A Novel Peptide Reagent for Investigating Disulfide-Coupled Folding Intermediates of Mid-Size Proteins.

Investigations of protein folding have largely involved the use of disulfide-containing proteins, since the disulfide-coupled folding of proteins allows folding intermediates to be trapped and their conformations determined. However, studies of the folding mechanisms of mid-size proteins face several problems, one of which is that detecting folding intermediates is difficult. Therefore, to solve this issue, a novel peptide reagent, maleimidohexanoyl-Arg5-Tyr-NH2, was designed and applied to the detection of folding intermediates of model proteins. BPTI was chosen as a model small protein to estimate the ability of the novel reagent to detect folding intermediates. In addition, a precursor protein (prococoonase) of Bombyx mori cocoonase was used as a model mid-size protein. Cocoonase is classified as a serine protease and has a high homology with trypsin. We recently found that the propeptide sequence of prococoonase (proCCN) is important for the folding of cocoonase. However, it was difficult to study the folding pathway of proCCN since the folding intermediates could not be separated on a reversed-phase HPLC (RP-HPLC). Therefore, to separate the folding intermediates by RP-HPLC, the novel labeling reagent was used to accomplish this for proCCN. The results indicated that the peptide reagent allowed the intermediates to be captured, separated on SDS-PAGE, and analyzed by RP-HPLC without the occurrence of undesirable disulfide-exchange reactions during the labeling reactions. The peptide reagent reported herein is a practical tool for investigating the mechanisms of disulfide-coupled folding of mid-size proteins.

The Cell Adhesion Activity of the Joining Peptide of Proopiomelanocortin.

Proopiomelanocortin (POMC) is a precursor protein of several peptide hormones, such as ACTH and ß-endorphin. Almost all of the peptide hormones in POMC have been drastically investigated in terms of their biological activities. However, the biological activity of the joining peptide region (JP) in POMC is unknown. Therefore, to explore the biological activity of JP, sequence analyses of mammalian POMC were performed. We found an -Arg-Gly-Asp- (RGD) motif in several mammalian species, such as porcine, suggesting that JP has cell adhesion activity. To validate this hypothesis, the cell adhesion activities of the synthetic porcine JP peptides were examined using 293T cells. Cell adhesions were observed in a concentration-dependent manner of the JP peptides. In addition, the JP peptide competitively inhibited cell adhesion to the POMC-coated plates. Moreover, the cell adhesion activity of the joining peptide was inhibited by the addition of EDTA, indicating that the JP peptide mediates the cell adhesion activity via a receptor protein, integrin. Interestingly, a human JP peptide, which possesses an -Arg-Ser-Asp- (RSD) sequence in place of the RGD sequence, exhibited a higher ability in the cell adhesion activity than that of the porcine JP peptide, suggesting that the cell adhesion activity of the joining peptide is developed during the molecular evolution of POMC. In conclusion, our results reveal that the joining peptide in POMC plays an important role during cell adhesion and provide useful information related to signal transduction of nerve peptide hormones derived from POMC.

The Molecular Basis of Heat-Stable Enterotoxin for Vaccine Development and Cancer Cell Detection.

Heat-stable enterotoxin (STa) produced by Enterotoxigenic E. coli is responsible for causing acute diarrhea in infants in developing countries. However, the chemical synthesis of STa peptides with the native conformation and the correct intra-molecular disulfide bonds is a major hurdle for vaccine development. To address this issue, we herein report on the design and preparation of STa analogs and a convenient chemical method for obtaining STa molecules with the correct conformation. To develop an STa vaccine, we focused on a structure in a type II ß-turn in the STa molecule and introduced a D-Lys residue as a conjugation site for carrier proteins. In addition, the -Glu-Leu- sequence in the STa molecule was replaced with a -Asp-Val- sequence to decrease the toxic activity of the peptide to make it more amenable for use in vaccinations. To solve several issues associated with the synthesis of STa, such as the formation of non-native disulfide isomers, the native disulfide pairings were regioselectively formed in a stepwise manner. A native form or topological isomer of the designed STa peptide, which possesses a right-handed or a left-handed spiral structure, respectively, were synthesized in high synthetic yields. The conformation of the synthetic STa peptide was also confirmed by CD and NMR spectroscopy. To further utilize the designed STa peptide, it was labeled with fluorescein for fluorescent detection, since recent studies have also focused on the use of STa for detecting cancer cells, such as Caco-2 and T84. The labeled STa peptide was able to specifically and efficiently detect 293T cells expressing the recombinant STa receptor (GC-C) protein and Caco-2 cells. The findings reported here provide an outline of the molecular basis for using STa for vaccine development and in the detection of cancer cells.

The propeptide sequence assists the correct folding required for the enzymatic activity of cocoonase.

Cocoonase, a protein that is produced by the silkworm (Bombyx mori), is thought to specifically digest the sericin protein of the cocoon and has a high homology with trypsin. Similar to trypsin, cocoonase is folded as an inactive precursor protein which is activated by releasing the propeptide moiety. However, the mechanism responsible for the activation of its catalytic structure has not yet been determined in detail. Therefore, to investigate the activation and folding mechanism of cocoonase, recombinant cocoonase (CCN) and prococoonase (proCCN) were over-expressed in E. coli cells. Both recombinant proteins (proCCN and CCN) were expressed as inclusion bodies in E. coli cells and their folding was examined under several sets of conditions. After the refolding reactions, both of the recombinant proteins were present as the oxidized soluble forms. The proCCN protein was then auto-processed to release the propeptide region for activation. Interestingly, the CCN (CCN∗) derived from the refolded proCCN showed a much stronger protease activity than the refolded CCN from the reduced CCN in a protease assay using Bz-Arg-OEt as a substrate. In addition, the secondary structure of the refolded CCN protein was similar to that of the CCN∗ protein, as evidenced by CD measurements. These results suggest that the CCN protein becomes trapped in a molten globule-like state without the assistance of the propeptide region during the folding process. We therefore conclude that the propeptide region of CCN kinetically accelerates the folding of CCN to adopt the correct conformation of cocoonase at the final step of the folding pathway.

Genome-wide meta-analysis between renal overload type and renal underexcretion type of clinically defined gout in Japanese populations.

Despite progress in understanding of the genetic basis of gout, the precise factors affecting differences in gout susceptibility among different gout subtypes remain unclear. Using clinically diagnosed gout patients, we conducted a genome-wide meta-analysis of two distinct gout subtypes: the renal overload type and the renal underexcretion type. We provide genetic evidence at a genome-wide level of significance that supports a positive association between ABCG2 dysfunction and acquisition of the renal overload type.

Two-year outcomes of low-exposure extended-release tacrolimus and mycophenolate mofetil regimen in de novo kidney transplantation: A multi-center randomized controlled trial.

BACKGROUND: Once-daily extended-release tacrolimus (TACER) is commonly administered following kidney transplantation (KTx); however, its optimal dosage remains unknown. METHODS: In this multi-center, randomized controlled trial, 62 living donor KTx recipients were assigned to either standard-exposure (SE; n = 32) or low-exposure (LE; n = 30) TACER (Graceptor®, Astellas Pharm Inc.) groups. All patients received basiliximab and mycophenolate mofetil (MMF). The primary outcomes were acute rejection, graft/patient survival, and the secondary outcomes were incidence of cytomegalovirus infection, and de novo donor-specific antibodies (dnDSA) production. RESULTS: The tacrolimus trough level and estimated area under the blood concentration-time curve (eAUC) were significantly higher in SE than in LE (SE vs. LE; 1 year: 5.0 ± 0.9 ng/ml and 206.9 ± 26.8 ng h/ml vs. 3.4 ± 1.0 ng/ml and 153.9 ± 26.4 ng h/ml; 2 years: 4.8 ± 1.0 ng/ml and 204.9 ± 30.1 ng h/ml vs. 3.8 ± 0.9 ng/ml and 164.4 ± 27.0 ng h/ml). In contrast, the dosage and eAUC of MMF did not differ between groups. Two-year graft and patient survival rates were 100% in both groups, and acute rejection rates were 0% and 10% in the SE and LE, respectively (p = 0.11). The mean estimated glomerular filtration rates did not differ between the groups. Cytomegalovirus infection was slightly lower in the LE (SE: 12.5% and LE: 6.7%, p = 0.37). In the LE, four cases of dnDSA were noted within 2 years of transplantation; no case was observed in the SE (p = 0.034). CONCLUSIONS: Although the LE TACER regimen showed similar rates of acute rejection, as well as graft and patient survival compared with SE after KTx, LE was significantly more associated with dnDSA. Further investigation of its long-term effect on graft survival is warranted. (University Hospital Medical Information Network Clinical Trials Registry ID: UMIN000033089).

Influence of Graft Ureter Length, a Donor-Related Factor, on Urinary Tract Infections After Living-Donor Kidney Transplantation: A Single-Center Analysis of 211 Cases.

Urinary tract infection (UTI) occurs in 25% of recipients of living-donor kidney transplantation (LDKT). Female sex, age, and anatomical abnormalities have been reported as recipient-related risk factors for UTI after LDKT; few studies have reported donor-related factors. We retrospectively examined UTI occurrence within 5 years of transplantation in recipients (n = 211) who underwent LDKT at our hospital between April 2011 and April 2021. All nephrectomies were performed using a retroperitoneal pure laparoscopic approach. The ureter was dissected at the lower level of the common iliac artery and trimmed to the shortest length, enough to reach the bladder using extra vesicular ureterocystoneostomy with a 3 cm submucosal tunnel. Twenty-nine recipients (13.7%) developed UTI within 5 years, and the median time to onset was 40.0 days. After adjusting for the well-known factors, including recipient sex, graft ureter length was an independent factor for UTI occurrence (HR 1.25, 95% CI 1.02∼1.53, p = 0.028) in the multivariate Cox regression analysis. The long ureter is usually trimmed, and the widest part is used for anastomosis, which may increase the possibility of reflux from the bladder to the ureter in the standard technique. The ureter length may be associated with the incidence of UTI after LDKT.

Degradation-Suppressed Cocoonase for Investigating the Propeptide-Mediated Activation Mechanism.

Cocoonase is folded in the form of a zymogen precursor protein (prococoonase) with the assistance of the propeptide region. To investigate the role of the propeptide sequence on the disulfide-coupled folding of cocoonase and prococoonase, the amino acid residues at the degradation sites during the refolding and auto-processing reactions were determined by mass spectrometric analyses and were mutated to suppress the numerous degradation reactions that occur during the reactions. In addition, the Lys8 residue at the propeptide region was also mutated to estimate whether the entire sequence is absolutely required for the activation of cocoonase. Finally, a degradation-suppressed [K8D,K63G,K131G,K133A]-proCCN protein was prepared and was found to refold readily without significant degradation. The results of an enzyme assay using casein or Bz-Arg-OEt suggested that the mutations had no significant effect on either the enzyme activity or the protein conformation. Thus, we, herein, provide the non-degradative cocoonase protein to investigate the propeptide-mediated protein folding of the molecule. We also examined the catalytic residues using the degradation-suppressed cocoonase. The point mutations at the putative catalytic residues in cocoonase resulted in the loss of catalytic activity without any secondary structural changes, indicating that the mutated residues play a role in the catalytic activity of this enzyme.

Substrate-induced product-release mechanism of lipocalin-type prostaglandin D synthase.

Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = âˆ¼0.6 µM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 µM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.

Comparative study of a novel selective urate reabsorption inhibitor "dotinurad" among patient groups with different stages of renal dysfunction.

BACKGROUND: Dotinurad is a selective urate reabsorption inhibitor (SURI), which selectively inhibits URAT1 to lower serum uric acid levels in patients with hyperuricemia. Herein, the effects of dotinurad were compared among patient groups with different stages of renal dysfunction. METHODS: Patient data from four clinical trials were pooled and divided into four groups according to the stage of renal dysfunction to compare the effects of dotinurad at different stages. The grouping (stages G1-G3b) was based on the estimated glomerular filtration rate (eGFR) of the patients. In addition, patient data from a long-term study (34 or 58 weeks) were evaluated in the same manner. RESULTS: In the pooled analysis, the percentage of patients achieving a serum uric acid level of ≤ 6.0 mg/dL was 64.7-100.0% at a dose of 2 or 4 mg. In the long-term analysis, the percentage of patients achieving a serum uric acid level of ≤ 6.0 mg/dL was 60.0-100.0% at a dose of 2 or 4 mg. Although the outcomes in stage G3b were worse due to higher baseline serum uric acid levels, satisfactory outcomes were observed in all stages. Even in stages G3a and G3b, when renal function declined, the eGFR remained constant throughout the dose period. CONCLUSION: The efficacy of dotinurad was confirmed in hyperuricemic patients with normal renal function (stage G1) and mild to moderate renal dysfunction (stage G2-G3b). Dotinurad was found to be effective in the treatment of hyperuricemia in patients with mild to moderate renal dysfunction.

Long-term impact of baseline serum uric acid levels on living kidney donors: a retrospective study.

BACKGROUND: Preoperative characteristics of living kidney donors are commonly considered during donor selection and postoperative follow-up. However, the impact of preoperative uric acid (UA) levels is poorly documented. The aim of this study was to evaluate the association between preoperative serum UA levels and post-donation long-term events and renal function. METHODS: This was a single-center retrospective analysis of 183 living kidney donors. The donors were divided into high (≥5.5 mg/dl) and low (< 5.5 mg/dl) UA groups. We analyzed the relationship between preoperative UA levels and postoperative estimated glomerular filtration rate (eGFR), as well as adverse events (cardiovascular events and additional prescriptions for hypertension, gout, dyslipidemia, and diabetes mellitus), over 5 years after donation. RESULTS: In total, 44 donors experienced 52 adverse events over 5 years. The incidence of adverse events within 5 years was significantly higher in the high UA group than in the low UA group (50% vs. 24%, p = 0.003); this was true even after the exclusion of hyperuricemia-related events (p = 0.047). UA emerged as an independent risk factor for adverse events (p = 0.012). Donors with higher UA levels had lower eGFRs after donation, whereas body mass index, hemoglobin A1c, blood pressure, and low-density lipoprotein cholesterol did not have any impact on the eGFR. CONCLUSIONS: The findings suggest that preoperative UA levels should be considered during donor selection and postoperative follow-up.

Uric acid-lowering effect of dotinurad, a novel selective urate reabsorption inhibitor, in hypertensive patients with gout or asymptomatic hyperuricemia: a pooled analysis of individual participant data in phase II and III trials.

Background: Hyperuricemia is a risk factor for the development of hypertension and is comorbid in many hypertensive patients. According to Japanese hypertension management guidelines published in 2019, a target serum uric acid level of ≤6.0 mg/dL is recommended in hypertensive patients with gout or asymptomatic hyperuricemia. Dotinurad is a novel uric acid-lowering drug classified as a selective urate reabsorption inhibitor. A pooled analysis was performed on the uric acid-lowering effect of dotinurad in 222 hypertensive patients with gout or asymptomatic hyperuricemia in four clinical trials (NCT02344862, NCT02416167, NCT03100318, NCT03372200). Moreover, we analyzed the long-term uric acid-lowering effect of dotinurad in 154 hypertensive patients with gout or asymptomatic hyperuricemia (NCT03006445).Results: In the pooled analysis, the percent change in the decrease of serum uric acid with the use of dotinurad was 42.17 ± 12.42% at a dose of 2 mg and 60.42 ± 8.03% at a dose of 4 mg; the percentage of patients who achieved a serum uric acid level of ≤6.0 mg/dL was 82.8% and 100.0%. The long-term uric acid-lowering effect of dotinurad showed almost the same results. In this study, the concomitant use of diuretics or angiotensin II receptor blockers affected the uric acid-lowering effect of dotinurad at only a dose of 2 mg in the pooled analysis.Conclusions: In the pooled analysis, dotinurad lowered serum uric acid levels. Dotinurad has an achievement rate of over 80% for serum uric acid level of ≤6.0 mg/dL in both analyses, and will be clinically useful for the management of hyperuricemic states in hypertensive patients.

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients.

OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.

Topological Regulation of the Bioactive Conformation of a Disulfide-Rich Peptide, Heat-Stable Enterotoxin.

Heat-stable enterotoxin (STa) produced by enterotoxigenic E. coli causes acute diarrhea and also can be used as a specific probe for colorectal cancer cells. STa contains three intra-molecular disulfide bonds (C1-C4, C2-C5, and C3-C6 connectivity). The chemical synthesis of STa provided not only the native type of STa but also a topological isomer that had the native disulfide pairings. Interestingly, the activity of the topological isomer was approximately 1/10-1/2 that of the native STa. To further investigate the bioactive conformation of this molecule and the regulation of disulfide-coupled folding during its chemical syntheses, we examined the folding mechanism of STa that occurs during its chemical synthesis. The folding intermediate of STa with two disulfide bonds (C1-C4 and C3-C6) and two Cys(Acm) residues, the precursor peptide, was treated with iodine to produce a third disulfide bond under several conditions. The topological isomer was predominantly produced under all conditions tested, along with trace amounts of the native type of STa. In addition, NMR measurements indicated that the topological isomer has a left-handed spiral structure similar to that of the precursor peptide, while the native type of STa had a right-handed spiral structure. These results indicate that the order of the regioselective formation of disulfide bonds is important for the regulation of the final conformation of disulfide-rich peptides in chemical synthesis.

[Necrotic Thymoma;Report of a Case].

A 36-year-old man, who had never been detected abnormalities on an annual chest X-ray check up, presented with a sudden onset of right-sided chest pain and fever. Contrast-enhanced computed tomography showed an anterior mediastinal mass with necrosis or hemorrhage and right pleural effusion. Neither computed tomography-guided biopsy nor video-assisted thoracic surgery (VATS) yielded definitive histological diagnosis due to insufficiency of the sample. For diagnosis and treatment, we performed thymectomy. Histopathologically, the tumor was almost entirely necrotic with few viable tumor cells on periphery. A diagnosis of B2 thymoma was rendered.

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout.

OBJECTIVE: The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout. METHODS: We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia). RESULTS: This new approach enabled us to identify two novel gout loci (rs7927466 of CNTN5 and rs9952962 of MIR302F) and one suggestive locus (rs12980365 of ZNF724) at the genome-wide significance level (p<5.0×10-8). The present study also identified the loci of ABCG2, ALDH2 and SLC2A9. One of them, rs671 of ALDH2, was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three 'gout vs AHUA GWAS'-specific loci (CNTN5, MIR302F and ZNF724) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level. CONCLUSIONS: This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals.

Prediction model of compensation for contralateral kidney after living-donor donation.

BACKGROUND: Compensation of contralateral kidney function after living-donor kidney donation is well known, and many predictive factors have been proposed. However, no prediction model has been proposed. This study was performed to establish a tool with which to estimate the degree of compensation of the contralateral kidney after living-donor kidney donation. METHODS: We retrospectively analyzed 133 living donors for renal transplantation in our institution. We defined a favorable compensation as a post-donation estimated glomerular filtration rate (eGFR) at 1 year (calculated by the Chronic Kidney Disease Epidemiology Collaboration equation) of > 60% of the pre-donation eGFR. We analyzed the living donors' clinical characteristics and outcomes. RESULTS: The median (range) donor age was 59 (24-79) years, median (range) body mass index was 22.9 (16.8-32.7) kg/m2, and median (range) body surface area was 1.6 (1.3-2.0) m2. All donors were Japanese, and 73% of the donors were biologically related. The median (range) donor pre-donation eGFR was 108.7 (82-144) ml/min/1.73 m2, and the median (range) post-donation eGFR at 1 year was 86.9 (43-143) ml/min/1.73 m2. Eighty-six percent of donors had compensatory hypertrophy. In the univariate analysis, age, female sex, history of hypertension, body surface area, and pre-donation eGFR were significantly associated with hypertrophy (p < 0.05). In the multivariate analysis, age, female sex, history of hypertension, and ratio of the remnant kidney volume to body weight were significantly associated with hypertrophy (p < 0.05). Based on these results, we created a compensation prediction score (CPS). The median (range) CPS was 8.7 (1.1-17.4). Receiver operating characteristic analysis showed strong diagnostic accuracy for predicting favorable compensation (area under the curve, 0.958; 95% confidence interval, 0.925-0.991, p < 0.001). The optimal cut-off value of the CPS was 5.0 (sensitivity, 92.0%; specificity, 89.5%). The CPS had a strong positive correlation with the post-donation eGFR (R = 0.797, p < 0.001). CONCLUSION: The CPS might be useful tool with which to predict a favorable compensation of the contralateral kidney and remnant kidney function. If the CPS is low, careful management and follow-up might be necessary. Further investigations are needed to validate these findings in larger populations.

Factors related to suboptimal recovery of renal function after living donor nephrectomy: a retrospective study.

BACKGROUND: The renal function of the remaining kidney in living donors recovers up to 60~70% of pre-donation estimated-glomerular filtration rate (eGFR) by compensatory hypertrophy. However, the degree of this hypertrophy varies from donor to donor and the factors related to it are scarcely known. METHODS: We analyzed 103 living renal transplantations in our institution and divided them into two groups: compensatory hypertrophy group [optimal group, 1-year eGFR ≥60% of pre-donation, n = 63] and suboptimal compensatory hypertrophy group (suboptimal group, 1-year eGFR < 60% of pre-donation, n = 40). We retrospectively analyzed the factors related to suboptimal compensatory hypertrophy. RESULTS: Baseline eGFRs were the same in the two groups (optimal versus suboptimal: 82.0 ± 13.1 ml/min/1.73m2 versus 83.5 ± 14.8 ml/min/1.73m2, p = 0.588). Donor age (optimal versus suboptimal: 56.0 ± 10.4 years old versus 60.7 ± 8.7 years old, p = 0.018) and uric acid (optimal versus suboptimal: 4.8 ± 1.2 mg/dl versus 5.5 ± 1.3 mg/dl, p = 0.007) were significantly higher in the suboptimal group. The rate of pathological chronicity finding on 1-h biopsy (ah≧1 ∩ ct + ci≧1) was much higher in the suboptimal group (optimal versus suboptimal: 6.4% versus 25.0%, p = 0.007). After the multivariate analysis, the pathological chronicity finding [odds ratio (OR): 4.8, 95% confidence interval (CI): 1.3-17.8, p = 0.021] and uric acid (per 1.0 mg/dl, OR: 1.5, 95% CI: 1.1-2.2, p = 0.022) were found to be independent risk factors for suboptimal compensatory hypertrophy. CONCLUSION: Chronicity findings on baseline biopsy and higher uric acid were associated with insufficient recovery of the post-donated renal function.

Stepwise dose increase of febuxostat is comparable with colchicine prophylaxis for the prevention of gout flares during the initial phase of urate-lowering therapy: results from FORTUNE-1, a prospective, multicentre randomised study.

OBJECTIVES: To determine whether febuxostat with stepwise dose increase is as useful as colchicine prophylaxis in reducing gout flares during the initial introduction of urate-lowering therapy in patients with gout in comparison with febuxostat with no dose titration. METHODS: In this prospective, multicentre, randomised open-label comparative study, patients were randomised to group A (stepwise dose increase of febuxostat from 10 to 40 mg/day), group B (fixed-dose febuxostat 40 mg/day plus colchicine 0.5 mg/day) or group C (fixed-dose febuxostat 40 mg/day) and observed for 12 weeks. Gout flare was defined as non-steroidal anti-inflammatory drug use for gout symptoms. RESULTS: A total of 255 patients were randomised, and 241 patients were treated. Among the treated patients, gout flares were experienced by 20/96 (20.8%) in group A, 18/95 (18.9%) in group B and 18/50 (36.0%) in group C. The incidence of flare was significantly lower in groups A and B than that in group C (P=0.047 and P=0.024, respectively), although the differences were not significant after correction for multiple comparisons. No significant difference was noted between the incidence of gout flare in groups A and B. CONCLUSIONS: Our data suggested that stepwise dose increase of febuxostat and low-dose colchicine prophylaxis effectively reduced gout flares in comparison with fixed-dose febuxostat alone. Stepwise dose increase of febuxostat may be an effective alternative to low-dose colchicine prophylaxis during the introduction of urate-lowering therapy. TRIAL REGISTRATION NUMBER: UMIN 000008414.

Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice.

PURPOSE: The concentration-sensitive sodium channel (NaC) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that NaC contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether NaC is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between NaC gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. METHODS: LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. NaC protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of NaC and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. RESULTS: The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the NaC protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of NaC decreased in the lung, as well as those for α-, ß-, and γ-ENaC during ALI. Thus, NaC expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. CONCLUSION: Our results suggest that suppression of the gene expression of NaC is involved in the development of pulmonary edema in ALI.