Suchai Sritippayawan

Identification of human urinary trefoil factor 1 as a novel calcium oxalate crystal growth inhibitor

Previous research on proteins that inhibit kidney stone formation has identified a relatively small number of well-characterized inhibitors. Identification of additional stone inhibitors would increase understanding of the pathogenesis and pathophysiology of nephrolithiasis. We have combined conventional biochemical methods with recent advances in mass spectrometry (MS) to identify a novel calcium oxalate (CaOx) crystal growth inhibitor in normal human urine. Anionic proteins were isolated by DEAE adsorption and separated by HiLoad 16/60 Superdex 75 gel filtration. A fraction with potent inhibitory activity against CaOx crystal growth was isolated and purified by anion exchange chromatography. The protein in 2 subfractions that retained inhibitory activity was identified by matrix-assisted laser desorption/ionization-time-of-flight MS and electrospray ionization-quadrupole-time-of-flight tandem MS as human trefoil factor 1 (TFF1). Western blot analysis confirmed the mass spectrometric protein identification. Functional studies of urinary TFF1 demonstrated that its inhibitory potency was similar to that of nephrocalcin. The inhibitory activity of urinary TFF1 was dose dependent and was inhibited by TFF1 antisera. Anti-C-terminal antibody was particularly effective, consistent with our proposed model in which the 4 C-terminal glutamic residues of TFF1 interact with calcium ions to prevent CaOx crystal growth. Concentrations and relative amounts of TFF1 in the urine of patients with idiopathic CaOx kidney stone were significantly less (2.5-fold for the concentrations and 5- to 22-fold for the relative amounts) than those found in controls. These data indicate that TFF1 is a novel potent CaOx crystal growth inhibitor with a potential pathophysiological role in nephrolithiasis.

Pharmacokinetics of Colistin Methanesulfonate and Formed Colistin in End-Stage Renal Disease Patients Receiving Continuous Ambulatory Peritoneal Dialysis

ABSTRACT Colistin, administered intravenously as its inactive prodrug colistin methanesulfonate (CMS), is increasingly used as last-line therapy to combat multidrug-resistant Gram-negative bacteria. CMS dosing needs to be adjusted for renal function. The impact of continuous ambulatory peritoneal dialysis (CAPD) on the pharmacokinetics of both CMS and colistin has not been studied. No CMS dosing recommendations are available for patients receiving CAPD. Eight CAPD patients received a single intravenous CMS dose (150 mg colistin base activity [CBA]) over 30 min. Serial blood and dialysate samples, and cumulative urine where applicable, were collected over 25 h. CMS and colistin concentrations were determined by high-performance liquid chromatography. Population pharmacokinetic modeling and Monte Carlo simulations were conducted. The total body clearance of CMS (excluding CAPD clearance) was 1.77 liters/h (44%) [population mean (between-subject variability)], while CAPD clearance was 0.088 liter/h (64%). The population mean terminal half-life of CMS was 8.4 h. For colistin, the total clearance/fraction of CMS metabolized to colistin (fm) (excluding CAPD clearance) was 2.74 liters/h (50%), the CAPD clearance was 0.101 liter/h (34%), and the mean terminal half-life was 13.2 h. Monte Carlo simulations suggested a loading dose of 300 mg CBA on day 1 and a maintenance dose of either 150 mg or 200 mg CBA daily to achieve a target average steady-state plasma colistin concentration of 2.5 mg/liter. Clearance by CAPD was low for both CMS and formed colistin. Therefore, CMS doses should not be increased during CAPD. Modeling and simulation enabled us to propose the first evidence-based CMS dosage regimen for CAPD patients.

Evidence suggesting a genetic contribution to kidney stone in northeastern Thai population

Genetic factor may play a role in the pathogenesis of kidney stone that is found in the northeastern (NE) Thai population. Herein, we report initial evidence suggesting genetic contribution to the disease in this population. We examined 1,034 subjects including 135 patients with kidney stone, 551 family members, and 348 villagers by radiography of kidney–ureter–bladder (KUB) and other methods, and also analyzed stones removed by surgical operations. One hundred and sixteen of 551 family members (21.05%) and 23 of the 348 villagers (6.61%) were affected with kidney stone. The relative risk (λR) of the disease among family members was 3.18. Calcium stones (whewellite, dahllite, and weddellite) were observed in about 88% of stones analyzed. Our data indicate familial aggregation of kidney stone in this population supporting that genetic factor should play some role in its pathogenesis. Genetic and genomic studies will be conducted to identify the genes associated with the disease.

Extracorporeal clearance of colistin methanesulphonate and formed colistin in end-stage renal disease patients receiving intermittent haemodialysis: implications for dosing

OBJECTIVES Colistin, administered intravenously as its inactive prodrug colistin methanesulphonate (CMS), is being increasingly used. However, there is very limited information available on the impact of haemodialysis (HD) on the pharmacokinetics of CMS and formed colistin. PATIENTS AND METHODS A single 30 min intravenous dose of CMS (150 mg of colistin base activity) was administered to 10 patients undergoing HD. HD was performed from 1.5 to 5.5 h after the start of the CMS infusion. Serial blood samples were collected over 50 h, additional blood samples pre- and post-dialysis membrane at three timepoints during HD, dialysate samples at four timepoints during HD, and a cumulative urine sample over 24 h. CMS and colistin were determined by HPLC. Population modelling and determination of HD clearance by multiple methods was conducted. RESULTS The average amount of CMS recovered in the dialysate was 30.6% of the dose administered. The concentrations of CMS and colistin in the plasma and the amounts of CMS recovered in the dialysate were well described by the population disposition model. The clearance of CMS by dialysis as estimated by population analysis based on systemic plasma concentrations and amounts in the dialysate was 4.26 L/h (26% coefficient of variation). The dialysis clearance determined from the pre- and post-membrane plasma concentrations was 5.67 L/h (21%) for CMS and 3.99 L/h (44%) for colistin. Thus, CMS clearance by dialysis from trans-cartridge extraction was ∼30% higher than when calculated based on the amount in dialysate, suggesting adsorption to the membrane. CONCLUSIONS Due to the extensive removal of CMS by dialysis, HD should be conducted at the end of a dosing interval and a supplemental dose should be administered.

Prothrombin Haplotype Associated With Kidney Stone Disease in Northeastern Thai Patients

OBJECTIVE To evaluate genetic variations associated with kidney stone disease in Northeastern Thai patients. METHODS Altogether, 67 single nucleotide polymorphisms (SNP) distributed within 8 candidate genes, namely TFF1, S100A8, S100A9, S100A12, AMBP, SPP1, UMOD, and F2, which encode stone inhibitor proteins, including trefoil factor 1, calgranulin (A, B, and C), bikunin, osteopontin, tamm-Horsfall protein, and prothrombin, respectively, were initially genotyped in 112 individuals each and in additional subjects to consist of 164 patients and 216 control subjects in total. RESULTS We found that minor allele and homozygous genotype frequencies of 8 of 10 SNPs distributed within the F2 gene were significantly higher in the control group than in the patient group. Two F2 haplotypes were found to be dually associated with kidney stone risk, one (TGCCGCCGCG) with increased disease risk and the other (CGTTCCGCTA) with decreased disease risk. However, these 2 haplotypes were associated with the disease risks in only the female, not the male, group. CONCLUSIONS The results of our study indicate that genetic variation of F2 is associated with kidney stone risk in Northeastern Thai female patients.

Association between Human Prothrombin Variant (T165M) and Kidney Stone Disease

We previously reported the association between prothrombin (F2), encoding a stone inhibitor protein - urinary prothrombin fragment 1 (UPTF1), and the risk of kidney stone disease in Northeastern Thai patients. To identify specific F2 variation responsible for the kidney stone risk, we conducted sequencing analysis of this gene in a group of the patients with kidney stone disease. Five intronic SNPs (rs2070850, rs2070852, rs1799867, rs2282687, and rs3136516) and one exonic non-synonymous single nucleotide polymorphism (nsSNP; rs5896) were found. The five intronic SNPs have no functional change as predicted by computer programs while the nsSNP rs5896 (c.494 C>T) located in exon 6 results in a substitution of threonine (T) by methionine (M) at the position 165 (T165M). The nsSNP rs5896 was subsequently genotyped in 209 patients and 216 control subjects. Genotypic and allelic frequencies of this nsSNP were analyzed for their association with kidney stone disease. The frequency of CC genotype of rs5896 was significantly lower in the patient group (13.4%) than that in the control group (22.2%) (P = 0.017, OR 0.54, 95% CI 0.32–0.90), and the frequency of C allele was significantly lower in the patient group (36.1%) than that in the control group (45.6%) (P = 0.005, OR 0.68, 95% CI 0.51–0.89). The significant differences of genotype and allele frequencies were maintained only in the female group (P = 0.033 and 0.003, respectively). The effect of amino-acid change on UPTF1 structure was also examined by homologous modeling and in silico mutagenesis. T165 is conserved and T165M substitution will affect hydrogen bond formation with E180. In conclusion, our results indicate that prothrombin variant (T165M) is associated with kidney stone risk in the Northeastern Thai female patients.

Correlation between genotypes of F2 rs5896 (p.Thr165Met) polymorphism and urinary prothrombin fragment 1

resolution melting (PCR-HRM) analysis and confirmed by DNA sequencing. Subjects that were taking medications that could cause the development of stones or that could affect mineral metabolism were excluded. To avoid potential effects of renal disease, subjects with dipstick proteinuria or glucosuria above trace levels were also excluded. Urine specimens were collected in the presence of 1 M sodium azide and were determined to be free of blood, haemoglobin, and nitrites using Combur Test M (Roche Diagnostics, Mannheim, Germany). One hundred millilitre aliquots of urine specimens were centrifuged at 3500 g (Beckman JA-14; Beckman Coulter, Inc. Brea, CA, USA) for 10 min, concentrated through a 3000 NMWL Amicon Ultra-15 Centrifugal Filter Device (Millipore Corporation, Billerica, MA, USA), and dialyzed using a 10,000 MWCO dialysis tube (SnakeSkin; Thermo Fisher Scientific, Inc., Waltham, MA, USA), with stirring at 4 °C for 20 h against water and with three changes of the bath. Urine samples were then lyophilized and reconstituted in rehydration buffer (8 M urea and 2% CHAPS). Protein concentration was determined by Bradford protein assay and proteins were then separated by electrophoresis on polyacrylamide gel. The electrophoretic mobility pattern and relative quantities of UPTF1 in the concentrated urine samples from each group were loaded with equal amounts of urine creatinine (50 μg) and examined by Western blot analysis using sheep anti-human prothrombin fragment 1 (CL20111AP; Cedarlane Laboratories Ltd, Burlington, NC, USA) and donkey anti-sheep IgG-HRP (sc-2473; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) [6]. Chemiluminescent signals generated by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc., Waltham, MA, USA) were detected using a G:BOX chemiluminescence imaging system (Syngene, Cambridge, UK). Specific band intensities of UPTF1 from Urinary prothrombin fragment 1 (UPTF1) is an F1 activation peptide of human prothrombin [1]. This 31-kDa glycoprotein, which was originally referred to as crystal matrix protein, was found to be the major protein incorporated within calcium oxalate (CaOx) crystals generated from human urine in vitro [2]. This protein is a potent inhibitor of CaOx crystal growth and aggregation in undiluted human urine and in inorganic conditions [3, 4]. A single nucleotide polymorphism (SNP) (rs5896: NM_000506.4:c.494C>T; NP_000497.1:p.Thr165Met) that is a genetic variation of F2 gene encoding human prothrombin was reported to be associated with kidney stone disease (KSD) risk in a group of female patients from Northeastern Thailand [5]. We hypothesised that this F2 variation correlates with human urinary prothrombin level. The aim of this study was to investigate the association between this genetic variation and UPTF1 protein in normal female subjects. Twenty-four hour urine samples were collected from five female subjects in each of the SNP rs5896 TT, TC, and CC genotype groups. The subjects’ DNA samples were genotyped by polymerase chain reaction-high

Loss-of-function mutations of SCN10A encoding Na V 1.8 α subunit of voltage-gated sodium channel in patients with human kidney stone disease

Human kidney stone disease (KSD) causes significant morbidity and public health burden worldwide. The etiology of KSD is heterogeneous, ranging from monogenic defects to complex interaction between genetic and environmental factors. However, the genetic defects causing KSD in the majority of affected families are still unknown. Here, we report the discovery of mutations of SCN10A, encoding NaV1.8 α subunit of voltage-gated sodium channel, in families with KSD. The region on chromosome 3 where SCN10A locates was initially identified in a large family with KSD by genome-wide linkage analysis and exome sequencing. Two mutations (p.N909K and p.K1809R) in the same allele of SCN10A co-segregated with KSD in the affected family. Additional mutation (p.V1149M) of SCN10A was identified in another affected family, strongly supporting the causal role of SCN10A for KSD. The amino acids at these three positions, N909, K1809, and V1149, are highly conserved in vertebrate evolution, indicating their structural and functional significances. NaV1.8 α subunit mRNA and protein were found to express in human kidney tissues. The mutant proteins expressed in cultured cells were unstable and causing reduced current density as analyzed by whole-cell patch-clamp technique. Thus, loss-of-function mutations of SCN10A were associated with KSD in the families studied.