Differential impact of glomerular and tubule-interstitial histological changes on kidney outcome between non-proteinuric and proteinuric diabetic nephropathy.

BACKGROUND: Studies on kidney function and histological findings in diabetic nephropathy (DN) with low urinary protein (UP) are few. We examined the differential impact of histological changes on kidney outcomes between non-proteinuric and proteinuric DN. METHODS: Patients diagnosed with DN by renal biopsy during 1981-2014 were divided into non-proteinuric (UP ≤ 0.5 g/day) and proteinuric (UP > 0.5 g/day) DN. The Cox proportional hazard model was used to examine the association of glomerular lesions (GLs) and interstitial fibrosis and tubular atrophy (IFTA) with end-stage kidney disease (ESKD) development after adjusting for relevant confounders. RESULTS: The non-proteinuric and proteinuric DN groups included 197 and 199 patients, respectively. During the 10.7-year median follow-up period, 16 and 83 patients developed ESKD in the non-proteinuric and proteinuric DN groups, respectively. In the multivariable Cox hazard model, hazard ratios (HRs) [95% confidence intervals (CIs)] of GL and IFTA for ESKD in proteinuric DN were 2.94 [1.67-5.36] and 3.82 [2.06-7.53], respectively. Meanwhile, HRs [95% CIs] of GL and IFTA in non-proteinuric DN were < 0.01 [0-2.48] and 4.98 [1.33-18.0], respectively. IFTA was consistently associated with higher incidences of ESKD regardless of proteinuria levels (P for interaction = 0.49). The prognostic impact of GLs on ESKD was significantly decreased as proteinuria levels decreased (P for interaction < 0.01). CONCLUSIONS: IFTA is consistently a useful predictor of kidney prognosis in both non-proteinuric and proteinuric DN, while GLs are a significant predictor of kidney prognosis only in proteinuric DN.

The association of 5-year therapeutic responsiveness with long-term renal outcome in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most common type of primary glomerulonephritis. Since most patients have a relatively benign renal prognosis, long-term follow-up is required. During such a long course of disease, relapse of IgAN is occasionally observed after upper respiratory tract infection or without any trigger. However, little is known about the impact of relapse on long-term renal outcomes. METHODS: In this retrospective cohort study of biopsy-proven primary IgAN, we analyzed the association of 5-year therapeutic responsiveness (relapse) with the subsequent development of end-stage kidney disease (ESKD) using a 5-year landmark analysis (Cox model) and explored predictors of relapse from histological and clinical data at baseline. RESULTS: Among 563 patients from the exploratory cohort, most relapses (13.7%) occurred within 5 years after treatment. Using 5-year landmark analysis, among 470 patients, 79 developed ESKD during a median follow-up period of 155 months. Even after adjustment for clinicopathological relevant confounders, hazard ratios (95% confidence intervals) in the relapse and non-responder groups compared with the remission group were 2.86 (1.41-5.79) and 2.74 (1.48-5.11), respectively. Among 250 patients who achieved remission within 5 years, proteinuria, eGFR, mesangial hypercellularity, endocapillary hypercellularity, segmental sclerosis, and crescent, but not interstitial fibrosis/tubular atrophy, were independent predictors of 5-year relapse in multivariable logistic regression analysis, CONCLUSIONS: Both relapsers and non-responders had similarly strong association with ESKD in patients with IgAN. We also confirmed the predictors of relapse 5 years after renal biopsy, which may guide the treatment strategies for patients with IgAN who occasionally relapse after remission.

Association of initial prednisolone dose with remission, relapse, and infectious complications in adult-onset minimal change disease.

BACKGROUND: A dose of 0.5-1 mg/kg/day of prednisolone (PSL) is administered for the initial treatment of minimal change disease (MCD). However, little is known about the optimal PSL dose for the initial treatment of MCD. METHODS: We conducted a retrospective multicenter cohort study of treatment-naive adult patients with MCD diagnosed by renal biopsy from 1981 to 2015 in whom PSL monotherapy was performed as the initial treatment. The exposure of interest was an initial median PSL dose of < 0.63 mg/kg/day (Group L) compared to ≥ 0.63 mg/kg/day (Group H). Cumulative remission and relapse after remission were compared between these groups using Cox regression adjusted for baseline characteristics. RESULTS: Ninety-one patients met the inclusion criteria. During a median follow-up of 2.98 years, 87 (95.6%) patients achieved complete remission, and 47.1% relapsed after remission. There was no significant difference in the remission rate between the groups at 4 weeks of follow-up (66.7 vs. 82.6%). The median time to remission in Group L was comparable to that in Group H (17.0 vs. 14.0 days). A multivariable Cox hazard model revealed that the initial PSL dose was not a significant predictor of remission. The cumulative steroid doses at 6 months, 1 year, and 2 years after treatment initiation were significantly lower in Group L than in Group H. CONCLUSION: The initial PSL dose was not associated with time to remission, remission rate, time to relapse, or relapse rate. Therefore, a low initial steroid dose may be sufficient to achieve remission.

Microscopic hematuria is a risk factor for end-stage kidney disease in patients with biopsy-proven diabetic nephropathy.

INTRODUCTION: There are fewer reports about whether the presence of hematuria affects the progression of chronic kidney disease in patients with diabetic nephropathy. We analyzed whether microscopic hematuria in diabetic nephropathy is a risk factor for end-stage kidney disease (ESKD). RESEARCH DESIGN AND METHODS: The present study was a retrospective cohort study of patients with biopsy-proven diabetic nephropathy. We recruited 397 patients with diabetic nephropathy, which was confirmed by renal biopsy between June 1981 and December 2014 and followed them until October 2018 or death. Patients with microscopic hematuria before renal biopsy were defined as the hematuria group (n=91), and the remainder as the no-hematuria group (n=306). The main outcome was the occurrence of ESKD, which was defined by the requirement of permanent renal replacement therapies. RESULTS: The systolic and diastolic blood pressure, serum creatinine and proteinuria were significantly higher, and the estimated glomerular filtration rate was significantly lower in the hematuria group compared with the no-hematuria group. Pathological evaluations revealed that glomerular, tubulointerstitial and vascular lesions in the hematuria group were significantly more severe. During a median of 10.1 years, 44 and 52 patients developed ESKD in the hematuria group and the no-hematuria group, respectively. Survival analyses showed that the incidence of ESKD was significantly higher in the hematuria group compared with the no-hematuria group (log-rank, p<0.0001). The multivariable Cox proportional hazards models revealed a significant association between hematuria and the incidence of ESKD after adjusting for clinically relevant factors, including proteinuria and renal pathology (adjusted HR 1.64, 95% CI 1.03 to 2.60). The subgroups of men, proteinuria ≥0.5 g/day, and systolic blood pressure ≥132 mm Hg showed a stronger association between hematuria and ESKD than their opposing subgroups. CONCLUSIONS: Microscopic hematuria is a risk factor for ESKD in diabetic nephropathy, independent of proteinuria and renal pathology.

Construction of T-Motif-Based DNA Nanostructures through Enzymatic Reactions.

The most common way to fabricate DNA nanostructures is to mix individually synthesized DNA oligomers in one pot. However, if DNA nanostructures could be produced through enzymatic reactions, they could be applied in various environments, including in vivo. Herein, an enzymatic method developed to construct a DNA nanostructure from a simple motif called a T-motif is reported. A long, repeated structure was replicated from a circular template by rolling circle amplification and then cleaved into T-motif segments by restriction enzymes. These motifs have been successfully assembled into a ladder-like nanostructure without purification or controlled annealing. This approach is widely applicable to constructing a variety of DNA nanostructures through enzymatic reactions.

Acute kidney injury in a postpartum woman with paroxysmal nocturnal hemoglobinuria: A case report and literature review.

Paroxysmal nocturnal hemoglobinuria is a rare clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, hemoglobinuria, and inflammatory thrombotic state. Intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) can lead to acute and chronic renal injury through hemoglobin-mediated toxicity. A 32-year-old pregnant woman with myelodysplastic syndrome was admitted to our hospital with severe preeclampsia. Shortly after an urgent caesarean section, she became obtunded and showed signs of acute kidney injury (AKI) with anuria, severe intravascular hemolysis, and hypermagnesemia. She was diagnosed with PNH with a positive Ham test and flow cytometry analysis. Renal magnetic resonance imaging revealed decreased signal intensity in the renal cortex due to hemosiderin deposition. Hemodialysis, plasma exchange, and administration of corticosteroids ameliorated her clinical condition and renal function. This case illustrates that careful management is required to prevent postpartum AKI in pregnant women with PNH.

Mycobacterium wolinskyi Peritonitis after Peritoneal Catheter Embedment Surgery.

Mycobacterium wolinskyi belongs to the Mycobacterium smegmatis group, which comprises rapidly growing non-tuberculous mycobacteria. The number of case reports on M. wolinskyi infections associated with postoperative wounds has increased in recent years. We herein report a case of peritonitis due to M. wolinskyi after peritoneal catheter embedment surgery. Identification was achieved based on 16S ribosomal RNA and rpoB gene sequencing of the isolate. The patient recovered following catheter removal and treatment with levofloxacin and minocycline for one month.

Prognostic Value of Predialysis Indices for Technique Failure and Mortality in Peritoneal Dialysis Patients.

Technique failure remains a frequent cause of peritoneal dialysis (PD) withdrawal. Many post-commencement predictors of PD technique failure have been identified, while predialysis predictors have remained unclear. The aim of this study was to identify predialysis indices for technique failure in PD patients. We recruited 206 consecutive PD patients who were treated at Nara Medical University Hospital between 1 April 1997 and 31 December 2012. Forty-eight patients were excluded because of transition from hemodialysis (HD) or withdrawal from PD within 3 months, leaving 158 patients for analysis. Clinical characteristics and laboratory data from within 3 months preceding PD commencement were analyzed. The primary outcome was the composite of time to combined use of HD, transition to HD, and all-cause mortality within 2 years after PD commencement. During the study period, the primary outcome was observed in 50 patients. Using multivariate analysis, greater age (odds ratios (ORs) [95%CI], 3.08 [1.72-5.61]), anemia (ORs [95%CI], 2.12 [1.08-4.43]), overweight/obesity (ORs [95%CI], 2.09 [1.16-3.72]), and hypocalcemia (ORs [95%CI], 1.86 [1.04-3.35]) were independently associated with technique failure. Adding corrected calcium to the model incorporating age, body mass index, and hemoglobin significantly increased the c-statistic from 0.678 to 0.755 (P = 0.048) relative to the model incorporating age alone. The integrated discrimination improvement was 0.085 (95% CI 0.036-0.134, P < 0.001) and the continuous net reclassification improvement was 0.395 (95% CI 0.066-0.724, P = 0.02). In conclusion, the combination of predialysis indices comprising age, overweight/obesity, anemia, and corrected calcium could provide a significant predictive value for technique failure of PD.

Maternal undernutrition induces the expression of hypoxia-related genes in the fetal brain.

Maternal undernutrition during pregnancy is a risk factor for cerebrovascular and cardiovascular diseases in adulthood. Hypoxia-inducible factor 1 alpha (HIF1α) plays an essential role in cellular hypoxic responses, and its increased expression is associated with cerebrovascular and cardiovascular diseases. However, it is not known whether maternal undernutrition influences HIF1α expression in the fetal brain. We therefore analyzed the expression levels of HIF1α and its downstream genes in the fetal brain (day 17.5 of gestation, 1-2 days before birth). Maternal undernutrition did not noticeably affect the fetal body and brain weights. Both HIF1α mRNA and protein levels were increased in the brain under maternal undernutrition, despite the absence of hypoxia, as judged by the staining profile with hypoxyprobe-1 that identifies hypoxic cells. Importantly, maternal undernutrition caused the accumulation of HIF1α protein in oligodendrocyte precursor cells at the subventricular zone, a site of neurogenesis in the fetal brain. Maternal undernutrition also increased the mRNA level of mammalian target of rapamycin (mTOR), which could increase the level of HIF1α protein under normoxia. Furthermore, microarray analysis revealed that expression levels of mRNAs for 10 HIF1α downstream targets, including enolase 1 and hexokinase 1, were increased in the fetal brain under maternal undernutrition. Thus, the biochemical consequence of maternal undernutrition is similar to that of mild hypoxia. In conclusion, maternal undernutrition induces the expression of HIF1α in oligodendrocyte precursor cells at the subventricular zone, and it also induces the expression of hypoxia-related genes in the fetal brain probably via activation of the mTOR pathway.

Aberrant expression of hypoxia-inducible factor 1α in the fetal heart is associated with maternal undernutrition.

Maternal undernutrition during pregnancy is a risk factor that impairs fetal growth and causes cardiovascular diseases. However, the underlying mechanism is still unknown. In this study, we evaluated the effect of maternal undernutrition on the expression levels of transcription factors in the fetal heart. Female mice were given low protein or regular food from 2 weeks before mating and during their pregnancy. The fetal hearts were collected on day 17.5 of gestation, about 1-2 days before birth. Maternal undernutrition resulted in a significant increase in the relative heart weight (heart weight/body weight) in female fetuses, but not in male fetuses. Microarray analysis revealed that expression levels of mRNAs for 133 transcription factors were changed in the fetal heart under maternal undernutrition. Among them, we focused on hypoxia-inducible factor 1 alpha (HIF1α) that is involved in the pathogenesis of cardiovascular diseases on adulthood. Quantitative real-time PCR analysis showed that the expression level of HIF1α mRNA was increased about 1.3-fold in male fetal heart under maternal undernutrition, but remained unchanged in female heart. Moreover, maternal undernutrition increased the mRNA level of prolyl hydroxylase 1 (PHD1), which contributes to degradation of HIF1α, in male heart but not in female heart. Immunohistochemical analysis showed the accumulation of HIF1α protein in the fetal heart of both sexes under maternal undernutrition, without the induction of HIF1α mRNA expression in female heart. These results suggest that maternal undernutrition may induce HIF1α expression in the fetal heart through the distinct mechanisms depending on the sex.

Maternal undernutrition with vaginal inflammation impairs the neonatal oligodendrogenesis in mice.

Maternal undernutrition and infection during pregnancy may impair development of oligodendrocytes, thereby increasing risks of neuropsychiatric disorders of their children. We analyzed the effects of those risk factors on oligodendrogenesis in fetal and neonatal brains. Female mice were given low-protein or regular food for 2 weeks before their pregnancy. On the 14th day of pregnancy, they received a transvaginal injection of lipopolysaccharide to induce inflammation or control solution, consisting of four groups, depending on nutritional conditions with or without vaginal inflammation. We collected fetal brains on embryonic day (E) 17 for evaluating oligodendrocyte precursor cells (OPCs) and neonatal brains on postnatal day (P) 7 for evaluating mature oligodendrocytes. OPCs and mature oligodendrocytes were identified as positive immunostaining for oligodendrocyte-lineage transcription factor 2 and myelin basic protein, respectively. There was no difference in the number of OPCs in E17 brains among the four groups, suggesting that nutritional restriction with or without inflammation exerts no noticeable influence on the differentiation of OPCs. However, the number of mature oligodendrocytes was decreased in P7 brains obtained from nutrient-restricted mice with inflammation, suggesting that their combination impairs oligodendrogenesis in the neonatal brain. We also analyzed reactive astrocytes that express both glial fibrillary acidic protein and nestin for evaluating brain inflammation. The population of reactive astrocytes was increased in P7 brains derived from mice with LPS injection, irrespective of nutritional restriction, indicating that maternal vaginal inflammation induces neonatal brain inflammation. The maternal management of both nutrition and infection is crucial to prevent neuropsychiatric disorders of the children.

Identification of an Htm1 (EDEM)-dependent, Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) pathway in Saccharomyces cerevisiae: application of a novel assay for glycoprotein ERAD.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related alpha-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.

Identification and quantification of 1-nitropyrene metabolites in human urine as a proposed biomarker for exposure to diesel exhaust.

1-nitropyrene (1-NP) is one of the most abundant nitrated polycyclic aromatic hydrocarbons (NPAHs) in diesel exhaust particulate matter (DEP) and is a main contributor of direct-acting mutagenicity in DEP. Therefore, the metabolites of 1-NP are expected to be a biomarker for assessment of exposure to DEP. In this study, a highly specific and sensitive analytical method using liquid chromatography with tandem mass spectrometry (LC-MS/MS) was developed to determine urinary 1-NP metabolites. After enzymatic hydrolysis of the conjugated metabolites, the analytes were selectively extracted from the urine matrix with blue rayon. The eluate from the rayon was further purified on an acidic alumina cartridge. Hydroxy-N-acetyl- 1-aminopyrenes (6- and 8-OHNAAP) and hydroxy-1-nitropyrenes (3-, 6-, and 8-OHNP) in human urine were identified by their retention times and MS/MS spectra and quantified by using deuterated internal standards. 1-NP metabolites were quantified in urine from all healthy, nonoccupationally exposed subjects. 6-OHNAAP, 8-OHNAAP, 6-OHNP, and 8-OHNP (means of 117, 109, 203, and 137 pmol/mol creatinine, respectively) were the most abundant isomers in human urine. This report is the first to demonstrate the presence of OHNAAPs and OHNPs in human urine, in agreement with previous in vivo and in vitro studies that predicted that these metabolites should be excreted into human urine. This method for determining urinary 1-NP metabolites should be useful for the surveillance of exposure to NPAHs and DEP and will facilitate the study of cancer risk associated with these exposures.

Dual enzymatic properties of the cytoplasmic peptide: N-glycanase in C. elegans.

The endoplasmic reticulum-associated degradation (ERAD) of misfolded (glyco)proteins ensures that only functional, correctly folded proteins exit from the ER and that misfolded ones are degraded by the ubiquitin-proteasome system. During the degradation of misfolded glycoproteins, some of them are subjected to deglycosylation by the cytoplasmic peptide:N-glycanase (PNGase). The cytosolic PNGase is widely distributed throughout eukaryotes. Here we show that the nematode Caenorhabditis elegans PNG-1, the cytoplasmic PNGase orthologue in this organism, exhibits dual enzyme functions, not only as PNGase but also as an oxidoreductase (thioredoxin). Using an in vitro assay as well as an in vivo assay system in budding yeast, the N-terminal thioredoxin domain and the central transglutaminase domain were found to be essential for oxidoreductase activity and PNGase activity, respectively. Occurrence of a C. elegans mutation affecting a catalytic residue in the PNGase domain strongly suggests the functional importance of this protein in higher eukaryotes.

A cytoplasmic peptide: N-glycanase.

A cytoplasmic peptide:N-glycanase (PNGase) has been implicated in the proteasomal degradation of aberrant glycoproteins synthesized in the endoplasmic reticulum. The reaction is believed to be important for subsequent proteolysis by the proteasome since bulky N-glycan chains on misfolded glycoproteins may impair their efficient entry into the interior of the cylinder-shaped 20S proteasome, where the active sites of the proteases reside. The deglycosylation reaction by PNGase brings about two major changes on substrate proteins; one is a removal of N-glycan chains, and the other is the introduction of negative charge(s) into the core peptide by converting glycosylated asparagine residue(s) into aspartic acid residue(s). Therefore, PNGase action can be accurately monitored by detecting both changes using two different methods; that is, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for deglycosylation and isoelectric focusing for detection of introduction of negative charge(s) into core proteins. This chapter will describe the simple in vivo as well as in vitro assay method to detect PNGase activity.

The Png1-Rad23 complex regulates glycoprotein turnover.

Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1-Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.

Truncated Sla1 induces haploid meiosis through the Pat1-Mei2 system in fission yeast.

We previously reported that expression of Sla1DeltaC, a truncated form of Sla1, induces ectopic meiosis in heterothallic fission yeast and this was possibly due to the inhibition of Pat1 kinase by Sla1DeltaC. Here we found mei2 mRNA and the Mei2 protein accumulated and stability of the Mei2 protein increased when Sla1DeltaC was expressed. The former two results are considered to be the consequence of de-repression of Ste11, which is the transcription factor of mei2 and negatively regulated by Pat1 kinase. The latter result reflects the consequence of deregulation of Mei2 by Pat1 kinase. In addition, Ste11 accumulated in the nucleus when Sla1DeltaC was expressed. All these data consistently support the idea that the action of Sla1DeltaC is to inactivate Pat1 kinase.

Sla1, a Schizosaccharomyces pombe homolog of the human La protein, induces ectopic meiosis when its C terminus is truncated.

Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1DeltaC) induced ectopic sporulation in the ras1Delta strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1DeltaC is found throughout the cell, suggesting the cytoplasmic localization of Sla1DeltaC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1DeltaC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1DeltaC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1DeltaC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1DeltaC but not full-length Sla1. Thus, Sla1DeltaC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.