Water intake, baseline biopsy, and graft function after living donor kidney transplantation.

Increased water intake is recommended for kidney transplant recipients; however, its efficacy remains controversial. We hypothesized that pre-existing histological findings of the allograft might modulate the impact of water intake. We retrospectively analyzed 167 adults with living-donor kidney transplants (April 2011-May 2020; median observation period, 77 months) whose baseline biopsy data were available. We compared the chronic-change group (n = 38) with the control group (n = 129) to assess the impact of self-reported daily water intake on the estimated glomerular filtration rate (eGFR). The range distribution of water intake was as follows: - 1000 ml (n = 4), 1000-1500 ml (n = 23), 1500-2000 ml (n = 64), 2000-2500 ml (n = 57), 2500-3000 ml (n = 16), and 3000 - ml (n = 3). Donor age was significantly higher in the chronic-change group. In the control group, the ΔeGFR/year increase was correlated with water intake. However, the increase in the water intake of the chronic-change group significantly decreased ΔeGFR/year (1000-1500 ml: + 1.95 ml/min/1.73 m2 and > 2000 ml: - 1.92 ml/min/1.73 m2, p = 0.014). This study suggested a potential influence of increased water intake on recipients with marginal grafts in living donor kidney transplantation.

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.

Comparison of INTREPID® balanced and hybrid tips on anterior capsule rupture in ex vivo porcine eyes.

Phacoemulsification has emerged as the global standard for cataract surgery, and various novel methods, tools, and agents have promoted surgical efficiency and reduced complications. Conventionally, the phaco tip, which cleaves and aspirates the cataractous lens, has been mainly constructed of metal. In this study, the risk of anterior capsule rupture was evaluated under conditions of different power modes, longitudinal (Mode-L), torsional (Mode-T), or both (Mode-LT), and different aspiration powers (0 or 200 mmHg), using a traditional metal phaco tip (Group-M) or a new phaco tip with a high-strength polymer overmold on the needle edge (Group-P), which was developed to reduce the risk of capsule rupture. One hundred twenty porcine eyes were used for experiments within a setting of typical human physiological intraocular pressure. We found that Group-M showed capsule rupture with a smaller ultrasound power than did Group-P, regardless of power mode or aspiration power. In Group-M, there was no significant difference in risk of capsule rupture among power modes, however in Group-P, capsule rupture was least likely to occur with Mode-T. These results provide useful information for inexperienced ophthalmologists to improve surgical safety.

The Resistive Index by Doppler Ultrasonography as a Predictor of the Long-Term Outcomes After Kidney Transplantation.

BACKGROUND: Doppler ultrasonography (US) is a noninvasive examination for assessing graft function after kidney transplantation. Although Doppler US is routinely performed, only a few reports have investigated whether a high resistive index (RI) detected by Doppler US affects graft function and survival. We hypothesized that there is a relationship between a high RI and inferior outcomes after kidney transplantation. METHODS: We included 164 living kidney transplant patients treated between April 2011 and July 2019. We divided the patients into 2 groups according to RI (cut-off, 0.7) 1 year after transplantation. RESULTS: The recipient was significantly older in the high RI (≥0.7) group. Moreover, there were significant differences in the prevalence of pretransplant diabetes mellitus and the value of pretransplant hemoglobin A1c. Regarding long-term outcome, there was no significant difference in overall graft survival (5 years, 92.6% vs 91.8%; 10 years, 85.0% vs 67.9%; P = .64). On the other hand, the mortality was significantly worse in the high RI group (5 years, 99.1% vs 93.9%; 10 years, 96.4% vs 70.0%, P = .013). CONCLUSIONS: A high RI might predict mortality after kidney transplantation.

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.

Appointment Nonadherence and Graft Outcomes in Living Donor Kidney Transplantation.

BACKGROUND: Medication nonadherence is associated with worse graft outcomes but is hard to recognize in clinical settings due to its self-reporting nature. We hypothesized that appointment nonadherence might be associated with worse graft outcomes in living donor kidney transplantation. METHODS: We included 167 adult living-donor kidney transplants whose grafts survived >2 years from April 2011 to May 2020. Thirty-two cases of appointment nonadherence were identified and compared with the controls (n = 135). RESULTS: Younger age, male sex, higher body weight, and parent donor were significantly observed in the appointment nonadherence group. The appointment nonadherence group was significantly associated with worse graft survival (5 years: 82.3% vs 98.9%, P < .001, 10 years: 67.2% vs 89.6%, P < .001), de novo donor-specific antibody production, acute rejection, as well as the decline of graft function. Furthermore, appointment nonadherence had a 4-fold higher risk of graft loss after an adjustment with recipient age, sex, body weight, and donor type (adjusted hazard ratio: 3.93, 95% CI: 1.15-13.42, P = .029). CONCLUSIONS: Appointment nonadherence might be an alternative predictor for worse graft outcomes after living donor kidney transplantation.

A Case of Chronic Active Antibody-Mediated Rejection Caused by a Pre-Existing Anti-DQ Donor-Specific Antibody in a Systemic Lupus Erythematosus Recipient Without History of Sensitization: A Case Report.

BACKGROUND: Systemic lupus erythematosus (SLE) is reported to produce anti-HLA antibodies. We report a case of chronic active antibody-mediated rejection caused by pre-existing donor-specific antibody (DSA) in a patient with SLE without a history of sensitization. CASE REPORT: The case was a 29-year-old man with end-stage renal disease due to lupus nephritis. Cross-match with the mother was negative, but low titer anti-DQ DSA was detected, although he had no prior history of sensitization. After desensitization with rituximab and mycophenolate mofetil, a living donor kidney transplant was undergone, and his early postoperative period was uneventful. However, his renal function started to decline at 2 years post-transplant. Although there was no rejection on the biopsy at 2.5 years post-transplant, his renal function continued to decline after that. At 7 years, he had failed his graft due to chronic active antibody-mediated rejection. Retrospective analysis of human leukocyte antigen antibody tests revealed that anti-DQ DSA had disappeared at 1 year post-transplant, but high titer DSA was detected again with complement-binding capacity at 2 years and after that. CONCLUSION: Careful monitoring might be warranted in an SLE patient with pre-existing DSA, even though the titer was low and without any prior histories of sensitization events.

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.

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.

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.

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.

NMR resonance assignments of mouse lipocalin-type prostaglandin D synthase/prostaglandin J2 complex.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to produce PGD2, an endogenous somenogen, in the brains of various mammalians. We recently reported that various other PGs also bind to L-PGDS, suggesting that it could serve as an extracellular carrier for PGs. Although the solution and crystal structure of L-PGDS has been determined, as has the structure of L-PGDS complexed PGH2 analog, a structural analysis of L-PGDS complexed with other PGs is needed in order to understand the mechanism responsible for the PG trapping. Here, we report the nearly complete 1H, 13C, and 15N backbone and side chain resonance assignments of the L-PGDS/PGJ2 complex and the binding site for PGJ2 on L-PGDS.

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).

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.

Glecaprevir/Pibrentasvir and Renal Dysfunction in Deceased Donor Renal Transplantation: A Case Report.

BACKGROUND: Glecaprevir/pibrentasvir is a novel anti-hepatitis C virus (HCV) drug, and it is currently the only drug available for patients with severe renal impairment. Here we report a case with renal dysfunction after an administration of glecaprevir/pibrentasvir. CASE REPORT: The case was 66-year-old Japanese man who turned out to be HCV-positive 14 years ago at the time of his second deceased renal transplantation. He had no prior history of HCV treatment. HCV genotype was serogroup 1, and baseline HCV-RNA was 5.3 LOG IU/mL. Since glecaprevir/pibrentasvir became available, he started to take it for treatment of HCV. His immunosuppressants were tacrolimus (trough levels 4.3∼6.5 ng/mL) and 5 mg of prednisolone. His baseline renal function was serum creatinine (Cr) 2.1 mg/dL and urine protein (-). Shortly after starting glecaprevir/pibrentasvir, the serum Cr started to increase. Serum Cr reached up to 2.92 mg/dL and urine protein was (+) at day 36. Right pleural effusion was observed while cardiac function was normal. His liver function had been consistently normal. We concluded glecaprevir/pibrentasvir was the cause of renal dysfunction as no other drugs were added. Immediately after discontinuation of glecaprevir/pibrentasvir at day 36, serum Cr decreased to 1.9 mg/dL and urine protein turned negative at day 64. Although the patient completed a half course of glecaprevir/pibrentasvir, HCV-RNA turned to be negative at day 36. CONCLUSIONS: We experienced a case with renal dysfunction after the initiation of glecaprevir/pibrentasvir in deceased donor renal transplant recipient. Renal dysfunction caused by glecaprevir/pibrentasvir has not been reported so far.

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.

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.