Showing papers in "Journal of The American Society of Nephrology in 2019"

Advantages of Single-Nucleus over Single-Cell RNA Sequencing of Adult Kidney: Rare Cell Types and Novel Cell States Revealed in Fibrosis

Abstract: Background A challenge for single-cell genomic studies in kidney and other solid tissues is generating a high-quality single-cell suspension that contains rare or difficult-to-dissociate cell types and is free of both RNA degradation and artifactual transcriptional stress responses. Methods We compared single-cell RNA sequencing (scRNA-seq) using the DropSeq platform with single-nucleus RNA sequencing (snRNA-seq) using sNuc-DropSeq, DroNc-seq, and 10X Chromium platforms on adult mouse kidney. We validated snRNA-seq on fibrotic kidney from mice 14 days after unilateral ureteral obstruction (UUO) surgery. Results A total of 11,391 transcriptomes were generated in the comparison phase. We identified ten clusters in the scRNA-seq dataset, but glomerular cell types were absent, and one cluster consisted primarily of artifactual dissociation – induced stress response genes. By contrast, snRNA-seq from all three platforms captured a diversity of kidney cell types that were not represented in the scRNA-seq dataset, including glomerular podocytes, mesangial cells, and endothelial cells. No stress response genes were detected. Our snRNA-seq protocol yielded 20-fold more podocytes compared with published scRNA-seq datasets (2.4% versus 0.12%, respectively). Unexpectedly, single-cell and single-nucleus platforms had equivalent gene detection sensitivity. For validation, analysis of frozen day 14 UUO kidney revealed rare juxtaglomerular cells, novel activated proximal tubule and fibroblast cell states, and previously unidentified tubulointerstitial signaling pathways. Conclusions snRNA-seq achieves comparable gene detection to scRNA-seq in adult kidney, and it also has substantial advantages, including reduced dissociation bias, compatibility with frozen samples, elimination of dissociation-induced transcriptional stress responses, and successful performance on inflamed fibrotic kidney.

Projecting ESRD Incidence and Prevalence in the United States through 2030.

Abstract: Background Population rates of obesity, hypertension, diabetes, age, and race can be used in simulation models to develop projections of ESRD incidence and prevalence. Such projections can inform long-range planning for ESRD resources needs. Methods We used an open compartmental simulation model to estimate the incidence and prevalence of ESRD in the United States through 2030 on the basis of wide-ranging projections of population obesity and ESRD death rates. Population trends in age, race, hypertension, and diabetes were on the basis of data from the Centers for Disease Control and Prevention’s National Health and Nutrition Examination Survey and the US Census. Results The increase in ESRD incidence rates within age and race groups has leveled off and/or declined in recent years, but our model indicates that population changes in age and race distribution, obesity and diabetes prevalence, and ESRD survival will result in a 11%–18% increase in the crude incidence rate from 2015 to 2030. This incidence trend along with reductions in ESRD mortality will increase the number of patients with ESRD by 29%–68% during the same period to between 971,000 and 1,259,000 in 2030. Conclusions The burden of ESRD will increase in the United States population through 2030 due to demographic, clinical, and lifestyle shifts in the population and improvements in RRT. Planning for ESRD resource allocation should allow for substantial continued growth in the population of patients with ESRD. Future interventions should be directed to preventing the progression of CKD to kidney failure.

Deep learning-based histopathologic assessment of kidney tissue

Abstract: BACKGROUND: The development of deep neural networks is facilitating more advanced digital analysis of histopathologic images. We trained a convolutional neural network for multiclass segmentation of digitized kidney tissue sections stained with periodic acid-Schiff (PAS). METHODS: We trained the network using multiclass annotations from 40 whole-slide images of stained kidney transplant biopsies and applied it to four independent data sets. We assessed multiclass segmentation performance by calculating Dice coefficients for ten tissue classes on ten transplant biopsies from the Radboud University Medical Center in Nijmegen, The Netherlands, and on ten transplant biopsies from an external center for validation. We also fully segmented 15 nephrectomy samples and calculated the network's glomerular detection rates and compared network-based measures with visually scored histologic components (Banff classification) in 82 kidney transplant biopsies. RESULTS: The weighted mean Dice coefficients of all classes were 0.80 and 0.84 in ten kidney transplant biopsies from the Radboud center and the external center, respectively. The best segmented class was "glomeruli" in both data sets (Dice coefficients, 0.95 and 0.94, respectively), followed by "tubuli combined" and "interstitium." The network detected 92.7% of all glomeruli in nephrectomy samples, with 10.4% false positives. In whole transplant biopsies, the mean intraclass correlation coefficient for glomerular counting performed by pathologists versus the network was 0.94. We found significant correlations between visually scored histologic components and network-based measures. CONCLUSIONS: This study presents the first convolutional neural network for multiclass segmentation of PAS-stained nephrectomy samples and transplant biopsies. Our network may have utility for quantitative studies involving kidney histopathology across centers and provide opportunities for deep learning applications in routine diagnostics.

CKD: A Call for an Age-Adapted Definition.

Abstract: Current criteria for the diagnosis of CKD in adults include persistent signs of kidney damage, such as increased urine albumin-to-creatinine ratio or a GFR below the threshold of 60 ml/min per 1.73 m2 This threshold has important caveats because it does not separate kidney disease from kidney aging, and therefore does not hold for all ages. In an extensive review of the literature, we found that GFR declines with healthy aging without any overt signs of compensation (such as elevated single-nephron GFR) or kidney damage. Older living kidney donors, who are carefully selected based on good health, have a lower predonation GFR compared with younger donors. Furthermore, the results from the large meta-analyses conducted by the CKD Prognosis Consortium and from numerous other studies indicate that the GFR threshold above which the risk of mortality is increased is not consistent across all ages. Among younger persons, mortality is increased at GFR <75 ml/min per 1.73 m2, whereas in elderly people it is increased at levels <45 ml/min per 1.73 m2 Therefore, we suggest that amending the CKD definition to include age-specific thresholds for GFR. The implications of an updated definition are far reaching. Having fewer healthy elderly individuals diagnosed with CKD could help reduce inappropriate care and its associated adverse effects. Global prevalence estimates for CKD would be substantially reduced. Also, using an age-specific threshold for younger persons might lead to earlier identification of CKD onset for such individuals, at a point when progressive kidney damage may still be preventable.

Exostosin 1/Exostosin 2-Associated Membranous Nephropathy.

Abstract: BACKGROUND: In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain-containing 7A are target antigens in approximately 70% and 1%-5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown. METHODS: We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry. RESULTS: Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R-negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies. CONCLUSIONS: A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.

Computational segmentation and classification of diabetic glomerulosclerosis

Abstract: Background Pathologists use visual classification of glomerular lesions to assess samples from patients with diabetic nephropathy (DN). The results may vary among pathologists. Digital algorithms may reduce this variability and provide more consistent image structure interpretation. Methods We developed a digital pipeline to classify renal biopsies from patients with DN. We combined traditional image analysis with modern machine learning to efficiently capture important structures, minimize manual effort and supervision, and enforce biologic prior information onto our model. To computationally quantify glomerular structure despite its complexity, we simplified it to three components consisting of nuclei, capillary lumina and Bowman spaces; and Periodic Acid-Schiff positive structures. We detected glomerular boundaries and nuclei from whole slide images using convolutional neural networks, and the remaining glomerular structures using an unsupervised technique developed expressly for this purpose. We defined a set of digital features which quantify the structural progression of DN, and a recurrent network architecture which processes these features into a classification. Results Our digital classification agreed with a senior pathologist whose classifications were used as ground truth with moderate Cohen's kappa κ = 0.55 and 95% confidence interval [0.50, 0.60]. Two other renal pathologists agreed with the digital classification with κ1 = 0.68, 95% interval [0.50, 0.86] and κ2 = 0.48, 95% interval [0.32, 0.64]. Our results suggest computational approaches are comparable to human visual classification methods, and can offer improved precision in clinical decision workflows. We detected glomerular boundaries from whole slide images with 0.93±0.04 balanced accuracy, glomerular nuclei with 0.94 sensitivity and 0.93 specificity, and glomerular structural components with 0.95 sensitivity and 0.99 specificity. Conclusions Computationally derived, histologic image features hold significant diagnostic information that may augment clinical diagnostics.

Cellular Senescence in the Kidney

Abstract: Senescent cells have undergone permanent growth arrest, adopt an altered secretory phenotype, and accumulate in the kidney and other organs with ageing and injury. Senescence has diverse physiologic roles and experimental studies support its importance in nephrogenesis, successful tissue repair, and in opposing malignant transformation. However, recent murine studies have shown that depletion of chronically senescent cells extends healthy lifespan and delays age-associated disease-implicating senescence and the senescence-associated secretory phenotype as drivers of organ dysfunction. Great interest is therefore focused on the manipulation of senescence as a novel therapeutic target in kidney disease. In this review, we examine current knowledge and areas of ongoing uncertainty regarding senescence in the human kidney and experimental models. We summarize evidence supporting the role of senescence in normal kidney development and homeostasis but also senescence-induced maladaptive repair, renal fibrosis, and transplant failure. Recent studies using senescent cell manipulation and depletion as novel therapies to treat renal disease are discussed, and we explore unanswered questions for future research.

Sex-related disparities in CKD progression

Abstract: Background In the United States, incidence of ESRD is 1.5 times higher in men than in women, despite men’s lower prevalence of CKD. Prior studies, limited by inclusion of small percentages of minorities and other factors, suggested that men have more rapid CKD progression, but this finding has been inconsistent. Methods In our prospective investigation of sex differences in CKD progression, we used data from 3939 adults (1778 women and 2161 men) enrolled in the Chronic Renal Insufficiency Cohort Study, a large, diverse CKD cohort. We evaluated associations between sex (women versus men) and outcomes, specifically incident ESRD (defined as undergoing dialysis or a kidney transplant), 50% eGFR decline from baseline, incident CKD stage 5 (eGFR 2 ), eGFR slope, and all-cause death. Results Participants’ mean age was 58 years at study entry; 42% were non-Hispanic black, and 13% were Hispanic. During median follow-up of 6.9 years, 844 individuals developed ESRD, and 853 died. In multivariable regression models, compared with men, women had significantly lower risk of ESRD, 50% eGFR decline, progression to CKD stage 5, and death. The mean unadjusted eGFR slope was −1.09 ml/min per 1.73 m 2 per year in women and −1.43 ml/min per 1.73 m 2 per year in men, but this difference was not significant after multivariable adjustment. Conclusions In this CKD cohort, women had lower risk of CKD progression and death compared with men. Additional investigation is needed to identify biologic and psychosocial factors underlying these sex-related differences.

GFR Slope as a Surrogate End Point for Kidney Disease Progression in Clinical Trials: A Meta-Analysis of Treatment Effects of Randomized Controlled Trials.

Abstract: BACKGROUND: Surrogate end points are needed to assess whether treatments are effective in the early stages of CKD GFR decline leads to kidney failure, but regulators have not approved using differences in the change in GFR from the beginning to the end of a randomized, controlled trial as an end point in CKD because it is not clear whether small changes in the GFR slope will translate to clinical benefits METHODS: To assess the use of GFR slope as a surrogate end point for CKD progression, we performed a meta-analysis of 47 RCTs that tested 12 interventions in 60,620 subjects We estimated treatment effects on GFR slope (mean difference in GFR slope between the randomized groups), for the total slope starting at baseline, chronic slope starting at 3 months after randomization, and on the clinical end point (doubling of serum creatinine, GFR<15 ml/min per 173 m2, or ESKD) for each study We used Bayesian mixed-effects analyses to describe the association of treatment effects on GFR slope with the clinical end point and to test how well the GFR slope predicts a treatment's effect on the clinical end point RESULTS: Across all studies, the treatment effect on 3-year total GFR slope (median R2=097; 95% Bayesian credible interval [BCI], 078 to 100) and on the chronic slope (R2 096; 95% BCI, 063 to 100) accurately predicted treatment effects on the clinical end point With a sufficient sample size, a treatment effect of 075 ml/min per 173 m2/yr or greater on total slope over 3 years or chronic slope predicts a clinical benefit on CKD progress with at least 96% probability CONCLUSIONS: With large enough sample sizes, GFR slope may be a viable surrogate for clinical end points in CKD RCTs

Genome-Wide Association Study of Diabetic Kidney Disease Highlights Biology Involved in Glomerular Basement Membrane Collagen.

Abstract: Background Although diabetic kidney disease demonstrates both familial clustering and single nucleotide polymorphism heritability, the specific genetic factors influencing risk remain largely unknown. Methods To identify genetic variants predisposing to diabetic kidney disease, we performed genome-wide association study (GWAS) analyses. Through collaboration with the Diabetes Nephropathy Collaborative Research Initiative, we assembled a large collection of type 1 diabetes cohorts with harmonized diabetic kidney disease phenotypes. We used a spectrum of ten diabetic kidney disease definitions based on albuminuria and renal function. Results Our GWAS meta-analysis included association results for up to 19,406 individuals of European descent with type 1 diabetes. We identified 16 genome-wide significant risk loci. The variant with the strongest association (rs55703767) is a common missense mutation in the collagen type IV alpha 3 chain (COL4A3) gene, which encodes a major structural component of the glomerular basement membrane (GBM). Mutations in COL4A3 are implicated in heritable nephropathies, including the progressive inherited nephropathy Alport syndrome. The rs55703767 minor allele (Asp326Tyr) is protective against several definitions of diabetic kidney disease, including albuminuria and ESKD, and demonstrated a significant association with GBM width; protective allele carriers had thinner GBM before any signs of kidney disease, and its effect was dependent on glycemia. Three other loci are in or near genes with known or suggestive involvement in this condition (BMP7) or renal biology (COLEC11 and DDR1). Conclusions The 16 diabetic kidney disease-associated loci may provide novel insights into the pathogenesis of this condition and help identify potential biologic targets for prevention and treatment.

Mitochondria in Sepsis-Induced AKI.

Abstract: AKI is a common clinical condition associated with the risk of developing CKD and ESKD. Sepsis is the leading cause of AKI in the intensive care unit (ICU) and accounts for nearly half of all AKI events. Patients with AKI who require dialysis have an unacceptably high mortality rate of 60%-80%. During sepsis, endothelial activation, increased microvascular permeability, changes in regional blood flow distribution with resulting areas of hypoperfusion, and hypoxemia can lead to AKI. No effective drugs to prevent or treat human sepsis-induced AKI are currently available. Recent research has identified dysfunction in energy metabolism as a critical contributor to the pathogenesis of AKI. Mitochondria, the center of energy metabolism, are increasingly recognized to be involved in the pathophysiology of sepsis-induced AKI and mitochondria could serve as a potential therapeutic target. In this review, we summarize the potential role of mitochondria in sepsis-induced AKI and identify future therapeutic approaches that target mitochondrial function in an effort to treat sepsis-induced AKI.

An Organizational-Level Program of Intervention for AKI: A Pragmatic Stepped Wedge Cluster Randomized Trial.

Abstract: Background Variable standards of care may contribute to poor outcomes associated with AKI. We evaluated whether a multifaceted intervention (AKI e-alerts, an AKI care bundle, and an education program) would improve delivery of care and patient outcomes at an organizational level. Methods A multicenter, pragmatic, stepped-wedge cluster randomized trial was performed in five UK hospitals, involving patients with AKI aged ≥18 years. The intervention was introduced sequentially across fixed three-month periods according to a randomly determined schedule until all hospitals were exposed. The primary outcome was 30-day mortality, with pre-specified secondary endpoints and a nested evaluation of care process delivery. The nature of the intervention precluded blinding, but data collection and analysis were independent of project delivery teams. Results We studied 24,059 AKI episodes, finding an overall 30-day mortality of 24.5%, with no difference between control and intervention periods. Hospital length of stay was reduced with the intervention (decreases of 0.7, 1.1, and 1.3 days at the 0.5, 0.6, and 0.7 quantiles, respectively). AKI incidence increased and was mirrored by an increase in the proportion of patients with a coded diagnosis of AKI. Our assessment of process measures in 1048 patients showed improvements in several metrics including AKI recognition, medication optimization, and fluid assessment. Conclusions A complex, hospital-wide intervention to reduce harm associated with AKI did not reduce 30-day AKI mortality but did result in reductions in hospital length of stay, accompanied by improvements in in quality of care. An increase in AKI incidence likely reflected improved recognition.

Arterial Stiffness in the Heart Disease of CKD.

Abstract: CKD frequently leads to chronic cardiac dysfunction. This complex relationship has been termed as cardiorenal syndrome type 4 or cardio-renal link. Despite numerous studies and reviews focused on the pathophysiology and therapy of this syndrome, the role of arterial stiffness has been frequently overlooked. In this regard, several pathogenic factors, including uremic toxins (i.e., uric acid, phosphates, endothelin-1, advanced glycation end-products, and asymmetric dimethylarginine), can be involved. Their effect on the arterial wall, direct or mediated by chronic inflammation and oxidative stress, results in arterial stiffening and decreased vascular compliance. The increase in aortic stiffness results in increased cardiac workload and reduced coronary artery perfusion pressure that, in turn, may lead to microvascular cardiac ischemia. Conversely, reduced arterial stiffness has been associated with increased survival. Several approaches can be considered to reduce vascular stiffness and improve vascular function in patients with CKD. This review primarily discusses current understanding of the mechanisms concerning uremic toxins, arterial stiffening, and impaired cardiac function, and the therapeutic options to reduce arterial stiffness in patients with CKD.

Single-Cell RNA Sequencing Identifies Candidate Renal Resident Macrophage Gene Expression Signatures across Species.

Abstract: Background Resident macrophages regulate homeostatic and disease processes in multiple tissues, including the kidney. Despite having well defined markers to identify these cells in mice, technical limitations have prevented identification of a similar cell type across species. The inability to identify resident macrophage populations across species hinders the translation of data obtained from animal model to human patients. Methods As an entry point to determine novel markers that could identify resident macrophages across species, we performed single-cell RNA sequencing (scRNAseq) analysis of all T and B cell–negative CD45 + innate immune cells in mouse, rat, pig, and human kidney tissue. Results We identified genes with enriched expression in mouse renal resident macrophages that were also present in candidate resident macrophage populations across species. Using the scRNAseq data, we defined a novel set of possible cell surface markers (Cd74 and Cd81) for these candidate kidney resident macrophages. We confirmed, using parabiosis and flow cytometry, that these proteins are indeed enriched in mouse resident macrophages. Flow cytometry data also indicated the existence of a defined population of innate immune cells in rat and human kidney tissue that coexpress CD74 and CD81, suggesting the presence of renal resident macrophages in multiple species. Conclusions Based on transcriptional signatures, our data indicate that there is a conserved population of innate immune cells across multiple species that have been defined as resident macrophages in the mouse. Further, we identified potential cell surface markers to allow for future identification and characterization of this candidate resident macrophage population in mouse, rat, and pig translational studies.

Single-Cell RNA Profiling of Glomerular Cells Shows Dynamic Changes in Experimental Diabetic Kidney Disease

Abstract: Background Recent single-cell RNA sequencing (scRNA-seq) analyses have offered much insight into cell-specific gene expression profiles in normal kidneys. However, in diseased kidneys, understanding of changes in specific cells, particularly glomerular cells, remains limited. Methods To elucidate the glomerular cell–specific gene expression changes in diabetic kidney disease, we performed scRNA-seq analysis of isolated glomerular cells from streptozotocin-induced diabetic endothelial nitric oxide synthase (eNOS)–deficient (eNOS −/− ) mice and control eNOS −/− mice. Results We identified five distinct cell populations, including glomerular endothelial cells, mesangial cells, podocytes, immune cells, and tubular cells. Using scRNA-seq analysis, we confirmed the expression of glomerular cell–specific markers and also identified several new potential markers of glomerular cells. The number of immune cells was significantly higher in diabetic glomeruli compared with control glomeruli, and further cluster analysis showed that these immune cells were predominantly macrophages. Analysis of differential gene expression in endothelial and mesangial cells of diabetic and control mice showed dynamic changes in the pattern of expressed genes, many of which are known to be involved in diabetic kidney disease. Moreover, gene expression analysis showed variable responses of individual cells to diabetic injury. Conclusions Our findings demonstrate the ability of scRNA-seq analysis in isolated glomerular cells from diabetic and control mice to reveal dynamic changes in gene expression in diabetic kidneys, with variable responses of individual cells. Such changes, which might not be apparent in bulk transcriptomic analysis of glomerular cells, may help identify important pathophysiologic factors contributing to the progression of diabetic kidney disease.

Investigating the Relationship between Cerebral Blood Flow and Cognitive Function in Hemodialysis Patients.

Abstract: Background The immediate and longer-term effects of hemodialysis on cerebral circulation, cerebral structure, and cognitive function are poorly understood. Methods In a prospective observational cohort study of 97 adults (median age 59 years) receiving chronic hemodialysis, we used transcranial Doppler ultrasound to measure cerebral arterial mean flow velocity (MFV) throughout dialysis. Using a well validated neuropsychological protocol, we assessed cognitive function during and off dialysis and after 12 months of treatment. We also used brain magnetic resonance imaging (MRI) to assess atrophy, white matter hyperintensities (WMHs), and diffusion parameters, and tested correlations between MFV, cognitive scores, and changes on MRI. Results MFV declined significantly during dialysis, correlating with ultrafiltrate volumes. Percentage of decline in MFV correlated with intradialytic decline in cognitive function, including global function, executive function, and verbal fluency. At follow-up, 73 patients were available for repeat testing, 34 of whom underwent repeat MRI. In a subgroup of patients followed for 12 months of continued dialysis, percentage of decline in MFV correlated significantly with lower global and executive function and with progression of WMH burden (a marker of small vessel disease). Twelve of 15 patients who received renal transplants during follow-up had both early and follow-up off-dialysis assessments. After transplant, patients' memory (on a delayed recall test) improved significantly; increased fractional anisotropy of white matter (a measure of cerebral diffusion) in these patients correlated with improving executive function. Conclusions Patients undergoing hemodialysis experience transient decline in cerebral blood flow, correlating with intradialytic cognitive dysfunction. Progressive cerebrovascular disease occurred in those continuing dialysis, but not in transplanted patients. Cognitive function and cerebral diffusion improved after transplant.

Indoxyl Sulfate and p-Cresyl Sulfate Promote Vascular Calcification and Associate with Glucose Intolerance.

Abstract: Background Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. Methods To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. Results Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucometabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. Conclusions In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis.

Therapeutic Inhibition of VEGF Signaling and Associated Nephrotoxicities.

Abstract: Inhibition of vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling is a common therapeutic strategy in oncology, with new drugs continuously in development. In this review, we consider the experimental and clinical evidence behind the diverse nephrotoxicities associated with the inhibition of this pathway. We also review the renal effects of VEGF inhibition's mediation of key downstream signaling pathways, specifically MAPK/ERK1/2, endothelial nitric oxide synthase, and mammalian target of rapamycin (mTOR). Direct VEGFA inhibition via antibody binding or VEGF trap (a soluble decoy receptor) is associated with renal-specific thrombotic microangiopathy (TMA). Reports also indicate that tyrosine kinase inhibition of the VEGF receptors is preferentially associated with glomerulopathies such as minimal change disease and FSGS. Inhibition of the downstream pathway RAF/MAPK/ERK has largely been associated with tubulointerstitial injury. Inhibition of mTOR is most commonly associated with albuminuria and podocyte injury, but has also been linked to renal-specific TMA. In all, we review the experimentally validated mechanisms by which VEGFA-VEGFR2 inhibitors contribute to nephrotoxicity, as well as the wide range of clinical manifestations that have been reported with their use. We also highlight potential avenues for future research to elucidate mechanisms for minimizing nephrotoxicity while maintaining therapeutic efficacy.

Whole-Exome Sequencing Enables a Precision Medicine Approach for Kidney Transplant Recipients.

Abstract: Background Whole-exome sequencing (WES) finds a CKD-related mutation in approximately 20% of patients presenting with CKD before 25 years of age. Although provision of a molecular diagnosis could have important implications for clinical management, evidence is lacking on the diagnostic yield and clinical utility of WES for pediatric renal transplant recipients. Methods To determine the diagnostic yield of WES in pediatric kidney transplant recipients, we recruited 104 patients who had received a transplant at Boston Children’s Hospital from 2007 through 2017, performed WES, and analyzed results for likely deleterious variants in approximately 400 genes known to cause CKD. Results By WES, we identified a genetic cause of CKD in 34 out of 104 (32.7%) transplant recipients. The likelihood of detecting a molecular genetic diagnosis was highest for patients with urinary stone disease (three out of three individuals), followed by renal cystic ciliopathies (seven out of nine individuals), steroid-resistant nephrotic syndrome (nine out of 21 individuals), congenital anomalies of the kidney and urinary tract (ten out of 55 individuals), and chronic glomerulonephritis (one out of seven individuals). WES also yielded a molecular diagnosis for four out of nine individuals with ESRD of unknown etiology. The WES-related molecular genetic diagnosis had implications for clinical care for five patients. Conclusions Nearly one third of pediatric renal transplant recipients had a genetic cause of their kidney disease identified by WES. Knowledge of this genetic information can help guide management of both transplant patients and potential living related donors.

A Randomized Trial of Magnesium Oxide and Oral Carbon Adsorbent for Coronary Artery Calcification in Predialysis CKD.

Abstract: Background Developing strategies for managing coronary artery calcification (CAC) in patients with CKD is an important clinical challenge. Experimental studies have demonstrated that magnesium inhibits vascular calcification, whereas the uremic toxin indoxyl sulfate aggravates it. Methods To assess the efficacy of magnesium oxide (MgO) and/or the oral carbon adsorbent AST-120 for slowing CAC progression in CKD, we conducted a 2-year, open-label, randomized, controlled trial, enrolling patients with stage 3−4 CKD with risk factors for CAC (diabetes mellitus, history of cardiovascular disease, high LDL cholesterol, or smoking). Using a two-by-two factorial design, we randomly assigned patients to an MgO group or a control group, and to an AST-120 group or a control group. The primary outcome was percentage change in CAC score. Results We terminated the study prematurely after an interim analysis with the first 125 enrolled patients (of whom 96 completed the study) showed that the median change in CAC score was significantly smaller for MgO versus control (11.3% versus 39.5%). The proportion of patients with an annualized percentage change in CAC score of ≥15% was also significantly lower for MgO compared with control (23.9% versus 62.0%). However, MgO did not suppress the progression of thoracic aorta calcification. The MgO group’s dropout rate was higher than that of the control group (27% versus 17%), primarily due to diarrhea. The percentage change in CAC score did not differ significantly between the AST-120 and control groups. Conclusions MgO, but not AST-120, appears to be effective in slowing CAC progression. Larger-scale trials are warranted to confirm these findings.

Long-Term Outcomes after Acute Rejection in Kidney Transplant Recipients: An ANZDATA Analysis.

Abstract: Background Declining rates of acute rejection (AR) and the high rate of 1-year graft survival among patients with AR have prompted re-examination of AR as an outcome in the clinic and in trials. Yet AR and its treatment may directly or indirectly affect longer-term outcomes for kidney transplant recipients. Methods To understand the long-term effect of AR on outcomes, we analyzed data from the Australia and New Zealand Dialysis and Transplant Registry, including 13,614 recipients of a primary kidney-only transplant between 1997 and 2017 with at least 6 months of graft function. The associations between AR within 6 months post-transplant and subsequent cause-specific graft loss and death were determined using Cox models adjusted for baseline donor, recipient, and transplant characteristics. Results AR occurred in 2906 recipients (21.4%) and was associated with graft loss attributed to chronic allograft nephropathy (hazard ratio [HR], 1.39; 95% confidence interval [95% CI], 1.23 to 1.56) and recurrent AR beyond month 6 (HR, 1.85; 95% CI, 1.39 to 2.46). Early AR was also associated with death with a functioning graft (HR, 1.22; 95% CI, 1.08 to 1.36), and with death due to cardiovascular disease (HR, 1.30; 95% CI, 1.11 to 1.53) and cancer (HR, 1.35; 95% CI, 1.12 to 1.64). Sensitivity analyses restricted to subgroups with either biopsy-proven, antibody-mediated, or vascular rejection, or stratified by treatment response produced similar results. Conclusions AR is associated with increased risks of longer-term graft failure and death, particularly death from cardiovascular disease and cancer. The results suggest AR remains an important short-term outcome to monitor in kidney transplantation and clinical trials.

Evaluating Glomerular Filtration Rate Slope as a Surrogate End Point for ESKD in Clinical Trials : An Individual Participant Meta-Analysis of Observational Data

Abstract: BACKGROUND: Decline in eGFR is a biologically plausible surrogate end point for the progression of CKD in clinical trials. However, it must first be tested to ensure strong associations with clinical outcomes in diverse populations, including patients with higher eGFR. METHODS: To investigate the association between 1-, 2-, and 3-year changes in eGFR (slope) with clinical outcomes over the long term, we conducted a random effects meta-analysis of 3,758,551 participants with baseline eGFR≥60 ml/min per 1.73 m2 and 122,664 participants with eGFR<60 ml/min per 1.73 m2 from 14 cohorts followed for an average of 4.2 years. RESULTS: Slower eGFR decline by 0.75 ml/min per 1.73 m2 per year over 2 years was associated with lower risk of ESKD in participants with baseline eGFR≥60 ml/min per 1.73 m2 (adjusted hazard ratio, 0.70; 95% CI, 0.68 to 0.72) and eGFR<60 ml/min per 1.73 m2 (0.71; 95% CI, 0.68 to 0.74). The relationship was stronger with 3-year slope. For a rapidly progressing population with predicted 5-year risk of ESKD of 8.3%, an intervention that reduced eGFR decline by 0.75 ml/min per 1.73 m2 per year over 2 years would reduce the ESKD risk by 1.6%. For a hypothetical low-risk population with a predicted 5-year ESKD risk of 0.58%, the same intervention would reduce the risk by only 0.13%. CONCLUSIONS: Slower decline in eGFR was associated with lower risk of subsequent ESKD, even in participants with eGFR≥60 ml/min per 1.73 m2, but those with the highest risk would be expected to benefit the most.

Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury.

Abstract: Background After bilateral kidney ischemia/reperfusion injury (IRI), monocytes infiltrate the kidney and differentiate into proinflammatory macrophages in response to the initial kidney damage, and then transition to a form that promotes kidney repair. In the setting of unilateral IRI (U-IRI), however, we have previously shown that macrophages persist beyond the time of repair and may promote fibrosis. Methods Macrophage homing/survival signals were determined at 14 days after injury in mice subjected to U-IRI and in vitro using coculture of macrophages and tubular cells. Mice genetically engineered to lack Ccr2 and wild-type mice were treated ±CCR2 antagonist RS102895 and subjected to U-IRI to quantify macrophage accumulation, kidney fibrosis, and inflammation 14 and 30 days after the injury. Results Failure to resolve tubular injury after U-IRI results in sustained expression of granulocyte-macrophage colony-stimulating factor by renal tubular cells, which directly stimulates expression of monocyte chemoattractant protein-1 (Mcp-1) by macrophages. Analysis of CD45+ immune cells isolated from wild-type kidneys 14 days after U-IRI reveals high-level expression of the MCP-1 receptor Ccr2. In mice lacking Ccr2 and wild-type mice treated with RS102895, the numbers of macrophages, dendritic cells, and T cell decreased following U-IRI, as did the expression of profibrotic growth factors and proimflammatory cytokines. This results in a reduction in extracellular matrix and kidney injury markers. Conclusions GM-CSF-induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating immune cells and myofibroblasts, and promotes sustained inflammation and tubular injury with progressive interstitial fibrosis in the late stages of U-IRI.

Single-Cell Transcriptomic Map of the Human and Mouse Bladders

Abstract: Background Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. Methods We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. Results We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A + and HRH2 + interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1 + epithelial cells that may be involved with bladder emptying. We verify these TNNT1 + epithelial cells also occur in rat and mouse bladders. Conclusions This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

Effect of Canagliflozin on Renal and Cardiovascular Outcomes across Different Levels of Albuminuria: Data from the CANVAS Program

Abstract: BACKGROUND If SGLT2 inhibitors protect the kidneys by reducing albuminuria as hypothesized, people with type 2 diabetes mellitus (T2DM) with higher albuminuria should benefit more. METHODS We conducted a post-hoc analysis of data from the CANagliflozin cardioVascular Assessment Study (CANVAS) Program, which randomized 10,142 participants with T2DM and high cardiovascular risk to canagliflozin or placebo. We assessed effects of canagliflozin on renal, cardiovascular, and safety outcomes by baseline albuminuria. The trial included 2266 participants (22.3%) with moderately increased albuminuria (urinary albumin/creatinine ratio [UACR] 30-300 mg/g) and 760 (7.5%) with severely increased albuminuria (UACR >300 mg/g) at baseline. RESULTS Canagliflozin lowered albuminuria with greater proportional reductions in those with moderately and severely increased albuminuria (P heterogeneity<0.001). After week 13, canagliflozin slowed the annual loss of kidney function across albuminuria subgroups, with greater absolute reductions in participants with severely increased albuminuria (placebo-subtracted difference 3.01 ml/min per 1.73 m2 per year; P heterogeneity<0.001). Heterogeneity for the renal composite outcome of 40% reduction in eGFR, ESKD, or renal-related death was driven by lesser effects in participants with moderately increased albuminuria (P heterogeneity=0.03), but no effect modification was observed when albuminuria was fitted as a continuous variable (P heterogeneity=0.94). Cardiovascular and safety outcomes were mostly consistent across albuminuria levels including increased risks for amputation across albuminuria subgroups (P heterogeneity=0.66). Greater absolute risk reductions in the renal composite outcome were observed in participants with severely increased albuminuria (P heterogeneity=0.004). CONCLUSIONS The proportional effects of canagliflozin on renal and cardiovascular outcomes are mostly consistent across patients with different levels of albuminuria, but absolute benefits are greatest among those with severely increased albuminuria.

A Phase 3b, Randomized, Double-Blind, Placebo-Controlled Study of Sodium Zirconium Cyclosilicate for Reducing the Incidence of Predialysis Hyperkalemia.

Abstract: Background Patients with ESRD have minimal renal potassium excretion and, despite hemodialysis, often have persistent predialysis hyperkalemia. The DIALIZE study ({"type":"clinical-trial","attrs":{"text":"NCT03303521","term_id":"NCT03303521"}}NCT03303521) evaluated sodium zirconium cyclosilicate (SZC) in the management of hyperkalemia in hemodialysis patients.

Use of Highly Individualized Complement Blockade Has Revolutionized Clinical Outcomes after Kidney Transplantation and Renal Epidemiology of Atypical Hemolytic Uremic Syndrome

Abstract: Background Atypical hemolytic uremic syndrome (HUS) is associated with high recurrence rates after kidney transplant, with devastating outcomes. In late 2011, experts in France recommended the use of highly individualized complement blockade-based prophylaxis with eculizumab to prevent post-transplant atypical HUS recurrence throughout the country. Methods To evaluate this strategy's effect on kidney transplant prognosis, we conducted a retrospective multicenter study from a large French nationwide registry, enrolling all adult patients with atypical HUS who had undergone complement analysis and a kidney transplant since January 1, 2007. To assess how atypical HUS epidemiology in France in the eculizumab era evolved, we undertook a population-based cohort study that included all adult patients with atypical HUS (n=397) between 2007 and 2016. Results The first study included 126 kidney transplants performed in 116 patients, 58.7% and 34.1% of which were considered to be at a high and moderate risk of atypical HUS recurrence, respectively. Eculizumab prophylaxis was used in 52 kidney transplants, including 39 at high risk of recurrence. Atypical HUS recurred after 43 (34.1%) of the transplants; in four cases, patients had received eculizumab prophylaxis and in 39 cases they did not. Use of prophylactic eculizumab was independently associated with a significantly reduced risk of recurrence and with significantly longer graft survival. In the second, population-based cohort study, the proportion of transplant recipients among patients with ESKD and atypical HUS sharply increased between 2012 and 2016, from 46.2% to 72.3%, and showed a close correlation with increasing eculizumab use among the transplant recipients. Conclusions Results from this observational study are consistent with benefit from eculizumab prophylaxis based on pretransplant risk stratification and support the need for a rigorous randomized trial.

Effects of Nicotinamide and Lanthanum Carbonate on Serum Phosphate and Fibroblast Growth Factor-23 in CKD: The COMBINE Trial.

Abstract: Background Higher serum phosphate and fibroblast growth factor-23 (FGF23) levels may be modifiable to prevent cardiovascular disease in CKD. Short-term studies have reported modest efficacy in phosphate and FGF23 reduction with intestinal phosphate binders in CKD. Methods To investigate effects of lanthanum carbonate (LC; a phosphate binder) and/or nicotinamide (NAM; an inhibitor of active intestinal phosphate transport) on serum phosphate and FGF23 in stage 3b/4 CKD, we conducted a randomized trial among individuals with eGFR 20–45 ml/min per 1.73 m 2 to NAM (750 mg twice daily) plus LC (1000 mg thrice daily), NAM plus LC placebo, LC plus NAM placebo, or double placebo for 12 months. Dual primary end points were change from baseline in serum phosphate and intact FGF23 concentrations. Results Mean eGFR for the 205 participants was 32ml/min per 1.73 m 2 . At baseline, serum phosphate was 3.7 mg/dl and median FGF23 was 99 pg/ml (10th, 90th percentiles: 59, 205). Mean rates of change in phosphate increased slightly over 12 months in all groups and did not differ significantly across arms. Similarly, percent changes in FGF23 per 12 months increased for all arms except LC plus placebo, and did not differ significantly across arms. Gastrointestinal symptoms limited adherence. Adverse events rates were similar across arms. Conclusions LC and/or NAM treatment did not significantly lower serum phosphate or FGF23 in stage 3b/4 CKD over 12 months. Although these agents appeared safe, intestinal symptoms limited adherence. Reducing phosphate and FGF23 in nondialysis CKD will require new approaches.

Overall and Site-Specific Cancer Mortality in Patients on Dialysis and after Kidney Transplant

Abstract: Background Patients with ESRD have a substantially increased cancer risk, but few studies have examined the patterns of cancer mortality along a patient9s journey from dialysis to transplantation. Methods We identified all Australian patients on dialysis and patients with transplants from 1980 to 2014 from the Australia and New Zealand Dialysis and Transplant Registry. Using standardized mortality ratios (SMRs), we compared cancer mortality among patients on dialysis and patients with transplants versus the general population (overall and by age, sex, year, and site); we also performed a subgroup analysis excluding patients with preexisting cancers. Results We followed 52,936 patients on dialysis and 16,820 transplant recipients for 170,055 and 128,352 patient-years, respectively. There were 2739 cancer deaths among patients on dialysis and 923 cancer deaths among transplant recipients. Overall, cancer SMRs were 2.6 for patients on dialysis and 2.7 for transplant recipients. For patients on dialysis, SMRs were highest for multiple myeloma (30.5), testicular cancer (17.0), and kidney cancer (12.5); for transplant recipients, SMRs were highest for non-Hodgkin lymphoma (10.7), kidney cancer (7.8), and melanoma (5.8). Some 61.0% of patients on dialysis and 9.6% of transplant recipients who experienced cancer death had preexisting cancer. The SMRs for de novo cancer was 1.2 for patients on dialysis and 2.6 for transplant recipients. Conclusions Patients on dialysis and transplant recipients experienced >2.5-fold increased risk of cancer death compared with the general population. This increased risk was largely driven by preexisting cancers in patients on dialysis and de novo cancers in patients with transplants.