Tsukasa Takemura

The Membrane-bound Form of Heparin-binding Epidermal Growth Factor-like Growth Factor Promotes Survival of Cultured Renal Epithelial Cells

To understand whether expression of membrane-anchored heparin binding epidermal growth factor (proHB-EGF) is involved in renal epithelial cell survival, rat membrane-bound HB-EGF precursor was stably transfected into a renal epithelial cell line, NRK 52E cells (NRKproHB-EGF). When exposed to 10% fetal calf serum (FCS), there were no differences in growth rates among wild-type (WT), vector-transfected (NRKvector), and NRKproHB-EGF. However, when cells were grown in the presence of 1% FCS, the growth rate of NRKproHB-EGF was 65% faster. When confluent cell monolayers were exposed to H2O2 or etoposide, WT or NRKvectorexhibited significant apoptotic bodies and DNA laddering; in contrast, NRKproHB-EGF were resistant to both stimuli, as indicated by increased cell viability and marked decrease of apoptotic bodies and DNA laddering. When plated at high density onto plastic dishes without FCS, WT and NRKvector formed few attachments, did not proliferate, and underwent apoptosis. By day 3, no cells survived. Addition of exogenous recombinant HB-EGF (10−8 m) to WT or NRKvector increased cell survival by <10% and incubation with conditioned media of NRKproHB-EGF had no effect. In contrast, NRKproHB-EGF attached and formed epithelial colonies, although they did not proliferate. After 3 days, cell viability was 84% of the initial cell number plated, and no evidence of apoptosis was present. When plated in 10% FCS, NRKproHB-EGFattachment to plastic substratum at 1, 2, and 3 h was 250% greater than that of WT or NRKvector. Addition of exogenous recombinant human HB-EGF to WT or NRKvector increased attachment by <50%. When grown on poly(2-hydroxyethyl methacrylate) or in the presence of the integrin receptor-blocking peptide GRGDTP, neither WT nor NRKvector attached to the substratum or formed cell-cell attachments. Compared with WT or NRKvector, NRKproHB-EGF exhibited 300% greater cell viability on either poly(2-hydroxyethyl methacrylate)-coated dishes or in the presence of GRGDTP and formed cell clusters. When plated at low density (1 × 103 cells/1.5-cm dish) or at high density in the presence of an anti-HB-EGF blocking antibody, NRKproHB-EGF failed to form epithelial colonies. Addition of formalin fixed NRKproHB-EGF promoted EGF receptor tyrosine phosphorylation in quiescent A431 cells and stimulated DNA synthesis and prevented H2O2-induced apoptosis in renal epithelial cells. These results indicate that membrane-bound HB-EGF promotes renal epithelial cell survival, possibly by promoting cell-matrix and cell-cell interactions. The failure of either conditioned media or exogenous HB-EGF to reproduce these findings suggests that juxtacrine or tightly coupled paracrine interactions underlie this cytoprotection.

Identification of Two Novel Mutations in the CLCN5 Gene in Japanese Patients With Familial Idiopathic Low Molecular Weight Proteinuria (Japanese Dent's Disease)

Two Japanese patients, belonging to unrelated families, with idiopathic low-molecular-weight proteinuria (LMWP; Japanese Dents disease) showed novel mutations of the gene encoding renal-specific chloride channel 5 (CLC-5). Proteinuria was first noticed at the ages of 2 and 3 years in patients 1 and 2, respectively. During follow-up, marked increases in urinary ss(2)-microglobulin levels, hypercalciuria, and high levels of urinary excretion of growth hormone were observed in both patients. Nephrocalcinosis was detected in patient 2. Renal biopsy specimens from both patients showed minimal alterations in glomeruli and tubulointerstitium, except for mild mesangial proliferation in patient 2. DNA sequence analysis of the entire 2,238-bp coding region and exon-intron boundaries of the CLCN5 gene showed the presence of two novel mutations in exon 10, consisting of one missense mutation (I524K) in patient 1 and one nonsense mutation (R637X) in patient 2. DNA analysis and measurement of urinary ss(2)-microglobulin levels in family members indicated an X-linked mode of inheritance in patient 1 and sporadic occurrence in patient 2. These results have expanded our understanding of the association between idiopathic LMWP (Japanese Dents disease) and mutations of the CLCN5 gene.

The effect of Lactobacillus brevis KB290 against irritable bowel syndrome: a placebo-controlled double-blind crossover trial

BackgroundIrritable bowel syndrome (IBS) is a functional disorder of the digestive tract that causes chronic abdominal symptoms. We evaluated the effects of Lactobacillus brevis KB290 (KB290), which has been demonstrated to be effective at improving bowel movements and the composition of intestinal microflora, on IBS symptoms.MethodsWe performed a placebo control double-blind cross matched trial. Thirty-five males and females (aged 6 years and above) who had been diagnosed with IBS according to the Rome III criteria were divided into 2 groups, and after a 4-week pre-trial observation period, they were administered test capsules containing KB290 or placebo for 4 weeks (consumption period I). Then, the capsule administration was suspended for 4 weeks in both groups (washout period), before the opposite capsules were administered for a further 4 weeks (consumption period II). Fecal samples were collected on the first day of the pre-consumption observation period, the last day of consumption period I, the last day of the washout period, and the last day of consumption period II. In addition, the subjects’ IBS symptoms and quality of life (QOL) and any adverse events that they experienced were evaluated.ResultsNo significant difference in IBS symptoms was noted among the various periods. However, the mean QOL scores were improved during the test capsule consumption.The frequencies of watery and mushy feces were significantly lower in the test capsule consumption period than during the pre-consumption observation period, and the frequency of abdominal pain was significantly reduced in the test capsule consumption period compared with the other periods.The frequency of the genus Bifidobacterium was significantly higher, and that of the genus Clostridium was significantly lower, after the test capsule consumption than after the placebo consumption. The frequencies of the genera Lactobacillus, Bacteroides, and Enterococcus were also investigated, but no differences in their frequencies were detected between the placebo and test capsule consumption periods.ConclusionsProbiotics, the safety of which has been established, are used widely in various foods and can now be purchased readily. The results of the present study suggest that KB290 is useful for early intervention in IBS.

A boy with Japanese Dent's disease exhibiting abnormal calcium metabolism and osseous disorder of the spine: defective megalin expression at the brushborder of renal proximal tubules.

We encountered a 16-year-old boy with Japanese Dents disease who exhibited renal insufficiency and an osseous disorder of the spine. Proteinuria first was noted at the age of 2 years. At 13 years, the patient underwent analysis of the CLCN5 gene, which identified missense mutation (I524K) in exon 10. During follow-up, a marked increase in urinary beta2-microglobulin was associated with mild deterioration of renal function. At the age of 15 years, hypocalcemia (7.5 mg/dl) accompanied by an increased plasma concentration of alkaline phosphatase was first detected. At that time, plasma concentration of 25(OH)D3 and 1alpha25(OH)2D3 were low accompanied by a high plasma parathyroid hormone concentration. A renal biopsy specimen revealed tubulointerstitial alterations including mononuclear cell infiltration, partial fibrosis and focal glomerular sclerosis. Immunofluorescence revealed weak, discontinuous staining of megalin along the brushborder of renal proximal tubules. Western blotting demonstrated decreased urinary excretion of megalin. Thus, clinical manifestations and prognosis may vary in Japanese Dents disease. Reduced megalin expression may have disturbed calcium homeostasis, leading to osseous disorder in our patient.

A Japanese family with Alport syndrome associated with esophageal leiomyomatosis: genetic analysis of COL4A5 to COL4A6 and immunostaining for type IV collagen subtypes.

BACKGROUNDnIn some families, X-linked Alport syndrome (AS) is associated with diffuse leiomyomatosis. We describe clinical, pathologic and molecular-genetic findings in a Japanese family with this inheritance mode of AS in association with leiomyomatosis.nnnPATIENTnAS was diagnosed in a one-year-old boy with recurrent aspiration pneumonia caused by esophageal stenosis from leiomyomatosis. Diagnosis was confirmed by electron microscopy coupled with type IV collagen chain subtype staining in a renal biopsy specimen. His mother, who exhibited esophageal leiomyomatosis and is heterozygous for AS, showed a discontinuous staining pattern for collagen alpha5(IV) chain along the epidermal basement membrane in a skin biopsy specimen. Genetic analysis in the boy revealed the deletion of the first two exons of COL4A6 together with deletion of the 5 end of COL4A5. Despite administration of cyclosporin A, massive proteinuria has persisted in the boy, although renal function otherwise remains normal.nnnCONCLUSIONnIdentification of an AS patient during infancy is extremely rare. Clinical manifestations, including macroscopic hematuria, cataracts and leiomyomatosis caused by the large deletion involving COL4A5 to COL4A6, led to early presentation with AS.

Chronic renal insufficiency in a boy with cystic renal lymphangiectasia : morphological findings and long-term follow-up

Cystic renal lymphangiectasia (CRL) is a rare malformation of lymphatics that can present in childhood and adulthood. Symptoms and radiologic features are relatively well defined, but clinical evolution and prognosis remain unclear. We treated a boy with CRL who developed chronic renal insufficiency. The first manifestation was abdominal swelling associated with an umbilical hernia noted incidentally at 1.6 years. Computed tomography with intravenous contrast administration demonstrated perirenal cysts with fluid collection, suggesting CRL. Intractable ascites resisted pharmacologic treatments such as diuretics. After approximately 7 years, the ascites resolved spontaneously, but the perirenal cysts persisted. At 11 years, proteinuria was noted. A renal biopsy specimen showed interstitial abnormalities consistent with CRL, glomeruli showed a focal segmental mesangial increase. Proteinuria persisted despite administration of an angiotensin-converting enzyme inhibitor, increasing as obesity and hypertension worsened. Renal function gradually declined in the ensuing years. Polycythemia coexisted with a normal serum erythropoietin concentration. A follow-up renal biopsy specimen disclosed glomerular enlargement together with focal segmental mesangial expansion, suggesting obesity-related glomerulopathy. Our observation suggest that under some specific circumstances like our patient CRL may exacerbate. Management of complicating obesity and hypertension are likely to be important for maintaining normal renal function, especially in the diffuse bilateral type of CRL present in our patient.

Alport syndrome and benign familial hematuria (thin basement membrane disease) in two brothers of a family with hematuria

Alport syndrome (AS) and benign familial hematuria (BFH) are inherited disorders of the glomerular basement membrane, which are sometimes difficult to differentiate at the early stage without type IV collagen staining of the renal basement membrane. Previous studies have indicated that mutation of type IV collagen alpha4 gene may be responsible for both BFH and AS. We report here a Japanese family with consanguinity, in which autosomal-recessive AS and BFH were separately identified in two brothers on the basis of findings of electron microscopy and type IV collagen chain staining of the renal biopsy specimens. Their parents, being first cousins, paternal uncle and grandmothers were found to have hematuria. Our observations suggest that BFH patients were heterozygous carriers of autosomal-recessive AS.

A case of renal-coloboma syndrome associated with mental developmental delay exhibiting a novel PAX2 gene mutation.

A case of an adolescent male with renal-coloboma syndrome (RCS) showing developmental delay is described. Birth and perinatal histories were typical. Proteinuria was initially observed at the age of 7 years during an annual mass screening program for school children. His urine was checked periodically at a local hospital. Because of an increase in proteinuria, he was referred to our hospital for further clinical evaluation. Proteinuria was moderate, ranging from 1.0 to 1.5 g/day, and was coupled with mild renal dysfunction. At that time, he was found to have myopia associated with astigmatism. He exhibited mild developmental delay, assessed by a WISC-III test. A renal biopsy sample showed marked glomerular enlargement, collapse of glomerular capillaries, mesangial matrix expansion, and tubulointerstitial change, demonstrating typical histologic features of RCS. Approximately five years after starting follow-up, the patient had severe renal dysfunction. Furthermore, optic nerve coloboma was also evident. Genetic analysis of the patient revealed a novel heterozygous mutation in exon 3 of the PAX2 gene (P130H).

A boy with IgA nephropathy complicated by tubulointerstitial nephritis and uveitis (TINU) syndrome.

BACKGROUNDnAbnormal cellular and humoral immunity underlie both immunoglobulin A (IgA) nephropathy and tubulointerstitial nephritis and uveitis (TINU) syndrome. We encountered a teenage boy who developed TINU syndrome during the course of IgA nephropathy.nnnCASE REPORTn1 year after onset of IgA nephropathy following acute enteritis, a 14-year-old boy again experienced acute enteritis caused by Campylobacter jejuni, which was followed by TINU syndrome with prominent low-molecular-weight proteinuria. Renal histologic examination showed T-cell-dominant tubulointerstitial infiltration of marked immune cells including CD54-positive cells. Steroid therapy improved renal function, reversing aggravation of IgA nephropathy by TINU syndrome.nnnCONCLUSIONSnThe boys human leukocyte antigen profile suggested predisposition to these two diseases, triggered by which were intestinal infections. The enteritis probably induced abnormalities in cellular and humoral immunity. Low-molecular-weight proteinuria, which reflected our patients tubulointerstitial lesions, should call for consideration of TINU syndrome, including ophthalmologic assessment for possible uveitis.

Pediatric Left Renal Vein Entrapment Syndrome Diagnosed by 99mTc-Albumin-Conjugate Scintigraphy

Background/Aims: Procedures for diagnosis of left renal vein entrapment syndrome (LRVES) in children have been either invasive or limited in accuracy. We examined scintigraphy with 99mTc-diethylene triamine pentaacetic acid-conjugated human serum albumin (99mTc-HSA-D) scintigraphy in childhood LRVES, demonstrating selective left renal nuclides excretion. We also measured peak velocity using pulse Doppler ultrasonography, calculating pressure differences between inferior vena cava and left renal vein using a simplified Bernoulli equation. Methods: Thirteen patients provisionally diagnosed with LRVES by ultrasonography combined with other imaging such as magnetic resonance angiography and three-dimensional computer tomography (CT) were examined. Results: Four children showing repeated gross hematuria all showed pressure differences exceeding 3.0 mm Hg. Selective left renal albumin excretion was demonstrated by 99mTc-HSA-D scintigraphy. Single-photon emission CT also showed accumulation in a site consistent with the left renal pelvis. Among 9 children manifesting mainly orthostatic proteinuria, selective left renal albumin excretion examined by 99mTc-HSA-D scintigraphy was demonstrated only in those with proteinuria exceeding 1 g/g Cr after standing in a lordotic position. Pressure differences in patients with orthostatic proteinuria were unrelated to proteinuria severity. Conclusions: Combining pulse Doppler ultrasonography with 99mTc-HSA-D scintigraphy, both noninvasive and safe in children, may suffice for diagnosis of LRVES, especially with gross hematuria.