Metabolomics Study for Identification of Potential Biomarkers of Long-term Survival in Kidney Transplantation Recipients.

BACKGROUND: The recent progress and appropriate use of immunosuppressive drugs have considerably improved the short-term survival in kidney transplantation recipients (KTRs). The development of new strategies to improve long-term survival outcome after kidney transplantation is also becoming important. Although current diagnosis of allograft dysfunction relies on serum creatinine concentration and biopsy, they are nonspecific indicators of allograft function. Therefore, noninvasive, sensitive, and specific biomarkers for the prediction of long-term survival are needed. The aim of this study was to discover potential biomarkers for long-term survival in KTRs through the use of liquid chromatography-tandem mass spectrometry. METHODS: We used the metabolic approach to explore the change of metabolites in the serum of KTRs. Twenty-four KTRs with long-term good survival (LGS) and 10 KTRs with chronic antibody-mediated rejection (CAMR) were included in this study. After quantile normalization with chromatographic data, multivariate statistical analysis was performed. We attempted to analyze metabolic profiling with LGS and CAMR groups. RESULTS: The orthogonal partial least-squares discriminant analysis score plot showed a separation between 2 groups in the principal component. In the corresponding loading plot, 344 metabolites responsible for the separation observed in the score plot were identified (variable influence on projection ≥1.0). We then selected 54 metabolites to compare mass with charge by searching a web database, and 11 compounds were identified. CONCLUSIONS: We found metabolites in serum that differ in LGS and CAMR groups. Further studies are needed to figure out potential metabolomic biomarkers to predict long-term survival in KTRs.

Cystatin C-Based Equation for Predicting the Glomerular Filtration Rate in Kidney Transplant Recipients.

BACKGROUND: Precise monitoring of the glomerular filtration rate (GFR) is needed to estimate the allograft function in kidney transplant recipients (KTRs). The GFR is widely estimated with the use of formulas based on serum cystatin C (SCys) and serum creatinine (SCr) levels. We compared the efficacy of SCys-based equations with that of SCr-based equations to predict the allograft function. METHODS: We calculated the Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI Cr), CKD-EPI creatinine-cystatin C (CKD-EPI Cr/Cys), and CKD-EPI cystatin C (CKD-EP ICys) equations in 70 KTRs. The measured GFR (mGFR) was defined as the GFR estimated by technetium-99m-diethylene triamine pentaacetic acid (99mTc-DTPA) clearance. The accuracy and precision of the equations were compared with the mGFR. The performance characteristics of SCr and SCys were analyzed with the use of receiver operating characteristic (ROC) curves to ascertain the sensitivity and specificity at the cutoff value of <45 mL/min/1.73 m2 DTPA. RESULTS: Overall, MDRD and CKD-EPICys did not show significant differences from mGFR (P = .05 and P = .077, respectively), whereas CKD-EPI Cr and CKD-EPI Cr/Cys significantly underestimated mGFR (P < .001 and P = .005, respectively). In the subgroup of patients with mGFR <45 mL/min/1.73 m2, CKD-EPI Cys showed little bias (P = .122), whereas MDRD significantly underestimated mGFR (P = .037). The area under the ROC curve for predicting mGFR <45 mL/min/1.73 m2 was 0.80 for SCys, which was better than that for SCr at 0.763. CONCLUSIONS: Cystatin C-based equations showed better predictive performance of the allograft function than creatinine-based equations for the KTRs, including patients with lower GFR. Cystatin C level might be a good alternate measurement to monitor the allograft function.

Clinical outcomes in Asian elderly kidney transplant recipients: a multicenter cohort study.

BACKGROUND: The kidney transplantation rate in elderly patients is increasing rapidly. However, the clinical outcomes of kidney transplantation in elderly patients have not yet been thoroughly evaluated. METHODS: This multicenter cohort study included adult kidney transplant recipients (KTRs) admitted to five major tertiary hospitals in Korea between 1997 and 2012. A total of 3,565 adult participants were enrolled. Patient survival, allograft survival, and biopsy-proven acute rejection (BPAR) of 242 elderly recipients (≥ 60 years) were assessed and compared with those of a younger population. RESULTS: Patients were divided into five groups according to age at time of transplantation. The proportion of elderly patients was 6.7 % (mean age, 63.1 ± 2.7 years; n = 242). The numbers of male patients (69.4%), those with diabetes mellitus history (36.3%), and those with pretransplantation ischemic heart disease history (17.7%) were significantly higher in the elderly group than in the younger age groups. Elderly patients were more likely to receive a cadaveric kidney, and overall mortality rates were significantly higher in the elderly patients (1-year survival 93.3%, 5-year survival 91.3%). However, death-censored allograft survival rate and BPAR were not affected by patient age (P = .104 and .501, respectively). Among the elderly, BPAR and female donors were independent risk factors for allograft loss. CONCLUSION: The overall survival rate of the elderly KTRs was significantly lower than that of younger KTRs. However, the death-censored allograft survival rate did not differ between groups. Kidney transplantation should not be stagnated especially in elderly patients with end-stage renal disease.

Clinical impact of BK virus surveillance on outcomes in kidney transplant recipients.

BACKGROUND: The objective of this study was to investigate the clinical impact of BK virus surveillance on graft injury in kidney transplantation. METHODS: BK viremia in kidney transplant recipients was evaluated by use of plasma quantitative polymerase chain reaction. The prevalence of BK viremia and BK virus-associated nephropathy (BKVAN) and the clinical impact of BK viremia on graft outcomes were assessed. RESULTS: This study took place between January 2008 and June 2013. A total of 213 kidney transplant recipients were included. The prevalence of BK viremia and high BK viremia (≥1 × 10(4) copies/mL) was 66.7% (142/213) and 17.4% (37/213), respectively. A diagnosis of BKVAN was confirmed by means of allograft biopsy in 9 patients (4.2%). The estimated glomerular filtration rate after transplantation was similar in both the low BK viremia (<1 × 10(4) copies/mL) and non-BK viremia groups but was significantly lower in the high BK viremia group after 18 months. In receiver operating characteristic curve analysis, the area under the curve value of plasma polymerase chain reaction was 0.980. We found that a viral load >92,850 copies/mL was able to predict BKVAN with 89% sensitivity and 94.6% specificity. The risk factors for viral loads ≥1 × 10(4) copies/mL were cytomegalovirus infection, steroid pulse therapy, and acute rejection. CONCLUSIONS: High BK viremia was associated with poor graft function after kidney transplantation. The serial monitoring of BK viremia in kidney transplant recipients was helpful in predicting BKVAN and might prevent further progression.

Successful withdrawal of antiviral treatment in kidney transplant recipients with chronic hepatitis B viral infection.

BACKGROUND: The optimal duration of antiviral therapy for kidney transplant recipients (KTR) with chronic hepatitis B virus (HBV) infection remains unclear. We reported the long-term outcomes after withdrawal of antiviral agent in KTR with chronic HBV infection. METHODS: We retrospectively investigated the hepatitis B surface antigen (HBsAg)-positive KTR with antiviral agents between January 2002 and January 2012. Antiviral treatments were withdrawn in patients who met all of the following 7 criteria: (i) no clinical and histologic evidence of cirrhosis, (ii) normal liver biochemistry, (iii) negative for both HBV DNA and hepatitis B envelope antigen (HBeAg), (iv) no resistance to antiviral agent, (v) antiviral therapy > 9 months, (vi) maintenance dosage of immunosuppressant for > 3 months, and (vii) no history of acute rejection during recent 6 months. All patients were followed regularly at approximately 3-6 months for liver enzyme, viral markers, and HBV DNA level after antiviral withdrawal. RESULTS: Among a total of 445 KTR, 14 HBsAg-positive patients were included in this study. Antiviral agents were used, with lamivudine in 11 patients, and with adefovir, entecavir, and telbivudine in 3 patients, respectively. Discontinuation of antiviral agent was attempted in 6 (42.9%) of 14 patients who satisfied the criteria. The median duration of antiviral therapy before withdrawal was 14.3 months (range, 9-24 months). Four (66.7%) of 6 patients were successfully withdrawn and remained negative for HBV DNA for a median 60.5 months (range, 47-82 months). The baseline HBV DNA level was not related to maintenance of remission after withdrawal. Two reactivated patients resumed antiviral treatment immediately, with subsequent normalization of HBV DNA. During the follow-up, 1 patient developed hepatocellular carcinoma; however, no patient death or graft failure was reported for all HBsAg-positive KTR. CONCLUSIONS: Antiviral therapy can be discontinued successfully and safely in selected KTR with chronic HBV infection, after complete suppression of HBV and sufficient duration of antiviral therapy.

Proton pump inhibitors as a possible cause of vitiligo: an in vivo and in vitro study.

BACKGROUND: Vitiligo is an acquired depigmentation disorder of melanocytes. Recently, some clinical reports have suggested that proton pump inhibitors (PPIs) may worsen vitiligo, but their effects on melanocytes have yet to be elucidated. OBJECTIVE: We investigated the effect of PPIs on melanogenesis in vivo and in vitro. METHODS: We examined the effect of PPIs on melanogenesis in B16 murine melanoma cells by measuring melanin content and tyrosinase (TYR) activity. TYR and tyrosinase-related protein-1 (TRP-1) were monitored by western blotting. Finally, a PPI was applied to zebrafish embryos to investigate its in vivo effect on pigmentation. RESULTS: In agreement with our clinical experience of worsened vitiligo after PPI treatment, PPIs decreased both melanin content and TYR activity. Western blotting showed that PPIs decreased TYR and TRP-1 protein levels. In the zebrafish test, PPIs inhibited body pigmentation in a dose-dependent manner. CONCLUSION: These results suggest that the functional inhibition of melanization by PPIs may induce or aggravate vitiligo lesions in genetically predisposed patients.

Mizoribine versus mycophenolate mofetil in combination therapy with tacrolimus for de novo kidney transplantation: evaluation of efficacy and safety.

The present study compared the efficacy and safety of mizoribine (MZR) with mycophenolate mofetil (MMF) in kidney transplantation. This multicenter, randomized clinical trial. Employed doses of study drug tailored to the immunosuppressive need. The primary efficacy outcome was the incidence of biopsy-proven acute rejection episodes (BPAR). The safety of the study drug was assessed using the incidences of adverse events, drug discontinuations, and abnormal laboratory results. The 7 (6.4%) BPARs above grade II were observed in the MZR group noninferior to the 2 (1.8%) in the MMF group (95% confidence interval, -0.007-0.097 > noninferiority limit [-0.2]). BPAR was significantly decreased in the MZR group after the dose change (17/41 [41.4%] vs 8/69 [11.6%]; P < .0001) and the incidence of BPAR was similar between the MZR and MMF groups after the dose change (P = .592). The uric acid level was significantly elevated in the MZR group (P = .002). In conclusion, the efficacy and safety of MZR were similar and statistically noninferior to MMF in combination therapy with tacrolimus.

Long-term outcome of azathioprine versus mycophenolate mofetil in cyclosporine-based immunosuppression in kidney transplantation: 10 years of experience at a single center.

BACKGROUND: Mycophenolate mofetil (MMF) has been used worldwide as part of maintenance immunosuppression since initial large cyclosporine-based trials reported that compared with azathioprine (AZA), MMF reduced acute rejection episodes after renal transplantation. However, long-term benefits of MMF have not been established; the follow-up period of previous studies was within 5 years. The aim of this study was to compare the acute rejection rates, allograft function, and graft and patient survivals of these 2 drugs in conjunction with cyclosporine and steroids over a period of 10 years. METHODS: We reviewed recipients who had undergone kidney transplantation from January 1998 to January 2002. Eighty-six patients were divided into 2 groups (MMF = 43, AZA = 43). All patients received cyclosporine and steroids concomitantly as maintenance immunosuppressive therapy. RESULTS: Baseline characteristics were similar between the 2 groups except donor type. Multiple logistic regression analysis showed MMF therapy to reduce the acute rejection rate in the first 12 months after transplantation (relative risk [RR], 0.181; 95% confidence interval [CI], 0.035-0.936; P = .042). Cox proportional hazard analysis revealed MMF to was not associated with improved graft and patient survival. Graft function was comparable between the 2 groups over 10 years. No significant differences were observed in the incidence of serious infections or malignancy. CONCLUSIONS: Compared with AZA, MMF offered the clinical benefit of prevention of acute rejection episodes, but displayed similar effects on long-term graft and patient survivals in kidney transplant recipients undergoing cyclosporine-based immunosuppression.

Homocysteine induces COX-2 expression in macrophages through ROS generated by NMDA receptor-calcium signaling pathways.

Homocysteine (Hcy) at elevated levels is a putative risk factor for many cardiovascular disorders including atherosclerosis. In the present study, we investigated the effect of Hcy on the expression of cyclooxygenase (COX)-2 in murine macrophages and the mechanisms involved. Hcy increased the expression of COX-2 mRNA and protein in dose- and time-dependent manners, but did not affect COX-1 expression. Hcy-induced COX-2 expression was attenuated not only by the calcium chelators, EGTA and BAPTA-AM, but also by an antioxidant, N-acetylcysteine. Calcium chelators also attenuated Hcy-induced reactive oxygen species (ROS) production in macrophages, indicating that Hcy-induced COX-2 expression might be mediated through ROS generated by calcium-dependent signaling pathways. In another series of experiments, Hcy increased the intracellular concentration of calcium in a dose-dependent manner, which was attenuated by MK-801, an N-methyl-D-aspartate (NMDA) receptor inhibitor, but not by bicuculline, a gamma-aminobutyric acid receptor inhibitor. Molecular inhibition of NMDA receptor using small interfering RNA also attenuated Hcy-induced increases in intracellular calcium. Furthermore, both ROS production and Hcy-induced COX-2 expression were also inhibited by MK-801 as well as by molecular inhibition of NMDA receptor. Taken together, these findings suggest that Hcy enhances COX-2 expression in murine macrophages by ROS generated via NMDA receptor-mediated calcium signaling pathways.

Suppression of PU.1-linked TLR4 expression by cilostazol with decrease of cytokine production in macrophages from patients with rheumatoid arthritis.

BACKGROUND AND PURPOSE: The present study assessed the effects of cilostazol on LPS-stimulated TLR4 signal pathways in synovial macrophages from patients with rheumatoid arthritis (RA). These effects were confirmed in collagen-induced arthritis (CIA) in mice. EXPERIMENTAL APPROACH: Expression of TLR4, PU.1, NF-κB p65 and IκBα on synovial fluid macrophages from RA patients was determined by Western blotting, and cytokines were measured by ELISA. Anti-arthritic effects were evaluated in CIA mice. KEY RESULTS: Intracellular cAMP was concentration-dependently raised by cilostazol (1-100 µM). Cilostazol significantly suppressed LPS-stimulated increase of TLR4 expression by blocking PU.1 transcriptional activity in RA macrophages. In addition, cilostazol decreased LPS-induced myeloid differentiation factor 88 (MyD88) expression, but not that of TNF receptor-associated factor 6 (TRAF6). Cilostazol also suppressed IkBα degradation and NF-κB p65 nuclear translocation. Moreover, LPS-induced increase of cytokine production (TNF-α, IL-1ß) was inhibited by cilostazol, an effect which was accompanied by suppression of IκBα degradation, and NF-κB p65 nuclear translocation. However, expression of anti-inflammatory IL-10 was elevated by cilostazol and forskolin/IBMX. In mice with CIA, post-treatment with cilostazol (30 mg kg⁻¹ day⁻¹) decreased expression of TLR4 in knee joints in association with decreased recruitment of macrophages. Consequently, synovial inflammation, proteoglycan depletion and bone erosion were significantly inhibited by cilostazol treatment. CONCLUSIONS AND IMPLICATIONS: Cilostazol down-regulated LPS-stimulated PU.1-linked TLR4 expression and TLR4/MyD88/NF-κB signal pathways, and then suppressed inflammatory cytokine production in synovial macrophages from RA patients. Also cilostazol markedly inhibited the severity of CIA in mice.

Mycobacterium kansasii pericarditis in a kidney transplant recipient: a case report and comprehensive review of the literature.

Mycobacterium kansasii is the second most common non-tuberculous mycobacteria in kidney transplant recipients (KTRs) and has been reported to cause disseminated infection in KTRs. We report the first case to our knowledge of M. kansasii pericarditis after kidney transplantation in a 54-year-old man. The patient was admitted with a 2-month history of intermittent fever and myalgia, treated with oral prednisolone and mycophenolate mofetil prior to admission. Chest computed tomography showed enlarged mediastinal lymph node and small amount of pericardial effusion. Mediastinoscopic biopsy of mediastinal lymph node revealed reactive hyperplasia, without evidence of granuloma, but acid-fast bacilli stain of pericardial fluid reported positive finding and pericardial fluid culture identified M. kansasii. The patient has been treated successfully with rifabutin-based combination therapy. All available cases of M. kansasii infection in kidney transplant patients and M. kansasii pericarditis in human immunodeficiency virus-infected patients are comprehensively reviewed.

Selective activation of Akt1 by mammalian target of rapamycin complex 2 regulates cancer cell migration, invasion, and metastasis.

Mammalian target of rapamycin complex (mTORC) regulates a variety of cellular responses including proliferation, growth, differentiation and cell migration. In this study, we show that mammalian target of rapamycin complex 2 (mTORC2) regulates invasive cancer cell migration through selective activation of Akt1. Insulin-like growth factor-1 (IGF-1)-induced SKOV-3 cell migration was completely abolished by phosphatidylinositol 3-kinase (PI3K) (LY294002, 10 µM) or Akt inhibitors (SH-5, 50 µM), whereas inhibition of extracellular-regulated kinase by an ERK inhibitor (PD98059, 10 µM) or inhibition of mammalian target of rapamycin complex 1 (mTORC1) by an mTORC1 inhibitor (Rapamycin, 100 nM) did not affect IGF-1-induced SKOV-3 cell migration. Inactivation of mTORC2 by silencing Rapamycin-insensitive companion of mTOR (Rictor), abolished IGF-1-induced SKOV-3 cell migration as well as activation of Akt. However, inactivation of mTORC1 by silencing of Raptor had no effect. Silencing of Akt1 but not Akt2 attenuated IGF-1-induced SKOV-3 cell migration. Rictor was preferentially associated with Akt1 rather than Akt2, and over-expression of Rictor facilitated IGF-1-induced Akt1 activation. Expression of PIP3-dependent Rac exchanger1 (P-Rex1), a Rac guanosine exchange factor and a component of the mTOR complex, strongly stimulated activation of Akt1. Furthermore, knockdown of P-Rex1 attenuated Akt activation as well as IGF-1-induced SKOV-3 cell migration. Silencing of Akt1 or P-Rex1 abolished IGF-1-induced SKOV-3 cell invasion. Finally, silencing of Akt1 blocked in vivo metastasis, whereas silencing of Akt2 did not. Given these results, we suggest that selective activation of Akt1 through mTORC2 and P-Rex1 regulates cancer cell migration, invasion and metastasis.

The effect of the respiratory cycle on liver stiffness values as measured by transient elastography.

The findings of several studies suggest that liver stiffness values can be affected by the degree of intrahepatic congestion respiration influence intrahepatic blood volume and may affect liver stiffness. We evaluated the influence of respiration on liver stiffness. Transient elastography (TE) was performed at the end of inspiration and at the end of expiration in patients with chronic liver disease. The median values obtained during the inspiration set and during the expiration set were defined as inspiratory and expiratory liver stiffness, respectively. A total of 123 patients with chronic liver disease were enrolled (mean age 49years; 64.2% men). Liver cirrhosis coexisted in 29 patients (23.6%). Expiratory liver stiffness was significantly higher than inspiratory liver stiffness (8.7 vs 7.9kPa, P=0.001), while the expiratory interquartile range/median ratio (IQR ratio) did not differ from the inspiratory IQR ratio. Expiratory liver stiffness was significantly higher than inspiratory liver stiffness in 49 (39.8%) patients (HE group), expiratory liver stiffness was significantly lower than inspiratory stiffness in 15 (12.2%) patients, and there was no difference in 59 (48.0%) patients. Liver cirrhosis was more frequent in those who had a lower liver stiffness reading in expiration, and only the absence of liver cirrhosis was significantly associated with a higher reading in expiration in multivariate analysis. In conclusion, liver stiffness was significantly elevated during expiration especially in patients without liver cirrhosis. The effect of respiration should be kept in mind during TE readings.

Interleukin-18, transforming growth factor-ß, and vascular endothelial growth factor gene polymorphisms and susceptibility to primary glomerulonephritis.

Several studies have showed an association of gene polymorphisms with the development of glomerulonephritis (GN). We investigated the effects of gene polymorphisms on the development of GN by analyzing polymorphisms in the interleukin (IL)-18, transforming growth factor (TGF)-ß, and vascular endothelial growth factor (VEGF) genes in Korean patients with primary GN. The study included 146 normal subjects (controls) and 100 patients diagnosed with primary GN by kidney biopsy. The gene polymorphisms A-607C and G-137C in IL-18, C-509T and T869C in TGF-ß1, and C-2578A and C405G in VEGF were investigated in DNA extracted from peripheral blood. Significant differences were observed between the GN and control groups in the genotype and allele frequencies of A-607C IL-18 and C405G VEGF. The frequencies of the IL-18-607CC genotype [P = 0.001, odds ratio (OR) = 2.473] and the VEGF 405GG genotype (P = 0.001, OR = 2.473) were significantly increased in the GN group. The combination of IL-18-607CC+ and VEGF 405GG+ genotypes had a higher risk for developing GN in comparison with the combination of IL-18-607CC- and VEGF 405GG- genotypes (P < 0.001, OR = 8.642). In the haplotype analysis of the IL-18 gene, the CG haplotype was significantly more frequent in the GN group than the control group (61.5% vs 46.9%, P = 0.002). These results show that the -607CC genotype of the IL-18 gene and the 405GG genotype of the VEGF gene are associated with susceptibility to and the development of primary GN.