Neutrophil Activation and Neutrophil Extracellular Trap Formation in Dialysis Patients.

RATIONALE & OBJECTIVE: The removal of metabolic waste by passing blood through synthetic tubing and membranes generates an immune response, even with the most biocompatible materials available. We evaluated blood levels of neutrophil activation and cell death during dialysis to devise a set of markers by which future dialysis interventions might be measured for biocompatibility. STUDY DESIGN: Observational, case control. SETTING & PARTICIPANTS: 30 patients with end-stage kidney disease in Seattle, WA, evaluated during 30 dialysis procedures in out- and inpatient settings were compared with 27 healthy (negative) controls and 20 nondialysis patients with systemic lupus erythematosus as positive controls. PREDICTORS: Blood levels of neutrophil activation (calprotectin and peroxidase activity) and cell death (cell-free DNA and neutrophil extracellular traps) were assayed. OUTCOMES: Markers of neutrophil activation and cell death can be used to assess immune response during dialysis. ANALYTICAL APPROACH: Descriptive analysis and group comparisons. RESULTS: Intradialytic levels of neutrophil activation markers are higher than prehemodialysis levels (P < 0.05), demonstrating neutrophil activation during hemodialysis. Less neutrophil activation occurs with peritoneal dialysis (P < 0.05). Immunosuppressive treatment and anticoagulant therapy did not seem to affect the capacity of neutrophils to undergo activation with hemodialysis. Finally, levels of hemodialysis-induced neutrophil activation correlated with markers of endothelial activation (r = 0.44; P = 0.01). LIMITATIONS: Low sample size with heterogeneous patient cohort. CONCLUSIONS: Neutrophil activation occurs during hemodialysis, potentially contributing to endothelial inflammation and damage. Neutrophil activation markers are novel and sensitive measures of biocompatibility for improving dialysis.

Human Organ-Specific Endothelial Cell Heterogeneity.

The endothelium first forms in the blood islands in the extra-embryonic yolk sac and then throughout the embryo to establish circulatory networks that further acquire organ-specific properties during development to support diverse organ functions. Here, we investigated the properties of endothelial cells (ECs), isolated from four human major organs-the heart, lung, liver, and kidneys-in individual fetal tissues at three months' gestation, at gene expression, and at cellular function levels. We showed that organ-specific ECs have distinct expression patterns of gene clusters, which support their specific organ development and functions. These ECs displayed distinct barrier properties, angiogenic potential, and metabolic rate and support specific organ functions. Our findings showed the link between human EC heterogeneity and organ development and can be exploited therapeutically to contribute in organ regeneration, disease modeling, as well as guiding differentiation of tissue-specific ECs from human pluripotent stem cells.

Multicolor Flow Cytometry and Cytokine Analysis Provides Enhanced Information on Kidney Transplant Biopsies.

INTRODUCTION: Current processing of renal biopsy samples provides limited information about immune mechanisms causing kidney injury and disease activity. We used flow cytometry with transplanted kidney biopsy samples to provide more information on the immune status of the kidney. METHODS: To enhance the information available from a biopsy, we developed a technique for reducing a fraction of a renal biopsy sample to single cells for multicolor flow cytometry and quantitation of secreted cytokines present within the biopsy sample. As proof of concept, we used our technique with transplant kidney biopsy samples to provide examples of clinically relevant immune information obtainable with cytometry. RESULTS: A ratio of CD8+ to CD4+ lymphocytes greater than or equal to 1.2 in transplanted allografts is associated with rejection, even before it is apparent by microscopy. Elevated numbers of CD45 leukocytes and higher levels of interleukin (IL)-6, IL-8, and IL-10 indicate more severe injury. Antibody binding to renal microvascular endothelial cells can be measured and corresponds to antibody-mediated forms of allograft rejection. Eculizumab binding to endothelial cells suggests complement activation, which may be independent of bound antibody. We compared intrarenal leukocyte subsets and activation states to those of peripheral blood from the same donor at the time of biopsy and found significant differences; thus the need for new techniques to evaluate immune responses within the kidney. CONCLUSION: Assessment of leukocyte subsets, renal microvascular endothelial properties, and measurement of cytokines within a renal biopsy by flow cytometry enhance understanding of pathogenesis, indicate disease activity, and identify potential targets for therapy.

Identification and Quantitation of Leukocyte Populations in Human Kidney Tissue by Multi-parameter Flow Cytometry.

Inflammatory immune cells play direct pathological roles in cases of acute kidney injury (AKI) and chronic kidney disease (CKD). However, the identification and characterization of distinct populations of leukocytes in human kidney biopsies have been confounded by the limitations of immunohistochemical (IHC)-based techniques used to detect them. This methodology is not amenable to the combinations of multiple markers necessary to unequivocally define discrete immune cell populations. We have developed a multi-parameter, flow cytometric-based approach that addresses the need for panels of cell-specific markers in the identification of immune cell populations, allowing both the accurate detection and quantitation of leukocyte subpopulations from a single, clinical kidney biopsy specimen. In this approach, fresh human kidney tissue is dissociated into a single cell suspension followed by antibody-labeling and flow cytometric-based acquisition and analysis. This novel technique provides a major step forward in identifying and enumerating immune cell subpopulations in human kidney disease and is a powerful platform to complement traditional histopathological examinations of clinical kidney biopsies.

Plasma C4d+ Endothelial Microvesicles Increase in Acute Antibody-Mediated Rejection.

BACKGROUND: Antibody-mediated rejection (AMR) is a major cause of kidney allograft loss. Currently, AMR diagnosis relies on biopsy which is an invasive procedure. A noninvasive biomarker of acute AMR could lead to early diagnosis and treatment of this condition and improve allograft outcome. Microvesicles are membrane-bound vesicles released from the cell surface after injury. We hypothesized that because AMR is associated with allograft endothelial injury and C4d deposition, plasma microvesicles positive for endothelial (CD144) marker and C4d are increased in this condition. METHODS: We studied microvesicle concentration in the plasma of 95 kidney transplant patients with allograft dysfunction and compared with 23 healthy volunteers. Biopsy diagnosis and scoring was performed using Banff classification. RESULTS: In the 28 subjects with AMR, the density of C4d+/CD144+ microvesicles was on average 11-fold (P = 0.002) higher than transplant recipients with no AMR and 24-fold (P = 0.008) than healthy volunteers. Densities of C4d+ and C4d+/annexin V+ (C4d+/AVB+) microvesicles were also increased in AMR patients compared with no AMR and healthy subjects. C4d+/AVB+ microvesicles correlated with AMR biopsy severity. Nine patients with acute AMR that received treatment showed a mean 72% decrease (P = 0.01) in C4d+/CD144+ microvesicle concentration compared with pretreatment values. CONCLUSIONS: Quantification of plasma C4d+ microvesicles provides information about presence of AMR, its severity and response to treatment in transplant patients.

Development of a microphysiological model of human kidney proximal tubule function.

The kidney proximal tubule is the primary site in the nephron for excretion of waste products through a combination of active uptake and secretory processes and is also a primary target of drug-induced nephrotoxicity. Here, we describe the development and functional characterization of a 3-dimensional flow-directed human kidney proximal tubule microphysiological system. The system replicates the polarity of the proximal tubule, expresses appropriate marker proteins, exhibits biochemical and synthetic activities, as well as secretory and reabsorptive processes associated with proximal tubule function in vivo. This microphysiological system can serve as an ideal platform for ex vivo modeling of renal drug clearance and drug-induced nephrotoxicity. Additionally, this novel system can be used for preclinical screening of new chemical compounds prior to initiating human clinical trials.

A Novel Three-Dimensional Human Peritubular Microvascular System.

Human kidney peritubular capillaries are particularly susceptible to injury, resulting in dysregulated angiogenesis, capillary rarefaction and regression, and progressive loss of kidney function. However, little is known about the structure and function of human kidney microvasculature. Here, we isolated, purified, and characterized human kidney peritubular microvascular endothelial cells (HKMECs) and reconstituted a three-dimensional human kidney microvasculature in a flow-directed microphysiologic system. By combining epithelial cell depletion and cell culture in media with high concentrations of vascular endothelial growth factor, we obtained HKMECs of high purity in large quantity. Unlike other endothelial cells, isolated HKMECs depended on high vascular endothelial growth factor concentration for survival and growth and exhibited high tubulogenic but low angiogenic potential. Furthermore, HKMECs had a different transcriptional profile. Under flow, HKMECs formed a thin fenestrated endothelium with a functional permeability barrier. In conclusion, this three-dimensional HKMEC-specific microphysiologic system recapitulates human kidney microvascular structure and function and shows phenotypic characteristics different from those of other microvascular endothelial cells.

CMS-required visits adversely impact Seattle area hemodialysis program.

Chronic hemodialysis in Seattle developed with a highly patient-centric approach, providing patients the option of dialyzing in neighborhood units under the administration of a non-profit organization, the Northwest Kidney Center (NKC). This study examined the effects of a requirement in the 2009 Conditions for Coverage that in-center hemodialysis patients be visited quarterly during their treatment session. A retrospective analysis of quality outcome indicators for patients managed at the University of Washington Medical Center and dialyzing at NKC in 2008 and 2010 found that dialysis adequacy (eKt/V), phosphorus, albumin, and hemoglobin were not improved by quarterly dialysis visits. Instead, dialysis unit visits adversely impacted patient-nephrologist relationships and diverted nephrologists from other patient care activities.

Increased tubulointerstitial recruitment of human CD141(hi) CLEC9A(+) and CD1c(+) myeloid dendritic cell subsets in renal fibrosis and chronic kidney disease.

Dendritic cells (DCs) play critical roles in immune-mediated kidney diseases. Little is known, however, about DC subsets in human chronic kidney disease, with previous studies restricted to a limited set of pathologies and to using immunohistochemical methods. In this study, we developed novel protocols for extracting renal DC subsets from diseased human kidneys and identified, enumerated, and phenotyped them by multicolor flow cytometry. We detected significantly greater numbers of total DCs as well as CD141(hi) and CD1c(+) myeloid DC (mDCs) subsets in diseased biopsies with interstitial fibrosis than diseased biopsies without fibrosis or healthy kidney tissue. In contrast, plasmacytoid DC numbers were significantly higher in the fibrotic group compared with healthy tissue only. Numbers of all DC subsets correlated with loss of kidney function, recorded as estimated glomerular filtration rate. CD141(hi) DCs expressed C-type lectin domain family 9 member A (CLEC9A), whereas the majority of CD1c(+) DCs lacked the expression of CD1a and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), suggesting these mDC subsets may be circulating CD141(hi) and CD1c(+) blood DCs infiltrating kidney tissue. Our analysis revealed CLEC9A(+) and CD1c(+) cells were restricted to the tubulointerstitium. Notably, DC expression of the costimulatory and maturation molecule CD86 was significantly increased in both diseased cohorts compared with healthy tissue. Transforming growth factor-ß levels in dissociated tissue supernatants were significantly elevated in diseased biopsies with fibrosis compared with nonfibrotic biopsies, with mDCs identified as a major source of this profibrotic cytokine. Collectively, our data indicate that activated mDC subsets, likely recruited into the tubulointerstitium, are positioned to play a role in the development of fibrosis and, thus, progression to chronic kidney disease.

Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system.

Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.

Treatment of severe lactic acidosis during the pre-anhepatic stage of liver transplant surgery with intraoperative hemodialysis.

Severe uncompensated lactic acidosis manifesting during the pre-anhepatic stage of orthotopic liver transplant surgery is an uncommon event, but it poses serious concern because of the additional lactate production and impaired elimination by the liver that develops during the anhepatic and allograft reperfusion stages of the procedure. A man with end-stage liver disease secondary to hepatitis C and hemochromatosis and normal renal function, who developed severe lactic acidosis in the pre-anhepatic stage of liver transplantation, was treated successfully with intraoperative, continuous venovenous hemodialysis. Hemodialysis effectively corrected the patient's lactic acidosis and removed lactate, which contributed to hemodynamic stability during the anhepatic and graft reperfusion stages of his liver transplant surgery.

Proliferative potential of human kidney endothelial cells: bone marrow-derived cells may not be required for high proliferation.

BACKGROUND: Proliferative potential of a single cell, defined as the number of progeny it gives rise to, has been used to define a hierarchy of endothelial progenitor cells in blood. Cells with high proliferative potential are presumed to have greater capacity for endothelium repair. Based on results with commercially available endothelial cells, it has been proposed that a proliferative hierarchy of endothelial cells also exists within blood vessels. It is unknown whether such vessel-derived highly proliferative endothelial cells originate from the bone marrow or whether the supply of precursors is limited to pre-existing cells that reside within vessels. METHODS: In this study, we isolated normal human renal microvascular endothelial cells (RMEC) and larger cortical vessel endothelial cells (EC) by flow cytometry based on differential expression of human leucocyte antigen (HLA)-DR, and evaluated the proliferative potential of single cells. To determine if highly proliferative clones might derive from bone marrow recruits, HLA-DR expression on RMEC from transplanted kidneys was evaluated using antibodies that distinguish donor cells from recipient cells. RESULTS: We found the proliferative potential of kidney endothelial cells diverse and variable. Subcloning indicated that proliferative potential was determined by epigenetic events. In transplanted kidneys affected with a variety of different injuries, RMEC were donor derived. CONCLUSIONS: We conclude that endothelial cells of high proliferative potential exist within human renal blood vessels, even in individuals into their eighth decade of life, and that highly proliferative endothelial cells are unlikely to be bone marrow derived.

Higher levels of leflunomide are associated with hemolysis and are not superior to lower levels for BK virus clearance in renal transplant patients.

BACKGROUND AND OBJECTIVES: Leflunomide use in renal transplantation has been increasing. Outcome correlation and safety data are still to be refined. The goals of this study were to report one center's experience with leflunomide, specifically the correlation of leflunomide levels with the outcomes of BK nephropathy and the observed toxic effects during the treatment with leflunomide. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Leflunomide was used in 21 patients with BK nephropathy. These patients were divided into two groups on the basis of the leflunomide levels achieved: Low-level group (<40 microg/ml) and high-level group (>40 microg/ml). RESULTS: During 13 mo of follow-up, there was no difference in the rate of serum BK viral clearance between the groups. There were three graft losses in the low-level group and one in the high-level group; however, creatinine levels were higher at the time of starting leflunomide in the low-level group. Leflunomide was also used in six patients with chronic allograft injury. No graft loss was observed during the follow-up period of 16 mo. Treatment with leflunomide seemed to be associated with a new toxicity, hemolysis, seen in four of the 27 patients so treated. Patients with hemolysis had high leflunomide levels (81.4 +/- 14 microg/ml) and worsening allograft function. Two patients had histologic evidence of thrombotic microangiopathy, which led to graft loss in one patient. CONCLUSIONS: The clinical correlation between leflunomide levels and outcomes needs to be further refined. This study described a possible association of leflunomide with thrombotic microangiopathy, especially at higher levels.

Normal human kidney HLA-DR-expressing renal microvascular endothelial cells: characterization, isolation, and regulation of MHC class II expression.

Human, but not murine, renal peritubular and glomerular capillaries constitutively express class II major histocompatibility (MHC) proteins at high levels in normal human kidney. Expression of class II proteins on renal microvascular endothelial cells (RMEC) makes it available to circulating lymphocytes and imparts a surveillance capacity to RMEC for controlling inflammatory responses. In this report, the co-expression of HLA-DR and the endothelial marker CD31 are used to identify RMEC as a distinct population of cells within a standard renal biopsy using flow cytometry. A three-laser, multicolor flow cytometry analysis using Alexa dyes, developed for characterizing the expression of cell surface antigens, identifies RMEC as a population separate from HLA-DR-expressing leukocytes. HLA-DR RMEC co-express HLA-DP and HLA-DQ. RMEC also express the T cell costimulatory factor CD58 but not CD80, CD86, or CD40. On the basis of high HLA-DR expression, RMEC are isolated for culture using fluorescence-activated cell sorting and magnetic beads. Cultured RMEC require normal basal physiologic concentrations of gamma interferon (gammaIFN) to maintain HLA protein expression. This expression is regulated by CIITA, the MHC class II-specific transcription factor. Four tissue-specific promoters have been described for CIITA. In freshly isolated RMEC, RT-PCR and hybridization using specific oligonucleotide probes to CIITA promoter sequences identify only the statin-sensitive gammaIFN-induced promoter IV of CIITA. Therefore, the constitutive expression of HLA-DR on RMEC in normal human kidney is located in a position for immune surveillance, depends on basal physiologic concentrations of gammaIFN, and may be amenable to regulation with statins.