Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

SIGNIFICANCE STATEMENT: CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. BACKGROUND: Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. METHODS: To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. RESULTS: Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 ß than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CONCLUSIONS: CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Impact of antimetabolite discontinuation following cytomegalovirus or BK polyoma virus infection in kidney transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) and BK polyoma virus (BKV) infection following kidney transplantation have been associated with allograft dysfunction and allograft loss. Reduction in immunosuppression is a mainstay of management yet has been associated with increased risk of rejection. According to international consensus guidelines, one approach to management of these viral infections is to discontinue the antimetabolite. Little is known surrounding long-term outcomes in these patients, and it remains unclear if consideration should be given to resuming the antimetabolite as variable re-escalation strategies have been reported. The objective was to describe episodes of rejection and identify risk factors for rejection following antimetabolite withdrawal after CMV or BKV DNAemia in kidney transplant recipients. METHODS: This single-center, retrospective review evaluated adult kidney transplant recipients with a serum CMV or BKV DNA PCR ≥500 copies/ml who underwent antimetabolite discontinuation. The primary outcome assessed was the incidence of biopsy-proven acute rejection (BPAR). RESULTS: One hundred fifty-nine patients were included. Overall, 14 patients (8.8%) experienced BPAR at a median of 1.6 years after antimetabolite discontinuation. Compared to CMV, discontinuation after BKV DNAemia was associated with a higher incidence of BPAR. Characteristics observed more frequently in patients with BPAR included younger age, female sex, higher initial viral load, and development of de novo donor-specific antibody (DSA). CONCLUSION: These findings suggest that antimetabolite discontinuation after CMV or BKV DNAemia in kidney transplant recipients is a reasonable and safe approach. Further prospective studies investigating optimal immunosuppression management following CMV or BKV DNAemia in kidney transplant recipients are warranted.

Monocytes and Macrophages in Kidney Transplantation and Insights from Single Cell RNA-Seq Studies.

Single-cell RNA sequencing (scRNA-seq) is a powerful technology that allows for the identification of minority cell types in complex tissues, such as immune cells in the kidney. Previously, gene expression from infrequent cell types was missed using bulk RNA-sequencing methods due to an averaging effect. Additionally, scRNA-seq facilitates assignment of cell origin in a sample, a shortcoming of previous bulk sequencing technologies. Thus, scRNA-seq is ideal to study the immune cell landscape and the alloimmune response in the human kidney transplant. However, there are few studies published to date. Macrophages are known to play an important role in health and disease in the kidney. Furthermore, it is known that macrophages play key roles in rejection of the kidney transplant. The definition, ontogeny, and function of these cells is complex and nomenclature has evolved as new technologies have become available. In this review, an overview is provided of monocyte and macrophage nomenclature, ontogeny, and function, with a specific focus on kidney transplantation, and including novel scRNA-seq findings. scRNA-seq offers an unbiased transcriptional approach to defining macrophages and provides insights into macrophage ontogeny and function not possible with contemporary methods.

CAR-T therapy in solid organ transplant recipients with treatment refractory posttransplant lymphoproliferative disorder.

Chimeric antigen receptor T cells (CAR-T) are genetically modified T cells with a chimeric antigen receptor directed against a specific tumor-associated antigen like CD19 in lymphoma. CAR-T cells have shown encouraging activity against recurrent and refractory diffuse large B cell lymphomas (DLBCL). However concurrent use of immunosuppressive agents was prohibited in most CAR-T trials effectively excluding patients with prior solid organ transplantation (SOT) and posttransplant lymphoproliferative disorders (PTLD). We report the outcomes for three patients with PTLD refractory to immunochemotherapy 10-20 years after SOT who received CAR-T therapy between January 2018 and December 2019. One patient had an orthotopic heart transplant, the second had a deceased donor kidney transplant, and the third had a pancreas after kidney transplant (PAK). All patients developed complications of CAR-T therapy such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome, and acute kidney injury requiring renal replacement therapy in the two out of three patients. All patients expired after withdrawal of care due to lack of response to CAR-T therapy. In addition, the PAK patient developed acute pancreatitis after CAR-T therapy. This case series identifies the challenges of using CAR-T therapy to manage refractory PTLD in SOT recipients and its possible complications.

Discovery of driver non-coding splice-site-creating mutations in cancer.

Non-coding mutations can create splice sites, however the true extent of how such somatic non-coding mutations affect RNA splicing are largely unexplored. Here we use the MiSplice pipeline to analyze 783 cancer cases with WGS data and 9494 cases with WES data, discovering 562 non-coding mutations that lead to splicing alterations. Notably, most of these mutations create new exons. Introns associated with new exon creation are significantly larger than the genome-wide average intron size. We find that some mutation-induced splicing alterations are located in genes important in tumorigenesis (ATRX, BCOR, CDKN2B, MAP3K1, MAP3K4, MDM2, SMAD4, STK11, TP53 etc.), often leading to truncated proteins and affecting gene expression. The pattern emerging from these exon-creating mutations suggests that splice sites created by non-coding mutations interact with pre-existing potential splice sites that originally lacked a suitable splicing pair to induce new exon formation. Our study suggests the importance of investigating biological and clinical consequences of noncoding splice-inducing mutations that were previously neglected by conventional annotation pipelines. MiSplice will be useful for automatically annotating the splicing impact of coding and non-coding mutations in future large-scale analyses.

Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing To Define Immune Cell Chimerism in the Rejecting Kidney Transplant.

BACKGROUND: In solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells. METHODS: Whole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples. RESULTS: Analysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation. CONCLUSIONS: Analysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.

Cathepsin S and Protease-Activated Receptor-2 Drive Alloimmunity and Immune Regulation in Kidney Allograft Rejection.

Alloantigen presentation is an essential process in acute allorejection. In this context, we speculated on a pathogenic role of cathepsin S (Cat-S), a cysteine protease known to promote antigenic peptide loading into MHC class II and to activate protease-activated receptor (PAR)-2 on intrarenal microvascular endothelial and tubular epithelial cells. Single-cell RNA sequencing and immunostaining of human kidney allografts confirmed Cat-S expression in intrarenal mononuclear phagocytes. In vitro, Cat-S inhibition suppressed CD4 + T cell lymphocyte activation in a mixed lymphocyte assay. In vivo, we employed a mouse model of kidney transplantation that showed preemptive Cat-S inhibition significantly protected allografts from tubulitis and intimal arteritis. To determine the contribution of PAR-2 activation, first, Balb/c donor kidneys were transplanted into Balb/c recipient mice without signs of rejection at day 10. In contrast, kidneys from C57BL/6J donor mice revealed severe intimal arteritis, tubulitis, interstitial inflammation, and glomerulitis. Kidneys from Par2-deficient C57BL/6J mice revealed partial protection from tubulitis and lower intrarenal expression levels for Fasl, Tnfa, Ccl5, and Ccr5. Together, we conclude that Cat-S and PAR-2 contribute to immune dysregulation and kidney allograft rejection, possibly involving Cat-S-mediated activation of PAR-2 on recipient parenchymal cells in the allograft.

Impact of metformin on malignancy in solid organ transplantation.

Malignancy after solid organ transplant is a common occurrence that is associated with increased morbidity and mortality. Literature in the general diabetic population has identified possible antineoplastic properties of metformin. This retrospective study aims to determine if metformin results in a malignancy risk reduction in a cohort of diabetic kidney, liver, and heart transplant recipients. The population included transplant recipients without use of metformin at any time point (DMO arm, n = 147) and those with use of metformin (DMM arm, n = 172); the two arms were matched based on organ type and transplant date prior to application of exclusion criteria. Recipients with prior malignancy, malignancy before diabetes diagnosis, and metformin duration <30 days were excluded. The primary endpoint of malignancy first occurrence post-transplant was not found to be statistically significant at 1, 5, 10, and 15 years. In the subgroup of heart transplant recipients, there was a significant reduction in malignancy at 15 years post-transplant. Older age and Caucasian race were identified as significant risk factors for malignancy, while never smoker was a protective factor. Metformin use in this solid organ transplant cohort was not found to significantly reduce malignancy risk compared to use of other anti-diabetic agents.

Poor Outcomes With the Use of Checkpoint Inhibitors in Kidney Transplant Recipients.

BACKGROUND: Checkpoint inhibitors are now frequently used for oncologic conditions. The impact of these therapies in solid organ transplant recipients was not assessed in clinical trials. Subsequent case reports highlight the major detrimental interactions of checkpoint inhibitors and the high risk of allograft rejection with their use. Patient outcomes have not been assessed in long-term follow-up. METHODS: We conducted a retrospective review of kidney transplant recipients with metastatic cancer who received checkpoint inhibitors at a single center between April 2015 and May 2018. RESULTS: Six kidney transplant recipients with metastatic cancers that were not responding to first-line treatments met study criteria. These include 2 with squamous cell cancers, 2 with melanoma, 1 with renal cell cancer, and 1 with adenocarcinoma of the lung. Four patients received anti-programmed cell death protein-1 (PD-1) antibody and 2 received a combination of anticytotoxic T-lymphocyte-associated protein 4 and anti-PD-1 antibodies. Three out of 6 patients developed acute kidney injury. Two were biopsy-proven acute rejections with subsequent graft failures. The third was attributed to rejection, but improved after discontinuing the checkpoint inhibitor. Five out of 6 patients had cancer progression and only 1 patient had remission. CONCLUSIONS: Providers and patients need to be aware of the high risk of rejection and the poor remission rate with the use of checkpoint inhibitors in kidney transplant patients. More research is warranted to assess the optimal maintenance immunosuppression during the use of checkpoint inhibitor therapy that would not diminish the chances of remission.

Tacrolimus Concentration-to-Dose Ratios in Kidney Transplant Recipients and Relationship to Clinical Outcomes.

INTRODUCTION: One factor impacting tacrolimus interpatient variability is the presence of CYP3A5 polymorphisms. Low tacrolimus concentration-to-dose ratios (CDRs), or rapid metabolizers (RMs), have been associated with poor graft function outcomes and higher biopsy-proven acute rejection (BPAR) rates in a predominantly white population. Pretransplant CYP genotyping is not routinely conducted, and therefore only a small number of studies have assessed the use of tacrolimus CDRs as a surrogate for metabolism. We explored differences in outcomes between patients with low tacrolimus CDRs and high tacrolimus CDRs (i.e., nonrapid metabolizers [NRMs]) in a diverse patient population. OBJECTIVE: To determine the relationship between tacrolimus CDRs and graft and patient outcomes in kidney transplant recipients at a large transplant center between 2006 and 2016. METHODS: Inclusion criteria consisted of adult kidney transplant recipients who received rabbit antithymocyte globulin induction followed by a maintenance regimen of tacrolimus, mycophenolate, and prednisone. The primary end point was BPAR at 1 year. Secondary end points included graft survival, patient survival, and toxicities. Determination of clusters was conducted using the two-step cluster analysis with a defined two-cluster distribution. Kaplan-Meier survival curves were created using the log-rank test. RESULTS: The NRM cluster consisted of 322 patients with a mean CDR of 2.91 ng/ml/mg. The RM cluster consisted of 932 patients with a mean CDR of 1.14 ng/ml/mg. The BPAR at 1 year posttransplant was 3.7% in the NRM cluster and 3.6% in the RM cluster (p=0.95). Death at 5 years was higher in the NRM group compared with the RM group for unknown reasons (p=0.03). Differences in the incidence of posttransplant toxicities were not statistically significant at any time point, except for increased rates of cutaneous cancer at 5 years and cardiovascular disease overall in the NRM group. CONCLUSION: Tailoring tacrolimus therapy early posttransplant based on CDR is not supported by the findings in this study.

WNT pathway signaling is associated with microvascular injury and predicts kidney transplant failure.

Microvascular injury is associated with accelerated kidney transplant dysfunction and allograft failure. Molecular pathology can identify new mechanisms of microvascular injury while improving on the diagnostic and prognostic capabilities of traditional histology. We conducted a case-control study of archived kidney biopsy specimens stored up to 10 years with microvascular injury (n = 50) compared with biopsy specimens without histologic injury (n = 45) from patients of similar age, race, and sex. We measured WNT gene expression with a multiplex quantification platform by using digital barcoding, given the importance of WNT reactivation to the response to wounding in the kidney microvasculature and other compartments. Of 210 genes from a commercial WNT panel, 71 were associated with microvascular injury and 79 were associated with allograft failure, with considerable overlap of genes between each set. Molecular pathology identified 46 biopsy specimens with molecular evidence of microvascular injury; 18 (39%) were either C4d negative, donor-specific antibody negative, or had no microvascular injury by histology. The majority of cases with molecular evidence of microvascular injury had poor long-term outcomes. We identified novel WNT pathway genes associated with microvascular injury and allograft failure in residual clinical biopsy specimens obtained up to 10 years earlier. Further mechanistic studies may identify the WNT pathway as a new diagnostic and therapeutic target.

Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response.

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

Bringing Renal Biopsy Interpretation Into the Molecular Age With Single-Cell RNA Sequencing.

The renal biopsy provides critical diagnostic and prognostic information to clinicians including cases of acute kidney injury, chronic kidney disease, and allograft dysfunction. Today, biopsy specimens are read using a combination of light microscopy, electron microscopy, and indirect immunofluorescence, with a limited number of antibodies. These techniques all were perfected decades ago with only incremental changes since then. By contrast, recent advances in single-cell genomics are transforming scientists' ability to characterize cells. Rather than measure the expression of several genes at a time by immunofluorescence, it now is possible to measure the expression of thousands of genes in thousands of single cells simultaneously. Here, we argue that the development of single-cell RNA sequencing offers an opportunity to describe human kidney disease comprehensively at a cellular level. It is particularly well suited for the analysis of immune cells, which are characterized by multiple subtypes and changing functions depending on their environment. In this review, we summarize the development of single-cell RNA sequencing methodologies. We discuss how these approaches are being applied in other organs, and the potential for this powerful technology to transform our understanding of kidney disease once applied to the renal biopsy.

Biomarkers to detect rejection after kidney transplantation.

Detecting acute rejection in kidney transplantation has been traditionally done using histological analysis of invasive allograft biopsies, but this method carries a risk and is not perfect. Transplant professionals have been working to develop more accurate or less invasive biomarkers that can predict acute rejection or subsequent worse allograft survival. These biomarkers can use tissue, blood or urine as a source. They can comprise individual molecules or panels, singly or in combination, across different components or pathways of the immune system. This review highlights the most recent evidence for biomarker efficacy, especially from multicenter trials.

A novel missense mutation of Wilms' Tumor 1 causes autosomal dominant FSGS.

FSGS is a clinical disorder characterized by focal scarring of the glomerular capillary tuft, podocyte injury, and nephrotic syndrome. Although idiopathic forms of FSGS predominate, recent insights into the molecular and genetic causes of FSGS have enhanced our understanding of disease pathogenesis. Here, we report a novel missense mutation of the transcriptional regulator Wilms' Tumor 1 (WT1) as the cause of nonsyndromic, autosomal dominant FSGS in two Northern European kindreds from the United States. We performed sequential genome-wide linkage analysis and whole-exome sequencing to evaluate participants from family DUK6524. Subsequently, whole-exome sequencing and direct sequencing were performed on proband DNA from family DUK6975. We identified multiple suggestive loci on chromosomes 6, 11, and 13 in family DUK6524 and identified a segregating missense mutation (R458Q) in WT1 isoform D as the cause of FSGS in this family. The identical mutation was found in family DUK6975. The R458Q mutation was not found in 1600 control chromosomes and was predicted as damaging by in silico simulation. We depleted wt1a in zebrafish embryos and observed glomerular injury and filtration defects, both of which were rescued with wild-type but not mutant human WT1D mRNA. Finally, we explored the subcellular mechanism of the mutation in vitro. WT1(R458Q) overexpression significantly downregulated nephrin and synaptopodin expression, promoted apoptosis in HEK293 cells and impaired focal contact formation in podocytes. Taken together, these data suggest that the WT1(R458Q) mutation alters the regulation of podocyte homeostasis and causes nonsyndromic FSGS.

IgG4-related tubulointerstitial nephritis associated with chronic lymphocytic leukemia.

BACKGROUND: Tubulointerstitial nephritis (TIN) is typically seen in association with drug exposure and infection or in autoimmune diseases such as Sjogren's syndrome or systemic lupus erythematosis. The recently described IgG4-related systemic diseases can affect many organ systems including the kidney and typically respond to corticosteroid treatment. CASE: We present a case of IgG4-related TIN in a patient with concomitant chronic lymphocytic leukemia. To our knowledge, IgG4-related TIN has not been associated with any hematological disorder such as chronic lymphocytic leukemia.
 CONCLUSION: We propose that all kidney biopsies with significant plasma cell infiltrate should be stained for IgG4 as response to treatment is common.