Extreme Prematurity and Pulmonary Outcomes Program in Saitama: Protocol for a Prospective Multicenter Cohort Study in Japan.

BACKGROUND: Because of the improvements in survival rates for preterm infants, not only the rates of bronchopulmonary dysplasia (BPD) but also those of long-term respiratory complications of premature birth are increasing, resulting in financial and health burdens in developed countries. Thus far, the risk factors of respiratory morbidities in extremely preterm infants remain unknown. Furthermore, the definition and the predictive ability of BPD for long-term respiratory outcomes are yet to be determined. OBJECTIVE: The objective of our study, Extreme Prematurity and Pulmonary Outcomes Program in Saitama, is to develop the diagnostic criteria for BPD and to determine the prognostic factors contributing to the long-term pulmonary outcomes manifesting in extremely preterm infants. METHODS: The Extreme Prematurity and Pulmonary Outcomes Program in Saitama is an observational prospective cohort study performed by a consortium of six neonatal intensive care units (NICUs) in Saitama, Japan. The subjects included in this study are infants (from each clinical center) with gestational ages 22 to 27 weeks. The target is 400 subjects. This study aims to determine the definition of BPD and other perinatal factors that accurately predict the long-term pulmonary outcomes in survivors of extreme prematurity. Moreover, the association between BPD and postprematurity respiratory disease will be investigated using generalized linear models. RESULTS: The protocol and consent forms were evaluated and approved on September 5, 2019, by the Ethics Committee of Saitama Medical Center, Saitama Medical University. Enrollment began on April 1, 2020. It is expected to end on March 31, 2023. The follow-up for 1 year corrected age is expected to continue through the middle of 2024. CONCLUSIONS: The Extreme Prematurity and Pulmonary Outcomes Program in Saitama incorporates aspects of neonatal care in secondary- and tertiary-level NICUs to develop existing research studies on the definition of BPD, objective biomarkers, and outcome measures of respiratory morbidity in extremely preterm infants beyond NICU hospitalization, thereby leading to a novel understanding of the nature and natural history of BPD and potential mechanistic and therapeutic targets in at-risk subjects. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/22948.

Successful Total Pericardiectomy for Constrictive Pericarditis in the First Series of Japanese Patients With Mulibrey Nanism.

Patients with Mulibrey nanism (MUL) present with growth failure and multiple organ manifestations, and MUL is caused by mutations in TRIM37. In this article, we report on the first case series of Japanese patients with MUL who developed congestive heart failure due to constrictive pericarditis. Our case series suggests that early diagnosis and total pericardiectomy before adherence of the pericardium might provide clinical benefit and better prognosis for MUL.

A Rare Case of Meconium Periorchitis Diagnosed in Utero.

Meconium periorchitis is a rare disorder caused by fetal meconium peritonitis, with subsequent passage of meconium into the scrotum via a patent processus vaginalis. To date, clinical significance of meconium periorchitis for the prenatal diagnosis of meconium peritonitis and prediction for postnatal surgery remains to be determined. We present a clinical course of a fetus presenting with meconium periorchitis induced by meconium peritonitis. At 28 weeks' gestation, fetal ultrasonography indicated fetal ascites associated with bilateral hydrocele and peritesticular calcification without other signs of meconium peritonitis. The pregnancy was uneventful until delivery and the infant was delivered at 37 weeks' gestation. No abdominal distension was observed at birth, and radiography did not reveal any abdominal calcification except for scrotal calcification. Abdominal distension was observed 3 days after birth and laparotomy was performed. The diagnosis of meconium peritonitis was confirmed at surgery. Our case illustrated that careful examination of the scrotum during fetal life was helpful for prenatal diagnosis of meconium peritonitis as well as postnatal management.

Hyponatremia, hypophosphatemia, and hypouricemia in a girl with macrophage activation syndrome.

Macrophage activation syndrome, a life-threatening complication of rheumatic disorders, is accompanied by the overproduction of cytokines. We describe a girl with macrophage activation syndrome complicating systemic-onset juvenile arthritis who developed hyponatremia, hypophosphatemia, and hypouricemia associated with a high level of serum tumor necrosis factor alpha. Renal proximal tubule dysfunction was considered to be the cause, which may be attributable to tumor necrosis factor alpha.

Extracellular signal-regulated kinase inhibition slows disease progression in mice with polycystic kidney disease.

The expression of mitogen-activated protein kinases (MAPK) in DBA/2-pcy/pcy (pcy) mice, a murine model of polycystic kidney disease was investigated. Proliferating cell nuclear antigen-positive cells were recognized in cyst epithelium from embryonic day 14.5 to 25 wk of age. Extracellular signal-regulated kinase (ERK) was expressed in the renal tubules of control and pcy mice, but stronger immunostaining was observed in cyst epithelium. Phosphorylated ERK was detected only in pcy mice and was localized predominantly in the cysts. p38 MAPK (p38) was no longer expressed after birth in controls but was detected in the cyst epithelium and in occasional tubular cells of pcy mice at all stages examined. c-Jun N-terminal kinase (JNK) was expressed in all tubular segments of controls after neonatal day 7, whereas in pcy kidneys, tubules became positive for JNK after 8 wk, and the cysts expressed little JNK. Administration of an oral MAP/ERK kinase inhibitor, PD184352, 400 mg/kg per d, to 10-wk-old pcy mice daily for the first week and then every third day for 6 additional weeks significantly decreased BP, kidney weight, serum creatinine level, and water intake and significantly increased urine osmolality. The cystic index and expression of phosphorylated ERK and ERK were significantly lower in PD184352-treated pcy mice. These results demonstrate that the expression of MAPK is dysregulated in cyst epithelium and that inhibition of ERK slowed the progression of renal disease in pcy mice.

ERK and p38 mediate high-glucose-induced hypertrophy and TGF-beta expression in renal tubular cells.

We investigated the expression of ERK, p38 mitogen-activated protein kinase (p38), and JNK in renal tubules of diabetic rats following 3 wk after streptozotocin injection (DM). Although the expression of ERK was not different between controls and DM, phosphorylated ERK was expressed more intensely in DM. p38 And phosphorylated p38 were detected only in the diabetic kidney and were localized in all tubular segments. JNK and phosphorylated JNK were expressed similarly in controls and DM. Transforming growth factor (TGF)-beta was expressed in all tubular segments of DM, coinciding with the localization of p38. In LLC-PK1 cells, phosphorylation of ERK and p38 increased after 24- to 72-h exposure to high glucose (HG). Coincubation with a p38 inhibitor SB-203580 or a MEK inhibitor, PD-98059, suppressed the HG-induced increases in protein content, [3H]leucine incorporation, and the protein-to-DNA ratio. SB-203580 or PD-98059 also abolished the HG-stimulated expression of TGF-beta protein. These results demonstrate that ERK and p38 are activated in renal tubular cells of DM and may mediate HG-induced cellular hypertrophy and TGF-beta expression.

Eicosapentaenoic acid inhibits PDGF-induced mitogenesis and cyclin D1 expression via TGF-beta in mesangial cells.

Eicosapentaenoic acid (EPA), an omega-3 polyunsaturated fatty acid derived from fish oil, is efficacious in glomerular diseases where mesangial proliferation is a key event. We examined the mechanisms of action of EPA on platelet-derived growth factor (PDGF)-stimulated rat mesangial cell mitogenesis. EPA dose-dependently inhibited PDGF-stimulated [(3)H]-thymidine incorporation. PDGF-induced PDGF receptor autophosphorylation, an initial event for PDGF signaling, was not affected by 2 micro g/ml EPA. Similarly, PDGF-stimulated activation of extracellular signal-regulated kinase (ERK) was not altered. On the other hand, EPA inhibited cyclin-dependent kinase 4 (CDK4) activation and cyclin D1 protein induction, a critical step for G1/S progression. TGF-beta secretion assessed by ELISA and bioassay was increased by EPA at 18 h. Coincubation with anti-TGF-beta antibody inhibited the EPA-induced suppression of [(3)H]-thymidine incorporation and cyclin D1 expression. SB203580, an inhibitor of p38, a downstream kinase of TGF-beta, did not affect EPA's growth inhibitory effect. These results demonstrate that EPA inhibits PDGF-stimulated mesangial cell mitogenesis and cyclin D1 expression via TGF-beta.

The lack of cyclin kinase inhibitor p27(Kip1) ameliorates progression of diabetic nephropathy.

Cyclin kinase inhibitor p27(Kip(1)) (p27) has been shown to be upregulated in glomeruli of diabetic animals and mesangial cells cultured under high glucose. This study was an investigation of the role of p27 in the progression of diabetic nephropathy. Mice deficient in p27 (p27 -/-) and wild-type mice (p27 +/+) were studied 12 wk after diabetes induction by streptozotocin. Blood glucose and BP were comparable between diabetic p27 +/+ and p27 -/- mice. The kidney weight to body weight ratio and glomerular volume increased in diabetic p27 +/+ mice. In contrast, these parameters did not change in diabetic p27 -/- mice. Similarly, albuminuria developed in diabetic p27 +/+ mice but not in diabetic p27 -/- mice. The mesangial expansion was significantly milder in diabetic p27 -/- mice than that in diabetic p27 +/+ mice. These changes were associated with a similar increase in glomerular TGF-beta expression in diabetic p27 +/+ and p27 -/- mice. However, glomerular protein expression of fibronectin, a target of TGF-beta, increased only in diabetic p27 +/+ mice. In mesangial cells cultured from p27 +/+ mice, exposure to high glucose caused significant increases in total protein content and [(3)H]-leucine incorporation. On the other hand, high glucose caused a significant reduction in these parameters in cells from p27 -/- mice. Phosphorylation of 4E-BP1, the translation inhibitor, increased after exposure to high glucose in p27 +/+ cells. In p27 -/- cells, the level of phosphorylated 4E-BP1 was higher than that in control p27 +/+ cells and decreased under high glucose conditions. In conclusion, renal hypertrophy, glomerular hypertrophy, and albuminuria did not develop, and mesangial expansion was milder in diabetic p27 -/- mice despite glomerular TGF-beta upregulation. These results suggest that controlling p27 function may ameliorate diabetic nephropathy.

The herbal medicine Sairei-to inhibits proliferation of rat mesangial cells.

BACKGROUND: The herbal medicine Sairei-to is efficacious in renal diseases where mesangial proliferation is a key event. We examined whether Sairei-to inhibits proliferation of cultured rat mesangial cells and investigated its mechanism of action. METHODS: The effect of Sairei-to on [(3)H]-thymidine incorporation stimulated by serum was assessed. Cell cycle was analyzed by flowcytometry. Extracellular signal-regulated kinase (ERK) activity was determined by immunecomplex kinase assay. Tyrosine phosphorylation of cellular proteins, and phosphorylation of ERK and Raf-1 were analyzed by immunoblot. Cyclic AMP was measured by radioimmunoassay. RESULTS: Incubation of mesangial cells for 18 h with water-soluble but not insoluble fraction of Sairei-to inhibited serum-stimulated [(3)H]-thymidine incorporation. In subsequent experiments, water-soluble fraction, at a dose required for a half-maximal response (2 mg/ml), was used. Sairei-to inhibited S-phase entry stimulated by serum. Serum-induced tyrosine phosphorylation of p44 and p42 ERK was inhibited by Sairei-to, but that of other cellular proteins was not affected. Suppression of serum-stimulated ERK activation by Sairei-to was confirmed by immunecomplex kinase assay. Activation of Raf-1, an upstream activator of ERK, was also attenuated by Sairei-to. Incubation of cells with Sairei-to significantly increased the generation of cAMP. CONCLUSIONS: Sairei-to inhibits serum-induced DNA synthesis of rat mesangial cells by suppressing Raf-1/ERK cascade probably via cAMP.

MAP kinase in renal development.

BACKGROUND: Among mitogen-activated protein kinase (MAPK) family members, ERK promotes proliferation or differentiation, whereas JNK and p38 are thought to inhibit cell growth and induce apoptosis. During renal development, large-scale proliferation and apoptosis occur. We investigated the temporal and spatial expression patterns of MAPK and its phosphatase MKP-1 as well as the role of ERK and p38 during kidney development. METHODS: Western blot analysis and immunohistochemistry were performed in the developing and mature kidney of the rat. Rat metanephroi were cultured from 15-day-old embryos, and exposed to inhibitors of MEK, an activator of ERK, PD98059, U0126 or a p38 inhibitor, SB203580 24-120 h after the start of culture. Growth of metanephroi was measured by surface area and thymidine incorporation. Ureteric buds and glomeruli were identified by labelling with Dolichos biflorus lectin and peanut agglutinin, respectively. RESULTS: The expression of ERK and p38 was high in the developing kidney. On the other hand, JNK was expressed abundantly in the adult kidney. Immunohistochemical studies revealed that the spatial expression of ERK coincided with the maturation of the kidney. p38 and MKP-1 were expressed uniformly. Growth of metanephroi was significantly inhibited by SB203580 but not by PD98059 or U0126. Ureteric bud branching was not affected by SB203580 or MEK inhibitors. Glomerular number was markedly reduced by SB203580 and to a lesser extent by U0126. CONCLUSIONS: ERK, p38 and MKP-1 are strongly expressed in the developing kidney, and JNK is detected predominantly in the adult kidney. ERK appears to play a role in nephrogenesis and p38 in kidney growth and nephrogenesis.

ERK and p38 MAP kinase are required for rat renal development.

BACKGROUND: We previously demonstrated that extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein (MAP) kinase (p38) are strongly expressed in the embryonic kidney. In the present study, we investigated the role of ERK and p38 during kidney development. METHODS: Rat metanephroi were cultured from 15-day-old embryos, and exposed to inhibitors of MEK, an activator of ERK, PD98059 (300 micromol/L), U0126 (10 micromol/L), or a p38 inhibitor SB203580 (30 micromol/L) 24 to 120 hours after the start of culture. Growth of metanephroi was measured by surface area and thymidine incorporation. Ureteric buds and glomeruli were identified by labeling with Dolichos biflorus lectin and peanut agglutinin, respectively. PCNA staining and TUNEL assay were performed on kidney sections. The level of apoptosis was evaluated by examining DNA ladder formation. RESULTS: Growth of metanephroi was significantly inhibited by SB203580 but not by PD98059 or U0126. Ureteric bud branching was not affected by SB203580 or MEK inhibitors. Glomerular number was markedly reduced by SB203580 and to a lesser extent by U0126 (14 +/- 2 and 48 +/- 10% of controls, respectively). On histological examination, the number of tubuloglomerular structures was reduced in MEK inhibitor-treated metanephroi compared to controls. Very few mesenchymal condensates were observed in kidneys incubated with SB203580. PCNA-positive cells were reduced in SB203580-treated metanephroi compared to control and PD98059-treated kidneys. Apoptosis was increased in SB203580-treated kidneys and to a lesser extent in PD98059-treated cultures. CONCLUSIONS: Both ERK and p38 are required for renal development. ERK appears to play a role in nephrogenesis and p38 for kidney growth and nephrogenesis.

Expression of mitogen-activated protein kinases in human renal dysplasia.

BACKGROUND: We previously reported that the expression of mitogen-activated protein kinases (MAPKs) is developmentally regulated. Dysregulation of MAPKs may lead to kidney malformation. Thus, we investigated the expression of MAPKs in human renal dysplasia, one of the most common kidney malformations. METHODS: Prenatal (gestational ages 20 to 36 weeks, N = 6) and postnatal (2 years old, N = 1) dysplastic kidneys, and normal kidneys (gestational ages 19 to 34 weeks, N = 4) were examined. Immunohistochemical studies were performed using antibodies against extracellular signal-regulated kinase (ERK), p38 MAPK (p38), c-Jun N-terminal kinase (JNK), phospho-MAPKs (P-MAPKs), and proliferating cell nuclear antigen (PCNA). Apoptosis was detected by the TUNEL method. RESULTS: In dysplastic kidneys, proliferation was prominent in dysplastic tubules and also found in cyst epithelia. TUNEL staining was detected in dysplastic tubules and cysts, and occasionally in undifferentiated cells. p38 and anti-phospho-p38 (P-p38) were strongly expressed in dysplastic epithelia, but not detected in normal kidneys at any stage examined. On the other hand, JNK and P-JNK were positive in tubular epithelia of normal kidneys, whereas their expression was barely detectable in dysplastic tubules and cysts. ERK was expressed in all tubular segments, and P-ERK was detected in distal tubules and collecting ducts of normal kidneys. Dysplastic kidney epithelia stained exclusively positive for ERK and P-ERK. CONCLUSIONS: p38 is ectopically expressed, and JNK is down-regulated in dysplastic kidney epithelia. Furthermore, dysplastic epithelia are exclusively positive for ERK and P-ERK. Activated p38 and ERK may mediate hyperproliferation of dysplastic tubules resulting in cyst formation, whereas down-regulated JNK expression may be the cause or the result of an undifferentiated state of dysplastic epithelia.