Vitamin D Metabolites Before and After Kidney Transplantation in Patients Who Are Anephric.

RATIONALE & OBJECTIVE: Kidneys are vital for vitamin D metabolism and disruptions in both production and catabolism occur in chronic kidney disease. Although vitamin D activation occurs in numerous tissues, the kidneys are the most relevant source of circulating active vitamin D. This study investigates extrarenal vitamin D activation and the impact of kidney transplantation on vitamin D metabolism in patients who are anephric. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Adult patients with previous bilateral nephrectomy (anephric) not receiving active vitamin D therapy were evaluated at the time of (N=38) and 1 year after (N=25) kidney transplantation. Liquid chromatography-tandem mass spectrometry was used to measure vitamin D metabolites. Metabolic ratios were expressed CYP24A1 (24,25(OH)2D/25(OH)D) and CYP27B1 (1α,25(OH)2D/25(OH)D) as activities. Differences between evaluation timepoints were evaluated by paired Student's t-test or Wilcoxon matched-pairs signed-rank test. FINDINGS: At time of transplantation, 1α,25(OH)2D was detectable in all patients (4 to 36 pg/mL). There was a linear relationship between 25(OH)D and 1α,25(OH)2D-levels (r=0.58, p<0.001) with 25(OH)D explaining 34% of the variation in 1α,25(OH)2D-levels. There were no associations between 1α,25(OH)2D and biointact PTH or FGF23. One year after transplantation, 1α,25(OH)2D levels recovered (+205%) and CYP27B1 activity increased (+352%). Measures of vitamin D catabolism, 24,25(OH)2D and CYP24A1 activity increased 3-5 fold. Also at 12 months after transplantation, 1α,25(OH)2D was positively correlated with PTH (rho=0.603, p=0.04), but not with levels of 25(OH)D or FGF23. LIMITATIONS: Retrospective, observational study design with a small cohort size. CONCLUSIONS: Low-normal levels of 1α,25(OH)2D was demonstrated in anephric patients, indicating production outside of the kidneys. This extrarenal CYP27B1 activity may be more substrate-driven than hormonally regulated. Kidney transplantation seems to restore kidney CYP27B1 and CYP24A1 activity, as evaluated by vitamin D metabolic ratios, resulting in both increased vitamin D production and catabolism. These findings may have implications for vitamin D supplementation strategies in the setting of kidney failure and transplantation.

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.

The Dietary Fiber Inulin Slows Progression of Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD) in a Rat Model of CKD.

Chronic kidney disease (CKD)-mineral bone disorder (CKD-MBD) leads to fractures and cardiovascular disease. Observational studies suggest beneficial effects of dietary fiber on both bone and cardiovascular outcomes, but the effect of fiber on CKD-MBD is unknown. To determine the effect of fiber on CKD-MBD, we fed the Cy/+ rat with progressive CKD a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30% and ~15% of normal kidney function; CKD 4 and 5). We assessed CKD-MBD end points of biochemistry, bone quantity and quality, cardiovascular health, and cecal microbiota and serum gut-derived uremic toxins. Results were analyzed by two-way analysis of variance (ANOVA) to evaluate the main effects of CKD stage and inulin, and their interaction. The results showed that in CKD animals, inulin did not alter kidney function but reduced the increase from stage 4 to 5 in serum levels of phosphate and parathyroid hormone, but not fibroblast growth factor-23 (FGF23). Bone turnover and cortical bone parameters were similarly improved but mechanical properties were not altered. Inulin slowed progression of aorta and cardiac calcification, left ventricular mass index, and fibrosis. To understand the mechanism, we assessed intestinal microbiota and found changes in alpha and beta diversity and significant changes in several taxa with inulin, together with a reduction in circulating gut derived uremic toxins such as indoxyl sulfate and short-chain fatty acids. In conclusion, the addition of the fermentable fiber inulin to the diet of CKD rats led to a slowed progression of CKD-MBD without affecting kidney function, likely mediated by changes in the gut microbiota composition and lowered gut-derived uremic toxins. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Phenylacetylglutamine and trimethylamine N-oxide: Two uremic players, different actions.

BACKGROUND: Chronic kidney disease (CKD) patients exhibit a heightened cardiovascular (CV) risk which may be partially explained by increased medial vascular calcification. Although gut-derived uremic toxin trimethylamine N-oxide (TMAO) is associated with calcium-phosphate deposition, studies investigating phenylacetylglutamine's (PAG) pro-calcifying potential are missing. METHODS: The effect of TMAO and PAG in vascular calcification was investigated using 120 kidney failure patients undergoing living-donor kidney transplantation (LD-KTx), in an observational, cross-sectional manner. Uremic toxin concentrations were related to coronary artery calcification (CAC) score, epigastric artery calcification score, and markers of established non-traditional risk factors that constitute to the 'perfect storm' that drives early vascular aging in this patient population. Vascular smooth muscle cells were incubated with TMAO or PAG to determine their calcifying effects in vitro and analyse associated pathways by which these toxins may promote vascular calcification. RESULTS: TMAO, but not PAG, was independently associated with CAC score after adjustment for CKD-related risk factors in kidney failure patients. Neither toxin was associated with epigastric artery calcification score; however, PAG was independently, positively associated with 8-hydroxydeoxyguanosine. Similarly, TMAO, but not PAG, promoted calcium-phosphate deposition in vitro, while both uremic solutes induced oxidative stress. CONCLUSIONS: In conclusion, our translational data confirm TMAO's pro-calcifying effects, but both toxins induced free radical production detrimental to vascular maintenance. Our findings suggest these gut-derived uremic toxins have different actions on the vessel wall and therapeutically targeting TMAO may help reduce CV-related mortality in CKD.

The Dietary Fermentable Fiber Inulin Alters the Intestinal Microbiome and Improves Chronic Kidney Disease Mineral-Bone Disorder in a Rat Model of CKD.

Background: Dietary fiber is important for a healthy diet, but intake is low in CKD patients and the impact this has on the manifestations of CKD-Mineral Bone Disorder (MBD) is unknown. Methods: The Cy/+ rat with progressive CKD was fed a casein-based diet of 0.7% phosphate with 10% inulin (fermentable fiber) or cellulose (non-fermentable fiber) from 22 weeks to either 30 or 32 weeks of age (~30 and ~15 % of normal kidney function). We assessed CKD-MBD, cecal microbiota, and serum gut-derived uremic toxins. Two-way ANOVA was used to evaluate the effect of age and inulin diet, and their interaction. Results: In CKD animals, dietary inulin led to changes in microbiota alpha and beta diversity at 30 and 32 weeks, with higher relative abundance of several taxa, including Bifidobacterium and Bacteroides , and lower Lactobacillus . Inulin reduced serum levels of gut-derived uremic toxins, phosphate, and parathyroid hormone, but not fibroblast growth factor-23. Dietary inulin decreased aorta and cardiac calcification and reduced left ventricular mass index and cardiac fibrosis. Bone turnover and cortical bone parameters were improved with inulin; however, bone mechanical properties were not altered. Conclusions: The addition of the fermentable fiber inulin to the diet of CKD rats led to changes in the gut microbiota composition, lowered gut-derived uremic toxins, and improved most parameters of CKD-MBD. Future studies should assess this fiber as an additive therapy to other pharmacologic and diet interventions in CKD. Significance Statement: Dietary fiber has well established beneficial health effects. However, the impact of fermentable dietary fiber on the intestinal microbiome and CKD-MBD is poorly understood. We used an animal model of progressive CKD and demonstrated that the addition of 10% of the fermentable fiber inulin to the diet altered the intestinal microbiota and lowered circulating gut-derived uremic toxins, phosphorus, and parathyroid hormone. These changes were associated with improved cortical bone parameters, lower vascular calcification, and reduced cardiac hypertrophy, fibrosis and calcification. Taken together, dietary fermentable fiber may be a novel additive intervention to traditional therapies of CKD-MBD.

Indoxyl Sulphate Retention Is Associated with Microvascular Endothelial Dysfunction after Kidney Transplantation.

Kidney transplantation (KTx) is the preferred form of renal replacement therapy in chronic kidney disease (CKD) patients, owing to increased quality of life and reduced mortality when compared to chronic dialysis. Risk of cardiovascular disease is reduced after KTx; however, it is still a leading cause of death in this patient population. Thus, we aimed to investigate whether functional properties of the vasculature differed two years post-KTx (postKTx) compared to baseline (time of KTx). Using the EndoPAT device in 27 CKD patients undergoing living-donor KTx, we found that vessel stiffness significantly improved while endothelial function worsened postKTx vs. baseline. Furthermore, baseline serum indoxyl sulphate (IS), but not p-cresyl sulphate, was independently negatively associated with reactive hyperemia index, a marker of endothelial function, and independently positively associated with P-selectin postKTx. Finally, to better understand the functional effects of IS in vessels, we incubated human resistance arteries with IS overnight and performed wire myography experiments ex vivo. IS-incubated arteries showed reduced bradykinin-mediated endothelium-dependent relaxation compared to controls via reduced nitric oxide (NO) contribution. Endothelium-independent relaxation in response to NO donor sodium nitroprusside was similar between IS and control groups. Together, our data suggest that IS promotes worsened endothelial dysfunction postKTx, which may contribute to the sustained CVD risk.

Tacrolimus induces a pro-fibrotic response in donor-derived human proximal tubule cells dependent on common variants of the CYP3A5 and ABCB1 genes.

BACKGROUND: Common genetic variants of the enzymes and efflux pump involved in tacrolimus disposition have been associated with calcineurin inhibitor nephrotoxicity, but their importance is unclear because of the multifactorial background of renal fibrosis. This study explores the pro-fibrotic response of tacrolimus exposure in relation to the differential capacity for tacrolimus metabolism in proximal tubule cells (PTCs) with a variable (pharmaco)genetic background. METHODS: PTCs were obtained from protocol allograft biopsies with different combinations of CYP3A5 and ABCB1 variants and were incubated with tacrolimus within the concentration range found in vivo. Gene and protein expression, CYP3A5 and P-glycoprotein function, and tacrolimus metabolites were measured in PTC. Connective tissue growth factor (CTGF) expression was assessed in protocol biopsies of kidney allograft recipients. RESULTS: PTCs produce CTGF in response to escalating tacrolimus exposure, which is approximately 2-fold higher in cells with the CYP3A5*1 and ABCB1 TT combination in vitro. Increasing tacrolimus exposure results in relative higher generation of the main tacrolimus metabolite {13-O-desmethyl tacrolimus [M1]} in cells with this same genetic background. Protocol biopsies show a larger increase in in vivo CTGF tissue expression over time in TT vs. CC/CT but was not affected by the CYP3A5 genotype. CONCLUSIONS: Tacrolimus exposure induces a pro-fibrotic response in a PTC model in function of the donor pharmacogenetic background associated with tacrolimus metabolism. This finding provides a mechanistic insight into the nephrotoxicity associated with tacrolimus treatment and offers opportunities for a tailored immunosuppressive treatment.

Isolating mineralized bone and bone marrow mRNA from transiliac bone biopsies stored in a stabilizing solution: A comparative study.

The molecular mechanisms underlying metabolic bone diseases, including renal osteodystrophy, are poorly understood. Transcriptomics are increasingly used to characterize biological molecular networks and prove promising in identifying therapeutic targets and biomarkers. A reliable method for obtaining sufficient amounts of high quality RNA from human bone biopsies is a prerequisite for the implementation of molecular diagnostics in clinical research and practice. The present study aimed to develop a simple and adequate method for isolating bone and bone marrow mRNA from transiliac bone biopsies. Several storage, separation, and extraction procedures were compared. The procedure was optimized in pig samples and subsequently validated in human samples. Appropriate amounts of mineralized bone and bone marrow mRNA of moderate to high quality were obtained from transiliac bone biopsies that were immersed in the stabilizing solution Allprotect Tissue Reagent at room temperature for up to 3 days prior to freezing. After thawing, bone marrow and mineralized bone were separated by a multistep centrifugation procedure and subsequently disrupted and homogenized by a bead crusher. Appropriate separation of mineralized bone and bone marrow was confirmed by discriminatory gene expression profiles.

Lipid Profile Is Negatively Associated with Uremic Toxins in Patients with Kidney Failure-A Tri-National Cohort.

Patients with kidney failure (KF) have a high incidence of cardiovascular (CV) disease, partly driven by insufficient clearance of uremic toxins. Recent investigations have questioned the accepted effects of adverse lipid profile and CV risk in uremic patients. Therefore, we related a panel of uremic toxins previously associated with CV morbidity/mortality to a full lipid profile in a large, tri-national, cross-sectional cohort. Total, high-density lipoprotein (HDL), non-HDL, low-density lipoprotein (LDL), and remnant cholesterol, as well as triglyceride, levels were associated with five uremic toxins in a cohort of 611 adult KF patients with adjustment for clinically relevant covariates and other patient-level variables. Univariate analyses revealed negative correlations of total, non-HDL, and LDL cholesterol with all investigated uremic toxins. Multivariate linear regression analyses confirmed independent, negative associations of phenylacetylglutamine with total, non-HDL, and LDL cholesterol, while indole-3 acetic acid associated with non-HDL and LDL cholesterol. Furthermore, trimethylamine-N-Oxide was independently and negatively associated with non-HDL cholesterol. Sensitivity analyses largely confirmed findings in the entire cohort. In conclusion, significant inverse associations between lipid profile and distinct uremic toxins in KF highlight the complexity of the uremic milieu, suggesting that not all uremic toxin interactions with conventional CV risk markers may be pathogenic.

On Methods for the Measurement of the Apelin Receptor Ligand Apelin.

Apelin exists in many isoforms, both in the circulation and in specific tissues. Apelin peptides have a short half-life but preservation before measurement is scarcely studied. Reproducible mass spectrometry methods to specifically measure a broad range of apelinergic peptide isoforms are currently lacking. A sample protocol to conserve apelinergic peptides in the preanalytical phase and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to measure apelinergic isoforms was developed. Apelin was measured in plasma. For validation, human embryonic kidney (HEK) cells transfected with cDNA for preproapelin were used. Results were compared with a validated radioimmunoassay (RIA) method. Acidifying plasma to pH 2.5 improves post-sampling stability of apelin. HPLC-MS/MS was unable to detect apelin isoforms in plasma of healthy volunteers (n = 16) and chronic kidney disease patients (n = 4). RIA could detect apelin in concentrations between 71 and 263 fmol/l in 10 healthy volunteers. An optimized preanalytical protocol was developed. A sensitive and specific HPLC-MS/MS method failed to detect apelin in human plasma. Apelin-36 was detected in HEK cells transfected with cDNA for preproapelin. Currently, RIA with relatively selective antibodies is the best alternative for the measurement of apelin but novel sensitive and specific methods are needed.

Biological pathways and comparison with biopsy signals and cellular origin of peripheral blood transcriptomic profiles during kidney allograft pathology.

Kidney transplant injury processes are associated with molecular changes in kidney tissue, primarily related to immune cell activation and infiltration. How these processes are reflected in the circulating immune cells, whose activation is targeted by strong immunosuppressants, is poorly understood. To study this, we analyzed the molecular alterations in 384 peripheral blood samples from four European transplant centers, taken at the time of a kidney allograft biopsy, selected for their phenotype, using RNA-sequencing. In peripheral blood, differentially expressed genes in 136 rejection and 248 no rejection samples demonstrated upregulation of glucocorticoid receptor and nucleotide oligomerization domain-like receptor signaling pathways. Pathways enriched in antibody-mediated rejection (ABMR) were strongly immune-specific, whereas pathways enriched in T cell-mediated rejection were less immune related. In polyomavirus infection, upregulation of mitochondrial dysfunction and interferon signaling pathways was seen. Next, we integrated the blood results with transcriptomics of 224 kidney allograft biopsies which showed consistently upregulated genes per phenotype in both blood and biopsy. In single-cell RNASeq (scRNASeq) analysis of seven kidney allograft biopsies, the consistently overexpressed genes in ABMR were mostly expressed by infiltrating leukocytes in the allograft. Similarly, in peripheral blood scRNASeq analysis, these genes were overexpressed in ABMR in immune cell subtypes. Furthermore, overexpression of these genes in ABMR was confirmed in independent cohorts in blood and biopsy. Thus, our results highlight the immune activation pathways in peripheral blood leukocytes at the time of kidney allograft pathology, despite the use of current strong immunosuppressants, and provide a framework for future therapeutic interventions.

Circulating Donor-Specific Anti-HLA Antibodies Associate With Immune Activation Independent of Kidney Transplant Histopathological Findings.

Despite the critical role of cytokines in allograft rejection, the relation of peripheral blood cytokine profiles to clinical kidney transplant rejection has not been fully elucidated. We assessed 28 cytokines through multiplex assay in 293 blood samples from kidney transplant recipients at time of graft dysfunction. Unsupervised hierarchical clustering identified a subset of patients with increased pro-inflammatory cytokine levels. This patient subset was hallmarked by a high prevalence (75%) of donor-specific anti-human leukocyte antigen antibodies (HLA-DSA) and histological rejection (70%) and had worse graft survival compared to the group with low cytokine levels (HLA-DSA in 1.7% and rejection in 33.7%). Thirty percent of patients with high pro-inflammatory cytokine levels and HLA-DSA did not have histological rejection. Exploring the cellular origin of these cytokines, we found a corresponding expression in endothelial cells, monocytes, and natural killer cells in single-cell RNASeq data from kidney transplant biopsies. Finally, we confirmed secretion of these cytokines in HLA-DSA-mediated cross talk between endothelial cells, NK cells, and monocytes. In conclusion, blood pro-inflammatory cytokines are increased in kidney transplant patients with HLA-DSA, even in the absence of histology of rejection. These observations challenge the concept that histology is the gold standard for identification of ongoing allo-immune activation after transplantation.

Contemporary kidney transplantation has a limited impact on bone microarchitecture.

Bone microarchitecture is an important component of bone quality and disturbances may reduce bone strength and resistance to trauma. Kidney transplant recipients have an excess risk of fractures, and bone loss affecting both trabecular and cortical bone compartments have been demonstrated after kidney transplantation. The primary aim of this study was to investigate the impact of kidney transplantation on trabecular and cortical bone microarchitecture, assessed by histomorphometry and micro computed tomography (µCT). Iliac crest bone biopsies, analyzed by bone histomorphometry and µCT, were performed at time of kidney transplantation and 12 months post-transplantation in an unselected cohort of 30 patients. Biochemical markers of mineral metabolism and bone turnover were measured at both time-points. At 12 months post-transplantation, bone turnover was low in 5 (17%) and normal in 25 (83%) patients. By histomorphometry, bone remodeling normalized, with decreases in eroded perimeters (4.0 to 2.1%, p = 0.02) and number of patients with marrow fibrosis (41 to 0%, p < 0.001). By µCT, trabecular thickness (134 to 125 µM, p = 0.003) decreased slightly. Other parameters of bone volume and microarchitecture, including cortical thickness (729 to 713 µm, p = 0.73) and porosity (10.2 to 9.5%, p = 0.15), remained stable. We conclude that kidney transplantation with current immunosuppressive protocols has a limited impact on bone microarchitecture.

Determination of tacrolimus, three mono-demethylated metabolites and a M1 tautomer in human whole blood by liquid chromatography - tandem mass spectrometry.

The immunosuppressant tacrolimus is the primary drug used in kidney transplantation to prevent organ rejection. A sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to measure tacrolimus and its three known mono-demethylated metabolites 13-O-desmethyl tacrolimus (M1), 31-O-desmethyl tacrolimus (M2), 15-O-desmethyl tacrolimus (M3). By generating the metabolites to use as standards after incubation of tacrolimus with rat liver microsomes, we discovered multiple M1 peaks which we identified as two tautomers of M1. The M1 tautomer II was also successfully validated in this method. The separation and purification of the metabolites and tautomers were performed by semi-preparative liquid chromatography with UV-detection, while confirmation was done by UPLC-MS/MS and Nuclear Magnetic Resonance. For quantification an easy sample preparation was performed with zinc sulfate and acetonitrile as cell lyses and precipitation. Detection was performed in positive electrospray ionization. By better characterization of the metabolites and the tautomers, we could possibly explain insight into the clinical condition and thus adjust the immunosuppressant therapy individually per patient. Calibration curves were linear for all compounds. Precision was assessed according to the NCCLS EP5-T guideline, being below 15 % and mean recoveries were between 93 and 110 % for tacrolimus, its three metabolites and the M1 tautomer II. The validated method was successfully applied in a cohort of 20 patients after kidney transplantation.

Changes in kynurenine pathway metabolites after acute psychosocial stress in healthy males: a single-arm pilot study.

Chronic stress is associated with an increased conversion of tryptophan (TRP) into kynurenine (KYN). However, only a few studies investigated KYN pathway metabolite concentrations following acute stress in healthy subjects. We hypothesized that TRP/KYN metabolism changes following acute stress, and that KYN pathway metabolites are associated with cortisol and subjective stress responses. In a single-arm pilot study, we explored whether KYN pathway metabolites concentrations were altered after acute stress induced by the Maastricht Acute Stress Test in healthy males (n = 56, mean age: 27 (SD = 4.5) years, BMI: 23 (SD = 1.8) kg/m2). In particular, we examined whether concentrations of TRP decreased, and KYN, kynurenic acid (KYNA), and the ratio of KYN to TRP (KYN:TRP) increased after acute stress. Furthermore, we assessed whether cortisol and subjective stress responses correlated with KYN pathway metabolite measures after stress induction, based on both the area under the curve with respect to the ground (AUCg) as well as the incremental area under the curve (AUCi). Concentrations of TRP, KYN, KYNA, and KYN:TRP were significantly lower after stress induction compared to pre-stress induction (all p < 0.01). AUCi and AUCg reflecting cortisol and subjective stress responses did not correlate with AUCi and AUCg reflecting KYN pathway metabolite responses. These preliminary results indicate that KYN pathway metabolites are lower after acute psychosocial stress induction. Moreover, although chronic stress and subsequent prolonged elevated cortisol concentrations and subjective stress stimulate the conversion of TRP into KYN, acute stress is not associated with such conversion up to 35 minutes after stress induction.

Effect of Dietary Inulin Supplementation on the Gut Microbiota Composition and Derived Metabolites of Individuals Undergoing Hemodialysis: A Pilot Study.

OBJECTIVE: The prebiotic fiber inulin has been studied in individuals undergoing hemodialysis (HD) due to its ability to reduce gut microbiota-derived uremic toxins. However, studies examining the effects of inulin on the gut microbiota and derived metabolites are limited in these patients. We aimed to assess the impact of a 4-week supplementation of inulin on the gut microbiota composition and microbial metabolites of patients on HD. DESIGN AND METHODS: In a randomized, double-blind, placebo-controlled, crossover study, twelve HD patients (55 ± 10 y, 50% male, 58% Black American, BMI 31.6 ± 8.9 kg/m2, 33% diabetes mellitus) were randomized to consume inulin [10 g/d for females; 15 g/d for males] or maltodextrin [6 g/d for females; 9 g/d for males] for 4 weeks, with a 4-week washout period. We assessed the fecal microbiota composition, fecal metabolites (short-chain fatty acids (SCFA), phenols, and indoles), and plasma indoxyl sulfate and p-cresyl sulfate. RESULTS: At baseline, factors that explained the gut microbiota variability included BMI category and type of phosphate binder prescribed. Inulin increased the relative abundance of the phylum Verrucomicrobia and its genus Akkermansia (P interaction = 0.045). Inulin and maltodextrin resulted in an increased relative abundance of the phylum Bacteroidetes and its genus Bacteroides (P time = 0.04 and 0.03, respectively). Both treatments increased the fecal acetate and propionate (P time = 0.032 and 0.027, respectively), and there was a trend toward increased fecal butyrate (P time = 0.06). Inulin did not reduce fecal p-cresol or indoles, or plasma concentrations of p-cresyl sulfate or indoxyl sulfate. CONCLUSIONS: A 4-week supplementation of inulin did not lead to major shifts in the fecal microbiota and gut microbiota-derived metabolites. This may be due to high variability among participants and an unexpected increase in fecal excretion of SCFA with maltodextrin. Larger studies are needed to determine the effects of prebiotic fibers on the gut microbiota and clinical outcomes to justify their use in patients on HD.

Transcriptional Changes in Kidney Allografts with Histology of Antibody-Mediated Rejection without Anti-HLA Donor-Specific Antibodies.

BACKGROUND: Circulating donor-specific anti-HLA antibodies (HLA-DSAs) are often absent in serum of kidney allograft recipients whose biopsy specimens demonstrate histology of antibody-mediated rejection (ABMR). It is unclear whether cases involving ABMR histology without detectable HLA-DSAs represent a distinct clinical and molecular phenotype. METHODS: In this multicenter cohort study, we integrated allograft microarray analysis with extensive clinical and histologic phenotyping from 224 kidney transplant recipients between 2011 and 2017. We used the term ABMR histology for biopsy specimens that fulfill the first two Banff 2017 criteria for ABMR, irrespective of HLA-DSA status. RESULTS: Of 224 biopsy specimens, 56 had ABMR histology; 26 of these (46.4%) lacked detectable serum HLA-DSAs. Biopsy specimens with ABMR histology showed overexpression of transcripts mostly related to IFNγ-induced pathways and activation of natural killer cells and endothelial cells. HLA-DSA-positive and HLA-DSA-negative biopsy specimens with ABMR histology displayed similar upregulation of pathways and enrichment of infiltrating leukocytes. Transcriptional heterogeneity observed in biopsy specimens with ABMR histology was not associated with HLA-DSA status but was caused by concomitant T cell-mediated rejection. Compared with cases lacking ABMR histology, those with ABMR histology and HLA-DSA had higher allograft failure risk (hazard ratio [HR], 7.24; 95% confidence interval [95% CI], 3.04 to 17.20) than cases without HLA-DSA (HR, 2.33; 95% CI, 0.85 to 6.33), despite the absence of transcriptional differences. CONCLUSIONS: ABMR histology corresponds to a robust intragraft transcriptional signature, irrespective of HLA-DSA status. Outcome after ABMR histology is not solely determined by the histomolecular presentation but is predicted by the underlying etiologic factor. It is important to consider this heterogeneity in further research and in treatment decisions for patients with ABMR histology.

Antibody-mediated rejection with and without donor-specific anti-human leucocyte antigen antibodies: performance of the peripheral blood 8-gene expression assay.

BACKGROUND: Recently a peripheral blood 8-gene expression assay was developed for non-invasive detection of antibody-mediated rejection (ABMR) after kidney transplantation. Its value has not yet been evaluated in detail in clinical scenarios with different baseline disease probability [human leucocyte antigen donor-specific antibodies (HLA-DSA)-positive versus HLA-DSA-negative cases at the time of stable graft function versus graft dysfunction]. METHODS: Here we investigated the diagnostic accuracy of the 8-gene expression assay for histology of ABMR (ABMRh) with or without HLA-DSA in a cross-sectional cohort study of 387 blood samples with a concomitant graft biopsy. RESULTS: In patients with HLA-DSA (n = 64), the 8-gene expression assay discriminated DSA-positive ABMRh (DSAposABMRh) cases (n = 16) with good diagnostic performance {area under the receiver operating characteristic curve [AUROC] 83.1% [95% confidence interval (CI) 70.8-95.3]}. Also, in HLA-DSA-negative samples (n = 323), a clinically relevant diagnostic performance for DSAnegABMRh cases was found (n = 30) with an AUROC of 75.8% (95% CI 67.4-84.4). The 8-gene assay did not discriminate DSAposABMRh cases from DSAnegABMRh cases. There was a net benefit for clinical decision-making when adding the 8-gene expression assay to a clinical model consisting of estimated glomerular filtration rate, proteinuria, HLA-DSA and age. CONCLUSION: The 8-gene expression assay shows great potential for implementation in the clinical follow-up of high-risk HLA-DSA-positive patients and clinical relevance in HLA-DSA-negative cases.

Sevelamer Use in End-Stage Kidney Disease (ESKD) Patients Associates with Poor Vitamin K Status and High Levels of Gut-Derived Uremic Toxins: A Drug-Bug Interaction?

Gut microbial metabolism is not only an important source of uremic toxins but may also help to maintain the vitamin K stores of the host. We hypothesized that sevelamer therapy, a commonly used phosphate binder in patients with end-stage kidney disease (ESKD), associates with a disturbed gut microbial metabolism. Important representatives of gut-derived uremic toxins, including indoxyl sulfate (IndS), p-Cresyl sulfate (pCS), trimethylamine N-oxide (TMAO), phenylacetylglutamine (PAG) and non-phosphorylated, uncarboxylated matrix-Gla protein (dp-ucMGP; a marker of vitamin K status), were analyzed in blood samples from 423 patients (65% males, median age 54 years) with ESKD. Demographics and laboratory data were extracted from electronic files. Sevelamer users (n = 172, 41%) were characterized by higher phosphate, IndS, TMAO, PAG and dp-ucMGP levels compared to non-users. Sevelamer was significantly associated with increased IndS, PAG and dp-ucMGP levels, independent of age, sex, calcium-containing phosphate binder, cohort, phosphate, creatinine and dialysis vintage. High dp-ucMGP levels, reflecting vitamin K deficiency, were independently and positively associated with PAG and TMAO levels. Sevelamer therapy associates with an unfavorable gut microbial metabolism pattern. Although the observational design precludes causal inference, present findings implicate a disturbed microbial metabolism and vitamin K deficiency as potential trade-offs of sevelamer therapy.