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Background Improvement of long-term outcomes in kidney transplantation remains one of the most pressing challenges, yet drug development is stagnating. Human genetics offers an opportunity for much-needed target validation in transplantation. Conflicting data exist about the effect of transforming growth factor-beta 1 (TGF-β1) on kidney transplant survival, since TGF-β1 has pro-fibrotic and protective effects. We investigated the impact of a recently discovered functional TGFB1 polymorphism on kidney graft survival. Methods We performed an observational cohort study analysing recipient and donor DNA in 1271 kidney transplant pairs from the University Medical Centre Groningen in The Netherlands, and associated a low-producing TGFB1 polymorphism (rs1800472-C > T) with 5-, 10- and 15-year death-censored kidney graft survival. Results Donor genotype frequencies of rs1800472 in TGFB1 differed significantly between patients with and without graft loss (P = 0.014). Additionally, the low-producing TGFB1 polymorphism in the donor was associated with an increased risk of graft loss following kidney transplantation (hazard ratio = 2.12 for the T-allele; 95% confidence interval 1.18–3.79; P = 0.012). The incidence of graft loss within 15 years of follow-up was 16.4% in the CC-genotype group and 31.6% in the CT-genotype group. After adjustment for transplant-related covariates, the association between the TGFB1 polymorphism in the donor and graft loss remained significant. In contrast, there was no association between the TGFB1 polymorphism in the recipient and graft loss. Conclusions Kidney allografts possessing a low-producing TGFB1 polymorphism have a higher risk of late graft loss. Our study adds to a growing body of evidence that TGF-β1 is beneficial, rather than harmful, for kidney transplant survival.

Renal endothelial cells (ECs) play crucial roles in vasorelaxation, ultrafiltration, and selective transport of electrolytes and water, but also in leakage of the glomerular filtration barrier and inflammatory processes like complement activation and leukocyte recruitment. In addition, they are target cells for both cellular and antibody-mediated rejection in the transplanted kidney. To study the molecular and cellular processes underlying EC behavior in renal disease, well-characterized primary renal ECs are indispensible. In this report, we describe a straightforward procedure to isolate ECs from the perfusion fluid of human donor kidneys by a combination of negative selection of monocytes/macrophages, positive selection by CD31 Dynabeads, and propagation in endothelium-specific culture medium. Thus, we isolated and propagated renal ECs from 102 donor kidneys, representative of all blood groups and major human leukocyte antigen (HLA) class I and II antigens. The obtained ECs were positive for CD31 and von Willebrand factor, expressed other endothelial markers such as CD34, VEGF receptor-2, TIE2, and plasmalemmal vesicle associated protein-1 to a variable extent, and were negative for the monocyte marker CD14 and lymphatic endothelial marker podoplanin. HLA class II was either constitutively expressed or could be induced by interferon-y. Furthermore, as a proof of principle, we showed the diagnostic value of this renal endothelial biobank in renal endothelium-specific cross-matching tests for HLA antibodies.NEW & NOTEWORTHY We describe a new and widely accessible approach to obtain human primary renal endothelial cells in a \standardized fashion, by isolating from the perfusate of machine-perfused donor kidneys. Characterization of the cells showed a mixed population originating from different compartments of the kidney. As a proof of principle, we demonstrated a possible diagnostic application in an endothelium-specific cross-match. Next to transplantation, we foresee further applications in the field renal endothelial research.

Urticarial vasculitis (UV) is a clinicopathologic entity characterized by persistent urticarial lesions with biopsy features of vasculitis. Currently, only certain clinical features such as arthralgia and serum complement concentrations are used to identify UV patients at risk for an underlying systemic disease. Hypocomplementemic urticarial vasculitis (HUV) is in contrast to normocomplementemic urticarial vasculitis (NUV), strongly associated with underlying systemic disease, especially systemic lupus erythematosus (SLE). The aim of this study was to find specific histopathological features associated with HUV and underlying systemic disease in UV. In addition, the use of complement C4d deposition in skin biopsies was evaluated as a diagnostic adjunct for HUV- and UV-associated systemic disease. In this retrospective study, the clinical, histopathological, and immunohistological (C4d) features of 43 patients with UV were compared between HUV and NUV and analyzed for association with UV-associated systemic disease. Eight of 43 patients with UV (19%) had hypocomplementemia. Patients with HUV showed a significantly higher number of perivascular neutrophils and lower number of eosinophils compared to NUV. Of all histopathological features, alignment of neutrophils along the dermal-epidermal junction (DEJ) and dermal granular C4d deposition were found to be strongly associated with HUV and underlying SLE. This study shows that both the alignment of neutrophils along the DEJ and dermal C4d deposition are strongly associated with HUV and SLE. Therefore, these (immuno)histopathological features can be used as an easy diagnostic adjunct for early detection of underlying systemic disease in UV.

Item does not contain fulltext BACKGROUND: Pre-transplant donor-specific anti-human leucocyte antigen (HLA) antibodies (DSAs) are associated with impaired kidney graft survival while the clinical relevance of non-donor-specific anti-HLA antibodies (nDSAs) is more controversial. The aim of the present paired kidney graft study was to compare the clinical relevance of DSAs and nDSAs. METHODS: To eliminate donor and era-dependent factors, a post hoc paired kidney graft analysis was performed as part of a Dutch multicentre study evaluating all transplantations between 1995 and 2005 with available pre-transplant serum samples. Anti-HLA antibodies were detected with a Luminex single-antigen bead assay. RESULTS: Among 3237 deceased donor transplantations, we identified 115 recipient pairs receiving a kidney from the same donor with one recipient being DSA positive and the other without anti-HLA antibodies. Patients with pre-transplant DSAs had a significantly lower 10-year death-censored graft survival (55% versus 82%, P=0.0001). We identified 192 pairs with one recipient as nDSA positive (against Class I and/or II) and the other without anti-HLA antibodies. For the patients with nDSAs against either Class I or II, graft survival did not significantly differ compared with patients without anti-HLA antibodies (74% versus 77%, P = 0.79). Only in patients with both nDSAs Class I and II was there a trend towards a lower graft survival (58%, P = 0.06). Lastly, in a small group of 42 recipient pairs, 10-year graft survival in recipients with DSAs was 49% compared with 68% in recipients with nDSAs (P=0.11). CONCLUSION: This paired kidney analysis confirms that the presence of pre-transplant DSAs in deceased donor transplantations is a risk marker for graft loss, whereas nDSAs in general are not associated with a lower graft survival. Subgroup analysis indicated that only in broadly sensitized patients with nDSAs against Class I and II, nDSAs may be a risk marker for graft loss in the long term.

Introduction Various studies have reported the importance of complement regulators in preventing mesothelial damage during peritoneal dialysis (PD). Its assessment, however, is limited in clinical practice due to the lack of easy access to the peritoneal membrane. Recently, a soluble form of the complement regulatory protein CD59 (sCD59) has been described. We therefore aimed to investigate the role of sCD59 in PD. Methods Plasma sCD59 was measured in 48 PD patients, 41 hemodialysis patients, 15 non-dialysis patients with chronic kidney disease and 14 healthy controls by ELISA (Hycult; HK374-02). Additionally, sCD59 and sC5b-9 were assessed in the peritoneal dialysate. Results sCD59 and sC5b-9 were detectable in the peritoneal dialysate of all patients, and marginally correlated (r = 0.27, P = 0.06). Plasma sCD59 levels were significantly higher in PD patients than in patients with chronic kidney disease and healthy controls, but did not differ from hemodialysis patients. During follow-up, 19% of PD patients developed peritoneal membrane failure and 27% of PD patients developed loss of residual renal function. In adjusted models, increased sCD59 levels in the dialysate (HR 3.44, 95% CI 1.04–11.40, P = 0.04) and in plasma (HR 1.08, 95% CI 1.01–1.17, P = 0.04) were independently associated with the occurrence of peritoneal membrane failure. Higher plasma levels of sCD59 were also associated with loss of residual renal function (HR 1.10, 95% CI 1.04–1.17, P Conclusions Our study suggests that sCD59 has potential as a biomarker to predict peritoneal membrane function and loss of residual renal function in PD, thereby offering a tool to improve patient management. Graphic abstract

Rationale & Objective: Glomerular C4d (C4dG) as an indicator of the lectin pathway of complement activation in immunoglobulin A nephropathy (IgAN) has been associated with more severe kidney damage. Recent studies have suggested that vascular lesions in IgAN biopsy specimens with complement deposition are also associated with disease progression. We aimed to study the clinical significance of arteriolar C4d (C4dA) in IgAN kidney biopsy tissue.Study Design: Retrospective cohort study.Setting & Participants: Kidney biopsy specimens from 126 adults with IgAN diagnosed by Oxford classification criteria were stained using immunohistochemistry and classified according to C4dG and C4dA deposition. Additionally, vascular lesions including acute and chronic microangiopathy, arteriolar hyalinosis, and arterial intima fibrosis were characterized.Predictor: C4dA.Outcome: Progressive kidney disease, defined as a decline in estimated glomerular filtration rate by >= 50% or occurrence of kidney failure.Analytical Approach: The association of C4dA and C4dG with baseline clinical and histologic characteristics, as well as progressive kidney disease, were assessed with survival analysis using multivariable Cox regression analysis.Results: C4dA was identified in 21 (17%) patients and was associated with mean arterial pressure, arterial intima fibrosis, and chronic microangiopathy. C4dA was also significantly associated with C4dG and both were associated with progressive kidney disease. In regression analysis, C4dA remained significantly associated with progressive kidney disease after adjusting for other significant predictors, including baseline estimated glomerular filtration rate, mean arterial pressure, and the presence of crescents.Limitations: Findings based on the retrospective evaluation of a single center's experience, limited number of events, a small number of patients with a broad range of kidney disease stages, and use of immunohistochemistry rather than immunofluorescence to detect C4d.Conclusions: C4dA is a potential biomarker for disease progression in IgAN. It should be further investigated in larger cohorts to determine the value of C4dA in improving prediction of IgAN disease progression.

Background Complement-fixing antibodies against donor HLA are considered a contraindication for kidney transplant. A modification of the IgG single-antigen bead (SAB) assay allows detection of anti-HLA antibodies that bind C3d. Because early humoral graft rejection is considered to be complement mediated, this SAB-based technique may provide a valuable tool in the pretransplant risk stratification of kidney transplant recipients.Methods Previously, we established that pretransplant donor-specific anti-HLA antibodies (DSAs) are associated with increased risk for long-term graft failure in complement-dependent cytotoxicity crossmatch-negative transplants. In this study, we further characterized the DSA-positive serum samples using the C3d SAB assay.Results Among 567 pretransplant DSA-positive serum samples, 97 (17%) contained at least one C3d-fixing DSA, whereas 470 (83%) had non-C3d-fixing DSA. At 10 years after transplant, patients with C3d-fixing antibodies had a death-censored, covariate-adjusted graft survival of 60%, whereas patients with non-C3d-fixing DSA had a graft survival of 64% (hazard ratio, 1.02; 95% confidence interval, 0.70 to 1.48 for C3d-fixing DSA compared with non-C3d-fixing DSA; P=0.93). Patients without DSA had a 10-year graft survival of 78%.Conclusions The C3d-fixing ability of pretransplant DSA is not associated with increased risk for graft failure.

Background Kidneys derived from brain-dead (BD) donors have lower graft survival rates compared with kidneys from living donors. Complement activation plays an important role in brain death. The aim of our study was therefore to investigate the effect of C1-inhibitor (C1-INH) on BD-induced renal injury. Methods Brain death was induced in rats by inflating a subdurally placed balloon catheter. Thirty minutes after BD, rats were treated with saline, low-dose or high-dose C1-INH. Sham-operated rats served as controls. After 4 hours of brain death, renal function, injury, inflammation, and complement activation were assessed. Results High-dose C1-INH treatment of BD donors resulted in significantly lower renal gene expression and serum levels of IL-6. Treatment with C1-INH also improved renal function and reduced renal injury, reflected by the significantly lower kidney injury marker 1 gene expression and lower serum levels of lactate dehydrogenase and creatinine. Furthermore, C1-INH effectively reduced complement activation by brain death and significantly increased functional levels. However, C1-INH treatment did not prevent renal cellular influx. Conclusions Targeting complement activation after the induction of brain death reduced renal inflammation and improved renal function before transplantation. Therefore, strategies targeting complement activation in human BD donors might clinically improve donor organ viability and renal allograft survival.

The clinical significance of non‐HLA antibodies on renal allograft survival is a matter of debate, due to differences in reported results and lack of large‐scale studies incorporating analysis of multiple non‐HLA antibodies simultaneously. We developed a multiplex non‐HLA antibody assay against 14 proteins highly expressed in the kidney. In this study, the presence of pretransplant non‐HLA antibodies was correlated to renal allograft survival in a nationwide cohort of 4770 recipients transplanted between 1995 and 2006. Autoantibodies against Rho GDP‐dissociation inhibitor 2 (ARHGDIB) were significantly associated with graft loss in recipients transplanted with a deceased‐donor kidney (N = 3276) but not in recipients of a living‐donor kidney (N = 1496). At 10 years after deceased‐donor transplantation, recipients with anti‐ARHGDIB antibodies (94/3276 = 2.9%) had a 13% lower death‐censored covariate‐adjusted graft survival compared to the anti‐ARHGDIB‐negative (3182/3276 = 97.1%) population (hazard ratio 1.82; 95% confidence interval, 1.32‐2.53; P = .0003). These antibodies occur independently from donor‐specific anti‐HLA antibodies (DSA) or other non‐HLA antibodies investigated. No significant relations with graft loss were found for the other 13 non‐HLA antibodies. We suggest that pretransplant risk assessment can be improved by measuring anti‐ARHGDIB antibodies in all patients awaiting deceased‐donor transplantation., From a multicenter evaluation of kidney transplants, the authors report that the pretransplant presence of autoantibodies against ARHGDIB are associated with long‐term graft loss in recipients transplanted with a deceased donor kidney, independent from donor‐specific HLA antibodies.

Introduction: The majority of kidneys used for transplantation are retrieved from brain-dead organ donors. In brain death, the irreversible loss of brain functions results in hemodynamic instability, hormonal changes and immunological activation. Recently, brain death has been shown to cause activation of the complement system, which is adversely associated with renal allograft outcome in recipients. Modulation of the complement system in the brain-dead donor might be a promising strategy to improve organ quality before transplantation. This study investigated the effect of an inhibitory antibody against complement factor B on brain death-induced renal inflammation and injury. Method: Brain death was induced in male Fischer rats by inflating a balloon catheter in the epidural space. Anti-factor B (anti-FB) or saline was administered intravenously 20 min before the induction of brain death (n = 8/group). Sham-operated rats served as controls (n = 4). After 4 h of brain death, renal function, renal injury, and inflammation were assessed. Results: Pretreatment with anti-FB resulted in significantly less systemic and local complement activation than in saline-treated rats after brain death. Moreover, anti-FB treatment preserved renal function, reflected by significantly reduced serum creatinine levels compared to saline-treated rats after 4 h of brain death. Furthermore, anti-FB significantly attenuated histological injury, as seen by reduced tubular injury scores, lower renal gene expression levels (>75%) and renal deposition of kidney injury marker-1. In addition, anti-FB treatment significantly prevented renal macrophage influx and reduced systemic IL-6 levels compared to saline-treated rats after brain death. Lastly, renal gene expression of IL-6, MCP-1, and VCAM-1 were significantly reduced in rats treated with anti-FB. Conclusion: This study shows that donor pretreatment with anti-FB preserved renal function, reduced renal damage and inflammation prior to transplantation. Therefore, inhibition of factor B in organ donors might be a promising strategy to reduce brain death-induced renal injury and inflammation.

The pathophysiology of late kidney-allograft failure remains complex and poorly understood. Activation of filtered or locally produced complement may contribute to the progression of renal failure through tubular C5b-9 formation. This study aimed to determine urinary properdin and sC5b-9 excretion and assess their association with long-term outcome in renal transplant recipients (RTR). Methods: We measured urinary properdin and soluble C5b-9 in a well-defined cross-sectional cohort of RTR. Urinary specimens were taken from a morning urine portion, and properdin and sC5b-9 were measured using an enzyme-linked-immunosorbent assay (ELISA). Cox proportional hazard regression analyses were used to investigate prospective associations with death-censored graft failure. Results: We included 639 stable RTR at a median [interquartile range] 5.3 (1.8-12.2) years after transplantation. Urinary properdin and sC5b-9 excretion were detectable in 161 (27%) and 102 (17%) RTR, respectively, with a median properdin level of 27.6 (8.6-68.1) ng/mL and a median sC5b-9 level of 5.1 (2.8-12.8) ng/mL. In multivariable-adjusted Cox regression analyses, including adjustment for proteinuria, urinary properdin (HR, 1.12; 95% CI 1.02-1.28; P = 0.008) and sC5b-9 excretion (HR, 1.34; 95% CI 1.10-1.63; P = 0.003) were associated with an increased risk of graft failure. If both urinary properdin and sC5b-9 were detectable, the risk of graft failure was further increased (HR, 3.12; 95% CI 1.69-5.77; P < 0.001). Conclusions: Our findings point toward a potential role for urinary complement activation in the pathogenesis of chronic allograft failure. Urinary properdin and sC5b-9 might be useful biomarkers for complement activation and chronic kidney allograft deterioration, suggesting a potential role for an alternative pathway blockade in RTR.

Whereas regular allocation avoids unacceptable mismatches on the donor organ, allocation to highly sensitized patients within the Eurotransplant Acceptable Mismatch (AM) program is based on the patient's HLA phenotype plus acceptable antigens. These are HLA antigens to which the patient never made antibodies, as determined by extensive laboratory testing. AM patients have superior long‐term graft survival compared with highly sensitized patients in regular allocation. Here, we questioned whether the AM program also results in lower rejection rates. From the PROCARE cohort, consisting of all Dutch kidney transplants in 1995‐2005, we selected deceased donor single transplants with a minimum of 1 HLA mismatch and determined the cumulative 6‐month rejection incidence for patients in AM or regular allocation. Additionally, we determined the effect of minimal matching criteria of 1 HLA‐B plus 1 HLA‐DR, or 2 HLA‐DR antigens on rejection incidence. AM patients showed significantly lower rejection rates than highly immunized patients in regular allocation, comparable to nonsensitized patients, independent of other risk factors for rejection. In contrast to highly sensitized patients in regular allocation, minimal matching criteria did not affect rejection rates in AM patients. Allocation based on acceptable antigens leads to relatively low‐risk transplants for highly sensitized patients with rejection rates similar to those of nonimmunized individuals., The authors show that kidney allocation to highly sensitized patients based on proven acceptable HLA antigens results in a significantly lower incidence of rejection episodes when compared to allocation based on the avoidance of unacceptable HLA antigens only.

Background: Intravenous (IV) iron is widely used to treat anemia in chronic kidney disease patients. Previously, iron formulations were shown to induce immune activation in-vitro. The current study aimed to investigate the effect of IV iron on complement activation in-vivo, and whether this subsequently induces inflammation and/or oxidative stress. Methods: Two distinct patient groups were included: 51 non-dialysis and 32 dialysis patients. The non-dialysis group received iron sucrose or ferric carboxymaltose, based on physicians’ choice. Plasma samples were collected prior to and 1 h after completion of IV iron infusion. The dialysis group received iron sucrose exclusively. Plasma samples were collected at the start and end of two consecutive hemodialysis sessions, one with and one without IV iron. Finally, plasma levels of MBL, C1q, properdin, factor D, sC5b-9, MPO, PTX3 were assessed by ELISA. Results: In the non-dialysis group, sC5b-9 levels significantly increased after IV iron by 32%, while levels of factor D and MBL significantly dropped. Subgroup analysis demonstrated that iron sucrose induced complement activation whereas ferric carboxymaltose did not. In the dialysis group, levels of sC5b-9 significantly increased by 46% during the dialysis session with IV iron, while factor D levels significantly fell. Furthermore, the relative decrease in factor D by IV iron correlated significantly with the relative increase in sC5b-9 by IV iron. MPO levels rose significantly during the dialysis session with IV iron, but not in the session without iron. Moreover, the relative increase in MPO and sC5b-9 by IV iron correlated significantly. PTX3 levels were not affected by IV iron. Conclusions: Iron sucrose but not ferric carboxymaltose, results in complement activation possibly via the lectin and alternative pathway partially mediating oxidative stress but not inflammation. Conflict of Interest Statement: CG received speaking fees and research funding from Vifor Pharma.

Supplemental Digital Content is available in the text., Background. There is no consensus in the literature on the interpretation of single-antigen bead positive for a specific HLA antibody. Methods. To inform the debate, we studied the relationship between various single-antigen bead positivity algorithms and the impact of resulting donor-specific HLA antibody (DSA) positivity on long-term kidney graft survival in 3237 deceased-donor transplants. Results. First, we showed that the interassay variability can be greatly reduced when working with signal-to-background ratios instead of absolute median fluorescence intensities (MFIs). Next, we determined pretransplant DSA using various MFI cutoffs, signal-to-background ratios, and combinations thereof. The impact of the various cutoffs was studied by comparing the graft survival between the DSA-positive and DSA-negative groups. We did not observe a strong impact of various cutoff levels on 10-year graft survival. A stronger relationship between the cutoff level and 1-year graft survival for DSA-positive transplants was found when using signal-to-background ratios, most pronounced for the bead of the same HLA locus with lowest MFI taken as background. Conclusions. With respect to pretransplant risk stratification, we propose a signal-to-background ratio-6 (using the bead of the same HLA-locus with lowest MFI as background) cutoff of 15 combined with an MFI cutoff of 500, resulting in 8% and 21% lower 1- and 10-year graft survivals, respectively, for 8% DSA-positive transplants.

Over the last decade, striking progress has been made in the field of organ transplantation, such as better surgical expertise and preservation techniques. Therefore, organ transplantation is nowadays considered a successful treatment in end-stage diseases of various organs, e.g. the kidney, liver, intestine, heart, and lungs. However, there are still barriers which prevent a lifelong survival of the donor graft in the recipient. Activation of the immune system is an important limiting factor in the transplantation process. As part of this pro-inflammatory environment, the complement system is triggered. Complement activation plays a key role in the transplantation process, as highlighted by the amount of studies in ischemia-reperfusion injury (IRI) and rejection. However, new insight have shown that complement is not only activated in the later stages of transplantation, but already commences in the donor. In deceased donors, complement activation is associated with deteriorated quality of deceased donor organs. Of importance, since most donor organs are derived from either brain-dead donors or deceased after circulatory death donors. The exact mechanisms and the role of the complement system in the pathophysiology of the deceased donor have been underexposed. This review provides an overview of the current knowledge on complement activation in the (multi-)organ donor. Targeting the complement system might be a promising therapeutic strategy to improve the quality of various donor organs. Therefore, we will discuss the complement therapeutics that already have been tested in the donor. Finally, we question whether complement therapeutics should be translated to the clinics and if all organs share the same potential complement targets, considering the physiological differences of each organ.

Background Few studies have evaluated the effect of different immunosuppressive strategies on long-term kidney transplant outcomes. Moreover, as they were usually based on historical data, it was not possible to account for the presence of pretransplant donor-specific human-leukocyte antigen antibodies (DSA), a currently recognized risk marker for impaired graft survival. The aim of this study was to evaluate to what extent frequently used initial immunosuppressive therapies increase graft survival in immunological low-risk patients. Methods We performed an analysis on the PROCARE cohort, a Dutch multicentre study including all transplantations performed in the Netherlands between 1995 and 2005 with available pretransplant serum (n = 4724). All sera were assessed for the presence of DSA by a luminex single-antigen bead assay. Patients with a previous kidney transplantation, pretransplant DSA or receiving induction therapy were excluded from the analysis. Results Three regimes were used in over 200 patients: cyclosporine (CsA)/prednisolone (Pred) (n = 542), CsA/mycophenolate mofetil (MMF)/Pred (n = 857) and tacrolimus (TAC)/MMF/Pred (n = 811). Covariate-adjusted analysis revealed no significant differences in 10-year death-censored graft survival between patients on TAC/MMF/Pred therapy (79%) compared with patients on CsA/MMF/Pred (82%, P = 0.88) or CsA/Pred (79%, P = 0.21). However, 1-year rejection-free survival censored for death and failure unrelated to rejection was significantly higher for TAC/MMF/Pred (81%) when compared with CsA/MMF/Pred (67%, P Conclusion These results suggest that in immunological low-risk patients excellent long-term kidney graft survival can be achieved irrespective of the type of initial immunosuppressive therapy (CsA or TAC; with or without MMF), despite differences in 1-year rejection-free survival.

Chronic kidney disease is a major health concern, and experimental models bridging the gap between animal studies and clinical research are currently lacking. Here, we evaluated precision-cut kidney slices (PCKSs) as a potential model for renal disease. PCKSs were prepared from human cortical tissue obtained from tumor nephrectomies and cultured up to 96 hours. Morphology, cell viability, and metabolic functionality (ie, uridine 5'-diphospho-glucuronosyltransferase and transporter activity) were determined to assess the integrity of PCKSs. Furthermore, inflammatory and fibrosis-related gene expressions were characterized. Finally, to validate the model, renal fibrogenesis was induced using transforming growth factor beta 1 (TGF-beta 1). Preparation of PCKSs induced an inflammatory tissue response, whereas long-term incubation (96 hours) induced fibrogenesis as shown by an increased expression of collagen type 1A1 (COL1A1) and fibronectin 1 (FN1). Importantly, PCKSs remained functional for more than 48 hours as evidenced by active glucuronidation and phenolsulfonphthalein uptake. In addition, cellular diversity appeared to be maintained, yet we observed a clear loss of nephrin messenger RNA levels suggesting that our model might not be suitable to study the role of podocytes in renal pathology. Moreover, TGF-beta 1 exposure augmented fibrosis, as illustrated by an increased expression of multiple fibrosis markers including COL1A1, FN1, and alpha-smooth muscle actin. In conclusion, PCKSs maintain their renal phenotype during culture and appear to be a promising model to investigate renal diseases, for example, renal fibrosis. Moreover, the human origin of PCKSs makes this model very suitable for translational research.

Introduction: The complement system is essential for an adequate immune response. Much attention has been given to the role of complement in disease. However, to better understand complement in pathology, it is crucial to first analyze this system under different physiological conditions. The aim of the present study was therefore to investigate the inter-individual variation in complement activity and the influences of age and sex. Methods: Complement levels and functional activity were determined in 120 healthy volunteers, 60 women, 60 men, age range 20–69 year. Serum functional activity of the classical pathway (CP), lectin pathway activated by mannan (MBL-LP) and alternative pathway (AP) was measured in sera, using deposition of C5b-9 as readout. In addition, levels of C1q, MBL, MASP-1, MASP-2, ficolin-2, ficolin-3, C2, C4, C3, C5, C6, C7, C8, C9, factor B, factor D, properdin, C1-inhibitor and C4b-binding protein, were determined. Age- and sex-related differences were evaluated. Results: Significantly lower AP activity was found in females compared to males. Further analysis of the AP revealed lower C3 and properdin levels in females, while factor D concentrations were higher. MBL-LP activity was not influenced by sex, but MBL and ficolin-3 levels were significantly lower in females compared to males. There were no significant differences in CP activity or CP components between females and males, nevertheless females had significantly lower levels of the terminal components. The CP and AP activity was significantly higher in the elderly, in contrast to MBL-LP activity. Moreover, C1-inhibitor, C5, C8, and C9 increased with age in contrast to a decrease of factor D and C3 levels. In-depth analysis of the functional activity assays revealed that MBL-LP activity was predominantly dependent on MBL and MASP-2 concentration, whereas CP activity relied on C2, C1-inhibitor and C5 levels. AP activity was strongly and directly associated with levels of C3, factor B and C5. Conclusion: This study demonstrated significant sex and age-related differences in complement levels and functionality in the healthy population. Therefore, age and sex analysis should be taken into consideration when discussing complement-related pathologies and subsequent complement-targeted therapies. Copyright © 2018 Gaya da Costa, Poppelaars, van Kooten, Mollnes, Tedesco, Würzner, Trouw, Truedsson, Daha, Roos and Seelen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Background: Hemodialysis (HD) is a life-saving treatment for patients with end stage renal disease. However, HD patients have markedly increased rates of cardiovascular morbidity and mortality. Previously, a link between the complement system and cardiovascular events (CV-events) has been reported. In HD, systemic complement activation occurs due to blood-to-membrane interaction. We hypothesize that HD-induced complement activation together with inflammation and thrombosis are involved in the development of CV-events in these patients. Methods: HD patients were followed for the occurrence of CV-events during a maximum follow-up of 45 months. Plasma samples were collected from 55 patients at different time points during one HD session prior to follow-up. Plasma levels of mannose-binding lectin, properdin and C3d/C3 ratios were assessed by ELISA. In addition, levels of von Willebrand factor, TNF-α and IL-6/IL-10 ratios were determined. An ex-vivo model of HD was used to assess the effect of complement inhibition. Results: During median follow-up of 32 months, 17 participants developed CV-events. In the CV-event group, the C3d/C3-ratio sharply increased 30 min after the start of the HD session, while in the event-free group the ratio did not increase. In accordance, HD patients that developed a CV-event also had a sustained higher IL-6/IL-10-ratio during the first 60 min of the HD session, followed by a greater rise in TNF-α levels and von Willebrand factor at the end of the session. In the ex-vivo HD model, we found that complement activation contributed to the induction of TNF-α levels, IL-6/IL-10-ratio and levels of von Willebrand factor. Conclusions: In conclusion, these findings suggest that early intradialytic complement activation predominantly occurred in HD patients who develop a CV-event during follow-up. In addition, in these patients complement activation was accompanied by a pro-inflammatory and pro-thrombotic response. Experimental complement inhibition revealed that this reaction is secondary to complement activation. Therefore, our data suggests that HD-induced complement, inflammation and coagulation are involved in the increased CV risk of HD patients. info:eu-repo/semantics/publishedVersion

Significant advances have lead to a greater understanding of the role of the complement system within nephrology. The success of the first clinically approved complement inhibitor has created renewed appreciation of complement-targeting therapeutics. Several clinical trials are currently underway to evaluate the therapeutic potential of complement inhibition in renal diseases and kidney transplantation. Although, complement has been known to be activated during dialysis for over four decades, this area of research has been neglected in recent years. Despite significant progress in biocompatibility of hemodialysis (HD) membranes and peritoneal dialysis (PD) fluids, complement activation remains an undesired effect and relevant issue. Short-term effects of complement activation include promoting inflammation and coagulation. In addition, long-term complications of dialysis, such as infection, fibrosis and cardiovascular events, are linked to the complement system. These results suggest that interventions targeting the complement system in dialysis could improve biocompatibility, dialysis efficacy, and long-term outcome. Combined with the clinical availability to safely target complement in patients, the question is not if we should inhibit complement in dialysis, but when and how. The purpose of this review is to summarize previous findings and provide a comprehensive overview of the role of the complement system in both HD and PD.

The presence of donor‐specific anti‐HLA antibodies (DSAs) is associated with increased risk of graft failure after kidney transplant. We hypothesized that DSAs against HLA class I, class II, or both classes indicate a different risk for graft loss between deceased and living donor transplant. In this study, we investigated the impact of pretransplant DSAs, by using single antigen bead assays, on long‐term graft survival in 3237 deceased and 1487 living donor kidney transplants with a negative complement‐dependent crossmatch. In living donor transplants, we found a limited effect on graft survival of DSAs against class I or II antigens after transplant. Class I and II DSAs combined resulted in decreased 10‐year graft survival (84% to 75%). In contrast, after deceased donor transplant, patients with class I or class II DSAs had a 10‐year graft survival of 59% and 60%, respectively, both significantly lower than the survival for patients without DSAs (76%). The combination of class I and II DSAs resulted in a 10‐year survival of 54% in deceased donor transplants. In conclusion, class I and II DSAs are a clear risk factor for graft loss in deceased donor transplants, while in living donor transplants, class I and II DSAs seem to be associated with an increased risk for graft failure, but this could not be assessed due to their low prevalence., Via a retrospective multicenter study, the authors investigate the impact of pretransplant donor‐specific HLA antibodies on long‐term graft survival in deceased and living donor kidney transplantations.

BACKGROUND: Smoking is a risk factor for poor late outcomes in renal transplant recipients (RTR). Smoking exposure can be assessed by self-report and cotinine measurements. We investigated whether use of cotinine as a biomarker for smoking exposure can serve as an alternative for self-report and to compare associations of smoking exposure by self-report and cotinine with outcomes in RTR and assess dose dependency.METHODS: Renal transplant recipients were classified as never, former, light (≤10 cigarettes/day), and heavy smokers (>10 cigarettes/day) according to self-report and analogous categories for urine and plasma cotinine. First, we assessed agreement of self-reported smoking exposure with smoking exposure according urine and plasma cotinine. Second, we compared the associations with graft failure and mortality.RESULTS: Of 603 RTR (age 51.5 ± 12.1 years, 55% men), 36.0% RTR were never, 42.3% former, 10.6% light, and 11.1% heavy smokers according to self-report. The majority (98.6%) of never smokers had nondetectable cotinine. However, 14 and 13 RTR reporting no active smoking had respective urine or plasma cotinine consistent with active smoking. Cotinine-based measurements were dose-dependently associated with mortality and graft failure.CONCLUSIONS: Plasma and urine cotinine can serve as an alternative to self-report and were dose-dependently associated with poor late outcomes in RTR.

Summary A number of molecules have been shown recently to be involved in the pathogenesis and progression of immunoglobulin (Ig)A nephropathy (IgAN). Among these, we have selected C4d (complement lectin pathway involvement), CD3 (T cell marker, traducing interstitial inflammation), transglutaminase 2 (TGase-2, involved in tissue fibrosis development) and p-extracelluar-regulated kinase (ERK)1/2 (protein kinase intracellular signaling molecule) to perform a panel of immunohistological biomarkers and assess its predictive value for disease progression. Immunohistochemical staining of these biomarkers was performed in paraffin sections from 74 renal biopsy cases with the clinical diagnosis of IgAN. Association between score analysis of these parameters and disease course was assessed through univariate and multivariate analysis, including baseline clinical and histological data. Univariate analysis showed that glomerular C4d, tubulointerstitial TGase2 and CD3 scores were associated with baseline proteinuria and disease progression. Multivariate analysis showed that only baseline estimated glomerular filtration rate (eGFR), C4d and CD3 were associated independently with progressive kidney disease (decline of at least 50% in the eGFR or progression to end-stage renal disease (ESRD) during the follow-up period). Establishing an accurate prediction model for IgAN progression is still a matter of research in clinical nephrology. The complement system, particularly lectin pathway activation, and T cell activation, have been shown previously to be potential modifiers of the disease course. Here we show that the combination of two histological biomarkers (C4d and CD3) can be a powerful predictor of IgAN progression and a potential useful tool for the clinical approach of this disease.

Kidney allograft failure due to chronic injury/rejection remains the main cause of graft loss in renal transplant recipients (RTR). Here, we investigated whether specific biomarkers of extracellular matrix (ECM) turnover are associated with allograft function and chronic kidney disease (CKD) stage in RTR. Seventy-eight patients who attended the University Medical Center Groningen for a routine check-up after kidney transplantation were enrolled in the study. Plasma and/or 24h-urine samples were collected and specific matrix-metalloproteinase- generated neo-epitope fragments of collagens were measured by enzyme-linked immunosorbent assay. Our results demonstrated that urinary levels of C3M, a marker for collagen type III degradation, correlated with estimated glomerular filtration rate (eGFR; r = 0.58, p

Aims: The complement system, and especially C5a, plays an important role in the pathophysiology of renal diseases and post-transplant renal injury. The two receptors for C5a are C5a receptor (C5aR) and C5a-like-receptor-2 (C5L2). Only renal C5aR expression has been reported, although exact localization and alterations in expression after transplantation are unknown.Materials and results: Renal C5aR and C5L2 expression and localization were analyzed immunohistochemically. C5aR and C5L2 expression was analyzed in human kidney biopsies obtained from living donors and patients suffering from acute tubular necrosis, acute cellular and vascular rejection or IF/TA.C5aR was expressed in the thick ascending limb of Henle's loop and first part of the distal convoluted tubule (DCT). Under inflammatory conditions. C5aR was de novo expressed in proximal tubuli. C5L2 was expressed in the kidney and localized to DCT1, DCT2 and connecting tubule. Persistent distal tubular expression of both receptors was demonstrated after renal transplantation.Conclusions: This study shows distinct renal expression patterns for C5aR and C5L2. Our findings suggest a functional role for renal C5L2 rather than being a C5a decoy receptor. Future studies focusing on renal C5a-C5aR interaction should take differential C5aR and C5L2 expression into account, alongside abundant C5aR expression on infiltrating cells. (c) 2012 Elsevier Ltd. All rights reserved.

The complement system, and specifically C5a, is involved in renal ischemia-reperfusion (IR) injury. The two receptors for C5a, C5aR and C5L2, are expressed on leukocytes as well as in the kidney. Extensive evidence shows that C5aR inhibition protects kidneys from IR injury, but the role of C5L2 in IR injury has not been studied so far. Therefore, WT, C5aR−/− and C5L2−/− mice were subjected to 40 min of bilateral renal ischemia, followed by reperfusion for 1, 3 or 7 days. It was found that C5L2−/− mice were protected against IR injury, resulting in significant lower plasma creatinine and BUN levels, and reduced acute tubular necrosis. Next, an in vivo migration study, where WT, C5aR−/− and C5L2−/− mice were injected intraperitoneally with complement ligands, revealed that C5L2 is not involved in leukocyte migration. To investigate the contribution of renal-expressed C5L2 versus leukocyte-expressed C5L2 to renal IR injury, bone marrow chimeras were created. Our data show that renal-expressed C5L2 and leukocyte-expressed C5L2 mediate IR injury-induced renal dysfunction. Therefore, C5L2 is a functional receptor in renal IR injury rather than a simple decoy receptor. For that reason, next to C5aR, C5L2 is a potential target for intervention during renal IR injury.

Hemodialysis patients have higher rates of cardiovascular morbidity and mortality compared to the general population. Mannose-binding lectin (MBL) plays an important role in the development of cardiovascular disease. In addition, hemodialysis alters MBL concentration and functional activity. The present study determines the predictive value of MBL levels for future cardiac events (C-event), cardiovascular events (CV-event) and all-cause mortality in HD patients. We conducted a prospective study of 107 patients on maintenance hemodialysis. Plasma MBL, properdin, C3d and sC5b-9 was measured before and after one dialysis session. The association with future C-events, CV-events, and all-cause mortality was evaluated using Cox regression models. During median follow-up of 27 months, 36 participants developed 21 C-events and 36 CV-events, whereas 37 patients died. The incidence of C-events and CV-events was significantly higher in patients with low MBL levels (

Background. Kidneys derived from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Strikingly, early and profound serum levels of IL-6 in brain-dead donors are observed. IL-6 is the main regulator of the acute phase response (APR). The aim of this translational study was to investigate the expression of renal acute phase proteins (APPs) following brain death (BD) and to assess the association with renal allograft outcome after transplantation.Methods. BD was induced in rats by inflating a subdurally placed balloon catheter. Kidney biopsies were obtained from human living and brain-dead donors at donation, after cold preservation and reperfusion. In vitro, renal proximal tubular epithelial cells (HK-2 cells) were stimulated with IL-6.Results. Both in human and rat brain-dead donors, C3 and FBG expression was enhanced at donation compared to living donors and sham-operated animals. In human donors, no additional expression was found after cold ischaemia or reperfusion. C3 expression after reperfusion was independently associated with decreased short-term function after transplantation in grafts from brain-dead donors. In cultured HK-2 cells, C3 production was induced in the presence of IL-6.Conclusions. In conclusion, BD induces renal C3 and FBG expression. Moreover, C3 expression is associated with a worse allograft function early after transplantation. Therefore, targeting renal APPs in brain-dead donors, especially complement C3, may improve transplant outcome.

A randomized controlled trial was designed to compare various outcome variables of the retroperitoneal mini-open muscle splitting incision (MSI) technique and the transperitoneal hand-assisted laparoscopic technique (HAL) in performing living donor nephrectomies. Fifty living kidney donors were randomized to MSI or HAL. Primary endpoint was pain experience scored on a visual analogue scale (VAS). After MSI living donors indicated lower median (range) VAS scores at rest than HAL living donors on postoperative day 2.5 [10 (0-44) vs. 15 (0-70), P = 0.043] and day 3 [7 (0-28) vs. 10 (0-91), P = 0.023] and lower VAS scores while coughing on postoperative day 3 [20 (0-73) vs. 42 (6-86), P = 0.001], day 7 [8 (0-66) vs. 33 (3-76), P < 0.001] and day 14 [2 (0-17) vs. 12 (0-51), P = 0.009]. The MSI technique also resulted in reduced morphine requirement, better scores on three domains of the RAND-36, reduced costs and reduced CRP and IL-6 levels. The HAL technique was superior in operating time and postoperative decrease of hemoglobin level. The MSI technique is superior to the HAL technique in performing living donor nephrectomies with regard to postoperative pain experience. This study reopens the discussion of the way to go in performing the living donor nephrectomy.

Background: Urinary uromodulin (UMOD) predicts renal prognosis in native kidneys, but data are conflicting. We investigated its prognostic impact for graft failure (GF) in renal transplant recipients (RTR; n = 600). Methods: UMOD concentration was measured cross-sectionally in RTR at 6.0 years [2.6–11.4] post-transplant, in matched patients with native chronic kidney disease (CKD) and healthy subjects. In 59 cases, RTR allograft biopsies were reviewed. Results: During a follow-up of 5.3 years [4.5–5.7], GF had occurred in 7% of RTR. Median UMOD excretion (mg/24 h) was 20.4 in RTR, 11.6 in CKD and 5.7 in controls (p < 0.001). There was a curvilinear association between UMOD excretion and baseline renal function (p < 0.003) and death-censored GF, with 5.5, 11.5 and 4.0% of the cases in subsequent UMOD excretion tertiles, respectively (p = 0.002). On multivariate Cox regression analysis, hazard ratios for GF for the 1st and 3rd tertiles were 0.37 (p = 0.01) and 0.21 (p = 0.001), respectively. Interstitial fibrosis and tubular atrophy were more severe in the middle tertile (p = 0.007). Conclusions: Urinary UMOD is elevated in RTR and associated with graft function, morphology and outcome in a bimodal fashion. Dissection of the disparate mechanisms of GF prediction by urinary UMOD might provide new clues for its alleged pathogenetic significance in progressive renal function loss.

The epidermal growth factor (EGF) receptor and its ligands are crucially involved in the renal response to ischaemia. We studied the heparin binding-epidermal growth factor (HB-EGF), a major ligand for the EGF receptor, in experimental and human ischaemia/reperfusion injury (IRI). HB-EGF mRNA and protein expression was studied in rat kidneys and cultured human tubular (HK-2) cells that were subjected to IRI and in human donor kidneys during transplantation. The effect of EGF receptor inhibition was investigated in vivo and in vitro. Furthermore, urinary HB-EGF protein excretion was studied after renal transplantation. Finally, HB-EGF KO and WT mice were subjected to IRI to study the role of HB-EGF in renal injury. HB-EGF mRNA was significantly up-regulated in the early phase of IRI in rats, cells, and human donor biopsies. Treatment with PKI-166 reduces macrophage accumulation and interstitial alpha-SMA in the early phase of IRI in rats. In vitro, PKI-166 causes a marked reduction in HB-EGF-induced cellular proliferation. Urinary HB-EGF is increased after transplantation compared with control urines from healthy subjects. HB-EGF KO mice subjected to IRI revealed significantly less morphological damage after IRI, compared with WT mice. We conclude that IRI results in early induction of HB-EGF mRNA and protein in vivo and in vitro. Absence of HB-EGF and inhibition of the EGF receptor in the early phase of IRI has protective effects, suggesting a modulating role for HB-EGF. Copyright (C) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Chronic low-grade inflammation is involved in chronic transplant dysfunction after renal transplantation. Procalcitonin (PCT), known to reflect microbial inflammation, may also reflect ongoing noninfectious chronic low-grade inflammation in organ parenchyma, including transplanted kidneys. We aimed to compare predictive performance of plasma PCT for development of graft failure in renal transplant recipients (RTR) with that of high-sensitivity C-reactive protein (hsCRP), an established marker of systemic chronic low-grade inflammation. We included 575 RTR with functioning grafts for more than or equal to 1 year at a median (interquartile range) time of 6.1 (2.9-11.7) years posttransplant. PCT was determined using an ultrasensitive immunoluminometric assay and hsCRP using high-sensitivity enzyme-linked immunosorbent assay. Median (interquartile range) plasma PCT and hsCRP concentrations were 0.023 (0.017-0.036) ng/mL and 2.1 (0.8-4.9) mg/L, respectively. After a median (interquartile range) of 5.2 (4.5-5.7) years of follow-up, incidence of graft failure was 0.5%, 2.6%, and 18.5% according to increasing PCT tertiles (P

Background. Brain death and cold preservation are major alloantigen-independent risk factors for transplantation Outcome. The present study was conducted to assess the influence of these factors on transplantation-associated injury independently or in combination.Methods. Brain death was induced in F344 rats. Renal grafts were harvested after 6 hr and either directly transplanted in unilateral nephrectomized Lewis recipient or Subjected to 24 hr of cold preservation in University of Wisconsin solution before implantation. Allografts obtained from living donor rats were also subjected to cold preservation or not. DNA damage was assessed before implantation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Ten days after transplantation, renal histology was performed according to Banff '97 classification. The expressions of cytokines and adhesion molecules were analyzed by quantitative polymerase chain reaction.Results. Cold preservation significantly increased the number of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling positive cells in renal allografts. Ten days after transplantation, histology revealed a higher degree of tubulitis and vasculitis scores when the grafts were Subjected to cold storage. Vasculitis was aggravated when the graft was obtained from brain death (BD) donors. BD, but not cold preservation alone, was associated with papillary necrosis. This was more frequently observed after cold preservation. Immunohistology showed an increase in MHC class II+ cells after cold preservation. The combination of BD and cold preservation revealed a higher degree of VEGF and IL-10 expression.Conclusions. Our Study emphasizes that cold ischemia time should be limited when renal allografts from brain-dead donors are transplanted.

Background. Chronic transplant dysfunction (CTD) is the leading cause of long-term loss of the renal allograft. So far, no single test is available to reliably predict the risk for CTD. Monitoring of tryptophan (trp) metabolism through indoleamine 2.3-dioxygenase (IDO) has been previously proposed to predict acute rejection of human kidney transplants. Here, we investigate the potential of IDO/trp degradation along the kynurenine (kyn) pathway to predict the long-term outcome of human kidney transplantation. Methods. During the 2-year follow-up blood, urine, and kidney biopsies were collected from 48 renal transplant patients. Concentrations of kyn and trp in serum and urine were measured at 2 weeks, 6 months, and 2 years after transplantation. Kynurenine to tryptophan ratio was calculated as an estimate of trp degradation. To evaluate the histological changes and IDO expression, respectively, periodic acid schiff staining and immunohistochemistry for IDO were performed on biopsies taken at 6 months and 2 years. Results. Two years after transplantation, kyn/trp was increased in urine and decreased in serum as compared to 2-week values. In 2-year biopsies, IDO expression was mainly found in infiltrating inflammatory cells and in the glomeruli. The urine level of trp 2 weeks after transplantation predicted the serum creatinine 6 months and the estimated creatinine clearance 2 years after transplantation. Additionally, serum level of kyn 6 months after transplantation predicted the serum creatinine 2 years after transplantation. Conclusions. Early serum and urine levels of trp and kyn may offer a novel route for early detection of patients at risk for developing CTD.

Background. In the last few decades, strategies to improve allograft survival after kidney transplantation have been directed to recipient-dependent mechanisms of renal injury. In contrast, no such efforts have been made to optimize organ quality in the donor. Optimizing deceased donor kidney quality opens new possibilities to improve renal allograft outcome. Methods. A total of 554 kidney biopsies were taken from donation after brain death (DBD) and donation after cardiac death (DCD) kidneys before donation, after cold ischemia and after reperfusion. Healthy living donor kidney biopsies served as controls. Transcriptomics was performed by whole genome microarray analyses followed by functional pathway analyses. Results. Before organ retrieval and before cessation of blood circulation, metabolic pathways related to hypoxia and complement-and-coagulation cascades were the major pathways enhanced in DBD donors. Similar pathways were also enriched in DCD donors after the first warm ischemia time. Shortly after reperfusion of DCD grafts, pathways related to prolonged and worsening deprivation of oxygen were associated with delayed graft function in the recipient. Conclusion. In conclusion, this large deceased donor study shows enrichment of hypoxia and complement-and-coagulation pathways already in DBD donors before cessation of blood flow, before organ retrieval. Therefore, future intervention therapies should target hypoxia and complement-and-coagulation cascades in the donor to improve renal allograft outcome in the recipient.

Activation of the innate immunity through Toll-like receptors (TLRs) has been postulated to play an important role in the pathophysiology of renal allograft dysfunction. TLR2 and TLR4 dynamics in different human post-transplant pathological entities has never been studied. Therefore, we evaluated pre- and post-transplantation protein expression of TLR2 and TLR4 in human kidney biopsies. Human kidney biopsies obtained from living kidney donors and patients with acute tubular necrosis, acute cellular and vascular rejection and interstitial fibrosis/tubular atrophy (IF/TA) were used. Translating results from animal studies to the clinical situation is highly important considering the upcoming clinical studies with TLR inhibitors in human renal transplantation. Hence, the TLR2 and TLR4 expression in healthy mouse and rat kidneys was analyzed and compared with human kidneys. In healthy human kidneys, TLR2 is expressed on the endothelium and Bowman's capsule, while TLR4 is expressed on the endothelium only. No tubular staining was found for both receptors in human kidneys. In contrast to human biopsies, TLR2 and TLR4 expression in rodents was observed on tubular epithelial cells. In all acute rejection human biopsies, increased infiltration of TLR4(+) leukocytes was observed. In conclusion, a discrepancy exists between human and rodent renal TLR expression, which suggests careful attention when translating results from rodent studies to the human situation. Additionally, this study revealed human TLR2 and TLR4 expression dynamics in human biopsies pre- and post-transplantation.

Objective. To investigate the possible association of the mannose binding lectin (MBL) pathway of complement activation with different disease parameters and disease activity in patients with systemic lupus erythematosus (SLE). Methods. MBL genotype, MBL serum concentration, MBL complex activity and MBL pathway activity were assessed in 53 patients. The activity of the MBL–MASP complex was assessed on the basis of its ability to activate exogenous C4. For MBL pathway activity the formation of the terminal complex of complement activation (C5b-9) was measured. Results were analysed in relation to clinical variables and autoantibody profiles in these patients. Results. MBL complex activity and MBL pathway activity were both reduced in patients carrying MBL variant alleles. Anticardiolipin and anti-C1q autoantibodies were observed significantly more frequently in patients with MBL variant alleles. Furthermore, the presence of these autoantibodies was associated with a decreased MBL concentration and function. In contrast, anti-MBL autoantibodies were not found in patients with MBL variant alleles, possibly related to impaired binding of variant MBL to apoptotic material. Conclusion. In patients with SLE, a reduced functional activity of the MBL pathway of complement, in relation to expression of MBL variant alleles, is associated with increased levels of autoantibodies against cardiolipin and C1q, but not against MBL. We hypothesize that an enhanced production of autoantibodies may be related to disturbed clearance of apoptotic material due to impaired MBL function.

SUMMARY In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL–complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab′)2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.

SUMMARY Anti-C1q autoantibodies are present in the serum of patients with different autoimmune diseases such as systemic lupus erythematosus (SLE). The occurrence of these autoantibodies correlates with renal involvement. In the present study we examined whether injection of rabbit antimouse C1q antibodies in mice leads to deposition in kidneys. Injection of healthy mice with a single dose of rabbit IgG antimouse C1q antibodies resulted in deposition of both C1q and IgG anti-C1q in glomeruli. The pattern of deposition observed in the glomeruli of mice injected with antimouse C1q antibodies both at 24 h and 2 weeks was both glomerular basement membrane (GBM)-associated and mesangial. Injection of control IgG did not have a detectable effect on circulating C1q levels, and no deposition of either C1q or rabbit IgG was seen at 24 h. The deposition of rabbit antimouse C1q and C1q in glomeruli resulted in complement activation, as assessed by C3 deposition, and influx of leucocytes associated with albuminuria in some, but not all mice. In none of the control mice was albuminuria observed. This report is the first to show that anti-C1q antibodies deposit in the healthy glomerulus together with autologous C1q. This deposition is stable for at least 2 weeks, causes complement activation, leucocyte influx and can lead to mild albuminuria.

BACKGROUND: Little is known about optimal protein intake after transplantation. The aim of this study was to prospectively investigate associations of urinary urea excretion, a marker for protein intake, with graft failure and mortality in renal transplant recipients (RTR) and potential effect modification by body mass index (BMI) and estimated glomerular filtration rate (eGFR).METHODS: Urinary urea excretion was measured in repeated 24-hr urine collections between 6 and 18 months after transplantation.RESULTS: In total, 940 RTR were included. During 4.4 (2.3-7.8) years of follow-up for graft failure and 4.8 (2.5-8.3) years for all-cause mortality, 78 RTR developed graft failure and 158 RTR died. Urinary urea excretion was not associated with graft failure in the overall population, but was inversely associated with graft failure in RTR with BMI less than 25 kg/m (hazard ratio [HR], 0.64 [0.28-1.50] and 0.27 [0.09-0.83] for the second and third tertiles, respectively, P < 0.001), and in RTR with eGFR of 45 mL per min per 1.73 m or higher (HR, 0.34 [0.15-0.79], P = 0.015 and HR, 0.31 [0.11-0.86], P = 0.025 for the second and third tertiles, respectively), both independent of potential confounders. Compared to the first tertile, RTR in the second and third tertiles of urinary urea excretion were at a lower risk of all-cause mortality (HR, 0.47 [0.32-0.69]; P < 0.001 and HR, 0.42 [0.26-0.68]; P < 0.001, respectively), independent of potential confounders. Body mass index and eGFR did not influence this association.CONCLUSION: Urinary urea excretion, a marker for protein intake, was inversely related to graft failure in RTR with BMI less than 25 kg/m and in RTR with an eGFR of 45 mL per min per 1.73 m or higher. In addition, urinary urea excretion was inversely related to mortality.

Reznichenko A, Sinkeler SJ, Snieder H, van den Born J, de Borst MH, Damman J, van Dijk MC, van Goor H, Hepkema BG, Hillebrands JL, Leuvenink HG, Niesing J, Bakker SJ, Seelen M, Navis G. SLC22A2 is associated with tubular creatinine secretion and bias of estimated GFR in renal transplantation. Physiol Genomics 45: 201-209, 2013. First published January 22, 2013; doi:10.1152/physiolgenomics.00087.2012.-Genomewide association studies reported SLC22A2 variants to be associated with serum creatinine. As SLC22A2 encodes the organic cation transporter 2 (OCT2), the association might be due to an effect on tubular creatinine handling. To test this hypothesis we studied the association of SLC22A2 polymorphisms with phenotypes of net tubular creatinine secretion: fractional creatinine excretion (FEcreat) and bias of estimated glomerular filtration rate (eGFR). We also studied the association with end-stage renal disease (ESRD) and graft failure (GF) in renal transplant recipients. SLC22A2 single nucleotide polymorphisms (SNPs), rs3127573 and rs316009, were genotyped in 1,142 ESRD patients receiving renal transplantation and 1,186 kidney donors as controls. GFR was measured with I-125-iothalamate clearance. Creatinine clearance was also assessed. FEcreat was calculated from the simultaneous clearances of creatinine and I-125-iothalamate. Donor rs316009 was associated with FEcreat (beta -0.053, P = 0.024) and with estimated [modification of diet in renal disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI)] but not measured GFR. In line with this, donor rs316009 was associated with bias of the MDRD and CKD-EPI but not the Cockroft-Gault equation. Both SNPs were associated with ESRD: odds ratios [95% CI] 1.39 [1.16-1.67], P = 0.00065, and 1.23 [1.02-1.48], P = 0.042, for rs3127573 and rs316009, respectively. Neither SNP was associated with GF. Thus, SLC22A2 is associated with phenotypes of net tubular creatinine secretion and ESRD.

International audience; During proteinuria, renal tubular epithelial cells become exposed to ultrafiltrate-derived serum proteins, including complement factors. Recently, we showed that properdin binds to tubular heparan sulfates (HS). We now document that factor H also binds to tubular HS, although to a different epitope than properdin. Factor H was present on the urinary side of renal tubular cells in proteinuric, but not in normal renal tissues and colocalized with properdin in proteinuric kidneys. Factor H dose-dependently bound to proximal tubular epithelial cells (PTEC) in vitro. Preincubation of factor H with exogenous heparin and pretreatment of PTECs with heparitinase abolished the binding to PTECs. Surface plasmon resonance experiments showed high affinity of factor H for heparin and HS (K(D) values of 32 and 93 nm, respectively). Using a library of HS-like polysaccharides, we showed that chain length and high sulfation density are the most important determinants for glycosaminoglycan-factor H interaction and clearly differ from properdin-heparinoid interaction. Coincubation of properdin and factor H did not hamper HS/heparin binding of one another, indicating recognition of different nonoverlapping epitopes on HS/heparin by factor H and properdin. Finally we showed that certain low anticoagulant heparinoids can inhibit properdin binding to tubular HS, with a minor effect on factor H binding to tubular HS. As a result, these heparinoids can control the alternative complement pathway. In conclusion, factor H and properdin interact with different HS epitopes of PTECs. These interactions can be manipulated with some low anticoagulant heparinoids, which can be important for preventing complement-derived tubular injury in proteinuric renal diseases.

Contains fulltext : 108225.pdf (Publisher’s version ) (Closed access) BACKGROUND: Interstitial fibrosis and tubular atrophy (IF/TA) is an important cause of renal function loss and ischaemia-reperfusion (I/R) injury is considered to play an important role in its pathophysiology. The aim of the present study was to investigate the role of a disintegrin and metalloproteinase 17 (ADAM17) in human renal allograft disease and in experimental I/R injury of the kidney. METHODS: We studied the expression of ADAM17 messenger RNA (mRNA) in IF/TA and control kidneys by reverse transcription-polymerase chain reaction and in situ hybridization. Moreover, we assessed ADAM17-mediated heparin-binding epidermal growth factor (HB-EGF) shedding in immortalized human cells. Finally, we studied the effect of pharmacological ADAM17 inhibition in a model of renal I/R injury in rats. RESULTS: ADAM17 mRNA was up-regulated in IF/TA when compared to control kidneys. In normal kidneys, ADAM17 mRNA was weakly expressed in proximal tubules, peritubular capillaries, glomerular endothelium and parietal epithelium. In IF/TA, tubular, capillary and glomerular ADAM17 expression was strongly enhanced with de novo expression in the mesangium. In interstitial fibrotic lesions, we observed co-localization of ADAM17 with HB-EGF protein. In vitro, inhibition of ADAM17 with TNF484 resulted in a dose-dependent reduction of HB-EGF shedding in phorbol 12-myrisate 13-acetate-stimulated cells and non-stimulated cells. In vivo, ADAM17 inhibition significantly reduced the number of glomerular and interstitial macrophages at Day 4 of reperfusion. CONCLUSIONS: In conclusion, HB-EGF co-expresses with ADAM17 in renal interstitial fibrosis, suggesting a potential interaction in IF/TA. Targeting ADAM17 to reduce epidermal growth factor receptor phosphorylation could be a promising way of intervention in human renal disease. 01 mei 2012

Kidneys recovered from brain-dead donors have inferior outcomes after transplantation compared to kidneys from living donors. Since complement activation plays an important role in renal transplant related injury, targeting complement activation in brain-dead donors might improve renal function after transplantation.Brain death (BD) was induced in Fisher rats by inflation of an epidurally placed balloon catheter and ventilated for 6 h. BD animals were treated with soluble complement receptor 1 (sCR1) 1 h before or 1 h after BD. Kidney transplantation was performed and 7 days after transplantation animals were sacrificed. Plasma creatinine and urea were measured at days 0, 1, 3, 5 and 7 after transplantation.Renal function was significantly better at day 1 after transplantation in recipients receiving a sCR1 pre-treated donor kidney compared to recipients of a non-treated donor graft Also treatment with sCR1, 1 h after the diagnosis of BD, resulted in a better renal function after transplantation. Gene expression of IL-6, IL-1 beta and TGF-beta were significantly lower in renal allografts recovered from treated donors.This study shows that targeting complement activation, during BD in the donor, leads to an improved renal function after transplantation in the recipient. (C) 2011 Elsevier B.V. All rights reserved.

Context Transplantation improves health-related quality of life in patients with end-stage renal disease. However, primarily because of adverse effects of medication, among other gastrointestinal symptoms, health-related quality of life is not completely restored to normal. Although many patients have various gastrointestinal symptoms only a small proportion may be reported spontaneously. Objective To evaluate the prevalence of gastrointestinal symptoms in kidney transplant recipients, also the difference between spontaneously reported symptoms and symptoms elicited by specific questioning was assessed. The burden of these symptoms in daily life also was analyzed. Design A single-center, sequential, mixed method study to assess the difference between spontaneous patient reports of gastrointestinal symptoms and active screening by a questionnaire in kidney transplant patients. Patients In February 2008, patients received a questionnaire on gastrointestinal symptoms; notes in medical records were consulted for patients scoring less than 100. In June 2008, those patients received a second, extended questionnaire aimed to assess the burden of gastrointestinal symptoms in daily life. Results Ninety-two of 513 patients eventually proved to have gastrointestinal symptoms. Completed questionnaires were compared with notes in the patients' files of the past year. A total of 51 of these 92 patients appeared to have not mentioned their gastrointestinal symptoms during the outpatient clinic visits. Of these 51 patients, 37 reported a significant impact of gastrointestinal symptoms on daily life. Conclusions The silent sufferer exists. Specific questioning helps to improve communication concerning bothersome gastrointestinal symptoms. To assess the burden of these symptoms, a validated questionnaire should be developed.