Chiara Divella

Therapeutic Targeting of Classical and Lectin Pathways of Complement Protects from Ischemia-Reperfusion-Induced Renal Damage

Ischemia-reperfusion injury is the major cause of delayed graft function in transplanted kidneys, an early event significantly affecting long-term graft function and survival. Several studies in rodents suggest that the alternative pathway of the complement system plays a pivotal role in renal ischemia-reperfusion injury. However, limited information is currently available from humans and larger animals. Here we demonstrated that 30 minutes of ischemia resulted in the induction of C4d/C1q, C4d/MLB, and MBL/MASP-2 deposits in a swine model of ischemia-reperfusion injury. The infusion of C1-inhibitor led to a significant reduction in peritubular capillary and glomerular C4d and C5b-9 deposition. Moreover, complement-inhibiting treatment significantly reduced the numbers of infiltrating CD163(+), SWC3a(+), CD4a(+), and CD8a(+) cells. C1-inhibitor administration led to significant inhibition of tubular damage and tubular epithelial cells apoptosis. Interestingly, we report that focal C4d-deposition colocalizes with C1q and MBL at the peritubular and glomerular capillary levels also in patients with delayed graft function. In conclusion, we demonstrated the activation and a pathogenic role of classical and lectin pathways of complement in a swine model of ischemia-reperfusion-induced renal damage. Therefore, inhibition of these two pathways might represent a novel therapeutic approach in the prevention of delayed graft function in kidney transplant recipients.

Human renal stem/progenitor cells repair tubular epithelial cell injury through TLR2-driven inhibin-A and microvesicle-shuttled decorin

Acute kidney injury (AKI) is emerging as a worldwide public health problem. Recent studies have focused on the possibility of using human adult renal stem/progenitor cells (ARPCs) to improve the repair of AKI. Here we studied the influence of ARPCs on the healing of cisplatin-injured renal proximal tubular epithelial cells. Tubular, but not glomerular, ARPCs provided a protective effect promoting proliferation of surviving tubular cells and inhibiting cisplatin-induced apoptosis. The recovery effect was specific to tubular ARPCs, occurred only after damage sensing, and was completely cancelled by TLR2 blockade on tubular ARPCs. Moreover, tubular, but not glomerular, ARPCs were resistant to the apoptotic effect of cisplatin. Tubular ARPCs operate mainly through the engagement of TLR2, the secretion of inhibin-A protein, and microvesicle-shuttled decorin, inhibin-A, and cyclin D1 mRNAs. These factors worked synergistically and were essential to the repair process. The involvement of tubular ARPC-secreted inhibin-A and decorin mRNA in the pathophysiology of AKI was also confirmed in transplant patients affected by delayed graft function. Hence, identification of this TLR2-driven recovery mechanism may shed light on new therapeutic strategies to promote the recovery capacity of the kidney in acute tubular damage. Use of these components, derived from ARPCs, avoids injecting stem cells.

IL‐17 Expression by Tubular Epithelial Cells in Renal Transplant Recipients with Acute Antibody‐Mediated Rejection

Acute rejection is still a common complication of kidney transplantation. IL‐17 is known to be associated with allograft rejection but the cellular source and the role of this cytokine remains unclear. We investigated IL‐17 graft expression in renal transplant recipients with acute antibody‐mediated rejection (ABMR), acute T‐cell‐mediated rejection (TCMR), interstitial fibrosis and tubular atrophy (IFTA) and acute tubular damage due to calcineurin‐inhibitor toxicity (CNI). In acute ABMR, tubular IL‐17 protein expression was significantly increased compared to TCMR, where most of the IL‐17+cells were CD4+graft infiltrating lymphocytes, IFTA and CNI control groups. The tubular expression of IL‐17 in acute ABMR colocalized with JAK2 phosphorylation and peritubular capillaries C4d deposition. In addition, IL‐17 tubular expression was directly and significantly correlated with the extension of C4d deposits. In cultured proximal tubular cells, C3a induced IL‐17 gene and protein expression along with an increased in JAK2 phosphorylation. The inhibition of JAK2 abolished C3a‐induced IL‐17 expression. The use of steroids and monoclonal antibodies reduced IL‐17 expression, JAK2 phosphorylation and C4d deposition in acute ABMR patients. Our data suggest that tubular cells represent a significant source of IL‐17 in ABMR and this event might be mediated by the complement system activation featuring this condition.

Endothelial-to-mesenchymal transition and renal fibrosis in ischaemia/reperfusion injury are mediated by complement anaphylatoxins and Akt pathway

BACKGROUND Increasing evidence demonstrates a phenotypic plasticity of endothelial cells (ECs). Endothelial-to-mesenchymal transition (EndMT) contributes to the development of tissue fibrosis. However, the pathogenic factors and signalling pathways regulating this process in ischaemia/reperfusion (I/R) injury are still poorly understood. METHODS We investigated the possible role of complement in the induction of this endothelial dysfunction in a swine model of renal I/R injury by using recombinant C1 inhibitor in vivo. RESULTS Here, we showed that I/R injury reduced the density of renal peritubular capillaries and induced tissue fibrosis with generation of CD31(+)/α-SMA(+) and CD31(+)/FPS-1(+) cells indicating EndMT. When we inhibited complement, the process of EndMT became rare, with preserved density of peritubular capillaries and significant reduction in renal fibrosis. When we activated ECs by anaphylatoxins in vitro, C3a and C5a led to altered endothelial phenotype with increased expression of fibroblast markers and decrease expression of specific endothelial markers. The activation of Akt pathway was pivotal for the C3a and C5a-induced EndMT in vitro. In accordance, inhibition of complement in vivo led to the abrogation of Akt signalling, with hampered EndMT and tissue fibrosis. CONCLUSIONS Our data demonstrate a critical role for complement in the acute induction of EndMT via the Akt pathway. Therapeutic inhibition of these systems may be essential to prevent vascular damage and tissue fibrosis in transplanted kidney.

T helper 1, 2 and 17 cell subsets in renal transplant patients with delayed graft function

Ischemia‐reperfusion injury (IRI) in kidney transplantation is the major cause of delayed graft function (DGF), an event associated with an increased risk of acute rejection. The aim of this study was to evaluate T helper (Th) cell phenotype in renal transplants with DGF. T‐bet (Th1), GATA‐3 (Th2) and IL‐17 (Th17) protein expression was investigated in pretransplant biopsies, DGF and acute tubular damage (ATD) caused by calcineurin‐inhibitor toxicity. Intracytofluorimetric analysis of IFN‐γ, IL‐4 and IL‐17 was performed to analyze Th1, Th2 and Th17 responses in peripheral blood mononuclear cells of recipients with early graft function (EGF) and DGF, before (T0) and 24 h after transplantation (T24). In pretransplant biopsies, T‐bet+, GATA‐3+ and IL‐17+ cells were barely detectable. In DGF, T‐bet+ and IL‐17+ cells were significantly increased compared with pretransplant and ATD. More than 90% of T‐bet+ and less then 5% of IL‐17+ cells were CD4+. GATA‐3+ cells were increased to a lower extent. T‐bet+/GATA‐3+ cell ratio was significantly higher in DGF. Peripheral CD4+ IFN‐γ/IL‐4 ratio was significantly decreased in DGF, while CD4+/IL‐17+ cells did not differ between T0 and T24 in DGF. Our data suggest that DGF is characterized by a prevalent Th1 phenotype within the graft. This event might represent a link between DGF and acute rejection.

Mutational Spectrum of the C1 Inhibitor Gene in a Cohort of Italian Patients with Hereditary Angioedema: Description of Nine Novel Mutations

Hereditary angioedema (HAE) is an autosomal dominant disease due to mutations in the C1 inhibitor gene (C1NH) that affects protein synthesis (HAE type I) or function (HAE type II). In 45 subjects affected by HAE diagnosed through clinical features and C1 inhibitor deficiency from the south of Italy (38 with type I and 7 with type II HAE), the whole C1NH coding region was screened for mutations by direct DNA sequencing. A severity score based on clinical manifestation, age at disease onset, and need for long‐term prophylaxis was used to investigate possible genotype‐phenotype correlations. A series of 22 different mutations was identified: nine missense (40.9%), five nonsense (22.7%), six frameshift (27.3), one small deletion (4.5%), and one splicing defect (4.5%). Nine C1NH mutations have not been previously described. No correlation was found between C1 inhibitor function level and severity score or age at first attack. Moreover, there was no correlation between different types of mutations and clinical phenotype. The number of different mutations identified highlights the heterogeneity of C1 inhibitor deficiency and supports the hypothesis that HAE clinical phenotype is not strictly related to the type of mutation but rather depends on unknown factors.

Complement Modulation of Anti-Aging Factor Klotho in Ischemia/Reperfusion Injury and Delayed Graft Function.

Klotho is an anti‐aging factor mainly produced by renal tubular epithelial cells (TEC) with pleiotropic functions. Klotho is down‐regulated in acute kidney injury in native kidney; however, the modulation of Klotho in kidney transplantation has not been investigated. In a swine model of ischemia/reperfusion injury (IRI), we observed a remarkable reduction of renal Klotho by 24 h from IRI. Complement inhibition by C1‐inhibitor preserved Klotho expression in vivo by abrogating nuclear factor kappa B (NF‐kB) signaling. In accordance, complement anaphylotoxin C5a led to a significant down‐regulation of Klotho in TEC in vitro that was NF‐kB mediated. Analysis of Klotho in kidneys from cadaveric donors demonstrated a significant expression of Klotho in pre‐implantation biopsies; however, patients affected by delayed graft function (DGF) showed a profound down‐regulation of Klotho compared with patients with early graft function. Quantification of serum Klotho after 2 years from transplantation demonstrated significant lower levels in DGF patients. Our data demonstrated that complement might be pivotal in the down‐regulation of Klotho in IRI leading to a permanent deficiency after years from transplantation. Considering the anti‐senescence and anti‐fibrotic effects of Klotho at renal levels, we hypothesize that this acquired deficiency of Klotho might contribute to DGF‐associated chronic allograft dysfunction.

The Anti-Fibrotic Effect of Mycophenolic Acid–Induced Neutral Endopeptidase

Mycophenolic acid (MPA) appears to have anti-fibrotic effects, but the molecular mechanisms underlying this are unknown. We prospectively studied 35 stable kidney transplant recipients maintained on cyclosporine and azathioprine. We converted 20 patients from azathioprine to enteric-coated mycophenolate sodium (EC-MPS) and continued the remaining 15 patients on azathioprine. Exploratory mRNA expression profiling, performed on five randomly selected EC-MPS patients, revealed significant upregulation of neutral endopeptidase (NEP), which is an enzyme that degrades angiotensin II. We confirmed these microarray data by measuring levels of NEP expression in all subjects; in addition, we found that NEP gene expression correlated inversely with proteinuria. In an additional 33 patients, glomerular and tubular NEP protein levels from renal graft biopsies were significantly higher among the 13 patients receiving cyclosporine + EC-MPS than among the 12 patients receiving cyclosporine + azathioprine or 8 patients receiving cyclosporine alone. Glomerular NEP expression inversely correlated with glomerulosclerosis and proteinuria, and tubular NEP expression inversely correlated with interstitial fibrosis. Incubation of human proximal tubular cells with MPA increased NEP gene expression in a dose- and time-dependent manner. Moreover, MPA reduced angiotensin II-induced expression of the profibrotic factor plasminogen activator inhibitor-1, and a specific NEP inhibitor completely reversed this effect. Taken together, our data suggest that MPA directly induces expression of neutral endopeptidase, which may reduce proteinuria and slow the progression of renal damage in kidney transplant recipients.

Immunohistochemical characterization of glomerular and tubulointerstitial infiltrates in renal transplant patients with chronic allograft dysfunction

BACKGROUND The term chronic allograft nephropathy (CAN) was deleted in the Eighth Banff Classification and two new categories were introduced: chronic T-cell-mediated rejection (CTMR) and chronic active humoral rejection (CAHR). The aim of this study was to revise our CAN cases diagnosed in the last 4 years, analyse allograft survival rates and identify types of infiltrating cells in the different settings. METHODS Seventy-nine patients with biopsy-proven CAN were examined and classified into four groups according to the Banff 2005 criteria: CTMR, CAHR, chronic calcineurin inhibitor toxicity (CNITOX) and interstitial fibrosis and tubular atrophy not otherwise specified (NOS). CD4, CD8, CD20, CD68, CD103, Foxp3 and IL-17 protein expression and C4d deposits were investigated. RESULTS We diagnosed 20 CTMR, 13 CAHR, 28 CNITOX, and 18 NOS. Death-censored graft survival at 4 years from renal biopsy was worse in CAHR compared with the other types of chronic injury. Glomerular CD8(+) cells were increased in CTMR vs CNITOX and NOS. Interstitial CD4(+) and CD8(+) cells were increased in CTMR vs CNITOX. CD68(+) cells in glomerular and peritubular capillaries were higher in CAHR vs CNITOX, CTMR and NOS. CD103(+) cells were higher in cases with tubulitis than in those without. T regulatory and T helper 17 cells were rarely observed in the different settings. CONCLUSIONS Graft survival was worse in patients with CAHR. The presence of any grade transplant glomerulopathy and chronic allograft vasculopathy are poorer prognostic factors. Infiltrating CD8(+), CD103(+) and CD4(+) cells may help to differentiate CTMR from other types of chronic injury, thus improving diagnostic/prognostic features of biopsy in patients with chronic allograft dysfunction.

Local synthesis of interferon-alpha in lupus nephritis is associated with type I interferons signature and LMP7 induction in renal tubular epithelial cells

IntroductionType I interferons are pivotal in the activation of autoimmune response in systemic lupus erythematous. However, the pathogenic role of interferon-alpha in patients affected by lupus nephritis remains uncertain. The aim of our study was to investigate the presence of a specific interferon signature in lupus nephritis and the effects of interferon-alpha at renal level.MethodsWe performed immunohistochemical analysis for MXA-protein and in situ hybridization to detect interferon-alpha signature and production in human lupus nephritis. Through microarray studies, we analyzed the gene expression profile of renal tubular epithelial cells, stimulated with interferon-alpha. We validated microarray results through real-time polymerase chain reaction, flow cytometry on renal tubular epithelial cells, and through immunohistochemical analysis and confocal microscopy on renal biopsies.ResultsType I interferons signature was characterized by MXA-specific staining in renal tubular epithelial cells; in addition, in situ hybridization showed that renal tubular epithelial cells were the major producers of interferon-alpha, indicating a potential autocrine effect. Whole-genome expression profile showed interferon-alpha induced up-regulation of genes involved in innate immunity, protein ubiquitination and switching to immunoproteasome. In accordance with the in vitro data, class IV lupus nephritis showed up-regulation of the immunoproteasome subunit LMP7 in tubular epithelial cells associated with type I interferon signature.ConclusionsOur data indicate that type I interferons might have a pathogenic role in lupus nephritis characterized by an autocrine effect of interferon-alpha on renal tubular epithelial cells. Therefore we hypothesize that inhibition of type I interferons might represent a therapeutic target to prevent tubulo-interstitial damage in patients with lupus nephritis.