Urine CXCL10 as a biomarker in kidney transplantation.

PURPOSE OF REVIEW: Urine CXCL10 is a promising biomarker for posttransplant renal allograft monitoring but is currently not widely used for clinical management. RECENT FINDINGS: Large retrospective studies and data from a prospective randomized trial as well as a prospective cohort study demonstrate that low urine CXCL10 levels are associated with a low risk of rejection and can exclude BK polyomavirus replication with high certainty. Urine CXCL10 can either be used as part of a multiparameter based risk assessment tool, or as an individual biomarker taking relevant confounders into account. A novel Luminex-based CXCL10 assay has been validated in a multicenter study, and proved to be robust, reproducible, and accurate. SUMMARY: Urine CXCL10 is a well characterized inflammation biomarker, which can be used to guide performance of surveillance biopsies. Wide implementation into clinical practice depends on the availability of inexpensive, thoroughly validated assays with approval from regulatory authorities.

Automated Urinary Chemokine Assays for Noninvasive Detection of Kidney Transplant Rejection: A Prospective Cohort Study.

RATIONALE & OBJECTIVE: Prior studies have demonstrated the diagnostic potential of urinary chemokines C-X-C motif ligand 9 (CXCL9) and CXCL10 for kidney transplant rejection. However, their benefit in addition to clinical information has not been demonstrated. We evaluated the diagnostic performance for detecting acute rejection of urinary CXCL9 and CXCL10 when integrated with clinical information. STUDY DESIGN: Single-center prospective cohort study. SETTING & PARTICIPANTS: We analyzed 1,559 biopsy-paired urinary samples from 622 kidney transplants performed between April 2013 and July 2019 at a single transplant center in Belgium. External validation was performed in 986 biopsy-paired urinary samples. TESTS COMPARED: We quantified urinary CXCL9 (uCXCL9) and CXCL10 (uCXCL10) using an automated immunoassay platform and normalized the values to urinary creatinine. Urinary chemokines were incorporated into a multivariable model with routine clinical markers (estimated glomerular filtration rate, donor-specific antibodies, and polyoma viremia) (integrated model). This model was then compared with the tissue diagnosis according to the Banff classification for acute rejection. OUTCOME: Acute rejection detected on kidney biopsy using the Banff classification. RESULTS: Chemokines integrated with routine clinical markers had high diagnostic value for detection of acute rejection (n=150) (receiver operating characteristic area under the curve 81.3% [95% CI, 77.6-85.0]). The integrated model would help avoid 59 protocol biopsies per 100 patients when the risk for rejection is predicted to be below 10%. The performance of the integrated model was similar in the external validation cohort. LIMITATIONS: The cross-sectional nature obviates investigating the evolution over time and prediction of future rejection. CONCLUSIONS: The use of an integrated model of urinary chemokines and clinical markers for noninvasive monitoring of rejection could enable a reduction in the number of biopsies. Urinary chemokines may be useful noninvasive biomarkers whose use should be further studied in prospective randomized trials to clarify their role in guiding clinical care and the use of biopsies to detect rejection after kidney transplantation. PLAIN-LANGUAGE SUMMARY: Urinary chemokines CXCL9 and CXCL10 have been suggested to be good noninvasive biomarkers of kidney transplant rejection. However, defining a context of use and integration with clinical information is necessary before clinical implementation can begin. In this study, we demonstrated that urinary chemokines CXCL9 and CXCL10, together with clinical information, have substantial diagnostic accuracy for the detection of acute kidney transplant rejection. Application of urinary chemokines together with clinical information may guide biopsy practices following kidney transplantation and potentially reduce the need for kidney transplant biopsies.

Urine CXCL10 to Assess BK Polyomavirus Replication After Kidney Transplantation.

BACKGROUND: Urine CXCL10 is a biomarker for renal allograft inflammation induced by rejection, urinary tract infection, or BK polyomavirus (BKPyV) replication. This study aimed to compare urine CXCL10 levels in different stages of BKPyV reactivation and to investigate urine CXCL10 as a biomarker for BKPyV replication. METHODS: We included 763 urine samples (235 patients) from an interventional, randomized trial obtained in the context of regular screening for urine CXCL10 levels. All urine samples had a complete urine sediment analysis, no rejection episode noted within 30 d before urine collection, and a urine decoy cell analysis was conducted within ±3 d. RESULTS: Urine CXCL10 levels were 2.31 ng/mmol in samples without BKPyV viruria, slightly rose to 4.35 ng/mmol with BKPyV viruria, and then markedly increased to 16.42 ng/mmol when decoy cells were detectable, but still in the absence of BKPyV DNAemia ( P < 0.001). The highest urine CXCL10 values were observed in samples with BKPyV DNAemia (median 42.59 ng/mmol). The area under the curve of urine CXCL10 levels to detect ≥3 decoy cells was 0.816. At a CXCL10 cutoff of 3 ng/mmol, the negative predictive value was 97%. The area under the curve of urine CXCL10 levels to detect BKPyV DNAemia was 0.882, with a negative predictive value of 99% at a CXCL10 cutoff of 3 ng/mmol. CONCLUSIONS: Urine CXCL10 levels are already significantly elevated in BKPyV viruria (especially with decoy cell shedding) and further increase with BKPyV DNAemia. Low urine CXCL10 values can rule out the presence of ≥3 decoy cells and BKPyV DNAemia with high certainty.

Serum and urinary galectin-9 and C-X-C motif chemokine ligand 10.

BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by a type I interferon (IFN) signature, and traditional methods for its measurement like gene expression analysis are cumbersome for routine use. Thus, we aimed to study galectin-9 as a biomarker and compared it with a validated marker, C-X-C motifchemokine ligand 10(CXCL-10). METHODS: Ninety-seven patients with SLE (26 years; 89 females) were included and stratified based on renal involvement and activity into - active (SLEDAI > 4) renal (35), active non-renal (32) and inactive renal subgroups (30) along with 20 healthy controls (HC, 25 years; 15 females). The median disease duration was 24 months (6-48), and SLEDAI 2K was 9 (2-15). Serum and urine galectin-9 and CXCL-10 levels were measured by ELISA. Urine levels were normalized with spot urine creatinine values. Follow-up serum and urine galectin-9 levels were measured for those in the active renal group at 6 months. RESULTS: Patients with SLE had higher serum galectin-9 (5.6 vs 1.7 µg/mL, p = .0001) but not urine galectin-9 (0.52 vs 0.32 µg, p = .7) levels as compared to HC. Serum galectin-9 but not urine galectin-9 was higher in patients with active as compared to inactive lupus (12.9 - active renal, 16.7 - active non-renal vs 3.57 µg/mL, p = .04 and .005). Serum CXCL-10 (0.16 vs 0.05, p = .01) and urine CXCL-10 (0 vs 0, p = .01) were both significantly higher in the SLE group as compared with HC. Serum but not urine CXCL-10 was higher in the active as compared to inactive lupus (0.2 - active renal, 0.3 - active non-renal vs 0.08 µg/mL, p = .9 and .02). Serum galectin-9 showed a modest correlation with CXCL-10 0.4 (0.2-0.6), whereas none was found between their urine levels.Serum galectin-9 and CXCL-10 showed a moderate positive correlation with SLEDAI 2K. Serum galectin-9 showed a greater AUC than CXCL-10 (0.77 vs 0.67) in differentiating active from inactive SLE, and both tested together had the best AUC of 0.82. However, urinary levels had no association with SLEDAI 2K or renal SLEDAI. In a subset of patients with active renal disease, serum galectin-9 but not urine levels declined significantly after 6 months. CONCLUSION: Serum galectin-9 is a good marker of lupus activity; however, it does not differentiate between active renal and active non-renal disease. It performs slightly better than CXCL-10. Urinary galectin-9 does not reflect renal activity.

Inflammatory markers in saliva and urine reflect disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Laboratory tests of blood and sometimes urine are used to diagnose and to monitor disease activity (DA) in SLE. Clinical practice would be simplified if non-invasive urine and salivary tests could be introduced as alternatives to blood samples. We therefore explored the levels of innate immunity-related biomarkers in matched serum, urine and saliva samples from patients with SLE. METHODS: A total of 84 patients with SLE selected to represent high and low general DA, and 21 controls were included. All participants underwent a thorough clinical examination. General DA and renal DA were measured. The levels of colony-stimulating factor (CSF)-1, interleukin (IL)-34, tumour necrosis factor (TNF)-α, interferon-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, calprotectin, macrophage inflammatory protein (MIP)-1α and MIP-1ß were analysed by immunoassays and related to DA. RESULTS: CSF-1, TNF-α, IP-10 and MCP-1 in saliva, serum and urine, as well as calprotectin in saliva and urine were increased in patients with SLE as compared with controls (p<0.05). TNF-α, IP-10 and MCP-1 in saliva, serum and urine, and CSF-1 in saliva and serum distinguished patients with SLE from controls (area under the curve >0.659; p<0.05 for all). CSF-1 in serum and urine, and calprotectin in saliva and urine, as well as TNF- α, IP-10 and MCP-1 in urine correlated positively with measures of general DA (p<0.05). Patients with SLE with active renal disease presented elevated levels of TNF-α, IP-10 and MCP-1 in urine and CSF-1 and IP-10 in serum as compared with patients with SLE with non-active renal disease. CONCLUSIONS: Our investigation demonstrates that saliva is a novel alternative body fluid, with potential for surveillance of general DA in patients with SLE, but urine is more informative in patients with SLE with predominantly renal DA.

Association Between Graft Function and Urine CXCL10 and Acylcarnitines Levels in Kidney Transplant Recipients.

OBJECTIVE: To evaluate post-transplantation graft functions noninvasively by using urine C-X-C motif chemokine 10 (CXCL10) and metabolome analysis. METHODS: The 65 living-donor kidney-transplant recipients in our cohort underwent renal biopsy to investigate possible graft dysfunction. The patients were divided into 2 groups, according to pathology reports: chronic allograft dysfunction (CAD; n = 18) and antibody-mediated/humoral allograft rejection (AMR; n = 16). The control group was composed of renal transplant recipients with stable health (n = 33). We performed serum creatinine, blood urea nitrogen (BUN), cystatin C, urine protein, CXCL10, and metabolome analyses on specimens from the patients. RESULTS: BUN, creatinine, cystatin C, urine protein, leucine + isoleucine, citrulline, and free/acetyl/propionyl carnitine levels were significantly higher in patients with CAD and AMR, compared with the control individuals. CXCL10 levels were significantly elevated in patients with AMR, compared with patients with CAD and controls. CXCL10 (AUC = 0.771) and cystatin C (AUC = 0.746) were significantly higher in the AMR group, compared with the CAD group (P<.02). CONCLUSIONS: CXCL10 and metabolome analyzes are useful for evaluation of graft functions. Also, CXCL10 might be useful as a supplementary noninvasive screening test for diagnosis of allograft rejection.

Advantages of plasmatic CXCL-10 as a prognostic and diagnostic biomarker for the risk of rejection and subclinical rejection in kidney transplantation.

This study evaluate the potential of plasmatic CXCL-10 (pCXCL-10) as a pre&post transplantation prognostic and diagnostic biomarker of T-cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR) and subclinical rejection (SCR) risk in adult kidney recipients considering BKV and CMV infections as possible clinical confounder factors. Twenty-eight of 100 patients included experienced rejection (TCMR:14; ABMR:14); 8 SCR; 13 and 16 were diagnosed with BKV and CMV infection, respectively. Pre-transplantation pCXCL-10 was significantly increased in TCMR and ABMR and post-transplantation in TCMR, ABMR and SCR compared with nonrejectors. All CMV+ patients showed pCXCL-10 levels above the cutoff values established for rejection whereas the 80% of BKV+ patients showed pCXCL-10 concentration < 100 pg/mL. pCXCL-10 could improve pre-transplantation patient stratification and immunosuppressive treatment selection according to rejection risk; and after kidney transplantation could be a potential early prognostic biomarker for rejection. Clinical confounding factor in BKV+ and particularly in CMV+ patients must be discarded.

Urinary Cell mRNA Profiles Predictive of Human Kidney Allograft Status.

Immune monitoring of kidney allograft recipients and personalized therapeutics may help reach the aspirational goal of "one transplant for life." The invasive kidney biopsy procedure, the diagnostic tool of choice, has become safer and the biopsy classification more refined. Nevertheless, biopsy-associated complications, interobserver variability in biopsy specimen scoring, and costs continue to be significant concerns. The dynamics of the immune repertoire make frequent assessments of allograft status necessary, but repeat biopsies of the kidney are neither practical nor safe. To address the existing challenges, we developed urinary cell mRNA profiling and investigated the diagnostic, prognostic, and predictive accuracy of absolute levels of a hypothesis-based panel of mRNAs encoding immunoregulatory proteins. Enabled by our refinements of the PCR assay and by investigating mechanistic hypotheses, our single-center studies identified urinary cell mRNAs associated with T cell-mediated rejection, antibody-mediated rejection, interstitial fibrosis and tubular atrophy, and BK virus nephropathy. In the multicenter National Institutes of Health Clinical Trials in Organ Transplantation-04, we discovered and validated a urinary cell three-gene signature of T-cell CD3 ε chain mRNA, interferon gamma inducible protein 10 (IP-10) mRNA, and 18s ribosomal RNA that is diagnostic of subclinical acute cellular rejection and acute cellular rejection and prognostic of acute cellular rejection and graft function. The trajectory of the signature score remained flat and below the diagnostic threshold for acute cellular rejection in the patients with no rejection biopsy specimens, whereas a sharp rise was observed during the weeks before the biopsy specimen that showed acute cellular rejection. Our RNA sequencing and bioinformatics identified kidney allograft biopsy specimen gene signatures of acute rejection to be enriched in urinary cells matched to acute rejection biopsy specimens. The urinary cellular landscape was more diverse and more enriched for immune cell types compared with kidney allograft biopsy specimens. Urinary cell mRNA profile-guided clinical trials are needed to evaluate their value compared with current standard of care.

Urinary C-X-C Motif Chemokine 10 Is Related to Acute Graft Lesions Secondary to T Cell- and Antibody-Mediated Damage.

BACKGROUND Non-invasive biomarkers of graft rejection are needed to optimize the management and outcomes of kidney transplant recipients. Urinary excretion of IFN-g-related chemokine CXCL10 is clearly associated with clinical and subclinical T cell-mediated graft inflammation, but its relationship with antibody-mediated damage has not been fully addressed. Further, the variables influencing levels of urinary CXCL10 excretion are unknown. MATERIAL AND METHODS A total of 151 kidney graft biopsies (92 surveillance and 59 indication biopsies) and 151 matched urine samples obtained before biopsy were prospectively analyzed. T cell-mediated rejection (TCMR) and antibody-mediated rejection (AbMR) were defined according to the 2017 Banff classification criteria. Urinary CXCL10 levels were measured by ELISA and corrected by urinary creatinine. RESULTS Banff scores 't', 'i', 'g', and 'ptc' were significantly related to urinary CXCL10 levels. Multivariate analysis showed that 't' (ß=0.107, P=0.001) and 'ptc' (ß=0.093, P=0.002) were significantly associated with urinary CXCL10. Donor-specific antibodies (DSAs) were related to the high excretion of urinary CXCL10 at 1 year after transplantation (odds ratio [OR] 17.817, P=0.003). Urinary CXCL10 showed good discrimination ability for AbMR (AUC-ROC 0.760, P=0.001). The third tertile of urinary CXCL10 remained significantly associated with AbMR (OR 4.577, 95% confidence interval 1.799-11.646, P=0.001) after multivariate regression analysis. CONCLUSIONS DSA was the only variable clearly related to high urinary CXCL10 levels. Urinary CXCL10 is a good non-invasive candidate biomarker of AbMR and TCMR, supplying information independent of renal function and other variables normally used to monitor kidney transplants.

Biomarker implementation: Evaluation of the decision-making impact of CXCL10 testing in a pediatric cohort.

BACKGROUND: Children are at high risk for subclinical rejection, and kidney biopsy is currently used for surveillance. Our objective was to test how novel rejection biomarkers such as urinary CXCL10 may influence clinical decision-making to indicate need for a biopsy. METHODS: A minimum dataset for standard decision-making to indicate a biopsy was established by an expert panel and used to design clinical vignettes for use in a survey. Pediatric nephrologists were recruited to review the vignettes and A) estimate rejection risk and B) decide whether to biopsy; first without and then with urinary CXCL10/Cr level. Accuracy of biopsy decisions was then tested against the biopsy results. IRA was assessed by Fleiss Kappa (κ) for binary choice and ICC for probabilities. RESULTS: Eleven pediatric nephrologists reviewed 15 vignettes each. ICC of probability assessment for rejection improved from poor (0.28, P < .01) to fair (0.48, P < .01) with addition of CXCL10/Cr data. It did not, however, improve the IRA for decision to biopsy (K = 0.48 and K = 0.43, for the comparison). Change in clinician estimated probability of rejection with additional CXCL10/Cr data was correlated with CXCL10/Cr level (r2  = 0.7756, P < .0001). Decision accuracy went from 8/15 (53.3%) cases to 11/15 (73.3%) with CXCL10/Cr, although improvement did not achieve statistical significance. Using CXCL10/Cr alone would have been accurate in 12/15 cases (80%). CONCLUSION: There is high variability in decision-making on biopsy indication. Urinary CXCL10/Cr improves probability estimates for risk of rejection. Training may be needed to assist nephrologists in better integrate biomarker information into clinical decision-making.

Deciphering the Prognostic and Predictive Value of Urinary CXCL10 in Kidney Recipients With BK Virus Reactivation.

BK virus (BKV) replication increases urinary chemokine C-X-C motif ligand 10 (uCXCL10) levels in kidney transplant recipients (KTRs). Here, we investigated uCXCL10 levels across different stages of BKV replication as a prognostic and predictive marker for functional decline in KTRs after BKV-DNAemia. uCXCL10 was assessed in a cross-sectional study (474 paired urine/blood/biopsy samples and a longitudinal study (1,184 samples from 60 KTRs with BKV-DNAemia). uCXCL10 levels gradually increased with urine (P-value < 0.0001) and blood BKV viral load (P < 0.05) but were similar in the viruria and no BKV groups (P > 0.99). In viremic patients, uCXCL10 at biopsy was associated with graft functional decline [HR = 1.65, 95% CI (1.08-2.51), P = 0.02], irrespective of baseline eGFR, blood viral load, or BKVN diagnosis. uCXL10/cr (threshold: 12.86 ng/mmol) discriminated patients with a low risk of graft function decline from high-risk patients (P = 0.01). In the longitudinal study, the uCXCL10 and BKV-DNAemia trajectories were superimposable. Stratification using the same uCXCL10/cr threshold at first viremia predicted the subsequent inflammatory response, assessed by time-adjusted uCXCL10/cr AUC (P < 0.001), and graft functional decline (P = 0.03). In KTRs, uCXCL10 increases in BKV-DNAemia but not in isolated viruria. uCXCL10/cr is a prognostic biomarker of eGFR decrease, and a 12.86 ng/ml threshold predicts higher inflammatory burdens and poor renal outcomes.

Acute Kidney Injury in Children after Hematopoietic Cell Transplantation Is Associated with Elevated Urine CXCL10 and CXCL9.

Acute kidney injury (AKI) is nearly universally associated with worse outcomes, especially among children after hematopoietic stem cell transplant (HCT). Our objective was to examine urinary immune biomarkers of AKI after HCT to provide insights into novel mechanisms of kidney injury in this population. Studying patients undergoing allogeneic HCT provides a unique opportunity to examine immune markers of AKI because the risk of AKI is high and the immune system newly develops after transplant. Children (>2 years old) and young adults undergoing their first allogeneic HCT and enrolled in a prospective, observational cohort study at 2 large children's hospitals had urine collected pre-HCT and monthly for the first 4 months after HCT. Urine samples at each monthly time point were assayed for 8 immune-related biomarkers. AKI was defined as a 1.5-fold increase in the monthly serum creatinine value, which was recorded ±1 day from when the research urine sample was obtained, as compared with the pre-HCT baseline. Generalized estimating equation regression analysis evaluated the association between the monthly repeated measures (urinary biomarkers and AKI). A total of 176 patients were included from 2 pediatric centers. Thirty-six patients from 1 center were analyzed as a discovery cohort and the remaining 140 patients from the second center were analyzed as a validation cohort. AKI rates were 18% to 35% depending on the monthly time point after HCT. Urine CXCL10 and CXCL9 concentrations were significantly higher among children who developed AKI compared with children who did not (P < .01) in both cohorts. In order to gain a better understanding of the cellular source for these biomarkers in the urine, we also analyzed in vitro expression of CXCL10 and CXCL9 in kidney cell lines after stimulation with interferon-γ and interferon-α. HEK293-epithelial kidney cells demonstrated interferon-induced expression of CXCL10 and CXCL9, suggesting a potential mechanism driving the key finding. CXCL10 and CXCL9 are associated with AKI after HCT and are therefore promising biomarkers to guide improved diagnostic and treatment strategies for AKI in this high-risk population.

Interferon-Inducible Protein 10 and Disease Activity in Systemic Lupus Erythematosus and Lupus Nephritis: A Systematic Review and Meta-Analysis.

There is increasing evidence of a correlation between interferon-inducible protein 10 (IP-10) and disease activity of systemic lupus erythematosus (SLE) and lupus nephritis (LN). We conducted a comprehensive search on IP-10 using MEDLINE, Scopus, and Cochrane electronic databases from the beginning to the end of December 2017. All studies that compared serum and/or urine IP-10 between active SLE/LN patients and any control groups were identified and included in this systematic review and meta-analysis. The mean difference (MD) of IP-10 level among active SLE and LN patients, as well as the correlation of IP-10 with disease activity, were meta-analyzed using a random-effects model. From 23 eligible studies, 15 provided adequate data for meta-analysis. Serum IP-10 was significantly elevated in patients with active SLE compared to non-active SLE patients (MD 356.5 pg/mL, 95% CI 59.6 to 653.4, p = 0.019). On the other hand, the levels of serum IP-10 was not different between active LN and non-active LN. However, serum IP-10 was positively correlated with disease activity like SLE disease activity index (SLEDAI) (pooled r = 0.29, 95% CI 0.22 to 0.35, p < 0.001). Furthermore, urine IP-10 tended to be higher in patients with active LN compared to non-active LN patients but this did not reach statistical significance (MD 3.47 pg/mgCr × 100, 95% CI -0.18 to 7.12, p = 0.06). Nevertheless, urine IP-10 was positively correlated with renal SLEDAI (pooled r = 0.29, 95% CI 0.05 to 0.50, p = 0.019). In conclusion, serum and urine IP-10 levels may be useful in monitoring the disease activity of SLE and LN. Serum IP-10 was correlated with systemic disease whereas urine IP-10 was a useful biomarker for detecting active LN.

Identification of inflammatory markers suitable for non-invasive, repeated measurement studies in biobehavioral research: A feasibility study.

INTRODUCTION: Studying the role of the immune system in the interaction between mental and physical health is challenging. To study individuals with an intensive, longitudinal study design that requires repetitive sampling in their daily life, non-invasive sampling techniques are a necessity. Urine can be collected in a non-invasive way, but this may be demanding for participants and little is known about fluctuation of inflammatory markers in urine over time. The aim of this study was to investigate the feasibility of non-invasive sampling, and to explore intra-individual differences in inflammatory markers in urine. MATERIALS & METHODS: Ten healthy individuals collected 24-hour urine for 63 consecutive days. In a pilot analysis, 39 inflammatory markers were examined for detectability in urine, stability over time and under storage conditions, and daily fluctuations. Multiplex analyses were used to quantify levels of eight selected markers: C-reactive protein (CRP), Fractalkine, Interleukin-1 receptor-antagonist (IL-1RA), interferon-α (IFNα), interferon-γ (IFNγ), Interferon gamma-induced protein 10 (IP10), Macrophage inflammatory protein-1ß (MIP-1ß), and Vascular Endothelial Growth Factor (VEGF). Cross-correlations were calculated between the overnight and 24-hour samples were calculated, to examine whether 24-hour urine could be replaced by the overnight portion for better feasibility. We examined intra- and interindividual differences in the levels of inflammatory markers in urine and the fluctuations thereof. RESULTS: This study showed that levels of selected inflammatory markers can be detected in urine. Cross-correlation analyses showed that correlations between levels of inflammatory markers in the night portion and the 24-hour urine sample varied widely between individuals. In addition, analyses of time series revealed striking inter- and intra-individual variation in levels of inflammatory markers and their fluctuations. CONCLUSION: We show that the assessment of urinary inflammatory markers is feasible in an intensive day-to-day study in healthy individuals. However, 24-hour urine cannot be replaced by an overnight portion to alleviate the protocol burden. Levels of inflammatory markers show substantial variation between and within persons.

Urinary levels of CCL2 and CXCL10 chemokines as potential biomarkers of ongoing pathological processes in kidney allograft: an association with BK virus nephropathy.

INTRODUCTION: Early prognostic markers that identify high­risk kidney transplant recipients may lead to optimization of immunosuppressive therapy and improved long­term outcomes. OBJECTIVES: The aim of this study was to assess whether the measurement of urinary concentrations of CCL2 and CXCL10 chemokines can be a valuable noninvasive tool for identifying ongoing pathological processes in a kidney allograft. PATIENTS AND METHODS: The study included 40 patients who underwent a protocol biopsy within 1­year post kidney transplant. The urinary concentrations of CCL2 and CXCL10 with reference to creatinine in urine were assayed in all patients. On the basis of biopsy results, a study group was selected (n = 25), including patients with a diagnosis of interstitial fibrosis and tubular atrophy grades II to III (n = 16), BK virus (BKV) nephropathy (n = 4), or mild inflammatory lesions fulfilling the criteria for mild rejection processes or borderline lesions (n = 11). Patients with normal biopsy results were included in a control group (n = 15). RESULTS: The ratio of CCL2 to creatinine (CCL2:Cr) was a significant independent predictor of BKV ephropathy (odds ratio, 1.1; 95% CI, 1.0-1.2; P = 0.04). The CXCL10:Cr ratio was not found to be an independent predictor of BKV nephropathy (odds ratio, 1.3; 95% CI, 0.99-1.71; P = 0.06). CONCLUSIONS: The CCL2:Cr and CXCL10:Cr ratios may predict BKV nephropathy. The diagnostic value of CCL2 and CXCL10 in BKV infection should be further evaluated.

Soluble TNF-R1, VEGF and other cytokines as markers of disease activity in systemic lupus erythematosus and lupus nephritis.

BACKGROUND: Current non-invasive methods of assessing disease activity in systemic lupus erythematosus (SLE) are of limited sensitivity and specificity. Testing includes acute phase markers, autoantibodies and complement levels. Although measurements of dsDNA antibodies and complement C3/C4 levels are routine, they remain of limited value. Improved blood and urine markers may help in early detection of flare, distinction between flare and chronic damage, and monitoring response to therapy. METHODS: A total of 87 patients with SLE were tested for the following cytokines in serum and urine: monocyte chemoattractant protein 1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), soluble tumour necrosis factor receptor 1 (sTNF-R1), interferon-inducible protein 10 (IP-10), monocyte inhibitory protein 1α (MIP-1α) and vascular endothelial growth factor (VEGF). Patients attending the Lupus Unit at St Thomas' Hospital, London, UK were divided into active lupus nephritis (LN), inactive LN and non-renal SLE groups based on their renal pathology and SLE disease activity index (SLEDAI). Cytokine testing was performed using the FIDIS multiplex bead assay. RESULTS: The mean level of serum sTNF-R1 was higher in the active LN group compared with both inactive LN and non-renal SLE groups ( p < 0.001). For urine measurements there were significant differences between active LN and non-renal SLE for VEGF ( p = 0.016), after statistical correction for multiple testing. Both urinary and serum sTNF-R1 and IP-10 levels correlated with SLEDAI scores ( p < 0.001), while serum VEGF correlated weakly with SLEDAI ( p = 0.025). The optimum combination for differentiating active from inactive LN patients was serum VEGF, sTNF-R1, MCP-1 and glomerular filtration rate plus urinary sTNF-R1 and protein-creatinine ratio. CONCLUSION: These results indicate that for active LN, sTNF-R1 could be a useful serum cytokine marker, with potential for VEGF in the urine. This study has confirmed the ability of the multiplex bead technique to detect cytokines in a good analytical range, including very low and high levels, in both serum and urine. Combining serum and urine markers provided additional sensitivity in distinguishing active from inactive LN.

Multicentre randomised controlled trial protocol of urine CXCL10 monitoring strategy in kidney transplant recipients.

INTRODUCTION: Subclinical inflammation is an important predictor of death-censored graft loss, and its treatment has been shown to improve graft outcomes. Urine CXCL10 outperforms standard post-transplant surveillance in observational studies, by detecting subclinical rejection and early clinical rejection before graft functional decline in kidney transplant recipients. METHODS AND ANALYSIS: This is a phase ii/iii multicentre, international randomised controlled parallel group trial to determine if the early treatment of rejection, as detected by urine CXCL10, will improve kidney allograft outcomes. Incident adult kidney transplant patients (n~420) will be enrolled to undergo routine urine CXCL10 monitoring postkidney transplant. Patients at high risk of rejection, defined as confirmed elevated urine CXCL10 level, will be randomised 1:1 stratified by centre (n=250). The intervention arm (n=125) will undergo a study biopsy to check for subclinical rejection and biopsy-proven rejection will be treated per protocol. The control arm (n=125) will undergo routine post-transplant monitoring. The primary outcome at 12 months is a composite of death-censored graft loss, clinical biopsy-proven acute rejection, de novo donor-specific antibody, inflammation in areas of interstitial fibrosis and tubular atrophy (Banff i-IFTA, chronic active T-cell mediated rejection) and subclinical tubulitis on 12-month surveillance biopsy. The secondary outcomes include decline of graft function, microvascular inflammation at 12 months, development of IFTA at 12 months, days from transplantation to clinical biopsy-proven rejection, albuminuria, EuroQol five-dimension five-level instrument, cost-effectiveness analysis of the urine CXCL10 monitoring strategy and the urine CXCL10 kinetics in response to rejection therapy. ETHICS AND DISSEMINATION: The study has been approved by the University of Manitoba Health Research Ethics Board (HS20861, B2017:076) and the local research ethics boards of participating centres. Recruitment commenced in March 2018 and results are expected to be published in 2023. De-identified data may be shared with other researchers according to international guidelines (International Committee of Medical Journal Editors [ICJME]). TRIAL REGISTRATION NUMBER: NCT03206801; Pre-results.

Identification of Low-Abundance Urinary Biomarkers in Lupus Nephritis Using Electrochemiluminescence Immunoassays.

OBJECTIVE: To investigate the utility of a sensitive platform using electrochemiluminescence (ECL) for the identification of low-abundance urinary protein biomarkers in lupus nephritis (LN). METHODS: Forty-eight urine samples were obtained from subjects in 2 independent cohorts, each consisting of 3 groups (matched for age, sex, and race) of 8 patients with active LN (renal Systemic Lupus Erythematosus Disease Activity Index [SLEDAI] >0), 8 patients with inactive SLE (renal SLEDAI 0), and 8 healthy controls. Samples were tested using a preexisting 40-plex ECL panel. A custom 5-plex ECL panel was then developed for further validation studies and used to test 140 urine samples (from 44 patients with active LN, 41 patients with inactive SLE, 28 healthy controls, and 27 patients with other kidney diseases). RESULTS: Levels of 17 urinary proteins were elevated (P < 0.05 by 2-tailed Mann-Whitney U test) in samples from patients with active LN compared to samples from patients with inactive SLE and healthy controls in cohort 1, while 9 were similarly elevated in cohort 2. Of these, interleukin-7 (IL-7), IL-12p40, IL-15, interferon-γ-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC) were chosen for further validation. These 5 proteins were undetectable by enzyme-linked immunosorbent assay (ELISA). Hence, a custom 5-plex ECL panel was developed and used to validate the results from the initial 40-plex screening panel. Urinary IL-7, IL-12p40, IL-15, IP-10, and TARC levels were again significantly elevated in patients with active LN compared to those with inactive SLE and healthy controls, and correlated well with the renal SLEDAI and physician's global assessment of disease activity (R > 0.67, P < 0.05). All 5 urinary proteins were more frequently elevated in LN compared to controls with other chronic kidney diseases, although overall group differences attained significance only for urinary IL-7 and IL-15. CONCLUSION: Urinary levels of IL-7, IL-12p40, IL-15, IP-10, and TARC are potentially useful diagnostic tools in LN. The use of ECL assays may allow detection of urinary biomarkers that are below ELISA detection limits.

Evidence for a role of angiotensin converting enzyme 2 in proteinuria of idiopathic nephrotic syndrome.

Introduction: Renin angiotensin system (RAS) plays a role in idiopathic nephrotic syndrome (INS). Most studies investigated only the classical RAS axis. Therefore, the aims of the present study were to evaluate urinary levels of RAS molecules related to classical and to counter-regulatory axes in pediatric patients with INS, to compare the measurements with levels in healthy controls and to search for associations with inflammatory molecules, proteinuria and disease treatment. Subjects and methods: This cross-sectional study included 31 patients with INS and 19 healthy controls, matched for age and sex. Patients and controls were submitted to urine collection for measurement of RAS molecules [Ang II, Ang-(1-7), ACE and ACE2] by enzyme immunoassay and cytokines by Cytometric Bead Array. Findings in INS patients were compared according to proteinuria: absent (<150 mg/dl, n = 15) and present (≥150 mg/dl, n = 16). Results: In comparison to controls, INS patients had increased Ang II, Ang-(1-7) and ACE, levels while ACE2 was reduced. INS patients with proteinuria had lower levels of ACE2 than those without proteinuria. ACE2 levels were negatively correlated with 24-h-proteinuria. Urinary concentrations of MCP-1/CCL2 were significantly higher in INS patients, positively correlated with Ang II and negatively with Ang-(1-7). ACE2 concentrations were negatively correlated with IP-10/CXCL-10 levels, which, in turn, were positively correlated with 24-h-proteinuria. Conclusion: INS patients exhibited changes in RAS molecules and in chemokines. Proteinuria was associated with low levels of ACE2 and high levels of inflammatory molecules.