Paola Pontrelli

Mitochondrial dysregulation and oxidative stress in patients with chronic kidney disease

BackgroundChronic renal disease (CKD) is characterized by complex changes in cell metabolism leading to an increased production of oxygen radicals, that, in turn has been suggested to play a key role in numerous clinical complications of this pathological condition. Several reports have focused on the identification of biological elements involved in the development of systemic biochemical alterations in CKD, but this abundant literature results fragmented and not exhaustive.ResultsTo better define the cellular machinery associated to this condition, we employed a high-throughput genomic approach based on a whole transcriptomic analysis associated with classical molecular methodologies. The genomic screening of peripheral blood mononuclear cells revealed that 44 genes were up-regulated in both CKD patients in conservative treatment (CKD, n = 9) and hemodialysis (HD, n = 17) compared to healthy subjects (HS, n = 8) (p < 0.001, FDR = 1%). Functional analysis demonstrated that 11/44 genes were involved in the oxidative phosphorylation system. Western blotting for COXI and COXIV, key constituents of the complex IV of oxidative phosphorylation system, performed on an independent testing-group (12 healthy subjects, 10 CKD and 14 HD) confirmed an higher synthesis of these subunits in CKD/HD patients compared to the control group. Only for COXI, the comparison between CKD and healthy subjects reached the statistical significance. However, complex IV activity was significantly reduced in CKD/HD patients compared to healthy subjects (p < 0.01). Finally, CKD/HD patients presented higher reactive oxygen species and 8-hydroxydeoxyguanosine levels compared to controls.ConclusionTaken together these results suggest, for the first time, that CKD/HD patients may have an impaired mitochondrial respiratory system and this condition may be both the consequence and the cause of an enhanced oxidative stress.

CD2AP mutations are associated with sporadic nephrotic syndrome and focal segmental glomerulosclerosis (FSGS)

BACKGROUND CD2-associated protein (CD2AP) is a crucial protein for the slit-diaphragm assembly and function. In spite of the fact that CD2AP knockout causes nephrotic syndrome in mice and the heterozygous +/- mouse is prone to proteinuria, little is known about the relevance of this molecule in human renal pathology. METHODS A total of 80 Italian patients with idiopathic nephrotic syndrome were enrolled and screened for changes in the CD2AP gene. A normal control group of 200 healthy donors was also studied. The coding region of the CD2AP gene was analysed by polymerase chain reaction, denaturing high-performance liquid chromatography and sequencing. Peripheral blood mononuclear cells from patients with CD2AP mutations and from healthy donors were isolated by the Ficoll-Hypaque gradient, and the CD2/CD2AP interaction was studied on T-lymphocytes by confocal laser scanning microscopy analysis. The expression levels of CD2AP, nephrin and podocin proteins were evaluated by indirect immunofluorescence on renal biopsies from a patient with p.delGlu525 mutation and from control subjects. Moreover, the effect of the p.K301M mutation on cell viability was evaluated by flow cytometry and annexin V/propidium iodide staining. RESULTS Three heterozygous mutations (c.904A>T; c.1120A>G; c.1573delAGA) producing respectively aminoacidic changes (p.K301M, p.T374A) or a deletion in functional domains (p.delGlu525) were found in three unrelated patients. One (p.K301M) produced a lysine to methionine change in the third interactive SH3 domain (position 301) and resulted in the defective CD2-CD2AP interaction and clustering; the other (c.1573delAGA) caused the deletion of the glutamic acid in position 525 in the COOH-terminal region of binding with nephrin and was associated with down-modulation of CD2AP, podocin and nephrin glomerular expression. CONCLUSIONS Our findings suggest that CD2AP mutations modify the interaction with CD2 in lymphocytes and alter the composition of the renal slit diaphragm.

Ischemia-Reperfusion Induces Glomerular and Tubular Activation of Proinflammatory and Antiapoptotic Pathways: Differential Modulation by Rapamycin

Ischemia-reperfusion (I-R) injury in transplanted kidney, a key pathogenic event of delayed graft function (DGF), is characterized by tubular cell apoptosis and interstitial inflammation. Akt-mammalian target of rapamycin-S6k and NF-kappaB-inducing kinase (NIK)-NF-kappaB axis are the two main signaling pathways regulating cell survival and inflammation. Rapamycin, an immunosuppressive drug inhibiting the Akt axis, is associated with a prolonged DGF. The aim of this study was to evaluate Akt and NF-kappaB axis activation in patients who had DGF and received or not rapamycin and in a pig model of I-R and the role of coagulation priming in this setting. In graft biopsies from patients who were not receiving rapamycin, phosphorylated Akt increased in proximal tubular, interstitial, and mesangial cells with a clear nuclear translocation. The same pattern of activation was observed for S6k and NIK. However, in rapamycin-treated patients, a significant reduction of S6k but not Akt and NIK activation was observed. A time-dependent activation of phosphatidylinositol 3-kinase, Akt, S6k, and NIK was observed in the experimental model with the same pattern reported for transplant recipients who did not receive rapamycin. Extensive interstitial and glomerular fibrin deposition was observed both in pig kidneys upon reperfusion and in DGF human biopsies. It is interesting that the activation of both Akt and NIK-NF-kappaB pathways was induced by thrombin in cultured proximal tubular cells. In conclusion, the data suggest that (1) coagulation may play a pathogenic role in I-R injury; (2) the Akt axis is activated after I-R, and its inhibition may explain the prolonged DGF observed in rapamycin-treated patients; and (3) NIK activation in I-R and DGF represents a proinflammatory, rapamycin-insensitive signal, potentially leading to progressive graft injury.

Protease-Activated Receptor-2 Expression in IgA Nephropathy: A Potential Role in the Pathogenesis of Interstitial Fibrosis

An increasing body of evidence suggests that proteases may play a key role in the pathogenesis of tissue fibrosis. Protease-activated receptor-2 (PAR-2) is cleaved and activated by trypsin-like proteolytic enzymes, including tryptase and activated coagulation factor X (FXa). Both these soluble mediators have been demonstrated, directly or indirectly, at the interstitial level in progressive renal diseases, including IgA nephropathy (IgAN). PAR-2 mRNA and protein levels were investigated by RT-PCR and immunohistochemistry, respectively, in 17 biopsies from IgAN patients and 10 normal kidneys. PAR-2 expression was also evaluated, by RT-PCR and western blotting, in cultured human mesangial and proximal tubular cells. Finally, gene expression of plasminogen activator inhibitor-1 (PAI-1) and TGF-beta, two powerful fibrogenic factors, was evaluated in FXa-, trypsin-, and PAR-2 activating peptide-stimulated human proximal tubular cells by Northern blot. In normal kidneys, PAR-2 gene expression was barely detectable, whereas in IgAN biopsies the mRNA levels for this protease receptor were strikingly increased and directly correlated with the extent of interstitial fibrosis. Immunohistochemical staining demonstrated that PAR-2 protein expression in IgAN biopsies was mainly localized in the proximal tubuli and within the interstitial infiltrate. Proximal tubular cells in culture expressed PAR-2. Activation of this receptor by FXa in tubular cells induced a striking increase in intracellular calcium concentration. In addition, incubation of both cell lines with trypsin, FXa, or PAR-2 activating peptide caused a marked upregulation of PAI-1 gene expression that was not counterbalanced by an increased expression of plasminogen activators. Finally, PAR-2 activation induced a significant upregulation of TGF-beta gene and protein expression in both mesangial and tubular cells. On the basis of our data, we can suggest that PAR-2 expressed by renal resident cells and activated by either mast cell tryptase or FXa may induce extracellular matrix deposition modifying the PAI-1/PA balance and inducing TGF-beta expression. These molecular mechanisms may underlie interstitial fibrosis in IgAN.

Sirolimus interferes with iron homeostasis in renal transplant recipients.

Background. Sirolimus is an immunosuppressive drug whose use is frequently associated with anemia. A pathogenic link between sirolimus-induced anemia and the appearance of an inflammatory state was recently suggested. Because inflammation-related anemia is characterized by a functional iron deficiency, we investigated whether sirolimus may influence iron homeostasis and serum levels of hepcidin, a key mediator of inflammation-related anemia. Methods. To this purpose, 42 consecutive transplanted patients with biopsy-proven chronic allograft nephropathy were randomized (2:1 ratio) to receive either a 40% cyclosporine reduction (group A, 14 patients) or immediate cyclosporine withdrawal and sirolimus introduction (group B, 28 patients). Hemoglobin levels and iron status were evaluated 6 months before and after randomization. Results. The two groups had similar hemoglobin levels and iron status at baseline. We did not observe any significant change in hemoglobin and iron status in group A patients after randomization. On the contrary, we observed a significant reduction of hemoglobin without any change of red blood cell count after sirolimus introduction, with a significant reduction of mean corpuscular volume and mean corpuscular hemoglobin. Serum iron and transferrin saturation (TSAT) levels were markedly reduced after the switch, while ferritin serum concentrations remained stable. Although sirolimus-induced anemia was recently suggested to resemble inflammation-related anemia, hepcidin serum levels were similar in the two groups after randomization. None of group A and eight of group B patients presented a TSAT <20 and were given iron supplementation after randomization, in all of them oral iron therapy did not influence either hemoglobin or serum iron levels. Conclusion. We demonstrated that sirolimus-induced anemia is independent of the drug antiproliferative effect and does not present the features of inflammation-related anemia. This event may be due to the direct influence of sirolimus on iron homeostasis.

Protease-activated receptor 1 and plasminogen activator inhibitor 1 expression in chronic allograft nephropathy : The role of coagulation and fibrinolysis in renal graft fibrosis

BACKGROUND Chronic allograft nephropathy (CAN), the major cause of renal graft failure, frequently displays extensive interstitial fibrin deposition. Little is known in regard to the cause of the altered coagulation/fibrinolysis balance and its relevance in the pathogenesis of CAN. Thrombin, present within the fibrin clots, can interact with a specific receptor, protease-activated receptor 1 (PAR-1), and modulate a variety of cell functions. On the other hand, the derangement of the fibrinolytic system may directly affect extracellular matrix (ECM) degradation. METHODS In the present study, we investigated, by in situ hybridization, PAR-1 gene expression and the mRNA levels for tissue factor and plasminogen activator inhibitor 1 (PAI-1), two key regulatory molecules of coagulation and fibrinolysis, in 16 CAN biopsies and in 10 normal human kidney grafts. The thrombin-induced transforming growth factor beta (TGF-beta) gene and protein expression in proximal tubular cells (PTC) was investigated by Northern blotting and ELISA, respectively. RESULTS Fibrin deposits, absent in normal grafts, were observed in the interstitial space and arterial wall of CAN. Tissue factor gene expression was not increased either at the vascular or at the interstitial level in CAN. On the contrary, PAI-1 gene expression, barely detectable in control tissue, was strikingly increased in CAN, with a distribution resembling the pattern of fibrin deposition. Note that PAI-1 gene expression was directly correlated with the degree of interstitial fibrosis. In addition, fibrin deposits were strictly associated with a marked increase of PAR-1 gene expression in endothelial cells and PTC. The tubular expression of PAR-1 was significantly higher in Banff grade II-III than in grade I. In vitro, incubation of PTC with thrombin caused a significant up-regulation of TGF-beta gene expression, followed by an increased TGF-beta release into the supernatant. Interestingly, urine from CAN patients contained significantly higher levels of TGF-beta. CONCLUSIONS Fibrin deposits in CAN may result from the increased expression of PAI-1 and the subsequent inhibition of fibrinolysis. The reduced fibrinolysis may cause, in turn, a decreased ECM turnover. Finally, thrombin, preserved in the active form within the fibrin clots, may interact with PAR-1 highly expressed on PTC and induce an up-regulation of ECM deposition in a TGF-beta-dependent manner.

Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3−/− mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3−/− mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re‐expression of Timp3 in Timp3−/− mesangial cells rescued the expression of Foxo1 and its targets, and decreased STAT1 expression to control levels; abolishing STAT1 expression led to a rescue of FoxO1, evoking a role of STAT1 in linking Timp3 deficiency to FoxO1. Studies on kidney biopsies from patients with diabetic nephropathy confirmed a significant reduction in TIMP3, FoxO1 and FoxO1 target genes involved in autophagy compared to controls, while STAT1 expression was strongly increased.

Sirolimus and proteinuria in renal transplant patients: evidence for a dose-dependent effect on slit diaphragm-associated proteins.

Background. The mechanisms underlying the development of proteinuria in renal-transplant recipients converted from calcineurin inhibitors to sirolimus are still unknown. Methods. This is a single-center cohort study. One hundred ten kidney transplant recipients converted from calcineurin inhibitors to sirolimus in the period from September 2000 to December 2005 were included in the study. All patients underwent a graft biopsy before conversion (T0) and a second protocol biopsy 2 years thereafter (T2), according to our standard clinical protocol. On the basis of the changes observed in proteinuria between T0 and T2 (median 70%), the patients were divided into two groups: group I (<70%) and group II (>70%). The authors blinded the sirolimus blood trough levels. We investigated in vivo the effects of sirolimus on nephrin, podocin, CD2ap, and actin protein expression. Slit diaphragm (SD)-associated protein expressions were evaluated in T0 and T2 biopsies. The same analysis was performed in cultured human podocytes treated with different doses of sirolimus (5, 10, 20, and 50 ng/mL). Results. The SD protein expression in group II T2 biopsies was significantly reduced compared with the T0 biopsies and with T2 group I biopsies. In addition, sirolimus blood trough levels directly and significantly correlated with the SD protein expression at T2 graft biopsies. Group II patients presented significantly higher sirolimus blood levels than group I. In vitro study confirmed that sirolimus effect on podocytes was dose dependent. Conclusions. Our data suggest that sirolimus-induced proteinuria may be a dose-dependent effect of the drug on key podocyte structures.

RAPAMYCIN INHIBITS PAI-1 EXPRESSION AND REDUCES INTERSTITIAL FIBROSIS AND GLOMERULOSCLEROSIS IN CHRONIC ALLOGRAFT NEPHROPATHY

Background. Chronic allograft nephropathy (CAN) is characterized by deposition of extracellular matrix (ECM) in all renal compartments. PAI-1 seems to play a pivotal role in ECM turnover in CAN. Rapamycin has been shown to improve long-term graft survival in patients with CAN. The aim of the study was to evaluate the molecular mechanisms underlying the beneficial effects of rapamycin on CAN progression at glomerular and tubulointerstitial level. Methods. After a biopsy-proven CAN diagnosis (T0), 18 patients on calcineurin inhibitors (CNI) were randomly assigned in a 2:1 ratio to continue CNI (6 patients) or to receive rapamycin (RAPA; 12 patients). After 2 years of treatment (T24), all patients underwent a second renal biopsy. Morphometric analysis was conducted at T0 and at T24. PAI-1 expression was evaluated at T0 and T24 by immunohistochemistry. We evaluated the effect of rapamycin on PAI-1 gene expression in cultured proximal tubular cells incubated with CD40L or thrombin, two potential CAN pathogenic mediators. Results. The RAPA group showed a significant regression of glomerulosclerotic lesions and only a 26% increase in interstitial fibrosis after 2 years compared to baseline, whereas the CNI group showed progression of glomerulosclerosis and 112% increase in fibrosis. Glomerular and tubulointerstitial PAI-1 expression was reduced compared to the baseline in the RAPA group, while they were unchanged in the CNI group. In vitro data showed that rapamycin significantly reduced PAI-1 gene expression induced by both CD40L and thrombin in proximal tubular epithelial cells. Conclusions. These data suggest that rapamycin may modulate ECM deposition in CAN reducing PAI-1 expression.

Altered modulation of WNT-beta-catenin and PI3K/Akt pathways in IgA nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common form of primary glomerulonephritis worldwide. The basic defect lies within the IgA immune system and in peripheral blood leukocytes, rather than local kidney abnormalities. To define the intracellular mechanisms leading to the disease, we conducted a microarray study to identify genes and pathways differentially modulated in peripheral blood leukocytes isolated from 12 IgAN patients and 8 healthy controls. The genes whose expression discriminated between the IgAN patients and controls were primarily involved in canonical WNT-beta-catenin and PI3K/Akt pathways. We also tested peripheral blood mononuclear cells and their subpopulations isolated from an independent group of IgAN patients and healthy controls. There were low protein levels of inversin and PTEN, key regulators of WNT-beta-catenin and PI3K/Akt, in IgAN patients, suggesting hyperactivation of these pathways. Also, there were increased phospho-Akt protein levels and nuclear beta-catenin accumulation with an enhanced peripheral blood mononuclear cell proliferation rate. Subpopulation analysis uncovered a major irregularity of WNT signaling in monocytes. Hence, hyperactivation of these pathways may provide insight into mechanisms contributing to the pathogenesis of IgAN.