Michelle Lubetzky

Urinary Cell Levels of mRNA for OX40, OX40L, PD-1, PD-L1 or PD-L2 and Acute Rejection of Human Renal Allografts

Background. The positive costimulatory proteins OX40 and OX40L and negative regulatory proteins programmed death (PD)-1, PD ligand 1, and PD ligand 2 have emerged as significant regulators of acute rejection in experimental transplantation models. Methods. We obtained 21 urine specimens from 21 renal allograft recipients with graft dysfunction and biopsy-confirmed acute rejection and 25 specimens from 25 recipients with stable graft function and normal biopsy results (stable). Urinary cell levels of mRNAs were measured using real-time quantitative polymerase chain reaction assays, and the levels were correlated with allograft status and outcomes. Results. Levels of OX40 mRNA (P<0.0001, Mann-Whitney test), OX40L mRNA (P=0.0004), and PD-1 mRNA (P=0.004), but not the mRNA levels of PD ligand 1 (P=0.08) or PD ligand 2 (P=0.20), were significantly higher in the urinary cells from the acute rejection group than the stable group. Receiver operating characteristic curve analysis demonstrated that acute rejection is predicted with a sensitivity of 95% and a specificity of 92% (area under the curve=0.98, 95% confidence interval 0.96–1.0, P<0.0001) using a combination of levels of mRNA for OX40, OX40L, PD-1, and levels of mRNA for the previously identified biomarker Foxp3. Within the acute rejection group, levels of mRNA for OX40 (P=0.0002), OX40L (P=0.0004), and Foxp3 (P=0.04) predicted acute rejection reversal, whereas only OX40 mRNA levels (P=0.04) predicted graft loss after acute rejection. Conclusion. A linear combination of urinary cell levels of mRNA for OX40, OX40L, PD-1, and Foxp3 was a strong predictor of acute rejection in human renal allograft biopsies. This prediction model should be validated using an independent cohort of renal allograft recipients.

Early Readmission After Kidney Transplantation: Examination of Discharge-level Factors

Background Early rehospitalization after kidney transplantation (KTx) is common and is considered a quality metric. Recipient and donor risk factors for early readmission after KTx are well studied. Little data exist on discharge-level factors associated with readmission. Methods We performed a single-center, retrospective cohort study between 2011 and 2015 of adult KTx recipients to examine readmission indication, risk factors, and opportunities for reduction. Results Of 462 KTxs, 145 (31.4%) were readmitted within 30 days of discharge. The primary reason for readmission was surgery-site specific in 30 cases (20.7%). Of 115 recipients with nonsurgical indications for readmission 25 (21.7%) were related to infection, 24 (20.9%) graft dysfunction, 25 (21.7%) gastrointestinal, 25 (21.7%) metabolic, and 16 (13.9%) other reasons. On multivariate analysis significant independent predictors of early readmission were electrolyte abnormalities on the day of discharge (odds ratio [OR], 1.77; 95% confidence interval [95% CI], 1.17-2.69), 3 or more comorbidities (OR, 2.01; 95% CI, 1.04-3.86), delayed graft function at the time of discharge (OR, 1.65; 95% CI, 1.00-2.70), and post-KTx hospitalization complication (OR, 1.70; 95% CI, 1.10-2.61). Among 11.7% of patients, readmission may have been attenuated by addressing the medical issue before discharge from index hospitalization. In 28.3% of patients, readmission rates may have been reduced with continued management as an outpatient or provision of observational or same-day diagnostic resources. Conclusions Specific discharge level factors correlate with readmission irrespective of comorbidities and transplant complications. These findings may have important implications on discharge practice by aiding to identify which KTx recipients could be targeted for enhanced care transitions. Overall, potential opportunities for readmission reduction exist on multiple process levels.

HIV-infected kidney graft recipients managed with an early corticosteroid withdrawal protocol: clinical outcomes and messenger RNA profiles.

Background The outcome of HIV-infected kidney transplant recipients managed with an early corticosteroid withdrawal protocol is not known. Methods Eleven consecutive HIV-infected patients with undetectable plasma HIV RNA and more than 200/mm3 CD4+ T cells underwent deceased-donor (n=8) or living-donor (n=3) kidney transplantation at our center. All were managed with an early corticosteroid withdrawal protocol; 9 of 11 received antithymocyte globulin and 2 received basiliximab induction. We analyzed patient and graft outcomes, acute rejection rate, HIV progression, BKV replication, infections, and urinary cell mRNA profiles. Results The median (range) follow-up was 44.5 (26–73) months. The incidence of acute rejection was 9% at 1 year and the patient and allograft survival rates were 100% and 91%, respectively. Estimated glomerular filtration rate at 1 year (mean±SD) was 78±39 mL/min/1.73 m2. Plasma HIV RNA was undetectable at 24 months and none had BKV replication. Six of the 11 kidney recipients developed eight infections requiring hospitalization. Urinary cell levels of mRNA for complement components and complement regulatory proteins, cell lineage–specific proteins CD3, CD4, CD8, CTLA4, Foxp3, chemokine IP-10, cytotoxic perforin and granzyme B, and BKV VP1 mRNA were not different (P>0.05) between HIV-infected patients and HIV-negative recipients (n=22) with stable graft function and normal biopsy results. Conclusion An early steroid withdrawal regimen with antithymocyte globulin induction was associated with excellent graft and patient outcomes in HIV-infected recipients of kidney allografts. Their urinary cell mRNA profiles are indistinguishable from those of HIV-negative patients with stable graft function and normal biopsy results.

Severe hyperosmolarity and hypernatremia in an adipsic young woman.

BACKGROUNDnCombined deficits in arginine vasopressin secretion (AVP) and thirst sensation can result in life threatening hyperosmolality and hypernatremia. Complications include seizures, profound volume contraction and renal failure. Fortunately, this is an uncommon clinical condition, with approximately 70 cases reported in the literature over the past 47 years [1]. Defects in AVP secretion and/or synthesis produce central diabetes insipidus (DI), polyuria with polydipsia, hypernatremia and hyperosmolality. Most awake and alert patients with an intact thirst stimulus will drink themselves back to a normal serum sodium and osmolality. However, if there is concomitant destruction of the osmoreceptors that regulate thirst, osmolal and volume homeostasis cannot be maintained. The relationships between urine osmolarity and serum osmolarity and plasma vasopressin levels are vital for distinguishing a reset osmostat from central DI.nnnMETHODSnAfter obtaining approval from our institutional review board, we retrospectively reviewed the medical record of a 37-year-old patient who presented to our institution with a serum sodium of 176 mEq/l.nnnRESULTSnAdmission laboratory examination revealed: hemoglobin 12.8 g/dl; white blood cell count 4.7 × 103/µl, with a normal differential; random serum glucose 91 mg/dl ; sodium 176 mEq/l; plasma osmolality 366 mOsm/kg; BUN 33 mg/dl; serum creatinine 1 mg/dl; calcium 9.5 mg/dl; urine specific gravity 1.032; and urine osmolality 1,172 mOsm/kg. An MRI with contrast of the sella/ pituitary revealed an enhancing mass centered within the suprasellar cistern and anterior third ventricle, measuring 3.0 × 3.9 × 3.4 cm. The lesion appeared to involve the hypothalamus and displaced the optic chiasm inferiorly. Evaluation of pituitary function revealed normal serum levels of thyroid stimulating hormone, AM cortisol, luteinizing hormone, follicle stimulating hormone and prolactin. Figure 1 illustrates the relationship between measured serum AVP levels and serum osmolality. Figure 2 shows the relationship between measured urine and serum osmolality. If the serum AVP levels were not available, it would appear as though the patient had a reset osmostat. The kidneys appear to appropriately generate maximally concentrated urine at a serum osmolality above 348 but are unable to below this value.nnnCONCLUSIONSnWhen compared with the normal curve, our patients AVP levels were lower than expected for the corresponding osmolality. This pattern is consistent with a partial central DI. She does not have a reset osmostat. In the presence of significant volume contraction and a reduced GFR, her kidneys produced more concentrated urine despite markedly decreased central vasopressin production. As the volume contraction abated and the GFR improved, polyuria recurred, despite persistent hyperosmolarity and hypernatremia.

Impact of Cold Ischemia Time in Kidney Transplants From Donation After Circulatory Death Donors

Background Deceased-donor kidneys are exposed to ischemic events from donor instability during the process of donation after circulatory death (DCD). Clinicians may be reluctant to transplant DCD kidneys with prolonged cold ischemia time (CIT) for fear of an additional deleterious effect. Methods We performed a retrospective cohort study examining US registry data between 1998 and 2013 of adult first-time kidney-only recipients of paired kidneys (derived from the same donor transplanted into different recipients) from DCD donors. Results On multivariable analysis, death-censored graft survival (DCGS) was comparable between recipients of kidneys with higher CIT relative to paired donor recipients with lower CIT when the CIT difference was 1 hour or longer (adjusted hazard ratio, [aHR], 1.02; 95% confidence interval [CI], 0.88-1.17; n = 6276), 5 hours or longer (aHR, 0.98; 95% CI, 0.80-1.19; n = 3130), 10 hours or longer (aHR, 1.15; 95% CI, 0.82-1.60; n = 1124) or 15 hours (aHR, 1.15; 95% CI, 0.66-1.99; n = 498). There was a higher rate of primary non function in the long CIT groups for delta 1 hour or longer (0.89% vs 1.63%; P = 0.006), 5 hours (1.09% vs 1.67%, P = 0.13); 10 hours (0.53% vs 1.78%; P = 0.03), and 15 hours (0.40% vs 1.61%; P = 0.18), respectively. Between each of the 4 delta CIT levels of shorter and longer CIT, there was a significantly and incrementally higher rate of delayed graft function in the long CIT groups for delta 1 hour or longer (37.3% vs 41.7%; P < 0.001), 5 hours (35.9% vs 42.7%; P < 0.001), 10 hours (29.4% vs 44.2%, P < 0.001), and 15 hours (29.6% vs 46.1%, P < 0.001), respectively. Overall patient survival was comparable with delta CITs of 1 hour or longer (aHR, 0.96; 95% CI, 0.84-1.08), 5 hours (aHR, 1.01; 95% CI, 0.85-1.20), and 15 hours (aHR, 1.27; 95% CI, 0.79-2.06) but not 10 hours (aHR, 1.47; 95% CI, 1.09-1.98). Conclusions These results suggest that in the setting of a prior ischemic donor event, prolonged CIT has limited bearing on long-term outcomes.

Urine biomarkers informative of human kidney allograft rejection and tolerance

We developed urinary cell messenger RNA (mRNA) profiling to monitor in vivo status of human kidney allografts based on our conceptualization that the kidney allograft may function as an in vivo flow cell sorter allowing access of graft infiltrating cells to the glomerular ultrafiltrate and that interrogation of urinary cells is informative of allograft status. For the profiling urinary cells, we developed a two-step preamplification enhanced real-time quantitative PCR (RT-QPCR) assays with a customized amplicon; preamplification compensating for the low RNA yield from urine and the customized amplicon facilitating absolute quantification of mRNA and overcoming the inherent limitations of relative quantification widely used in RT-QPCR assays. Herein, we review our discovery and validation of urinary cell mRNAs as noninvasive biomarkers prognostic and diagnostic of acute cellular rejection (ACR) in kidney allografts. We summarize our results reflecting the utility of urinary cell mRNA profiling for predicting reversal of ACR with anti-rejection therapy; differential diagnosis of kidney allograft dysfunction; and noninvasive diagnosis and prognosis of BK virus nephropathy. Messenger RNA profiles associated with human kidney allograft tolerance are also summarized in this review. Altogether, data supporting the idea that urinary cell mRNA profiles are informative of kidney allograft status and tolerance are reviewed in this report.

Influence of Cold Ischemia Time in Kidney Transplants From Small Pediatric Donors

Background Clinicians may be reluctant to transplant small pediatric kidneys that have prolonged cold ischemia time (CIT) for fear of an additional deleterious effect because pediatric grafts are thought to be more sensitive to ischemia. We aimed to assess the risks associated with transplantation of small pediatric kidneys with prolonged CIT. Methods We performed a retrospective cohort study examining US registry data between 1998 and 2013 of adult first-time kidney-only recipients of small pediatric kidneys from donors weighing 10 to 20 kg, stratified by CIT levels of 0 to 18 (n = 1413), 19 to 30 (n = 1116), and longer than 30 (n = 338) hours. Results All-cause graft survival by CIT groups at 1-year was 92%, 88%, and 89%, respectively. 1-year risk-adjusted graft survival hazard ratios were significantly higher with CIT of 19 to 30 hours (adjusted hazard ratios, 1.37; 95% confidence interval, 1.04-1.81) and somewhat higher with CIT greater than 30 hours (adjusted hazard ratios, 1.24; 95% confidence interval, 0.82-1.88) relative to recipients with CIT 0 to 18 hours. There was little variation in the effect of CIT on graft survival when restricted to single kidney transplants only and no significant interaction of CIT category and single kidney transplantation (P = 0.93). Conclusions Although prolonged CIT is associated with lower early graft survival in small pediatric donor kidney transplants, absolute decreases in 1-year graft survival rates were 3% to 4%.

Gut microbiota dysbiosis and diarrhea in kidney transplant recipients

Posttransplant diarrhea is associated with kidney allograft failure and death, but its etiology remains unknown in the majority of cases. Because altered gut microbial ecology is a potential basis for diarrhea, we investigated whether posttransplant diarrhea is associated with gut dysbiosis. We enrolled 71 kidney allograft recipients for serial fecal specimen collections in the first 3 months of transplantation and profiled the gut microbiota using 16S ribosomal RNA (rRNA) gene V4‐V5 deep sequencing. The Shannon diversity index was significantly lower in 28 diarrheal fecal specimens from 25 recipients with posttransplant diarrhea than in 112 fecal specimens from 46 recipients without posttransplant diarrhea. We found a lower relative abundance of 13 commensal genera (Benjamini‐Hochberg adjusted P ≤ .15) in the diarrheal fecal specimens including the same 4 genera identified in our prior study. The 28 diarrheal fecal specimens were also evaluated by a multiplexed polymerase chain reaction (PCR) assay for 22 bacterial, viral, and protozoan gastrointestinal pathogens, and 26 specimens were negative for infectious etiologies. Using PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) to predict metagenomic functions, we found that diarrheal fecal specimens had a lower abundance of metabolic genes. Our findings suggest that posttransplant diarrhea is not associated with common infectious diarrheal pathogens but with a gut dysbiosis.

Single nucleotide variant counts computed from RNA sequencing and cellular traffic into human kidney allografts

Advances in bioinformatics allow identification of single nucleotide polymorphisms (variants) from RNA sequence data. In an allograft biopsy, 2 genomes contribute to the RNA pool, 1 from the donor organ and the other from the infiltrating recipients cells. We hypothesize that imbalances in genetic variants of RNA sequence data of kidney allograft biopsies provide an objective measure of cellular infiltration of the allograft. We performed mRNA sequencing of 40 kidney allograft biopsies, selected to represent a comprehensive range of diagnostic categories. We analyzed the sequencing reads of these biopsies and of 462 lymphoblastoid cell lines from the 1000 Genomes Project, for RNA variants. The ratio of heterozygous to nonreference genome homozygous variants (Het/Hom ratio) on all autosomes was determined for each sample, and the estimation of stromal and immune cells in malignant tumors using expression data (ESTIMATE) score was computed as a complementary estimate of the degree of cellular infiltration into biopsies. The Het/Hom ratios (P = .02) and the ESTIMATE scores (P < .001) were associated with the biopsy diagnosis. Both measures correlated significantly (r = .67, P < .0001), even though the Het/Hom ratio is based on mRNA sequence variation, while the ESTIMATE score uses mRNA expression. Het/Hom ratio and the ESTIMATE score may offer unbiased and quantitative parameters for characterizing cellular traffic into human kidney allografts.

DIFFERENTIAL REGULATION OF POSITIVE OR NEGATIVE COSTIMULATORY MOLECULES DURING ACUTE REJECTION OF HUMAN RENAL ALLOGRAFTS: 1025

C. Afaneh1, M. Lubetzky2, T. Muthukumar3, R. Ding4, S. Seshan5, C. Snopkowski4, V. Sharma6, D. Dadhania3, M. Suthanthiran6 1Surgery, NY Presbyterian Hospital -Weill Cornell Medical College, New York/NY/UNITED STATES OF AMERICA, 2Nephrology, NY Presbyterian HospitalWeill Cornell Medical Center, New York/ UNITED STATES OF AMERICA, 3Medicine/nephrology, Cornell University, New York/UNITED STATES OF AMERICA, 4Nephrology, Cornell University, New York/UNITED STATES OF AMERICA, 5Pathology, Cornell University, New York/UNITED STATES OF AMERICA, 6, Weill Cornell Medical College, New York/NY/UNITED STATES OF AMERICA