Quantification of treprostinil concentration in rat and human using a novel validated and rapid liquid chromatography-tandem mass spectrometry method: Experimental and clinical applications in ischemia-reperfusion injury.

Treprostinil (Remodulin®) is a Food and Drug Administration (FDA) approved prostacyclin analog to treat pulmonary arterial hypertension. Recently, treprostinil has been investigated to reduce ischemia-reperfusion injury (IRI) during transplantation, which currently has no approved treatment. A validated analytical method is necessary to measure treprostinil concentrations in biological specimens. Here, a novel, sensitive, and specific method to measure treprostinil concentrations in rat serum, human serum, and human plasma has been developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Biological samples were processed by protein precipitation before chromatography and 6-keto Prostaglandin F1α-d4 was used as an internal standard. A gradient method was established with a total run time of 4 min. The assay was linear over the range of 0.25-75.0 ng/ml with accuracy (92.97-107.87 %), intra-assay precision (1.16-3.34 %), and inter-assay precision (1.11-4.58 %) in all biological matrices, which are within FDA acceptance criteria. No significant variation in treprostinil or 6-keto Prostaglandin F1α-d4 concentrations were observed under the investigated storage conditions. This novel, sensitive, and specific LC/MS-MS method is cost-effective and suitable for measuring treprostinil concentrations in animal studies and human biological samples for clinical applications.

Unexpected Medical Conditions Discovered During Live Donor Kidney Evaluation: Single Center Study.

OBJECTIVES: Living donor kidney transplantation (LDKT) is the preferred method of treatment for patients with end-stage kidney disease. Potential living kidney donors (PLKD) are evaluated through a thorough medical, psychological and surgical work-up to ensure successful transplantation with minimal risks to all parties involved. The transplant center at Rhode Island Hospital has noticed an increasing number of PLKDs excluded from donation due to conditions newly diagnosed during the screening process. Our objective is to understand the local trends underlying the high PLKD exclusion rates in the context of newly diagnosed conditions, age, race, and sex of the excluded donors. STUDY DESIGN AND METHODS: Our study is a retrospective electronic medical record review of the 429 PLKDs screened at Rhode Island Hospital Kidney Transplant Center between December 2012 and April 2023. Age, race, gender, relationship to recipient, and reasons for exclusion were collected from the medical record for each PLKD. CONCLUSION: 115 of the 429 total PLKDs screened were excluded for newly diagnosed conditions, the most common of which were renal issues (49%), diabetes mellitus (33%), and hypertension (13%), with many comorbid diagnoses. While these donors were able to receive proper treatment after their diagnosis, the earliest intervention possible yields the best prognosis. The high prevalence of treatable yet undiagnosed conditions raise many public health concerns, such as primary care gaps or discontinuous healthcare, and increases awareness about the importance of follow-up care for the excluded PLKDs.

Oral Nicotinamide for Actinic Keratosis Prevention in Kidney Transplant Recipients: A Pilot Double-Blind, Randomized, Placebo-Controlled Trial.

BACKGROUND: Oral nicotinamide (NAM) has shown promise in preventing actinic keratoses (AKs) in trials based outside of the United States. We assessed the efficacy of oral NAM supplementation in kidney transplant recipients with a history of keratinocyte carcinoma. MATERIAL AND METHODS: Patients enrolled in a 2-week run-in phase, during which NAM 1000 mg was taken twice daily. After a washout period, patients who tolerated the run-in phase were randomized to NAM 500 mg twice daily or placebo. At baseline, 4, 8, and 12 months, dermatologists conducted full-body skin exams to document area-specific AKs. Routine lab work was collected to ensure the stability of renal allograft function. RESULTS: The dosage was reduced from 1000 to 500 mg due to gastrointestinal symptoms in the run-in phase. Patients were randomized to NAM (n = 10) or placebo (n = 11). At 12 months, mean AK count was 30.8 (95% CI -11.7-73.4) for NAM and 26.6 (95% CI 10.8-42.5) for placebo. The difference in percent AK count change at 12 months compared with baseline was 259.8% (95% CI -385.9 to 905.5) for NAM and 72.4% (95% CI -118.6 to 263.5) for placebo. The between-group difference in percent AK change was not significant (P = .38). There was no attrition in the placebo group and 40% attrition in the NAM arm. DISCUSSION: Nicotinamide did not decrease AK development among kidney transplant recipients. Limitations include drug tolerability, small sample size, and single-center trial nature.

Campylobacter Infection-Associated Acute Pancreatitis in a Renal Transplant Recipient.

Immunocompromised individuals (patients with cancer, diabetes, HIV/AIDS, transplant recipients) and pregnant women are at greater risk of complicated foodborne illness than the general population. Though rare, Campylobacter enteritis-associated acute pancreatitis has not been reported in an immunocompromised host to our knowledge. Herein, we describe a case of Campylobacter infection-associated pancreatitis in a renal transplant recipient. This case highlights the need for food safety education for the immunocompromised, emphasizes the role of health care providers in encouraging adherence to food safety guidelines, and stresses the need to maintain broad infectious differentials for immunocompromised patient populations, even for conditions which are not commonly associated with an infectious etiology.

Obstructive Uropathy due to Bilateral Sliding Hernia in a Renal Transplant Patient with Incidental RCC in Native Kidney.

BACKGROUND: Ureteral obstruction is a common complication after kidney transplantation. Ureteral obstruction caused by inguinal hernia, however, is a rare complication of transplantation and requires urgent surgical repair to prevent allograft loss.  Case presentation: A 58-year-old man presented with allograft dysfunction 18-years after renal transplant. He was compliant with medications and given the long duration of allograft survival, a primary renal etiology was suspected. Thus, the initial work-up included allograft biopsy that was unremarkable. Three months later, worsening allograft function prompted further evaluation. At this time, allograft ultrasound and computed tomography led to the diagnosis of ureteral obstruction due to uretero-inguinal herniation of left kidney transplant secondary to bilateral sliding inguinal hernias. The patient was also found to have incidental renal cell carcinoma of the left native kidney. A percutaneous nephrostomy tube was placed and then followed by surgical repair with ureteral reimplantation, herniorrhaphy with mesh, and left native nephrectomy. CONCLUSIONS: Mechanical obstruction can occur years after kidney transplantation. Even though it is uncommon, ureteral obstruction due to inguinal herniation is critical. Early detection of this complication and surgery can salvage the allograft and prolong function. ABBREVIATIONS: RCC: renal cell carcinoma; PCN: Percutaneous Nephrostomy; ACKD: Acquired Cystic Kidney Disease.

Genetic evaluation of living kidney donor candidates: A review and recommendations for best practices.

The growing accessibility and falling costs of genetic sequencing techniques has expanded the utilization of genetic testing in clinical practice. For living kidney donation, genetic evaluation has been increasingly used to identify genetic kidney disease in potential candidates, especially in those of younger ages. However, genetic testing on asymptomatic living kidney donors remains fraught with many challenges and uncertainties. Not all transplant practitioners are aware of the limitations of genetic testing, are comfortable with selecting testing methods, comprehending test results, or providing counsel, and many do not have access to a renal genetic counselor or a clinical geneticist. Although genetic testing can be a valuable tool in living kidney donor evaluation, its overall benefit in donor evaluation has not been demonstrated and it can also lead to confusion, inappropriate donor exclusion, or misleading reassurance. Until more published data become available, this practice resource should provide guidance for centers and transplant practitioners on the responsible use of genetic testing in the evaluation of living kidney donor candidates.

Osteomalacia and Renal Osteodystrophy.

Osteomalacia is defined by the undermineralization of newly formed bone due to a lack of available calcium, phosphorus, or vitamin D. Causative factors of osteomalacia include nutritional deficiency, diminished absorptive capabilities (often due to gastrointestinal disorders), and renal insufficiency. Renal osteodystrophy is a specific form of metabolic bone disease defined by the presence of osteomalacia and associated hyperparathyroidism secondary to a malfunction in, or absence of, renal parenchyma. This reduces the conversion of vitamin D to its active form, thus leading to a cascade of effects that negatively impact the stability of the skeleton. Osteomalacia occurs across a spectrum of severity and can produce severe consequences for specific populations, including patients with dietary, nutritional, and absorptive deficiencies. Renal osteodystrophy affects patients with chronic kidney disease, those undergoing dialysis, and renal transplant patients. Special considerations must be taken into account when assessing the bone health of patients fitting these criteria.

Vignette-Based Reflections to Inform Genetic Testing Policies in Living Kidney Donors.

Family history of kidney disease increases risk of end-stage kidney disease (ESKD) in donors. Pre-donation genetic testing is recommended in evaluation guidelines and regulatory policy. Collaborating across several institutions, we describe cases to illustrate the utility as well as practical issues in incorporating genetic testing in transplant protocols. Case 1 is from 2009, before pervasive genetic testing. A healthy 27-year-old Caucasian male had an uneventful donor evaluation for his mother, who had early onset ESKD of unclear cause. He participated in paired-exchange kidney donation, but developed progressive kidney disease and gout over the next 10 years. A uromodulin gene mutation (NM_003361.3(UMOD):c.377 G>A p.C126Y) was detected and kidney biopsy showed tubulointerstitial kidney disease. The patient subsequently required kidney transplantation himself. Case 2 was a 36-year-old African American female who had an uneventful kidney donor evaluation. She underwent gene panel-based testing to rule out ApolipoproteinL1 risk variants, for which was negative. Incidentally, a sickle-cell trait (NM_000518.5(HBB):c.20A>T p.Glu7Val) was noted, and she was declined for kidney donation. This led to significant patient anguish. Case 3 was a 26-year-old Caucasian female who underwent panel-based testing because the potential recipient, her cousin, carried a variant of uncertain significance in the hepatocyte nuclear factor-1-ß (HNF1B) gene. While the potential donor did not harbor this variant, she was found to have a likely pathogenic variant in complement factor I (NM_000204.4(CFI):c.1311dup:p.Asp438Argfs*8), precluding kidney donation. Our cases emphasize that while genetic testing can be invaluable in donor evaluation, transplant centers should utilize detailed informed consent, develop care pathways for secondary genetic findings, and share experience to develop best practices around genetic testing in donors.

Elevated Tacrolimus Levels at Time of Diagnosis of COVID-19 Compared to Baseline Among Hospitalized Organ Transplant Recipients.

BACKGROUND: The effect of COVID-19 on immunosuppressant drug levels in organ transplant recipients (OTRs) has not been adequately studied. OBJECTIVE: To study the effect of COVID-19 on tacrolimus trough levels (primary outcome) in OTRs and the association of the later with acute kidney injury, bacterial infection, and oxygen requirements. METHODS: We studied adult (>18-year-old) hospitalized OTRs with COVID-19, who were receiving tacrolimus between 3/1 and 12/16/2020. RESULTS: Among 30 OTRs, 67% were men, 90% had a kidney transplant. Median age was 60.5 (interquartile range [IQR]: 45-68) years, median time from transplant 36 (IQR: 20-84) months. Tacrolimus troughs were higher on admission for COVID-19 than baseline (average over 6 months prior) (P = .001). Eighteen patients (60%) had admission tacrolimus trough >10, 5 (17%) >20 ng/mL. Patients with diarrhea had borderline higher tacrolimus troughs, compared to those without diarrhea (P = .09). Organ transplant recipients with a tacrolimus trough >10 ng/mL were more likely to have elevated aspartate aminotransferase on admission (P = .01) and require supplemental oxygen. (P = .026). CONCLUSION AND RELEVANCE: Tacrolimus trough levels were elevated in most OTRs with COVID-19 at the time of hospital admission, compared to baseline. Potential mechanisms are diarrhea and hepatic involvement in COVID-19. In OTRs with COVID-19, including outpatients, immunosuppressant drug levels should be closely followed; management of immunosuppression should be individualized.

Relation of abnormal cardiac stress testing with outcomes in patients undergoing renal transplantation.

Cardiovascular risk stratification is often performed in patients considered for renal transplantation. In a single center, we sought to examine the association between abnormal stress testing with imaging and post-renal transplant major adverse cardiovascular events (MACE) using multivariable logistic regression. From January 2006 to May 2016 232 patients underwent renal transplantation and 59 (25%) had an abnormal stress test result. Compared to patients with a normal stress test, patients with an abnormal stress test had a higher prevalence of dyslipidemia, diabetes mellitus, obesity, coronary artery disease (CAD), and heart failure. Among those with an abnormal result, 45 (76%) had mild, 10 (17%) moderate, and 4 (7%) severe ischemia. In our cohort, 9 patients (3.9%) had MACE at 30-days post-transplant, 5 of whom had an abnormal stress test. The long-term MACE rate, at a median of 5 years, was 32%. After adjustment, diabetes (OR 2.37, 95% CI 1.12-5.00, p = 0.02), CAD (OR: 3.05, 95% CI 1.30-7.14, p = 0.01) and atrial fibrillation (OR: 5.86, 95% CI 1.86-18.44, p = 0.002) were independently associated with long-term MACE, but an abnormal stress test was not (OR: 0.83, 95% CI 0.37-1.92, p = 0.68). In conclusion, cardiac stress testing was not an independent predictor of long-term MACE among patients undergoing renal transplant.

Low-Dose Valganciclovir Prophylaxis Is Safe and Cost-Saving in CMV-Seropositive Kidney Transplant Recipients.

Introduction: Observational studies suggest that low-dose valganciclovir prophylaxis (450 mg daily for normal renal function) is as effective as and perhaps safer than standard-dose valganciclovir (900 mg daily) in preventing CMV infection among kidney transplant recipients. However, this practice is not supported by current guidelines due to concerns for breakthrough infection from resistant CMV, mainly in high-risk CMV donor-seropositive/recipient-seronegative kidney transplant recipients. Standard-dose valganciclovir is costly and possibly associated with higher incidence of neutropenia and BKV DNAemia. Our institution adopted low-dose valganciclovir prophylaxis for intermediate-risk (seropositive) kidney transplant recipients in January 2018. Research Question: To analyze the efficacy (CMV DNAemia), safety (BK virus DNAemia, neutropenia, graft loss, and death), and cost savings associated with this change. Design: We retrospectively compared the above outcomes between CMV-seropositive kidney transplant recipients who received low-dose and standard-dose valganciclovir, transplanted within our institution, between 1/19/2014 and 7/15/2019, using propensity score-adjusted competing risk analyses. We also compared cost estimates between the two dosing regimens, for 3 months of prophylaxis, and for different percentage of patient-weeks with normal renal function, using the current average wholesale price of valganciclovir. Results: We studied 179 CMV-seropositive kidney transplant recipients, of whom 55 received low-dose and 124 standard-dose valganciclovir. The majority received nonlymphocyte depleting induction (basiliximab). Low-dose valganciclovir was at least as effective and safe as, and more cost-saving than standard-dose valganciclovir. Conclusion: This single-center study contributes to mounting evidence for future guidelines to be adjusted in favor of low-dose valganciclovir prophylaxis in CMV-seropositive kidney transplant recipients.

Implementing a regional standardized BK polyomavirus screening protocol across eleven transplant centres.

BK polyomavirus (BKPyV) reactivation is regularly monitored after kidney transplant to prevent progression to BK associated nephropathy (BKAN). The New England BK Consortium, made up of 12 transplant centres in the northeastern United States, conducted a quality improvement project to examine adherence to an agreed upon protocol for BKPyV screening for kidney transplants performed in calendar years 2016-2017. In a total of 1047 kidney transplant recipients (KTR) from 11 transplant centres, 204 (19%) had BKPyV infection, defined as detection of BKPyV in plasma, with 41 (4%) KTR progressing to BKAN, defined by either evidence on biopsy tissues or as determined by treating nephrologists. BKPyV infection was treated with reduction of immune suppressants (RIS) in >70% of the patients in all but two centres. There was no graft loss because of BKAN during the two-year follow-up. There were nine cases of post-RIS acute rejection detected during this same period. Adherence to the protocol was low with 54% at 12 months and 38% at 24 months, reflecting challenges of managing transplant patients at all centres. The adherence rate was positively correlated to increased detection of BKPyV infection and was unexpectedly positively correlated to an increase in diagnosis of BKAN.

Treprostinil reduces mitochondrial injury during rat renal ischemia-reperfusion injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major factor contributing to acute kidney injury and it is associated with a high morbidity and mortality if untreated. Renal IRI depletes cellular and tissue adenosine triphosphate (ATP), which compromises mitochondrial function, further exacerbating renal tubular injury. Currently, no treatment for IRI is available. This study investigates the protective role of treprostinil in improving mitochondria biogenesis and recovery during rat renal IRI. METHODS: Male Sprague Dawley rats were randomly assigned to groups: control, sham, IRI-placebo or IRI-treprostinil and subjected to 45 min of bilateral renal ischemia followed by 1-72 h reperfusion. Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump. RESULTS: Treprostinil significantly reduced peak elevated serum creatinine (SCr) levels and accelerated normalization relative to IRI-placebo (p < 0.0001). Treatment with treprostinil also inhibited IRI-mediated renal apoptosis, mitochondrial oxidative injury (p < 0.05), and the release of cytochrome c (p < 0.01) vs. IRI-placebo. In addition, treprostinil preserved renal mitochondrial DNA copy number (p < 0.0001) and renal ATP levels (p < 0.05) to nearly those of sham-operated animals. Non-targeted semi-quantitative proteomics showed reduced levels of ATP synthase subunits in the IRI-placebo group which were restored to sham levels by treprostinil treatment (p < 0.05). Furthermore, treprostinil reduced renal IRI-induced upregulated Drp1 and pErk protein levels, and restored Sirt3 and Pgc-1α levels to baseline (p < 0.05). CONCLUSIONS: Treprostinil reduces mitochondrial-mediated renal apoptosis, inhibits mitochondria fission, and promotes mitochondria fusion, thereby accelerating mitochondrial recovery and protecting renal proximal tubules from renal IRI. These results support the clinical investigation of treprostinil as a viable therapy to reduce renal IRI.

Coronavirus Disease 2019 in Kidney Transplant Recipients: Single-Center Experience and Case-Control Study.

BACKGROUND: Kidney transplant recipients (KTR) are considered high-risk for morbidity and mortality from coronavirus disease 2019 (COVID-19). However, some studies did not show worse outcomes compared to non-transplant patients and there is little data about immunosuppressant drug levels and secondary infections in KTR with COVID-19. Herein, we describe our single-center experience with COVID-19 in KTR. METHODS: We captured KTR diagnosed with COVID-19 between March 1, 2020 and May 18, 2020. After exclusion of KTR on hemodialysis and off immunosuppression, we compared the clinical course of COVID-19 between hospitalized KTR and non-transplant patients, matched by age and sex (controls). RESULTS: Eleven KTR were hospitalized and matched with 44 controls. One KTR and 4 controls died (case fatality rate: 9.1%). There were no significant differences in length of stay or clinical outcomes between KTR and controls. Tacrolimus or sirolimus levels were >10 ng/mL in 6 out of 9 KTR (67%). Bacterial infections were more frequent in KTR (36.3%), compared with controls (6.8%, P = .02). CONCLUSIONS: In our small case series, unlike earlier reports from the pandemic epicenters, the clinical outcomes of KTR with COVID-19 were comparable to those of non-transplant patients. Calcineurin or mammalian target of rapamycin inhibitor (mTOR) levels were high. Bacterial infections were more common in KTR, compared with controls.

Successful recovery from COVID-19 in three kidney transplant recipients who received convalescent plasma therapy.

Novel coronavirus disease 2019 (COVID-19) is a highly infectious, rapidly spreading viral disease that typically presents with greater severity in patients with underlying medical conditions or those who are immunosuppressed. We present a novel case series of three kidney transplant recipients with COVID-19 who recovered after receiving COVID-19 convalescent plasma (CCP) therapy. Physicians should be aware of this potentially useful treatment option. Larger clinical registries and randomized clinical trials should be conducted to further explore the clinical and allograft outcomes associated with CCP use in this population.

Treprostinil, a prostacyclin analog, ameliorates renal ischemia-reperfusion injury: preclinical studies in a rat model of acute kidney injury.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is a major factor causing acute kidney injury (AKI). No pharmacological treatments for prevention or amelioration of I/R-induced renal injury are available. Here we investigate the protective effects of treprostinil, a prostacyclin analog, against renal IRI in vivo. METHODS: Male Sprague Dawley rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion for 1-168 h. Treprostinil (100 ng/kg/min) or placebo was administered subcutaneously for 18-24 h before ischemia. RESULTS: Treatment with treprostinil both significantly reduced peak elevation and accelerated the return to baseline levels for serum creatinine and blood urea nitrogen versus I/R-placebo animals following IRI. I/R-treprostinil animals exhibited reduced histopathological features of tubular epithelial injury versus I/R-placebo animals. IRI resulted in a marked induction of messenger RNA coding for kidney injury biomarkers, kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin and for pro-inflammatory cytokines chemokine (C-C motif) ligand 2, interleukin 1ß, interleukin 6 and intracellular adhesion molecular 1 in animals treated with placebo only relative to sham controls. Upregulation of expression of all these genes was significantly suppressed by treprostinil. Treprostinil significantly suppressed the elevation in renal lipid peroxidation found in the I/R-placebo group at 1-h post-reperfusion. In addition, renal protein expression of cleaved poly(ADP-ribose) polymerase 1 and caspase-3, -8 and -9 in I/R-placebo animals was significantly inhibited by treprostinil. CONCLUSIONS: This study demonstrates the efficacy of treprostinil in ameliorating I/R-induced AKI in rats by significantly improving renal function early post-reperfusion and by inhibiting renal inflammation and tubular epithelial apoptosis. Importantly, these data suggest that treprostinil has the potential to serve as a therapeutic agent to protect the kidney against IRI in vivo.

Undiagnosed fibromuscular dysplasia in a deceased donor kidney transplant successfully treated with angioplasty.

A 41-year-old man with end-stage renal disease received a deceased donor kidney transplant without complication. Maintenance immunosuppression consisted of tacrolimus, mycophenolate and prednisone. Two months after transplantation, his creatinine did not improve beyond 2-2.3 mg/dL, which prompted allograft biopsy. His biopsy showed tubular epithelial injury without rejection, and given concern for possible calcineurin-inhibitor toxicity, his tacrolimus was changed to sirolimus. Renal function improved, but 1 month later he presented to the hospital with seizure activity, severe hypertension, acute kidney injury and MRI findings suggestive of posterior reversible encephalopathy syndrome. Blood pressure was difficult to control, which had not been the case in the immediate posttransplant period, and addition of lisinopril worsened his renal function. Transplant renal artery stenosis was suspected, and allograft ultrasound with doppler confirmed our suspicion. The patient underwent an angiogram, showing 60% stenosis of the mid-distal transplanted renal artery. Interventional radiology successfully stented this lesion, with subsequent improvement in allograft function and blood pressure control. He did not require further intervention in follow-up.