Proteinuria is accompanied by intratubular complement activation and apical membrane deposition of C3dg and C5b-9 in kidney transplant recipients.

Proteinuria predicts accelerated decline in kidney function in kidney transplant recipients (KTRs). We hypothesized that aberrant filtration of complement factors causes intraluminal activation, apical membrane attack on tubular cells, and progressive injury. Biobanked samples from two previous studies in albuminuric KTRs were used. The complement-activation split products C3c, C3dg, and soluble C5b-9-associated C9 neoantigen were analyzed by ELISA in urine and plasma using neoepitope-specific antibodies. Urinary extracellular vesicles (uEVs) were enriched by lectin and immunoaffinity isolation and analyzed by immunoblot analysis. Urine complement excretion increased significantly in KTRs with an albumin-to-creatinine ratio of ≥300 mg/g compared with <30 mg/g. Urine C3dg and C9 neoantigen excretion correlated significantly to changes in albumin excretion from 3 to 12 mo after transplantation. Fractional excretion of C9 neoantigen was significantly higher than for albumin, indicating postfiltration generation. C9 neoantigen was detected in uEVs in six of the nine albuminuric KTRs but was absent in non-albuminuric controls (n = 8). In C9 neoantigen-positive KTRs, lectin affinity enrichment of uEVs from the proximal tubules yielded signal for iC3b, C3dg, C9 neoantigen, and Na+-glucose transporter 2 but only weakly for aquaporin 2. Coisolation of podocyte markers and Tamm-Horsfall protein was minimal. Our findings show that albuminuria is associated with aberrant filtration and intratubular activation of complement with deposition of C3 activation split products and C5b-9-associated C9 neoantigen on uEVs from the proximal tubular apical membrane. Intratubular complement activation may contribute to progressive kidney injury in proteinuric kidney grafts.NEW & NOTEWORTHY The present study proposes a mechanistic coupling between proteinuria and aberrant filtration of complement precursors, intratubular complement activation, and apical membrane attack in kidney transplant recipients. C3dg and C5b-9-associated C9 neoantigen associate with proximal tubular apical membranes as demonstrated in urine extracellular vesicles. The discovery suggests intratubular complement as a mediator between proteinuria and progressive kidney damage. Inhibitors of soluble and/or luminal complement activation with access to the tubular lumen may be beneficial.

A comparison of four established GFR formulas to estimate measured GFR and changes in GFR in adult kidney transplant recipients.

The accurate assessment of glomerular filtration rate (GFR) is important in the follow-up of kidney transplant recipients in order to identify graft dysfunction. A number of formulas have been proposed to calculate GFR from endogenous plasma markers such as creatinine or cystatin C since measuring GFR using exogenous markers is troublesome. This study compares and evaluates the ability of four different GFR formulas to estimate kidney graft function and to detect changes in GFR in kidney transplant recipients. The study included patients from the prospective, multicenter CONTEXT trial in kidney transplant recipients. GFR was measured using plasma clearance of 51Cr-EDTA and estimated using the MDRD, CKD-EPI Creatinine, CKD-EPI Cystatin C and CKD-EPI Cystatin C + Creatinine equations at three (n = 83) and twelve (n = 65) months post-transplantation. For each formula mean bias, precision, and accuracy were evaluated. The MDRD equation had the lowest mean bias (0.2 ml/min/1.73 m2), whereas the CKD-EPI Cystatin C + Creatinine equation had the highest precision (8 ml/min/1.73 m2). Accuracy at three months were similar for all equations (P30 > 80%) except for the CKD-EPI Cystatin C equation, which performed poorer (P30 = 55%). None of the formulas evaluated avoided misclassification of changes in GFR. The most optimal combination of precision and accuracy suggests the use of CKD-EPI Creatinine + Cystatin C equation in kidney transplant recipients.

Dynamics of circulating dendritic cells and cytokines after kidney transplantation-No effect of remote ischaemic conditioning.

Inflammation resulting from ischaemia/reperfusion injury can cause kidney graft dysfunction, increase the risk of delayed graft function and possibly reduce long-term graft survival. Remote ischaemic conditioning may protect against ischaemia/reperfusion injury and mitigate the immunological response to the graft. We investigated the immunological effects of remote ischaemic conditioning on kidney transplantation from deceased donors in the randomized CONTEXT study. Three circulating dendritic cell (DC) subtypes identified in peripheral blood from kidney transplant recipients [myeloid DCs, plasmacytoid DCs and immunoglobulin-like transcript (ILT)3+ DCs] were measured at baseline, days 1, 3 and 5 and 1 and 3 months after transplantation. We also quantified 21 cytokines at baseline, days 1 and 5 and 3 months after transplantation. Neither DC counts nor cytokine levels differed between patients receiving remote ischaemic conditioning and controls; however, several parameters exhibited dynamic and parallel alterations in the two groups over time, reflecting the immunological response to the kidney transplantation and immunosuppression.

A new method of modelling early plasma creatinine changes predicts 1-year graft function after kidney transplantation.

BACKGROUND: Delayed graft function after renal transplantation is associated with inferior long-term outcome. To evaluate the impact of slow onset graft function, we aimed to model and correlate early changes in plasma creatinine (p-cr) with long-term graft function. MATERIALS: In a single centre observational study of 100 kidney transplants we identified all p-cr measurements from the time of transplantation until 30 days post-transplant or last post-transplant dialysis, and correlated this with estimated glomerular filtration rate (eGFR) 1 year after transplantation. The initial changes in p-cr were modelled for each patient using an exponential, logistic, or linear model, and the time to a 50% decrease in p-cr (tCr50) was estimated. RESULTS: Linear regression analysis showed a negative correlation between tCr50 and eGFR 1 year post-transplant (n = 96, r = -0.369, ß = -0.112, p = 0.0002). The correlation was maintained when corrected for the relevant recipient and donor characteristics. tCr50 correlated positively with the number of hospitalisation days, the number of graft ultrasound examinations, and the number of biopsies. CONCLUSIONS: A modelled time to a 50% decrease in p-cr predicts 1-year graft function. tCr50 may be a relevant surrogate endpoint in renal transplant studies aimed at improving long-term function by reducing the incidence of slow onset graft function.

Molecular Mechanisms of Renal Ischemic Conditioning Strategies.

Ischemia-reperfusion injury is the leading cause of acute kidney injury in a variety of clinical settings such as renal transplantation and hypovolemic and/or septic shock. Strategies to reduce ischemia-reperfusion injury are obviously clinically relevant. Ischemic conditioning is an inherent part of the renal defense mechanism against ischemia and can be triggered by short periods of intermittent ischemia and reperfusion. Understanding the signaling transduction pathways of renal ischemic conditioning can promote further clinical translation and pharmacological advancements in this era. This review summarizes research on the molecular mechanisms underlying both local and remote ischemic pre-, per- and postconditioning of the kidney. The different types of conditioning strategies in the kidney recruit similar powerful pro-survival mechanisms. Likewise, renal ischemic conditioning mobilizes many of the same protective signaling pathways as in other organs, but differences are recognized.

Correction: P-NGAL Day 1 predicts early but not one year graft function following deceased donor kidney transplantation-The CONTEXT study.

[This corrects the article DOI: 10.1371/journal.pone.0212676.].

ß-Trace Protein and ß2-Microglobulin do not Improve Estimation of Glomerular Filtration Rate in Kidney Transplant Recipients Compared With Creatinine and Cystatin C.

BACKGROUND: Reliable estimates of glomerular filtration rate (eGFR) are important for detecting changes in graft function in kidney transplant recipients. Current eGFR equations are based on plasma creatinine and/or cystatin C; however, these are associated with significant bias. This study investigated if equations based on ß-trace protein (BTP) and ß2-microglobulin (B2M) performed better than the 2021 Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations based on creatinine and cystatin C among kidney transplant recipients. METHODS: We included samples and data from the clinical trial CONTEXT. Glomerular filtration rate (GFR) was measured by plasma clearance of an exogenous marker. The eGFR was calculated using the CKD-EPI equations for estimating GFR from BTP and/or B2M and the 2021 CKD-EPI creatinine and creatinine-cystatin C equations. The GFR estimates were evaluated 3 (n = 82) and 12 (n = 64) months after transplant using mean bias, precision, and accuracy. Furthermore, we analyzed the ability of the equations to correctly classify the direction of changes in measured GFR from 3 to 12 months. RESULTS: Among the BTP- and B2M-based equations, the combined eGFR-BTP-B2M performed best with respect to precision (SD = 7.64 mL/min/1.73 m2) and accuracy (±10% from measured GFR = 36%). The eGFR-BTP-B2M and the eGFR-creatinine-cystatin C (2021) performed similarly when comparing precision, accuracy, and residuals (P = .481). The BTP- and/or B2M-based equations did not perform better than the eGFR-creatinine-cystatin C (2021) in correctly classifying the direction of changes in measured GFR from 3 to 12 months. CONCLUSIONS: ß-trace protein and/or B2M do not improve the estimation of GFR when compared with creatinine- and cystatin C-based 2021 CKD-EPI equations.

Endotrophin Levels Are Associated with Allograft Outcomes in Kidney Transplant Recipients.

Early prediction of kidney graft function may assist clinical management, and for this, reliable non-invasive biomarkers are needed. We evaluated endotrophin (ETP), a novel non-invasive biomarker of collagen type VI formation, as a prognostic marker in kidney transplant recipients. ETP levels were measured with the PRO-C6 ELISA in the plasma (P-ETP) of 218 and urine (U-ETP/Cr) of 172 kidney transplant recipients, one (D1) and five (D5) days, as well as three (M3) and twelve (M12) months, after transplantation. P-ETP and U-ETP/Cr at D1 (P-ETP AUC = 0.86, p < 0.0001; U-ETP/Cr AUC = 0.70, p = 0.0002) were independent markers of delayed graft function (DGF) and P-ETP at D1 had an odds ratio of 6.3 (p < 0.0001) for DGF when adjusted for plasma creatinine. The results for P-ETP at D1 were confirmed in a validation cohort of 146 transplant recipients (AUC = 0.92, p < 0.0001). U-ETP/Cr at M3 was negatively associated with kidney graft function at M12 (p = 0.007). This study suggests that ETP at D1 can identify patients at risk of delayed graft function and that U-ETP/Cr at M3 can predict the future status of the allograft. Thus, measuring collagen type VI formation could aid in predicting graft function in kidney transplant recipients.

Improved GFR and renal plasma perfusion following remote ischaemic conditioning in a porcine kidney transplantation model.

Delayed graft function (DGF) complicates approximately 25% of kidney allografts donated after brain death (DBD). Remote ischaemic conditioning (rIC) involves brief, repetitive, ischaemia in a distant tissue in connection with ischaemia/reperfusion in the target organ. rIC has been shown to induce systemic protection against ischaemic injuries. Using a porcine kidney transplantation model with donor (63 kg) recipient (15 kg) size mismatch, we investigated the effects of recipient rIC on early renal plasma perfusion and GFR. Brain death was induced in donor pigs (n = 8) and kidneys were removed and kept in cold storage until transplantation. Nephrectomized recipient pigs were randomized to rIC (n = 8) or non-rIC (n = 8) with one kidney from the same donor in each group. rIC consisted of 4 × 5 min clamping of the abdominal aorta. GFR was significantly higher in the rIC group compared with non-rIC (7.2 ml/min vs. 3.4 ml/min; ΔGFR = 3.7 ml/min, 95%-CI: 0.3-7.2 ml/min, P = 0.038). Renal plasma perfusion in both cortex and medulla measured by dynamic contrast-enhanced magnetic resonance imaging (MRI) was significantly higher over time in the rIC group compared with non-rIC. This experimental study demonstrated a positive effect of rIC on early graft perfusion and function in a large animal transplantation model.

Pretransplant endotrophin predicts delayed graft function after kidney transplantation.

Delayed graft function after kidney transplantation is common and increases morbidity and health care costs. There is evidence that endotrophin, a specific fragment of pro-collagen type VI, promotes the inflammatory response in kidney diseases. We tested the hypothesis that pretransplant endotrophin in kidney transplant recipients may be associated with the risk of delayed graft function. Pretransplant plasma endotrophin was assessed using an enzyme-linked immunosorbent assay in three independent cohorts with 806 kidney transplant recipients. The primary outcome was delayed graft function, i.e., the necessity of at least one dialysis session within one-week posttransplant. In the discovery cohort median pretransplant plasma endotrophin was higher in 32 recipients (12%) who showed delayed graft function when compared to 225 recipients without delayed graft function (58.4 ng/mL [IQR 33.4-69.0]; N = 32; vs. 39.5 ng/mL [IQR 30.6-54.5]; N = 225; P = 0.009). Multivariable logistic regression, fully adjusted for confounders showed, that pretransplant plasma endotrophin as a continuous variable was independently associated with delayed graft function in both validation cohorts, odds ratio 2.09 [95% CI 1.30-3.36] and 2.06 [95% CI 1.43-2.97]. Pretransplant plasma endotrophin, a potentially modifiable factor, was independently associated with increased risk of delayed graft function and may be a new avenue for therapeutic interventions.

Elevated plasma free thiols are associated with early and one-year graft function in renal transplant recipients.

BACKGROUND: Reduced free thiols in plasma are indicative of oxidative stress, which is an important contributor to ischaemia-reperfusion injury (IRI) in kidney transplantation leading to kidney damage and possibly delayed graft function (DGF). In a post-hoc, exploratory analysis of the randomised controlled CONTEXT trial, we investigated whether higher (i.e. less oxidised) plasma levels of free thiols as a biomarker of reduced oxidative stress are associated with a better initial graft function or a higher GFR. METHODS: Free thiol levels were measured in plasma at baseline, 30 and 90 minutes after reperfusion of the kidney as well as at Day 1, Day 5 and twelve months after kidney transplantation in 217 patients from the CONTEXT study. Free thiol levels were compared to the kidney graft function measured as the estimated time to a 50% reduction in plasma creatinine (tCr50), the risk of DGF and measured GFR (mGFR) at Day 5 and twelve months after transplantation. RESULTS: Higher levels of free thiols at Day 1 and Day 5 are associated with higher mGFR at Day 5 (p<0.001, r2adj. = 0.16; p<0.001, r2adj. = 0.25), as well as with mGFR at twelve months (p<0.001, r2adj. = 0.20; p<0.001, r2adj. = 0.16). However, plasma levels of free thiols at 30 minutes and 90 minutes, but not Day 1, were significantly higher among patients experiencing DGF. CONCLUSION: Higher levels of plasma free thiols at Day 1 and Day 5, which are reflective of lower levels of oxidative stress, are associated with better early and late graft function in recipients of a kidney graft from deceased donors. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01395719.

Remote ischaemic conditioning and early changes in plasma creatinine as markers of one year kidney graft function-A follow-up of the CONTEXT study.

BACKGROUND: Ischaemia-reperfusion injury in kidney transplantation leads to delayed graft function (DGF), which is associated with reduced long term graft function. Remote ischaemic conditioning (RIC) improved early kidney graft function in a porcine model of donation after brain death and was associated with improved long-term cardiac outcome after myocardial ischaemia. This randomised, double-blinded trial evaluated the effect of RIC on kidney graft outcome in the first year, and examined the predictive value of a new measure of initial kidney graft function, i.e. the estimated time to a 50% reduction in plasma creatinine post-transplantation (tCr50). METHODS: A total of 225 patients undergoing deceased donor kidney transplantation were randomised to RIC or a sham procedure performed prior to kidney reperfusion. Up to four repetitive cycles of five minutes of leg ischaemia and five minutes of reperfusion were given. GFR, plasma creatinine, cystatin C and neutrophil gelatinase associated lipocalin (NGAL) were measured at three and twelve months and estimated GFR was calculated using four different equations. Other secondary outcomes were identified from patient files. RESULTS: RIC did not affect GFR or other outcomes when compared to the sham procedure at three or twelve months. tCr50 correlated with one year graft function (p<0.0001 for both mGFR and eGFR estimates). In contrast, DGF i.e. "need of dialysis the first week" did not correlate significantly with one year GFR. CONCLUSION: RIC during deceased donor kidney transplantation did not improve one year outcome. However, tCr50 may be a relevant marker for studies aiming to improve graft onset. TRIAL REGISTRATION: www.ClinicalTrials.gov Identifier: NCT01395719.

P-NGAL Day 1 predicts early but not one year graft function following deceased donor kidney transplantation - The CONTEXT study.

BACKGROUND: Early markers to predict delayed kidney graft function (DGF) may support clinical management. We studied the ability of four biomarkers (neutrophil gelatinase associated lipocalin (NGAL), liver-type fatty acid-binding protein (L-FABP), cystatin C, and YKL-40) to predict DGF after deceased donor transplantation, and their association with early graft function and GFR at three and twelve months. METHODS: 225 deceased donor kidney transplant recipients were included. Biomarkers were measured using automated assays or ELISA. We calculated their ability to predict the need for dialysis post-transplant and correlated with the estimated time to a 50% reduction in plasma creatinine (tCr50), measured glomerular filtration rate (mGFR) and estimated GFR (eGFR). RESULTS: All biomarkers measured at Day 1, except urinary L-FABP, significantly correlated with tCr50 and mGFR at Day 5. Plasma NGAL at Day 1 and a timed urine output predicted DGF (AUC = 0.91 and AUC 0.98). Nil or only weak correlations were identified between early biomarker levels and mGFR or eGFR at three or twelve months. CONCLUSION: High plasma NGAL at Day 1 predicts DGF and is associated with initial graft function, but may not prove better than P-creatinine or a timed urine output. Early biomarker levels do not correlate with one-year graft function. TRIAL REGISTRATION: ClinicalTrials.gov NCT01395719.

Remote ischaemic conditioning on recipients of deceased renal transplants, effect on immediate and extended kidney graft function: a multicentre, randomised controlled trial protocol (CONTEXT).

INTRODUCTION: Delayed graft function due to ischaemia-reperfusion injury is a frequent complication in deceased donor renal transplantation. Experimental evidence indicates that remote ischaemic conditioning (RIC) provides systemic protection against ischaemia-reperfusion injury in various tissues. METHODS AND ANALYSIS: 'Remote ischaemic conditioning in renal transplantation--effect on immediate and extended kidney graft function' (the CONTEXT study) is an investigator initiated, multicentre, randomised controlled trial investigating whether RIC of the leg of the recipient improves short and long-term graft function following deceased donor kidney transplantation. The study will include 200 kidney transplant recipients of organ donation after brain death and 20 kidney transplant recipients of organ donation after circulatory death. Participants are randomised in a 1:1 design to RIC or sham-RIC (control). RIC consists of four cycles of 5 min occlusion of the thigh by a tourniquet inflated to 250 mm Hg, separated by 5 min of deflation. Primary end point is the time to a 50% reduction from the baseline plasma creatinine, estimated from the changes of plasma creatinine values 30 days post-transplant or 30 days after the last performed dialysis post-transplant. Secondary end points are: need of dialysis post-transplant, measured and estimated-glomerular filtration rate (GFR) at 3 and 12 months after transplantation, patient and renal graft survival, number of rejection episodes in the first year, and changes in biomarkers of acute kidney injury and inflammation in plasma, urine and graft tissue. ETHICS AND DISSEMINATION: The study is approved by the local ethical committees and national data security agencies. Results are expected to be published in 2016. TRIAL REGISTRATION NUMBER: NCT01395719.