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ABSTRACTPodocyte injury leading to albuminuria is a characteristic feature of diabetic nephropathy (DN). Hyperglycemia and advanced glycation end products (AGEs) are major determinants of DN. However, the underlying mechanisms of podocyte injury remain poorly understood. The cytosolic protein TNFAIP2/M-Sec is required for tunneling nanotubes (TNTs) formation, which are membrane channels that transiently connect cells, allowing organelle transfer. Podocytes express TNFAIP2 and form TNTs, but the potential relevance of the TNFAIP2-TNT system in DN is unknown. We studied TNFAIP2 expression in both human and experimental DN and the renal effect of tnfaip2deletion in streptozotocin-induced DN. Moreover, we explored the role of the TNFAIP2-TNT system in podocytes exposed to diabetes-related insults. TNFAIP2 was overexpressed by podocytes in both human and experimental DN and exposre of podocytes to high glucose and AGEs induced the TNFAIP2-TNT system. In diabetic mice, tnfaip2deletion exacerbated albuminuria, renal function loss, podocyte injury, and mesangial expansion. Moreover, blockade of the autophagic flux due to lysosomal dysfunction was observed in diabetes-injured podocytes both in vitroand in vivoand exacerbated by tnfaip2deletion. TNTs allowed autophagosome and lysosome exchange between podocytes, thereby ameliorating AGE-induced lysosomal dysfunction and apoptosis. This protective effect was abolished by tnfaip2deletion, TNT inhibition, and donor cell lysosome damage. By contrast, Tnfaip2overexpression enhanced TNT-mediated transfer and prevented AGE-induced autophagy and lysosome dysfunction and apoptosis. In conclusion, TNFAIP2 plays an important protective role in podocytes in the context of DN by allowing TNT-mediated autophagosome and lysosome exchange and may represent a novel druggable target.Abbreviations:AGEs: advanced glycation end products; AKT1: AKT serine/threonine kinase 1; AO: acridine orange; ALs: autolysosomes; APs: autophagosomes; BM: bone marrow; BSA: bovine serum albumin; CTSD: cathepsin D; DIC: differential interference contrast; DN: diabetic nephropathy; FSGS: focal segmental glomerulosclerosis; HG: high glucose; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LMP: lysosomal membrane permeabilization; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PI3K: phosphoinositide 3-kinase; STZ: streptozotocin; TNF: tumor necrosis factor; TNFAIP2: tumor necrosis factor, alpha-induced protein 2; TNTs: tunneling nanotubes; WT: wild type.

Paracrine mediators released from endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis following ischemia. Recently, we demonstrated that microvesicles (MVs) derived from EPCs are able to activate an angiogenic program in quiescent endothelial cells by a horizontal transfer of RNA. In this study we aim to investigate whether EPC-derived MVs are able to induce neoangiogenesis and to enhance recovery in a murine model of hindlimb ischemia. Hindlimb ischemia was induced in severe combined immunodeficient (SCID) mice by ligation and resection of the left femoral artery and mice were treated with EPC-derived MVs (MVs), RNase-inactivated MVs (RnaseMVs), fibroblast-derived MVs or vehicle alone as control (CTL). Since MVs contained the angiogenic miR-126 and miR-296, we evaluated whether microRNAs may account for the angiogenic activities by treating mice with MVs obtained from DICER-knock-down EPC (DICER-MVs). The limb perfusion evaluated by laserdoppler analysis demonstrated that MVs significantly enhanced perfusion in respect to CTL (0.50±0.08 vs0.39±0.03, p < 0.05). After 7 days, immunohistochemical analyses on the gastrocnemius muscle of the ischemic hindlimb showed that MVs but not fibroblast-MVs significantly increased the capillary density in respect to CTL (MVs vs CTL: 24.7±10.3 vs13.5±6, p < 0.0001) and (fibroblast-MVs vs CTL: 10.2±3.4 vs 13.5±6, ns); RNaseMVs and DICER-MVs significantly reduced the effect of MVs (RNaseMVs vs CTL: 15.7±4.1 vs 13.5±6, ns) (MVs vs DICER-MVs 24.7±10.3 vs 18.1±5.8, p < 0.05), suggesting a role of RNAs shuttled by MVs. Morphometric analysis confirmed that MVs enhanced limb perfusion and reduced injury. The results of the present study indicate that treatment with EPC-derived MVs improves neovascularization and favors regeneration in severe hindlimb ischemia induced in SCID mice. This suggests a possible use of EPCs-derived MVs for treatment of peripheral arterial disease.

The development of proteinuria has been observed in kidney‐transplanted patients on m‐TOR inhibitor (m‐TORi) treatment. Recent studies suggest that m‐TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney‐transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit‐diaphragm. In a group of patients on ‘de novo’ m‐TORi‐treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m‐TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m‐TORi‐treatment, a protocol biopsy performed before introduction of m‐TORi was also available. The expression of nephrin in the pre‐m‐TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m‐TORi(s). Proteinuria increased after the m‐TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin. In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m‐TORi(s) may affect nephrin expression in kidney‐transplanted patients, consistently with the observation in vitroon cultured podocytes.

The development of proteinuria has been observed in kidney‐transplanted patients on m‐TOR inhibitor (m‐TORi) treatment. Recent studies suggest that m‐TORi(s) may alter the behavior and integrity of glomerular podocytes. We analyzed renal biopsies from kidney‐transplanted patients and evaluated the expression of nephrin, a critical component of the glomerular slit‐diaphragm. In a group of patients on ‘de novo’ m‐TORi‐treatment, the expression of nephrin within glomeruli was significantly reduced in all cases compared to pretransplant donor biopsies. Biopsies from control transplant patients not treated with m‐TORi(s) failed to present a loss of nephrin. In a group of patients subsequently converted to m‐TORi‐treatment, a protocol biopsy performed before introduction of m‐TORi was also available. The expression of nephrin in the pre‐m‐TORi biopsies was similar to that observed in the pretransplant donor biopsies but was significantly reduced after introduction of m‐TORi(s). Proteinuria increased after the m‐TORi inititiation in this group. However, in some cases proteinuria remained normal despite reduction of nephrin.In vitro, sirolimus downregulated nephrin expression by human podocytes. Our results suggest that m‐TORi(s) may affect nephrin expression in kidney‐transplanted patients, consistently with the observationin vitroon cultured podocytes.

Donor intra-islet endothelial cells contribute to neovascularization after transplantation. Several factors may interfere with this process and ultimately influence islet engraftment. Rapamycin, a central immunosuppressant in islet transplantation, is an mTOR inhibitor that has been shown to inhibit cancer angiogenesis. The aim of this study was to evaluate the effects of rapamycin on islet endothelium. Rapamycin inhibited the outgrowth of endothelial cells from freshly purified human islets and the formation of capillary-like structures in vitroand in vivoafter subcutaneous injection within Matrigel plugs into SCID mice. Rapamycin decreased migration, proliferation and angiogenic properties of human and mouse islet-derived endothelial cell lines with appearance of apoptosis. The expression of angionesis-related factors VEGF, αVβ3 integrin and thrombospondin-1 on islet endothelium was altered in the presence of rapamycin. On the other hand, rapamycin decreased the surface expression of molecules involved in immune processes such as ICAM-1 and CD40 and reduced the adhesion of T cells to islet endothelium. Our results suggest that rapamycin exerts dual effects on islet endothelium inducing a simultaneous inhibition of angiogenesis and a down-regulation of receptors involved in lymphocyte adhesion and activation.

Background & Aims T-helper (Th)1 cells play a central role in the pathogenesis of tissue damage in Crohn's disease (CD). Interleukin (IL)-12/STAT4 signaling promotes Th1 cell commitment in CD, but other cytokines are needed to maintain activated Th1 cells in the mucosa. In this study, we examined the expression and role of IL-21, a T-cell-derived cytokine of the IL-2 family; in tissues and cells isolated from patients with inflammatory bowel disease. Methods IL-21 was examined by Western blotting in whole mucosa and lamina propria mononuclear cells (LPMCs) from patients with CD, ulcerative colitis (UC), and controls. We also examined the effects of exogenous IL-12 on IL-21 production, as well as the effects of blocking IL-21 with an IL-21-receptor Ig fusion protein. Interferon (IFN)-@c was measured in the culture supernatants by enzyme-linked immunosorbent assay, and phosphorylated STAT4 and T-bet were examined by Western blotting. Results IL-21 was detected in all samples, but its expression was higher at the site of disease in CD in comparison with UC and controls. Enhanced IL-21 was seen in both ileal and colonic CD and in fibrostenosing and nonfibrostenosing disease. IL-12 enhanced IL-21 in normal lamina propria lymphocytes through an IFN-@c-independent mechanism, and blocking IL-12 in CD LPMCs decreased anti-CD3-stimulated IL-21 expression. Neutralization of IL-21 in CD LPMC cultures decreased phosphorylated STAT4 and T-bet expression, thereby inhibiting IFN-@c production. Conclusions Our data suggest that IL-21 contributes to the ongoing Th1 mucosal response in CD.

A report on our clinical experience based on 3 male patients who developed Churg-Strauss syndrome (CSS) after standard oral montelukast use. All patients affected by moderate asthma and chronic hyperplastic rhinosinusitis were treated with inhaled corticosteroids and ß2 agonists. Systemic corticosteroid treatment consisted in oral daily prednisone in case 1, in short courses of oral betamethasone in case 2, and in remote and isolated administrations of oral betamethasone and intramuscular methylprednisolone in case 3. Because of the improvement of the asthma symptoms after montelukast use, patient 1 decided to take half the dose of prednisone for 10 days and patient 2 decided to discontinue systemic and inhaled corticosteroids for 45 days. Overt CSS was heralded by vasculitic skin lesions and developed in each patient with severe organ damage, consisting in renal, myocardial and gastrointestinal involvement. Remission was obtained by standard CSS therapy after montelukast withdrawal.According to the unmasking hypothesis, antileukotriene treatment, by enabling the reduction in systemic corticosteroid therapy in case 1 and its discontinuation in case 2, might have only permitted the precipitation of the vasculitis. However antileukotriene-associated CSS reportedly occurred in systemic corticosteroid-naïve patients and relapsed in one patient after antileukotriene treatment. These observations lend support to the concept that the precipitation of the vasculitic phase may be associated with leukotriene modifier deleterious effects. In conclusion there is not enough evidence to prove that antileukotriene treatment plays a direct causative role in the pathogenesis of CSS. Further clinical and experimental research is required to clarify the antileukotriene associated CSS controversy.

Background Dialysis is often neglected in academic teaching. At the University of Torino, Italy, teaching Nephrology (4thyear of Medical School) consists of 21 hours of formal lessons, 10 hours/student of interactive lessons (4/10 dedicated to dialysis) and 10 optional lessons (3 regarding dialysis). Interactive and optional lessons widely employ computer assisted teaching. Aim of the study was to evaluate student satisfaction on this approach.Methods Student satisfaction was assessed on 4 sample lessons (166 students), by two short dedicated questionnaires (0–10 scale, open questions).Results High scores were given to the dialysis lessons (median 8/10). Computer assisted interface (median 8/10, range 6–10) was of help in check of knowledge in real time (86%), enhancing participation (61%); 62% suggest extending this experience to selected courses, 38% to all.Conclusions Medical students consider dialysis an important part of the academic teaching of Nephrology; new interfaces may help to enhance student satisfaction.

In normal conditions, human gut mucosa is infiltrated with a large number of mononuclear cells. This is a reflection of the fact that human intestine is continuously subjected to a massive stimulation by luminal antigens. This state of "physiological" inflammation is a tightly controlled phenomenon, as several mucosal cells interact to generate and maintain an appropriate local immune response. Changes in cell type number and/or function, including the release of soluble mediators, have been associated with the development of chronic inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease. Evidence also indicates that the type of inflammatory response occurring in the intestine of patients with CD differs from that in UC, and this probably reflects distinct pathways of immune activation. In CD mucosa, a Th1 response with high IL-12 and IFNγ production prevails, while in UC a humoral immunity appears to be predominant. Despite this, CD and UC share downstream inflammatory events, characterised by high levels of inflammatory cytokines, free radicals, matrix-degrading enzymes and growth factors.

In normal conditions, human gut mucosa is infiltrated with a large number of mononuclear cells. This is a reflection of the fact that human intestine is continuously subjected to a massive stimulation by luminal antigens. This state of "physiological" inflammation is a tightly controlled phenomenon, as several mucosal cells interact to generate and maintain an appropriate local immune response. Changes in cell type number and/or function, including the release of soluble mediators, have been associated with the development of chronic inflammatory diseases, such as Crohn's disease (CD) and ulcerative colitis (UC), the two major forms of inflammatory bowel disease. Evidence also indicates that the type of inflammatory response occurring in the intestine of patients with CD differs from that in UC, and this probably reflects distinct pathways of immune activation. In CD mucosa, a Th1 response with high IL-12 and IFNgamma production prevails, while in UC a humoral immunity appears to be predominant. Despite this, CD and UC share downstream inflammatory events, characterised by high levels of inflammatory cytokines, free radicals, matrix-degrading enzymes and growth factors.

Background.A loss of intestinal glycosaminoglycans (GAGs) has been shown in inflammatory bowel diseases (IBD). Since GAGs are involved in the regulation of renal protein filtration and GAGs disruption is associated with anionic proteinuria, we examined whether changes in the selectivity of renal protein filtration occur in IBD.
Methods.From 46 patients with IBD (17 with Crohn's disease (CD), and 29 with ulcerative colitis (UC)) and 21 healthy subjects, urine and serum samples were obtained. Albumin, total IgG and IgG₄ clearances were measured using sensitive methods. Serum p-ANCA and TNF-α were tested.
Results.Median IgG₄ clearance was 0.041 ml/ min/10^-3 in patients with UC and 0.10 ml/ min/10^-3 in CD patients, both significantly higher than in controls (0.03 ml/min/10^-3) (P&lt;0.03). IgG₄ clearance was above the upper normal limit in 9/17 CD (53%) and in 10/29 UC (34.5%). Eighteen of 19 patients showing abnormal IgG₄ clearance were taking mesalazine. In patients on maintenance oral mesalazine, IgG₄ clearance was higher than that in patients off treatment (0.12 vs 0.03 ml/min/10^-3, P=0.04). No clinical/laboratory sign of renal dysfunction was documented in patients with altered IgG₄ clearance and maintained on mesalazine treatment.
Conclusion.Renal protein charge permselectivity is impaired in 40% of patients with IBD with no overt proteinuria. Our data suggest that altered IgG₄ clearance may represent a subclinical marker of renal involvement in IBD.

A loss of intestinal glycosaminoglycans (GAGs) has been shown in inflammatory bowel diseases (IBD). Since GAGs are involved in the regulation of renal protein filtration and GAGs disruption is associated with anionic proteinuria, we examined whether changes in the selectivity of renal protein filtration occur in IBD.

SummaryThe occurrence of intestinal bacterial overgrowth in patients with Crohn's Disease (CD) has been described and antimicrobial treatment has been shown to be effective in reversing this condition. However, the mechanisms underlying the efficacy of antimicrobial therapy are still only partially known.The aim of the present study was to evaluate the effect of a non-absorbable antibiotic (rifaximin) in comparison to placebo on bacterial overgrowth in patients with CD.Methods Fourteen patients with inactive CD of the ileum and bacterial overgrowth, as assessed by the hydrogen breath test, were blindly allocated to receive rifaximin (1200 mg/day) or placebo t.i.d. for one week. A hydrogen breath test, and clinical and biochemical parameters were further performed 14 days and 30 days after starting treatment.Results After 14 days, the hydrogen breath test proved to be negative in seven out of seven patients treated with rifaximin (p < 0.05), and in two out of seven in the placebo group (p = ns). After 30 days, the hydrogen breath test was positive in all patients of the rifaximin and placebo group, respectively. No changes in the CDAI score were documented in any patients.Conclusions Short-term administration of rifaximin is effective in the therapy of bacterial overgrowth in patients with inactive CD of the ileum, thus suggesting that the control of luminal bacterial growth could be useful in the management of these patients. However, since we observed a decline with time in this positive effect, further studies are needed to identify the most appropriate therapeutic strategies.

The selectin class of adhesion molecules plays a critical role in facilitating leukocyte adhesion to and subsequent transmigration of endothelium. On this basis, selectins have been suggested to promote tumor cell attachment to endothelium, thereby facilitating metastasis of certain types of tumors, although direct evidence for such a role is lacking. To explore this hypothesis, two sets of transgenic mice were developed: TgnES, which constitutively expresses cell surface E-selectin in all tissues, under the control of the beta-actin promoter; and TgnEsol, which expresses truncated, soluble E-selectin in the liver, under the control of the alpha 1 antitrypsin promoter. B16F10 melanoma cells were stably transfected with alpha(1,3/1,4) fucosyltransferase-specific cDNA (B16F10ft), allowing them to express E-selectin ligands or with hygromycin resistance selection vector only B16F10hygro). Normal mice injected with B16F10ft and B16F10hygro and transgenic mice injected with B16F10hygro developed lung tumors exclusively. In contrast, TgnES mice injected with B16F10ft cells developed massive infiltrating liver tumors. B16F10ft cells injected into TgnEsol mice also formed liver tumors, but these grew more slowly, with a well-delineated, noninfiltrating distinct histologic pattern. These observations provide direct evidence that expression of E-selectin can redirect metastasis of tumor cells expressing appropriate ligands in vivo.

The six group B coxsackieviruses (CVBs) are highly prevalent human pathogens that cause viremia followed by involvement of different organs. Clinical and experimental evidence suggests that CVBs can induce kidney injury, but the susceptibility of human renal cells to these viruses is unknown. By using pure cultures of human glomerular and tubular cells, we demonstrated that all CVBs are capable of productively infecting renal cells of three different histotypes. Distinct pathogenic effects were observed. Proximal tubular epithelial cells and, to a lesser extent, glomerular podocytes were highly susceptible to CVBs; in both cases, infection led to cytolysis. In contrast, glomerular mesangial cells supported the replication of the six CVBs but failed to develop overt cytopathologic changes. Mesangial cells continued to produce infectious progeny for numerous serial subcultures (i.e., more than 50 days), especially with type 1, 3, 4, and 5 viruses. In the above cells, persistent infection induced the de novo synthesis of platelet-derived growth factor A/B and enhanced the release of transforming growth factor beta1/2. These two factors are important mediators of progression from glomerular inflammation to glomerulosclerosis. CVB replication appeared also to impair the phagocytic and contractile activity of mesangial cells. Loss of these properties--which are important in glomerular physiopathology--may contribute to the development of progressive nephropathy. The results show that CVBs induce distinct effects in different types of cultured renal cells and suggest that CVB infections may be associated with both acute and progressive renal injury.

We assessed the role of CD40-CD40L, cytotoxic T lymphocyte (CTL)A4/CD28-B7s, and CD2-CD48/CD58 lymphocyte costimulatory pathways in the development of mercury chloride (HgCl2)-induced autoimmune disease in mice, which is believed to be mediated by T helper (Th) subset Th2. Inhibition of CD40-CD40-L and CTLA4/CD28-B7s interactions by anti-CD40-L antibody and soluble CTLA4-immunoglobulin (Ig) fusion protein, respectively, abrogated the autoimmune disease without affecting interleukin 4 (IL-4) production, showing the importance of physical contact between T and B lymphocytes in the Th2-mediated process. In contrast, two anti-CD2 antibodies that have been shown to induce immunosuppression of Th1-mediated events exacerbated the autoantibody response and augmented IgG1, IgE, and IL-4 production, transforming a mild mesangial glomerulopathy into a severe systemic immune complex disease. These observations demonstrate that manipulation of lymphocyte accessory counterreceptor interactions may affect the course of Th2-associated autoimmune disease and suggest that signals resulting from CD2 engagement play an essential role in the regulation of the Th1-Th2 effector equilibrium.

CTLA-4/CD28-B7 and CD40-CD40L interactions constitute two key costimulatory pathways in lymphocyte signalling during experimental allograft rejection. Studies on the expression of these molecules in human transplant rejection are still lacking.

BACKGROUND & AIMS: Cell-mediated immunity is a feature of Crohn's disease (CD). The heterodimer interleukin (IL)-12, produced by phagocytes, induces T-cell cytokines, primarily interferon (IFN)-gamma. This study examined whether CD lamina propria mononuclear cells (LPMCs) express and release bioactive IL-12. METHODS: LPMCs were isolated from 13 patients with CD, 9 with ulcerative colitis (UC), and 13 controls. Messenger RNA for p40 and p35 IL-12 subunits was evaluated by reverse- transcription polymerase chain reaction. IL-12 was measured by enzyme- linked immunosorbent assay in LPMC culture supernatants. The INF-gamma- inducing effect of unstimulated LPMC supernatants was evaluated. RESULTS: Messenger RNA for both IL-12 subunits was detected in LPMCs of 11 of 13 patients with CD, 1 of 9 patients with UC, and 1 of 13 controls (P < 0.001). IL-12 was measured (10.5 +/- 2 pg/mL at 24 hours) in unstimulated CD LPMCs and was enhanced by pokeweed mitogen, lipopolysaccharide, and staphylococcal enterotoxin B. No IL-12 was detectable in 8 of 9 patients with UC and 12 of 13 control-unstimulated LPMCs. IL-12 induced by pokeweed mitogen and staphylococcal enterotoxin B in UC was lower than in CD and did not differ from controls. An IFN- gamma-inducing effect was restricted to unstimulated CD LPMC supernatants and was inhibited by an anti-IL-12 antibody in a dose- dependent fashion. CONCLUSIONS: IL-12 transcripts are expressed in CD intestinal tissues. CD LPMCs are up-regulated in their capability of releasing bioactive IL-12. Expression and release of bioactive IL-12 seem to differentiate CD from UC. (Gastroenterology 1997 Apr;112(4):1169-78)

CD5 is a 67-kD glycoprotein that is expressed on most T lymphocytes and on a subset of mature B cells. Although its physiologic function is unknown, several lines of evidence suggest that CD5 may play a role in the regulation of T cell activation and in T cell-antigen presenting cell interactions. Using a CD5-immunoglobulin fusion protein (CD5Rg, for receptorglobulin) we have uncovered a new CD5 ligand (CD5L) expressed on the surface of activated splenocytes. Stimulation of murine splenocytes with anti-CD3 and anti-CD28 antibodies induce transient expression of CD5L on B lymphocytes that lasts for approximately 72 h. Binding of CD5Rg to activated splenocytes is trypsin resistant and independent of divalent cations. However, it is pronase sensitive and dependent on N-linked glycosylation of CD5, since treatment of CD5Rg with PNGaseF on N-glycanase completely abrogates its ability to bind activated splenocytes. It addition to splenocytes, CD5L is expressed on activated murine T cell clones. Immunoprecipitation, antibody, and recombinant protein blocking studies indicate that CD5L is distinct from CD72, which has been proposed to be a CD5 ligand. To determine whether CD5-CD5L interaction might play a role in vivo, we tested the effect of CD5Rg in a murine model of antibody-mediated membranous glomerulonephritis. Injection of CD5Rg was found to abrogate development of the disease. Taken together, our results help identify a novel ligand of CD5 and propose a role for CD5 in the regulation of immune responses.