Role of Urinary Neutrophil Gelatinase-Associated Lipocalin for Predicting the Severity of Renal Functions in Patients With Autosomal-Dominant Polycystic Kidney Disease.

BACKGROUND: Autosomal-dominant polycystic kidney disease (ADPKD) has a feature of disruption of tubular integrity with increased cellular proliferation and apoptosis. There are several known tubular membrane proteins in the pathogenesis of ADPKD, and one of these proteins is the neutrophil gelatinase-associated lipocalin (NGAL). NGAL is a protein expressed on renal tubular cells of which production is markedly increased in response to harmful stimuli such as ischemia or toxicity. OBJECTIVE: We aim to study whether urinary NGAL levels could be used as a marker to identify the severity of ADPKD in patients. METHODS: Urinary NGAL levels were measured in 30 patients with ADPKD compared with 30 control patients who were matched by age, gender, and glomerular filtration rate (GFR). All patients with ADPKD were diagnosed by using both phenotypic and genotypic criteria, which showed that all cases of ADPKD were caused by PKD1 gene mutation. The urinary NGAL level was measured using The NGAL Test by Roche, with analytic range of 25-1000 ng/mL. RESULTS: In the ADPKD group, there was significant negative correlation between urinary NGAL and GFR (Pearson r = -0.472; P = .008) and significant positive correlation between urinary NGAL and serum creatinine (Pearson r = 0.718; P < .01). Elevated urinary NGAL was increased as GFR of ADPKD patients was decreased. CONCLUSION: Urinary NGAL might play role in the pathway of renal tubular damage in patients with ADPKD and might be useful in the prediction of the possibility to progress to chronic kidney disease in patients with ADPKD.

Potential impact of Thai Kidney Transplant program on immunosuppressive utilization: an analysis of the national transplant registry.

BACKGROUND: The Thai Kidney Transplant (TKT) program was launched in October 2008 to promote transplantation among previously disadvantaged populations, using fixed-rate provider payment. This study investigated if the introduction of this program could alter the natural practice trends of immunosuppressive drug use. METHODS: Data from the Thai Transplantation Registry were analyzed. The change in trend of immunosuppressive use was assessed using the multivariate adaptive regression splines (MARS) technique. RESULTS: During 1987-2012, 3975 kidney transplantations were done. The average age of patients was 42 years and 62% were male. Chronic glomerulonephritis accounted for one third of those with known causes of end-stage renal disease (ESRD). Eighty-six percent were on hemodialysis before transplantation. Prednisolone was used in 95.87% of all transplant recipients, whereas calcineurin inhibitors (CNIs), mycophenolates (MPAs), azathioprine (AZA), and mammalian target of rapamycin inhibitors (mTORis) were used in 95.67%, 64.22%, 12.25%, and 2.31%, respectively. Overall use after 2008 was decreased for AZA (18.16% to 3.40%) and mTORis (2.86% to 1.5%) but increased for MPAs (50.80% to 84.34%), CNIs (95.43% to 96.04%), and prednisolone (95.60% to 96.29%), as compared with before the program inception. The slopes of use trends of AZA, MPAs, and CNIs did not significantly marginally differ from their natural trends before the program inception (P = .496, .108, and .741, respectively). However, the natural increasing use trend of mTORis significantly changed to a decreasing pattern after the introduction of the TKT program (P = .018). CONCLUSION: Fixed-rate provider payment might interfere with the natural practice trends of immunosuppressive drug use.

Predictive model for the optimal glomerular filtration rate in living kidney transplant recipients.

BACKGROUND: Recipient glomerular filtration rate (GFR) after living kidney transplantation (KT) is influenced by many factors. Defining the appropriate level of recipient GFR post-KT is helpful. The aim of this study was to establish a predictive model to estimate the optimal recipient GFR at 1 week post-KT. METHODS: We retrospectively analyzed 211 living KTs without delayed or slow graft function. Estimated GFR was calculated using the Cockcroft-Gault (CG) formula. Donor kidney volume was obtained from routine computed tomographic angiography (CTA) by work station GE (AW 4.20) program. Multivariate analysis was carried out with automated backward selection to establish the predictive model. The bias, precision, and accuracy of our model were also determined by application of the model to another 37 living KTs. RESULTS: In multivariate analysis, the significant parameters to predict recipient GFR were donor age (P = .025) and kidney volume (P < .0001) and both were incorporated in the predictive model; predicted CG recipient GFR = 28.325 + (donor kidney volume x 0.282) - (0.297 x donor age). The correlation coefficient (R) is 0.5. Application to another group revealed that our model had high precision (14.45 mL/min), small positive bias (0.24 mL/min), and high percentage (81%) of predicted value, which was within 30% of the observed recipient GFR post-KT. CONCLUSION: Our predictive model included donor age and donor kidney volume and could be used to estimate the optimal recipient GFR post-KT. This could be helpful to identify early graft dysfunction and to make a decision if further invasive investigation such as allograft biopsy is necessary.

Clinicopathologic features and treatment response of early acute antibody-mediated rejection in Thai kidney transplant recipients: a single-center experience.

BACKGROUND: Acute antibody-mediated rejection (AMR) is a major cause of early kidney allograft dysfunction. This study was conducted to examine the clinicopathologic features and long-term outcomes of early AMR in our center. METHODS: We retrospectively reviewed all patients who underwent kidney transplantation between January 2005 and December 2012. Patients who had histopathologic features of AMR within 3 months after transplantation were enrolled. RESULTS: Of 444 patients, early acute AMR was diagnosed in 25 patients (5.36%). Seventeen patients (68%) were highly sensitized. Histological analysis revealed acute vascular rejection and thrombotic microangiopathy in 21 (84%) and 6 (24%) patients, respectively. Staining of C4d in peritubular capillaries was detected in 6/20 patients (12%). All patients received plasma exchange (PE) 1.5 blood volume for 1-5 sessions followed by intravenous immunoglobulin (IVIG) 2 g/kg. Sixteen patients (64%) received 1-2 doses of rituximab 375 mg/m(2). We repeated treatment with PE and IVIG in refractory cases. Allografts could be rescued in 20 patients (80%) whereas 5 patients (20%) lost their grafts. Kaplan-Meier survival analysis revealed lower cumulative graft survival in the early AMR group compared with patients without early AMR (1 year survival rate of 80% vs 96% and 3 survival of 64% vs 80%; P < .001). After median follow-up time of 25 months, 7/20 patients (33%) developed late AMR. CONCLUSION: ABMR is a serious early complication after KT. Early detection and intensive treatment is mandatory for salvaging the graft. After surpassing from early AMR, long-term close monitoring is also necessary.

Clinicopathologic characteristics and outcomes of late acute antibody-mediated rejection in Thai kidney transplant recipients: a single-center experience.

BACKGROUND: Late antibody-mediated rejection (ABMR) has worse prognosis than early ABMR. The objective of this study was to examine the clinical and pathological features of late acute ABMR in our experience. METHOD: We retrospectively reviewed all patients who underwent kidney transplantation (KT) between January 2001 and December 2012. Patients who had glomerulitis and/or peritubular capillaritis on kidney biopsy performed 6 months after KT were enrolled. RESULTS: Of 592 patients, late acute ABMR was diagnosed in 34 cases (5.74%) with a mean onset of 49.2 ± 30.2 months post-KT. Six patients (17.6%) had nonadherence. Allograft histopathology demonstrated concomitant transplant glomerulopathy in 23 patients (67.6%) and positive peritubular C4d staining in 25 patients (73.5%). Donor-specific antibody (DSA) was detected in 25 patients (73.5%). Anti-HLA class II antibody was more prevalent than class I (67.6% vs 20.6%; P = .003) and most of them were anti-HLA DQ. We prescribed intravenous immunoglobulin (IVIG) 1-2 g/kg for 30 patients (88.2%), plasma exchange (PE) for 27 patients (79.4%), and rituximab 375 mg/m(2) for 18 patients (52.9%). We repeated treatment with PE and IVIG in 12 refractory cases. For clinical outcome, 21 patients (61.7%) had deterioration of graft function; 9 of them (26.5%) eventually lost their graft. Thirteen patients (38.2%) had stable graft function. CONCLUSION: Late acute ABMR has unsatisfactory prognosis in spite of aggressive standard antihumoral treatment. Surveillance of late ABMR using DSA monitoring may be helpful in early detection and management.

The 37-year kidney transplantation experience at Siriraj Hospital.

With 37-years of experience, a total of 801 kidney transplantations (59.4% were deceased donors and 40.6% were living donors) performed at Siriraj hospital were reported. The point system parallel to OPTN/UNOS for waitlists was utilized. Most of the recipients of deceased donor kidney transplantations had 3 HLA mismatches. Due to the point allocation system, none of them had 6 HLA mismatches. Extended criteria donor comprised 7.8% of all deceased donors. Mean duration of dialysis prior to deceased donor transplant was 53 +/- 34 months. Delayed graft function (DGF) was found in 54% of deceased donor kidney transplantation and resulted in significantly higher rate of 1 year biopsy-proven acute rejection, longer duration of kidney transplant admission, higher admission cost and lower patient survival compared to those with immediate graft function. Most of living donor kidney transplant recipient had 1 haplotype match. Mean donor age was 35.9 +/- 9.8 years. 95.6% of the recipients were on hemodialysis prior to transplantation. The current standard regimen includes calcineurin inhibitor, Mycophenolic acid and prednisolone. Interleukin-2 receptor monoclonal antibody has been used in the high immunological risk or high risk for DGF recipients that were 50% of the recipients. There was no statistically significant difference in the biopsy-proven acute rejection (BPAR) free survival between deceased and living donor transplantation. Proportion of cases with the diagnosis of acute rejection according to Banff 2007 classification is as follows: 32.4% acute cellular rejection (ACR), 39.4% antibody-mediated rejection (AMR) and 21.1% mixed cellular and antibody-mediated rejection. Seventy two patients, 35 deceased donor and 37 living donor kidney transplant recipients, had biopsy-proven glomerular disease after transplantation which IgA nephropathy is the most common form of glomerulonephritis. Median graft survival was 7.6 and 13.2 years and median patient survival was 12.1 and 15.5 years for recipient of deceased and living donor transplant respectively. The follow up program of living donors was introduced in 2003 and there were not any donors who required renal replacement therapy.

Effect of posttransplant anti-HLA antibody on outcome of renal transplantation.

Pretransplant anti-HLA antibody has an impact on renal transplantation (RT) outcome. However, the role of posttransplant anti-HLA antibody in renal allograft outcome remains unclear. We conducted this study to determine whether posttransplant anti-HLA plays an important role in the outcome of renal allografts. Our investigation used a cross sectional design. Class I and II anti-HLA antibodies were obtained in 41 renal transplant patients. Patients had undergone either living-related (n = 15) or cadaveric (n = 26) RT. All patients had been transplanted for >6 months. The correlation of posttransplant class I and class II, anti-HLA antibodies with renal allograft function glomerular filtration rate (GFR) was analyzed. Patients displaying a GFR of >60 mL/min showed positive anti-HLA antibody status for class I (n = 2) and class II (n = 9). In contrast, those whose renal transplants showed a GFR <60 mL/min included three patients positive for HLA class I and 19 patients for HLA class II. Posttransplant class II anti-HLA antibody showed a negative correlation with GFR (r = -0.31, P = .03). Preliminary results indicated that class II posttransplant anti-HLA antibody might be one mechanism of chronic renal allograft rejection and may confirm the important role of HLA matching in renal transplantation outcome.

Abnormal kinetics of erythrocyte sodium lithium countertransport in renal transplant recipients.

Cardiovascular disease is now the most common cause of death in renal transplantation. Cyclosporine (CsA)-associated hypertension might be a major cause of cardiovascular risk factors. There is evidence suggesting that one mechanism of CsA toxicity might be mediated through alteration of membrane lipid peroxidation, which can activate cellular pathways. Erythrocyte sodium lithium countertransport (Na/Li CT) is a sensitive membrane protein that is abnormal in several hypertensive-related diseases. We have studied the kinetics of erythrocyte Na/Li CT in 38 renal transplant recipients. Group 1 (15 patients) received CsA, azathioprine, and prednisolone (C+A+P), Group 2 (15 patients) CsA and prednisolone (C+P), and Group 3 (8 patients) azathioprine and prednisolone (A+P). Compared with the normal subjects, the Michaelis constant for extracellular sodium (Km) of erythrocyte Na/Li CT was lower among the CsA-based regimen groups (C+A+P and C+P), but not the A+P group. The maximum velocity (Vmax)/Km ratio was also higher among the C+A+P and C+P groups than the A+P group. These abnormalities of Na/Li CT kinetics might be due to abnormalities of cell membrane functions, caused by immunosuppressive drugs, particularly CsA. Further studies involving the effect of CsA on the physiological function of membrane thiol proteins are required.

Erythrocyte sodium lithium countertransport in renal transplant recipients with mycophenolate mofetil and low-dose cyclosporine.

Hypertension, a common complication after renal transplantation, has many potential etiologies. Erythrocyte sodium lithium countertransport (Na/LiCT) is a sensitive membrane protein that has been observed to be abnormal in several hypertension-related diseases. We have shown that the kinetics of Na/LiCT were abnormal in renal transplant recipients treated with usual dose of cyclosporine (CsA). We postulated that CsA might be a cause of post-renal transplantation hypertension. There is evidence showing that the severity of CsA nephrotoxicity is dependent on the dose. Mycophenolate mofetil (MMF) may allow CsA dose reduction without increasing the risk of rejection. We studied the impact of CsA dose reduction in association with MMF on the kinetics of erythrocyte Na/LiCT in renal transplants. In 15 renal allograft recipients, 2 g/d MMF were introduced and the CsA dose reduced to reach whole-blood levels between 70 and 100 ng/mL within 1 month. CsA doses and levels, renal function parameters, blood pressure, and the kinetics of Na/LiCT were evaluated before and 6 months after CsA dose reduction. Overall, renal transplant recipients with usual doses of CsA showed a lower Km with a higher Vmax/Km ratio for erythrocyte Na/LiCT than normal controls (Km, 40 +/- 4 vs 74 +/- 11; P <.05; Vmax/Km, 10.2 +/- 1.7 vs 6.1 +/- 0.9; P <.05). After 6 months of CsA dose reduction, the Km and Vmax/Km of Na/LiCT were similar to those of normal controls (Km, 66 +/- 8 vs 74 +/- 11; P >.05; Vmax/Km, 5.7 +/- 1.2 vs 6.1 +/- 0.9; P >.05). These results demonstrate that reduction of CsA dose in combination with MMF may improve the kinetics of Na/LiCT and lessen the long-term side effects of CsA without increasing the risk of rejection.

Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.

A novel splice-acceptor site mutation (IVS13-2A>T) of polycystic kidney disease 1 (PKD1) gene resulting in an RNA processing defect with a 74-nucleotide deletion in exon 14 of the mRNA transcript.

Autosomal dominant polycystic kidney disease (ADPKD) occurs mainly from mutations of polycystic kidney disease 1 (PKD1) gene. A novel mutation of the PKD1 gene due to a nucleotide substitution in splice-acceptor site of IVS13 (AG->TG) was identified by analyses of PKD1-cDNA and genomic DNA. The IVS13-2A>T substitution resulted in an inactivation of this splice site and utilization of cryptic splice acceptor site in exon 14, causing a 74-nucleotide deletion of this exon in the PKD1-mRNA transcript. The abnormal transcript was present ectopically in the patients' lymphocytes. The partial deletion of PKD1-mRNA leads to frameshift translation and introduces a termination signal at codon 1075. The truncated protein with about one quarter of the full-length polycystin-1 is most likely inactive. Thus, the effect of this mutation would be "loss-of-function" type. Allele specific amplification (ASA) was developed to detect the mutation in DNA samples of other family members. The mutation was present in 11 affected but absent in 13 unaffected family members, corresponding to the results of linkage analysis. In addition, it was not observed in DNA samples of 57 unrelated healthy individuals. Hum Mutat 15:115, 2000.

Long RT-PCR Amplification of the entire coding sequence of the polycystic kidney disease 1 (PKD1) gene.

Characterization of mutations of the PKD1 gene has been limited by the fact that three-fourths of this gene at its 5' end is homologous to sequences of at least three other genes on the same chromosome. We have therefore developed a method of long reverse transcription PCR for selective amplification of the entire coding sequence of the PKD1 gene from its mRNA. A PCR primer specific to the sequence in the 3' unique region of the PKD1 gene was synthesized for use coupled with a primer binding to sequence in the homologous region at a distance of about 13.6 kb apart. The commercial availability of RNase H-free reverse transcriptase for long cDNA synthesis and of an enzyme mixture containing Taq and Pfu DNA polymerases for long-range PCR have made this development possible. The long PCR product was proven to be derived from PKD1-mRNA. The results clearly indicated that the long PCR product contained the coding sequence derived from PKD1-mRNA. To our knowledge, this is the first report of a procedure that can reproducibly isolate full-length PKD1 coding sequence from its mRNA transcript, which will prove useful for screening and characterization of mutations in the PKD1 gene.

Erythrocyte membrane thiol proteins associated with changes in the kinetics of Na/Li countertransport: a possible molecular explanation of changes in disease.

BACKGROUND: Abnormal erythrocyte Na/Li countertransport is associated with diseases such as essential hypertension and diabetic renal disease. Although it seems unlikely that Na/Li countertransport contributes to any disease process, it may be abnormal because of a change in the cell membrane that is part of the disease process. METHODS: We have shown that Na/Li countertransport kinetics are modified by two types of thiol group. One of these, which we have called 'type 1', is rapidly alkylated by N-ethylmaleimide to give a kinetic pattern similar to that in the above diseases. RESULTS: AtpH 6 and 2 degrees C, both N-ethylmaleimide and iodoacetamide cause the K(m) of Na/Li countertransport to decrease to completion in 300s, with 78% (SEM 6%) of the decrease occurring in 30s. Using these reaction conditions, N-ethylmaleimide reacted with a unique thiol group on a 33-kD protein, blocking its subsequent reaction with biotin maleimide. This 33-kD protein was present in rabbit erythrocytes, which have high levels of Na/Li countertransport, but absent from rat erythrocytes, which have no Na/Li countertransport. Iodoacetyl biotin labelled a 60-kD protein that was specifically blocked by iodoacetamide. CONCLUSION: We suggest that these proteins are members of a cluster of membrane proteins that can modify Na/Li countertransport and may have a functional role in the disease processes.

Lack of function of an N-ethylmaleimide-sensitive thiol protein in erythrocyte membrane of autosomal dominant polycystic kidney disease.

The polycystic kidney disease 1 (PKD1) gene product polycystin has been predicted to be an integral membrane protein involved in cell-cell and cell-matrix interactions. The erythrocyte membrane fluidity in autosomal dominant polycystic kidney disease (ADPKD) patients is increased, and this may be due to a membrane cytoskeletal abnormality. The abnormal erythrocyte sodium-lithium countertransport kinetics in-ADPKD are related to an altered thiol protein in the cytoskeleton. The possibility that a similar thiol protein abnormality causes the increased erythrocyte membrane fluidity in ADPKD was investigated. The membrane fluidity of intact erythrocytes from 12 ADPKD patients and 12 healthy control subjects was assessed from the fluorescence anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) and trimethylammonium-diphenyl-hexatriene (TMA-DPH). The effect on membrane fluidity of N-ethylmaleimide (NEM), cytochalasin D, heating at 48 degrees C for 20 min, or more specifically, liposomes containing antibodies to actin or ankyrin, was determined. In erythrocytes from healthy control subjects, the fluorescence anisotropy of DPH (mean +/- SEM: 0.223 +/- 0.001) was decreased after treatment with NEM (0.200 +/- 0.003, P < 0.001), cytochalasin D (0.206 +/- 0.006, P < 0.001), heating (0.199 +/- 0.002, P < 0.001), and antibodies to actin (0.194 +/- 0.002, P < 0.001) or ankyrin (0.196 +/- 0.002, P < 0.001). The TMA-DPH anisotropy (0.279 +/- 0.001) was also decreased after treatment with NEM (0.264 +/- 0.001, P < 0.001), cytochalasin D (0.264 +/- 0.001, P < 0.001), heating (0.265 +/- 0.001, P < 0.001), and antibodies to actin (0.262 +/- 0.002, P < 0.001) or ankyrin (0.262 +/- 0.002, P < 0.001). NEM had no additional effect on the other treatments, suggesting that its target thiol protein was associated with the cytoskeleton. In untreated erythrocytes from ADPKD patients, fluorescence anisotropies of both DPH and TMA-DPH were reduced, and none of the treatments altered the anisotropy of either DPH or TMA-DPH. In ADPKD, a cytoskeletal thiol protein is abnormal and possibly explains abnormal lipid bilayer properties and transport protein function in erythrocytes in this disease.

Insulin resistance in adult polycystic kidney disease.

Adult polycystic kidney disease (APKD) is a common hereditary disease with renal and extra-renal manifestations. There are at least three genes responsible for this disease. The polycystic kidney disease 1 (PKD1) gene product is a membrane protein involved in cell-cell and cell-matrix interactions and has a widespread tissue distribution. Abnormal membrane fluidity in erythrocytes from APKD patients is due to altered membrane proteins. Membrane fluidity of mononuclear cells is related to whole body insulin sensitivity. Insulin sensitivity might therefore be disturbed in APKD if the erythrocyte membrane abnormality is also present in other cells. Therefore, we investigated insulin sensitivity in 15 APKD patients and 20 normal subjects matched for age and sex. Insulin sensitivity was assessed by a short insulin tolerance test to derive the first-order rate constant for the disappearance of glucose (Kitt) and mononuclear leukocyte membrane fluidity was measured by fluorescence anisotropy. The Kitt value (% mmol.liter-1.min-1) was lower in APKD patients than in normal subjects [median (range) 2.2 (1.5 to 6.3) vs. 4.1 (2.0 to 5.4). P < 0.001]. Fasting plasma insulin concentrations were negatively correlated with the Kitt values (r = -0.66, P < 0.001). Core region anisotropy was significantly lower (higher fluidity) in leukocytes from APKD patients [mean (SEM) 0.164 (0.003) vs. 0.174 (0.001), P < 0.001]. Insulin sensitivity was positively correlated with the fluorescence anisotropy of the core region of leukocyte membranes (r = 0.81, P = 0.0001). In conclusion, APKD patients were insulin resistant and some patients were hyperinsulinemic, which may indicate increased cardiovascular risk. The cellular basis of the insulin resistance may be directly related to the proteins causing the disease or to the general change in membrane properties.