The miR-143/145 cluster induced by TGF-ß1 suppresses Wilms' tumor 1 expression in cultured human podocytes.

Transforming growth factor (TGF)-ß1 contributes to podocyte injury in various glomerular diseases, including diabetic kidney disease, probably at least in part by attenuating the expression of Wilms' tumor 1 (WT1). However, the precise mechanisms remain to be defined. We performed miRNA microarray analysis in a human podocyte cell line cultured with TGF-ß1 to examine the roles of miRNAs in podocyte damage. The microarray analysis identified miR-143-3p as the miRNA with the greatest increase following exposure to TGF-ß1. Quantitative RT-PCR confirmed a significant increase in the miR-143-3p/145-5p cluster in TGF-ß1-supplemented cultured podocytes and demonstrated upregulation of miR-143-3p in the glomeruli of mice with type 2 diabetes. Ectopic expression of miR-143-3p and miR-145-5p suppressed WT1 expression in cultured podocytes. Furthermore, inhibition of Smad or mammalian target of rapamycin signaling each partially reversed the TGF-ß1-induced increase in miR-143-3p/145-5p and decrease in WT1. In conclusion, TGF-ß1 induces expression of miR-143-3p/145-5p in part through Smad and mammalian target of rapamycin pathways, and miR-143-3p/145-5p reduces expression of WT1 in cultured human podocytes. miR-143-3p/145-5p may contribute to TGF-ß1-induced podocyte injury.NEW & NOTEWORTHY This study by miRNA microarray analysis demonstrated that miR-143-3p expression was upregulated in cultured human podocytes following exposure to transforming growth factor (TGF)-ß1. Furthermore, we report that the miR-143/145 cluster contributes to decreased expression of Wilms' tumor 1, which represents a possible mechanism for podocyte injury induced by TGF-ß1. This study is important because it presents a novel mechanism for TGF-ß-associated glomerular diseases, including diabetic kidney disease (DKD), and suggests potential therapeutic strategies targeting miR-143-3p/145-5p.

Otitis media with ANCA-associated vasculitis: A retrospective study of 30 patients.

OBJECTIVES: Otitis media with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (OMAAV) is a new category of otitis media in which cases of otitis media due to ANCA-associated vasculitis (AAV) are classified, regardless of ANCA variant or ANCA serotype. We aimed to describe the clinical features and course of patients with OMAAV and identify factors associated with hearing outcomes. METHODS: We retrospectively analysed 30 patients with OMAAV, classified based on the criteria proposed by the Japan Otological Society in 2016. RESULTS: Single-positive myeloperoxidase-ANCA, single-positive proteinase 3-ANCA, double-positive ANCA, and double-negative ANCA were identified in 47%, 33%, 7%, and 13% of the patients, respectively. All patients subjected to audiometry showed hearing impairments, and 85% were affected bilaterally. Mixed- and sensorineural-type hearing impairments were identified in 80% and 20% of impaired ears, respectively. Hypertrophic pachymeningitis (HPM) was identified in 37% of the patients. Immunosuppressive therapy was administered to 93% of patients, and the median air conduction hearing levels at pre- and post-treatment were 66.1 dB and 43.4 dB, respectively, indicating significant hearing improvements. HPM and a long interval between disease onset and treatment initiation were significantly correlated with poor hearing prognosis. CONCLUSIONS: OMAAV develops under any type of ANCA-serology and typically causes mixed or sensorineural bilateral hearing loss. The early initiation of immunosuppressive therapy and the absence of HPM were associated with good hearing outcomes.

Pregnancy outcomes in patients with systemic lupus erythematosus with or without a history of lupus nephritis.

BACKGROUND: Pregnancy is an important issue for many women with systemic lupus erythematosus (SLE). This study examined maternal and fetal outcomes among SLE women with or without a history of lupus nephritis (LN). METHODS: We retrospectively analyzed 98 pregnancies in 57 women previously diagnosed with SLE who gave birth at our hospital. RESULTS: There were 44 pregnancies in women with a history of LN and 54 pregnancies in those without. Fetal loss was observed in 16.1% of SLE pregnancies when excluding induced abortion, and preeclampsia and SLE flare were observed in 12.2 and 6.1% of SLE pregnancies, respectively. No significant differences were evident between women with or without LN in rate of fetal loss, preeclampsia or SLE flare. Women with a history of LN exhibited a significantly shorter duration of gestation (37.0 weeks vs. 38.4 weeks, P = 0.006) and lower birth weight (2484 g vs. 2746 g, P = 0.007) than those without LN. Multivariate analysis revealed glucocorticoid dose but not history of LN, as an independent risk factor for preterm delivery and low birth weight. CONCLUSION: This study was unable to conclude that a history of LN predicted pregnancy outcomes among SLE women. Instead, a higher dose of glucocorticoid at conception was unexpectedly associated with preterm delivery and low birth weight. Further studies are awaited to verify the relationship.

TGF-ß1 alters DNA methylation levels in promoter and enhancer regions of the WT1 gene in human podocytes.

AIM: Wilms' tumour 1 (WT1) is essential for normal podocyte function. Previous reports have demonstrated that the WT1 promoter is often methylated in cancers, leading to transcriptional silencing. Transforming growth factor-ß1 (TGF-ß1) is reported to down-regulate WT1 expression in podocytes. Based on the hypothesis that epigenetic modification plays a role in this process, we examined whether TGF-ß1 affects the methylation status of WT1 regulatory regions. METHODS: Conditional immortalized human podocytes were treated with TGF-ß1. A human renal proximal tubular epithelial cell line (HK2), which does not express WT1, was used as a control. The degree of DNA methylation of the WT1 promoter, 5' enhancer, intron 3 enhancer and 3' enhancer was determined using quantitative methylation-specific PCR, bisulfite sequencing and pyrosequencing. RESULTS: Both WT1 mRNA and protein expression were reduced by long-term treatment with TGF-ß1. The WT1 promoter was hypomethylated, and the 5' enhancer and intron 3 enhancer were substantially methylated in untreated podocytes. In contrast, in HK2 cells, the WT1 promoter was strongly methylated, and the 5' enhancer and intron 3 enhancer were less methylated than in untreated podocytes. TGF-ß1 tended to increase WT1 promoter methylation, tended to decrease 5' enhancer methylation and significantly decreased intron 3 enhancer methylation in podocytes. Methylation levels of the 3' enhancer did not differ among untreated cells, TGF-ß1-treated podocytes or HK2 cells. CONCLUSION: Our data suggest that the methylation pattern of the WT1 promoter and enhancers in human podocytes are distinctive from those in HK2. Furthermore, TGF-ß1 alters the methylation levels of the WT1 promoter and enhancers in human podocytes. This modification may be relevant to the attenuation of WT1 by TGF-ß1, which could contribute to podocyte injury.

Lineage tracing aged mouse kidneys shows lower number of cells of renin lineage and reduced responsiveness to RAAS inhibition.

Blocking the renin-angiotensin-aldosterone system (RAAS) remains a mainstay of therapy in hypertension and glomerular diseases. With the population aging, our understanding of renin-producing cells in kidneys with advanced age is more critical than ever. Accordingly, we administered tamoxifen to Ren1cCreERxRs-tdTomato-R mice to permanently fate map cells of renin lineage (CoRL). The number of Td-tomato-labeled CoRL decreased significantly in aged mice (24 mo of age) compared with young mice (3.5 mo of age), as did renin mRNA levels. To determine whether aged CoRL responded less to RAAS blockade, enalapril and losartan were administered over 25 days following uninephrectomy in young and aged mice. The number of CoRL increased in young mice in response to enalapril and losartan. However, this was significantly lower in aged mice compared with young mice due to limited proliferation, but not recruitment. Gene expression analysis of laser-captured CoRL showed a substantial increase in mRNA levels for proapoptotic and prosenescence genes, and an increase in a major prosenescence protein on immunostaining. These results show that CoRL are lower in aged mice and do not respond to RAAS inhibition to the same extent as young mice.

Renal outcomes in mixed proliferative and membranous lupus nephritis (Class III/IV + V): A long-term observational study.

OBJECTIVES: In this study, we aimed to assess the effect of combination of proliferative and membranous lesions (Class III + V or IV + V) on renal outcomes as an independent category distinct from Class III and IV. METHODS: We retrospectively analyzed 103 Japanese patients (14 male and 89 female) with Class III/IV LN, with or without Class V, who underwent renal biopsy and were treated at our institution. Renal endpoint was defined as doubling of serum creatinine or end-stage renal disease (ESRD). RESULTS: The number of patients in each group was as follows: pure Class III/IV, 81 patients and mixed Class III/IV + V, 22 patients. During a median follow-up period of 125.0 months, 10 patients developed renal endpoint: five had Class III/IV LN and five had a combination of Class III/IV + V. Kaplan-Meier analyses demonstrated that patients with mixed Class III/IV + V LN had significantly poorer renal outcomes than patients with Class III/IV LN. Multivariate Cox regression analyses identified serum creatinine, active and chronic lesions (A/C), and mixed Class III/IV + V) as independent risk factors for poor renal outcomes. CONCLUSIONS: This study demonstrated a combination of proliferative and membranous LN (ISN/RPS Class III/IV + V) predicts poor renal outcomes.

Expression of a novel stress-inducible protein, sestrin 2, in rat glomerular parietal epithelial cells.

Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury.

Nephrotic syndrome caused by immune-mediated acquired LCAT deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is an enzyme involved in maintaining cholesterol homeostasis. In familial LCAT deficiency (FLD), abnormal lipid deposition causes renal injury and nephrotic syndrome, frequently progressing to ESRD. Here, we describe a 63-year-old Japanese woman with no family history of renal disease who presented with nephrotic syndrome. The laboratory data revealed an extremely low level of serum HDL and undetectable serum LCAT activity. Renal biopsy showed glomerular lipid deposition with prominent accumulation of foam cells, similar to the histologic findings of FLD. In addition, she had subepithelial electron-dense deposits compatible with membranous nephropathy, which are not typical of FLD. A mixing test and coimmunoprecipitation study demonstrated the presence of an inhibitory anti-LCAT antibody in the patient's serum. Immunohistochemistry and immunofluorescence detected LCAT along parts of the glomerular capillary walls, suggesting that LCAT was an antigen responsible for the membranous nephropathy. Treatment with steroids resulted in complete remission of the nephrotic syndrome, normalization of serum LCAT activity and HDL level, and disappearance of foam cell accumulation in renal tissue. In summary, inhibitory anti-LCAT antibody can lead to glomerular lesions similar to those observed in FLD.

SIRPα signaling regulates podocyte structure and function.

Signal-regulatory protein-α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylation-dependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knockin mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα-mutant mice). Light microscopic examination revealed no apparent morphological abnormalities in the kidneys of the SIRPα-mutant mice. On the other hand, electron microscopic examination revealed abnormal podocytes with irregular major processes and wider and flattened foot processes in the SIRPα-mutant mice compared with their wild-type counterparts. Significantly impaired renal functions and slight albuminuria were demonstrated in the SIRPα-mutant mice. In addition, adriamycin injection induced massive albuminuria together with focal glomerulosclerosis in the SIRPα-mutant mice, while their wild-type counterparts were resistant to adriamycin-induced nephropathy. These data demonstrate that SIRPα is involved in the regulation of podocyte structure and function as a filtration barrier under both physiological and pathological conditions.

Hydronephrosis caused by a relapse of granulomatosis with polyangiitis (Wegener's).

We describe a case of relapsed granulomatosis with polyangiitis (Wegener's) (GPA) that presented with abdominal pain. (18)F-fluoro-2-deoxy-D: -glucose positron emission tomography (FDG-PET)/computed tomography (CT) clearly depicted an inflammation of the left peri-iliac arterial soft tissue, which was thought to be the cause of the ureteral obstruction and hydronephrosis. Our case shows that peri-iliac arterial inflammation occurs in GPA and causes hydronephrosis. In addition, FDG-PET/CT is a useful tool for management of this systemic inflammatory disease.

Primary central nervous system lymphoma in a patient with neuropsychiatric systemic lupus erythematosus receiving mycophenolate mofetil: A case report and literature review.

A 41-year-old woman with a 14-month history of systemic lupus erythematosus (SLE) presented with headache, aphasia, and agraphia. A laboratory examination revealed mild proteinuria, hypocomplementemia, and elevated anti-double-stranded DNA antibody levels. A cerebrospinal fluid analysis demonstrated elevated protein and interleukin-6 levels. Magnetic resonance imaging (MRI) of the brain identified multiple lesions suggestive of brain edemas and small haemorrhages. She was diagnosed as having neuropsychiatric lupus and lupus nephritis and received remission induction therapy with high-dose corticosteroid and intravenous cyclophosphamide. She achieved a complete remission, and treatment with mycophenolate mofetil (MMF) was initiated 3 months thereafter for remission maintenance. At 13 months after the exacerbation of SLE, she complained of headache and nausea. A gadolinium-enhanced MRI of the brain revealed a low-signal-intensity tumour with marginal ring enhancement of 50 mm in the left frontal lobe. The tumour was excised, and the histological diagnosis was diffuse large B-cell lymphoma with positive Epstein-Barr virus (EBV). MMF was discontinued. Remission induction therapy with rituximab, high-dose methotrexate, procarbazine, and vincristine was administered, and she achieved remission. Previous reports suggest that use of MMF is associated with primary central nervous system (CNS) lymphoma (PCNSL) in patients with lupus nephritis or other autoimmune diseases or in post-transplant patients. Our observation that PCNSL occurred after CNS involvement of SLE suggests that EBV and CNS inflammation arising from SLE might have contributed to the development of PCNSL.

Effects of proteasome inhibitors on rat renal fibrosis in vitro and in vivo.

AIM: Transforming growth factor-ß (TGF-ß) is involved in renal tubulointerstitial fibrosis. Recently, the ubiquitin proteasome system was shown to participate in the TGF-ß signalling pathway. The aim of this study was to examine the effects of proteasome inhibitors on TGF-ß-induced transformation of renal fibroblasts and tubular epithelial cells in vitro and on unilateral ureteral obstruction (UUO) in vivo. METHODS: Rat renal fibroblasts NRK-49F cells and tubular epithelial cells, NRK-52E, were treated with TGF-ß in the presence or absence of a proteasome inhibitor, MG132 or lactacystin. Rats were subjected to UUO and received MG132 i.p. for 7 days. RESULTS: In cultured renal cells, both MG132 and lactacystin inhibited TGF-ß-induced α-smooth muscle actin (α-SMA) protein expression according to both western blotting and immunofluorescent study results. MG132 also suppressed TGF-ß-induced mRNA expression of α-SMA and upregulation of Smad-response element reporter activity. However, MG132 did not inhibit TGF-ß-induced phosphorylation and nuclear translocation of Smad2. In contrast, MG132 increased the protein level of Smad co-repressor SnoN, demonstrating that SnoN is one of the target molecules by which MG132 blocks the TGF-ß signalling pathway. Although the proteasome inhibitor suppressed TGF-ß-induced transformation of cultured fibroblasts and tubular epithelial cells, MG132 treatment did not ameliorate tubulointerstitial fibrosis in the rat UUO model. CONCLUSION: Proteasome inhibitors attenuate TGF-ß signalling by blocking Smad signal transduction in vitro, but do not inhibit renal interstitial fibrosis in vivo.

Successful treatment of lipoprotein glomerulopathy in a daughter and a mother using niceritrol.

We report two patients, a daughter and a mother, with lipoprotein glomerulopathy (LPG) who were successfully treated with niceritrol. Both patients carried a mutation in the apolipoprotein E (apoE) gene known as ApoE Tokyo/Maebashi. The daughter was found to have proteinuria at the age of 4 years. Four years later, she was diagnosed as having LPG based on a renal biopsy. She was treated with several medications including pravastatin, ethyl icosapentate, enalapril, warfarin and cyclophosphamide, all of which failed to reduce her proteinuria. At the age of 17 years, she exhibited an increase in proteinuria and a decline in renal function, despite ongoing treatment with pravastatin and enalapril. After switching from pravastatin to niceritrol, a marked reduction in the proteinuria and an improvement in renal function were observed. Her mother was found to have proteinuria at the age of 57 years and was diagnosed as having LPG based on a renal biopsy. She was also treated with niceritrol, resulting in an improvement in her proteinuria and renal function. These cases suggest that niceritrol might be a useful therapeutic option for LPG.

CD44 impacts glomerular parietal epithelial cell changes in the aged mouse kidney.

CD44 contributes to the activation of glomerular parietal epithelial cells (PECs). Although CD44 expression is higher in PECs of healthy aged mice, the biological role of CD44 in PECs in this context remains unclear. Accordingly, young (4 months) and aged (24 months) CD44-/- mice were compared to age-matched CD44+/+ mice, both aged in a nonstressed environment. Parietal epithelial cell densities were similar in both young and aged CD44+/+ and CD44-/- mice. Phosphorylated ERK 1/2 (pERK) was higher in aged CD44+/+ mice. Vimentin and α-SMA, markers of changes to the epithelial cell phenotype, were present in PECs in aged CD44+/+ mice, but absent in aged CD44-/- mice in both outer cortical (OC) and juxtamedullary (JM) glomeruli. Because age-related glomerular hypertrophy was lower in CD44-/- mice, mTOR activation was assessed by phospho-S6 ribosomal protein (pS6RP) staining. Parietal epithelial cells and glomerular tuft staining for pS6RP was lower in aged CD44-/- mice compared to aged CD44+/+ mice. Podocyte density was higher in aged CD44-/- mice in both OC and JM glomeruli. These changes were accompanied by segmental and global glomerulosclerosis in aged CD44+/+ mice, but absent in aged CD44-/- mice. These results show that the increase in CD44 in PECs in aged kidneys contributes to several changes to the glomerulus during healthy aging in mice, and may involve ERK and mTOR activation.

A case of long-term dasatinib-induced proteinuria and glomerular injury.

A 52-year-old woman was diagnosed with chronic myeloid leukemia. Treatment with dasatinib, a second-generation Bcr-Abl tyrosine kinase inhibitor, was initiated, and complete cytogenetic remission was achieved. Two years later, proteinuria occurred, and the urinary protein level increased gradually in the next 3 years. Moreover, the serum creatinine level increased mildly during this period. The urinary protein level reached 2.18 g/gCr; hence, a renal biopsy was conducted. Light microscopy revealed mild proliferation of mesangial cells, and immunofluorescence analysis revealed IgG and C3 depositions in the mesangial area. Electron microscopy revealed electron-dense deposition in the paramesangial area, partial podocyte foot process effacement, and segmental endothelial cell swelling with a slight expansion of the subendothelial space. Dasatinib was discontinued, and within 3 weeks, the proteinuria disappeared, with improvements in her renal function. After switching to bosutinib, a new second-generation of tyrosine kinase inhibitor, the proteinuria remained negative. The rapid cessation of proteinuria following dasatinib discontinuation indicated that proteinuria was induced by the long-term administration of dasatinib. Proteinuria and renal function should be regularly monitored during dasatinib therapy.

Diaphragmatic hernia with ischemic bowel obstruction in pregnancy: report of a case.

Diaphragmatic hernia complicating pregnancy is rare and is associated with high morbidity and mortality, particularly if surgical intervention is delayed. We report a case of diaphragmatic hernia in a pregnant 25-year-old woman. The patient was referred to our hospital in respiratory distress in the 28th week of her pregnancy. Chest radiograph and computed tomography showed air-filled bowel loops in the left side of the chest, with a marked mediastinal shift. Immediately after an emergency caesarean section, the herniated abdominal viscera were reduced through the opening in the diaphragm. We resected the ischemic segment of ileum and repaired the diaphragmatic defect with interrupted sutures and a Gore-Tex sheet. She had an uneventful postoperative course and her baby boy also recovered well. We report this case to alert surgeons to the possibility of this rare surgical emergency during pregnancy.