a lesser extent to u-PA, decreased this fibrinolytic activity whether or not AA was added. Furthermore, AA and EPA were found to increase the activity of purified u-PA and t-PA. We conclude that human glomeruli release both t-PA and u-PA, and that this release is increased by calcium and alkaline pH. The polyunsaturated fatty acids enhanced the GFA, mainly by a stimulatory effect of PA activity rather than an increased release of PA from glomerular cells.

Isolated rat glomeruli contain an adenylate cyclase system. The amount of cyclic AMP formed increased progressively with incubation time. The rate of cyclic AMP formation increased linearly with glomerular protein concentration. This adenylate cyclase system was temperature and pH dependent. There was no evidence for saturation of the enzyme with substrate up to 10−2 M ATP. Adenylate cyclase was strikingly activated by fluoride (10−2 M). Purified bovine parathyroid hormone (PTH) and synthetic 1–34 bovine PTH fragment both stimulated adenylate cyclase activity: maximum activity was 3.9 to 5.4 basal activity and km close to 10−7 M. Epinephrine and isoproterenol produced a slight stimulation whereas salmon calcitonin, glucagon, norepinephrine and antidiuretic hormone were inactive. The demonstration of PTH activated adenylate cyclase in glomeruli raises the possibility of a role for this hormone in regulation of glomerular activity.

In vitro PG synthesis by glomeruli isolated from rats with glycerol-induced acute renal failure (ARF) was measured by radiometric high performance liquid chromatography after incubation with [14C]arachidonic acid and radioimmunoassay (RIA). The four PGs, 6-keto-PGF1 alpha, TXB2, PGF2 alpha, and PGE2 were each synthesized by glomeruli from both control and treated rats but the synthesis rates were greater after glycerol. This increase was not apparent 1 hour after injection but, at 24 hours, all PGs were produced in greater amounts by glomeruli of treated rats. Thus, we studied PGE2, PGE2 alpha, and TXB2 synthesis by glomeruli at various time intervals after induction of ARF using direct RIA, PGF 2 alpha and TXB2 synthesis were greater only at 24 hours and only in the presence of arachidonic acid, whereas PGE2 synthesis was greater at 24 hours, irrespective of arachidonic acid, but at 48 hours only with arachidonic acid. The stimulatory effect of arachidonic acid was always greater in glycerol-treated than in control rats for these three PGs in the later period, whereas a significant decrease for PGE2 was observed at 1 hour. The late increase in PG synthesis may be due to stimulation of the renin-angiotensin system since it was abolished in rats pretreated for 48 hours with captopril. A late increase in PG synthesis by the papilla of the treated rats also was observed. We concluded that any increase in the glomerular production of vasoconstrictor PGs could contribute to the maintenance of acute renal failure, whereas the early fall in the stimulatory effect of arachidonic acid on PGE2 synthesis could play a role in its initiation.

We have investigated in vitro prostaglandin synthesis by human isolated glomeruli and papillary homogenates and compared the results with those obtained in parallel studies using rat material. Prostaglandins were measured by two methods, namely radiometric high performance liquid chromatography after incubation with 14C arachidonic acid and radioimmunoassay. The relative abundance of various prostaglandins synthesized by glomeruli was different in man (6 keto PGF1 alpha greater than TXB2 greater than PGF2 alpha greater than PGE2) and in the rat (PGE2 greater than or equal to PGF2 alpha greater than TXB2 greater than 6 keto PGF1 alpha). Unidentified peaks eluting between 6 keto PGF1 alpha and TXB2 were observed only in rat glomeruli. These peaks were suppressed by indomethacin. Direct radioimmunoassay of prostaglandins in the incubation medium of human glomeruli confirmed the predominance of 6 keto PGF1 alpha synthesis and showed its stimulation by arachidonic acid, its progressive decrease with time and its linear relationship with glomerular protein at low concentrations. On the contrary, the profile of prostaglandin synthesis by the papilla was similar in man and in the rat, PGE2 and PGF2 alpha being the major products in both species. However, related to one mg of protein, papillary synthesis of these two prostaglandins was greater in the rat. These results show that PGI2 is the major prostaglandin synthesized in human glomeruli and suggest a role for this prostaglandin in glomerular physiology in man.

[ 125 I]angiotensin II binding to isolated rat renal glomeruli was clearly increased in the presence of either Ca 2 + or Mg 2 + (10 μM to 5mM for both). This effect was more marked at higher pH. In the absence of both Ca 2 + and Mg 2 + , [ 125 I]angiotensin II binding decreased with pH whereas at 2 mM of these two cations, the opposite was observed. There was a clear-cut interaction between the effects of pH and those of Ca 2 + or Mg 2 + . The increase in [ 125 I] angiotensin II binding was similar for the same molar concentrations of Ca 2 + or Mg 2 + . Effects of these two cations were additive at pH 8.5, but negatively interacted at pH 7.5. Mn 2 + had the same effects as Ca 2 + and Mg 2 + and interacted with them. Addition of ethyleneglycol bis ( β aminoethylether)- N , N ′tetraacetic acid or of ethylenediamine tetraacetic acid suppressed the stimulatory effects of Ca 2 + and Mg 2 + and decreased [ 125 I]angiotensin II binding even when these two cations were not introduced in the incubation milieu. The extent of increase in [ 125 I]angiotensin II binding at increasing concentrations of Ca 2 + or Mg 2 + was greater in the presence than in the absence of these chelating substances. Kinetics of [ 125 I]angiotensin II binding were modified in the presence of Ca 2 + and Mg 2 + . The slopes of the association curves were smaller and the heights of the equilibrium plateaus greater. Moreover the dissociation after addition of an excess of angiotensin II was slower and irregular and did not correspond to an exponential function. The constant of affinity estimated from association curves at two different hormonal concentrations or from binding experiments at equilibrium was unchanged. Addition of EDTA at equilibrium also produced dissociation of bound [ 12 5 I]angiotensin II whether or not Ca 2 + and Mg 2 + were present. Enhancement of [ 125 I]angiotensin II binding by Ca 2 + and Mg 2 + and interaction of these two cations with pH seem particular to the glomerular binding sites and suggest a role in the binding reaction for the electric charges of both angiotensin II and the glomerular structures.

The effects of Escherichia coli lipopolysaccharide (LPS) on adenylate cyclase have been tested using renal tubular membranes and renal glomeruli isolated from rats. E. Coli LPS did not stimulate glomerular and tubular basal adenylate cyclase activity whereas it was an activator in the presence of fluoride. The effect of E. Coli LPS was immediate but was greater after 20 min preincubation. Maximum stimulation of both glomerular and tubular fluoride sensitive adenylate cyclase occurred at 125 microgram/ml of E. Coli LPS with an apparent Km (dose corresponding to 50% of maximum stimulation) of 30 microgram/ml. Above 125 microgram/ml there was a decrease in adenylate cyclase activity. E. Coli LPS produced an increase in the maximum velocity of both enzymes but did not affect their affinity for adenosine triphosphate. E. Coli LPS did not potentiate the effect of parathyroid hormone on glomerular and tubular adenylate cyclase. The lipid A moiety which is common to all LPS whatever the original strain gave results similar to those obtained with the entire LPS. This effect was specific and did not depend on the phospholipidic structure in general since no activation was obtained in the presence of phosphatidylserine.

Renal transplantation was performed in five adult patients with thrombotic microangiopathy, three of whom had had a bilateral nephrectomy prior to transplantation. The graft remained functional in three patients 72, 18, and 12 months after transplantation. One patient developed a thrombosis of the renal artery and one patient died from infection. There was no clinical or histological evidence of recurrence of thrombotic microangiopathy in the five patients after transplantation. Immunological investigations were performed in four of five patients before transplantation: C3 and C1q levels were low in two patients; serum C3-splitting activity and circulating immune complexes were present in all four patients and remained unchanged on haemodialysis and/or after bilateral nephrectomy. Complement abnormalities and immune complexes were not detected in the three patients with successful renal transplantation.

Human glomeruli release fatty acids which stimulate thromboxane synthesis in platelets. The cell–free medium of isolated human glomeruli exhibited a procoagulant activity and stimulated thromboxane (TXB 2 ) synthesis in human platelets in a dose–dependent manner. The amount of TXB 2 measured was 16-fold higher than what could have been predicted (TXB 2 synthesized by the platelets under control conditions plus TXB 2 present in the glomerular supernatant). The lipid extract of the glomerular supernatant and its purified fraction including the fatty acids was still able to stimulate—although at a lesser degree—TXB 2 synthesis in platelets. Stimulation was abolished after treatment of this fraction by charcoal or albumin. Gas chromatography/mass spectrometry analysis demonstrated the presence in the purified glomerular fraction of several long–chain saturated or monoenoic fatty acids at a total concentration of 80 µM with the following order of abundance: stearic, palmitic, myristic and oleic acids. Addition to human platelets of these same exogenous synthetic acids resulted in a dose–dependent stimulation of TX synthesis. It was maximum with three or four fatty acids tested in combination, but still present with myristic acid used separately. Arachidonic acid was absent in the glomerular supernatant. Thus the stimulation observed could not be related to a greater availability of substrate. Fatty acids did not act on platelets through a non-specific detergent effect since addition of high doses of detergents inhibited TXB 2 formation in platelets. The combination of fatty acids from glomerular origin identified in the present study represents a novel factor involved in the control of intracapillary hemostasis, but different from the procoagulant activity common to many tissues. It is hypothesized that in vivo damage of the glomerular cells could release saturated and monoenoic fatty acids able to activate platelet functions independently of collagen, and thus result in the local accumulation of TXA 2 .