Marilena Gregorini

Hepatocyte growth factor/scatter factor released during peritonitis is active on mesothelial cells.

Peritonitis causes mesothelial detachment that may result in persistent peritoneal denudation and fibrosis. We investigated whether hepatocyte growth factor (HGF), a scatter factor that induces detachment from substrate and fibroblastic transformation of several cell types, is produced during peritonitis and is active on mesothelial cells. We studied 18 patients on peritoneal dialysis, 9 uncomplicated, 9 with peritonitis. HGF was measured in serum, peritoneal fluid, and supernatant of peripheral blood mononuclear cells and peritoneal mononuclear cells. Primary culture of human peritoneal mesothelial cells and the human mesothelial cell line MeT-5A were conditioned with recombinant HGF, serum, and peritoneal fluid. HGF levels were significantly higher in serum and peritoneal fluid of peritonitic than uncomplicated patients. Mononuclear cells of peritonitic patients produced more HGF than cells of uncomplicated patients. Recombinant HGF, serum, and peritoneal fluid of peritonitic patients caused mesothelial cell growth, detachment, transformation from epithelial to fibroblast-like shape, overexpression of vimentin, and synthesis of type I and III collagen. In conclusion, HGF released during peritonitis causes a change in mesothelial cell phenotype and function. HGF may affect the healing process facilitating repair through mesothelial cell growth, but may contribute to peritoneal fibrosis inducing cell detachment with mesothelial denudation and collagen synthesis.

The Ron proto-oncogene product is a phenotypic marker of renal oncocytoma

The proto-oncogene product Ron is the receptor for macrophage stimulating protein, a scatter factor that stimulates cell proliferation, prevents apoptosis, and induces an invasive cell phenotype. We investigated the expression of Ron, Ki-67 (proliferation index), p53, and bcl-2 (proapoptotic and antiapoptotic proteins, respectively) in 50 renal tumors (19 clear cell carcinomas, 18 oncocytomas, 7 papillary cell carcinomas, 5 chromophobe cell carcinomas, and 1 carcinoma with sarcomatoid areas). In addition, we studied Ron in normal kidney and in the renal carcinoma cell line Caki-1. Immunostaining and Western blot showed Ron in normal kidney and in all oncocytomas but never in renal cell carcinomas or in Caki-1. In addition, Western blot showed that Ron was expressed in phosphorylated, i.e., active, form. Bcl-2 was strongly expressed in oncocytomas, whereas Ki-67 and p53 were much less expressed in oncocytomas than in carcinomas. These results indicate in Ron a marker that differentiates oncocytoma from the other renal epithelial tumors. We therefore think that Ron may prove to be a new tool for a sound and precise diagnosis of oncocytoma, a benign tumor that cannot always be distinguished from carcinomas at histologic examination. The overexpression of bcl-2, but not p53 in oncocytoma, suggests that the MSP/Ron system sustains the growth of oncocytoma by opposing apoptosis.

Mesenchymal Stem Cells Infusion Prevents Acute Cellular Rejection in Rat Kidney Transplantation

Mesenchymal stem cells (MSC) are multipotent cells that differentiate into various mature cell lineages. MSC show immunomodulatory effects by inhibiting T-cell proliferation. We evaluated the effect of the infusion of MSC in rats experimental kidney transplantation. Sprague-Dawley transgenic rats (SD) able to express the green fluorescent protein (EGFP) were used as MSC donors. Syngeneic (Lewis to Lewis, n = 10) and allogeneic (Fischer to Lewis, n = 10) kidney transplantations were performed after bilateral nephrectomy. Five transplanted rats who received syngeneic grafts, were treated with 3 x 10(6) MSC (Gr B), while the other 5 did not received MSC (Gr A). Five rats with allogenic grafts received 3 x 10(6) MSC (Gr C) and another 5 did not receive MSC (Gr D). The MSC were infused directly into the renal artery of the graft. No immunosuppressive therapy was provided. The animals were killed after 7 days. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological analysis (ED1+ and CD8+) were performed on treated animals. MSC improved kidney function in Gr B and D vs Gr A and C. The tubular damage appeared to be less severe among Gr B and Gr D with respect to Gr A and C (P < .01). Vasculitis was more accentuated in Gr A and C (P < .01). MSCs reduced the inflammatory infiltrate; in Gr B and D, the number of ED1+ cells was lower than in Gr A and C (P < .005), which was also observed for CD8+ cells (P < .05). Our study demonstrated that the infusion of MSC attenuated histological damage from acute rejection by reducing the cellular infiltration.

Which Is the Most Suitable and Effective Route of Administration for Mesenchymal Stem Cell-Based Immunomodulation Therapy in Experimental Kidney Transplantation: Endovenous or Arterial?

Immunomodulating cell therapy represents a new perspective for the control of cellular immune responses that determine the occurrence of acute rejection (ACR) in allo-transplantation. Mesenchymal stem cells (MSC) demonstrate immunoregulatory effects by inactivating T-cell components that regulate tissue damage in transplantation models. The presumed mechanism of action is recruitment of cells by a cytokine network. The purpose of this study was to test which route of administration (intra-arterial vs intravenous) was the most effective route to achieve immunomodulating effects in experimental rat kidney transplantation. Transgenic Sprague-Dawley rats (SD) expressing the enhanced green fluorescent protein (EGFP) at the somatic level were used as MSC donors: Allogeneic Fischer to Lewis grafts (n = 4 per group) were performed in rats after bilateral nephrectomy. In Gr B, 3 x 10(6) MSCs were infused into the renal graft artery, whereas in Gr C, they were infused into the tail vein. The untreated Gr A were a control group. No immunosuppressive therapy was administered. The animals were sacrificed at day 7 postoperatively. Biochemical analysis for renal function, histological (Banff criteria) and immunohistological (anti-EGFP-Immunoglobulin) analysis were performed on the transplanted animals. In Gr B, functional recovery was more rapid (creatinine: Gr B vs Gr C, P < .05). The inflammatory infiltrate in the graft was less in Gr B vs Gr C, with preservation of tubules, arteries, and glomeruli (P < .01). Intra-arterial infusion of MSCs was more effective to control ACR.

KCNA1 and TRPC6 ion channels and NHE1 exchanger operate the biological outcome of HGF/scatter factor in renal tubular cells

Hepatocyte growth factor (HGF) is a glycoprotein that induces in vitro epithelial tubular cell growth, motility, scattering and branching morphogenesis. The cell machineries that account for HGF biological effects are still unclear. In previous study, we found that HGF upregulated in epithelial tubular cell line (HK2) 3 genes: potassium channel KCNA1, calcium channel (transient receptor potential channel, subfamily C, member 6, TRPC6) and Na+/H+ exchanger-1 (NHE1). In this study, we validated these results with reverse transcription PCR and WB analysis. To investigate whether KCNA1, TRPC6, NHE1 mediate the changes induced by HGF in HK2, we studied the effects of their inhibitors: 4-aminopyridine, charybdotoxin, dendrotoxin K inhibitors of KCNA1, lanthanum, N-(p-amylcinnamoyl) anthranilic acid inhibitors of TRPC6, 5-(N-ethyl-N-isopropyl)amiloride, cariporide inhibitors of NHE1. The inhibitors prevented HGF-induced growth, migration, cytoskeletal reorganization and tubulogenesis in HK2. These results indicate that KCNA1, TRPC6 and NHE1 are cell machineries that are exploited by HGF to effect its biological outcome in renal tubular cells.

Mesenchymal stromal cells improve renal injury in anti-Thy 1 nephritis by modulating inflammatory cytokines and scatter factors

MSC (mesenchymal stromal cells) can differentiate into renal adult cells, and have anti-inflammatory and immune-modulating activity. In the present study, we investigated whether MSC have protective/reparative effects in anti-Thy1 disease, an Ab (antibody)-induced mesangiolysis resulting in mesangioproliferative nephritis. We studied five groups of rats: (i) rats injected with anti-Thy1.1 Ab on day 0 (group A); (ii) rats injected with anti-Thy1.1 Ab on day 0+MSC on day 3 (group B); (iii) rats injected with anti-Thy1.1 Ab on day 0+mesangial cells on day 3 (group C); (iv) rats injected with saline on day 0+MSC on day 3 (group D); and (v) rats injected with saline on day 0 (group E). Rats were killed on days 1, 3, 7 and 14. MSC prevented the increase in serum creatinine, proteinuria, glomerular monocyte influx and glomerular histopathological injury. Furthermore, MSC suppressed the release of IL-6 (interleukin-6) and TGF-β (transforming growth factor-β), modulated glomerular PDGF-β (platelet-derived growth factor-β), and reset the scatter factors and their receptors, potentiating HGF (hepatocyte growth factor)/Met and inactivating MSP (macrophage-stimulating protein)/Ron (receptor origin nantaise). Few MSC were found in the kidney. These results indicate that MSC improve anti-Thy 1 disease not by replacing injured cells, but by preventing cytokine-driven inflammation and modulating PDGF-β and the scatter factors, i.e. systems that regulate movement and proliferation of monocytes and mesangial cells.

Severe Symptomatic Hyponatremia During Sibutramine Therapy: A Case Report

Sibutramine, a serotonin reuptake inhibitor, currently is used in treatment of obesity. The known side effects of sibutramine, ie, hypertension and tachycardia, depend on its adrenergic and serotoninergic effects. We describe a case of life-threatening hyponatremia associated with sibutramine use in an obese woman. We hypothesize that sibutramine, through its effect on neurotransmitters, may induce antidiuretic hormone secretion and lead to a syndrome of inappropriate antidiuretic hormone secretion. We advise careful monitoring of water-electrolytic balance during sibutramine therapy.

The role of therapeutic drug monitoring in the treatment of cytomegalovirus disease in kidney transplantation

Cytomegalovirus (CMV) infection is a common complication following solid organ transplantation that may severely affect the outcome of transplantation. Ganciclovir (GCV) and its prodrug valganciclovir are successfully used to prevent and treat CMV infection; however, in a small percentage of patients, CMV gene mutations may lead to drug resistance. GCV resistance is defined as increasing CMV viremia or progressive clinical disease during prolonged antiviral therapy, due to CMV gene mutation. This has emerged as a new challenge, especially because alternative drugs such as cidofovir and foscarnet have a number of important side effects. Here we report the case of a kidney transplanted patient who experienced life-threatening CMV disease, which initially appeared to be GCV-resistant, but was instead found to be associated with inadequate antiviral drug levels. The patient was then successfully treated by monitoring plasma GCV levels. We suggest using plasma GCV monitoring in the management of all cases of critical CMV disease, in which GCV resistance is suspected.

Mesenchymal stromal cells reset the scatter factor system and cytokine network in experimental kidney transplantation

BackgroundIn former studies we showed in a rat model of renal transplantation that Mesenchymal Stromal Cells (MSC) prevent acute rejection in an independent way of their endowing in the graft. In this study we investigated whether MSC operate by resetting cytokine network and Scatter Factor systems, i.e. Hepatocyte Growth Factor (HGF), Macrophage Stimulating Protein (MSP) and their receptors Met and RON, respectively.MethodsMSC were injected into the renal artery soon after reperfusion. Controls were grafted untreated and normal rats. Rats were sacrificed 7 days after grafting. Serum and renal tissue levels of IFN-γ, IL-1, IL-2, IL-4, IL-6, IL-10, MSP/RON, HGF/Met systems, Treg lymphocytes were investigated.ResultsIn grafted untreated rats IFN-γ increased in serum and renal tissue and IL-6 rose in serum. MSC prevented both the phenomena, increased IL-10 serum levels and Treg number in the graft. Furthermore MSC increased serum and tissue HGF levels, Met tubular expression and prevented the suppression of tubular MSP/RON expression.ConclusionsOur results demonstrate that MSC modify cytokine network to a tolerogenic setting, they suppress Th1 cells, inactivate monocytes/macrophage, recruit Tregs. In addition, MSC sustain the expression of the Scatter Factor systems expression, i.e. systems that are committed to defend survival and stimulate regeneration of tubular cells.

Mechanisms underlying sCD40 production in hemodialysis patients.

CD40 and its ligand (CD40L) regulate several cellular functions, including T and B-cell activation. The soluble form of CD40 (sCD40) antagonizes CD40/CD40L interaction. Patients undergoing hemodialysis (HD) present elevated sCD40 serum levels, which underlying molecular mechanisms are unknown. We studied sCD40 serum and urinary levels, CD40 membrane and gene expression and membrane shedding in HD, uremic not-HD patients (UR) and healthy subjects (N). We found that in HD sCD40 serum levels were higher than UR and N, being significantly elevated in anuric patients, and that sCD40 correlated to renal function in UR subjects, who presented also a reduced sCD40 urinary excretion rate. HD and UR presented reduced CD40 membrane and gene expression. The concentration of TNF-α converting enzyme (TACE), responsible for CD40 cleavage was not different between HD and N. Therefore the reduced renal clearance is the main cause of elevated sCD40 levels in HD. This finding could have relevant clinical implications.