Hiroki Kurahashi

Increased Water Intake Decreases Progression of Polycystic Kidney Disease in the PCK Rat

Renal enlargement in polycystic kidney disease (PKD) is caused by the proliferation of mural epithelial cells and transepithelial fluid secretion into the cavities of innumerable cysts. Arginine vasopressin (AVP) stimulates the proliferation of human PKD cells in vitro via cAMP-dependent activation of the B-Raf/MEK (MAPK/ERK kinase/extracellular signal-regulated kinase (ERK) pathway. ERK activity is elevated in cells that line the cysts in animals with PKD, and AVP receptor antagonists reduce ERK activity and halt disease progression. For suppression of the effect of AVP physiologically, water intake was increased in PCK rats, a model of PKD, and the effect on renal morphology, cellular mechanism, and function was determined. The addition of 5% glucose in the drinking water increased fluid intake approximately 3.5-fold compared with rats that received tap water. In PCK rats, increased water intake for 10 wk reduced urinary AVP excretion (68.3%), and urine osmolality fell below 290 mOsmol/kg. High water intake was associated with reduced renal expression of AVP V2 receptors (41.0%), B-Raf (15.4%), phosphorylated ERK (38.1%), and proliferating cell nuclear antigen-positive renal cells (61.7%). High water intake reduced the kidney/body weight ratio 28.0% and improved renal function. Taken together, these data demonstrate that water intake that is sufficient to cause persistent water diuresis suppresses B-Raf/MEK/ERK activity and decreases cyst and renal volumes in PCK rats. It is suggested that limiting serum AVP levels by increased water intake may be beneficial to some patients with PKD.

CDH1 Is a Specific Marker for Undifferentiated Spermatogonia in Mouse Testes

Abstract In the mammalian testis, spermatogenesis is initiated from a subset of stem cells belonging to undifferentiated type A spermatogonia. In spite of the biologic significance of undifferentiated type A spermatogonia, little is known about their behavior and properties because of a lack of specific cell surface markers. Here we show that CDH1 (previously known as E-cadherin) is expressed specifically in undifferentiated type A spermatogonia in the mouse testis. Histologic analysis showed that CDH1-positive cells had all the characteristics of undifferentiated type A spermatogonia. Whole-mount immunohistochemistry showed that CDH1-positive cells made clusters mainly comprising one, two, four, or eight cells. They survived after administration of the cytotoxic agent busulfan to mice, and then regenerated seminiferous epithelia. Transplantation experiments showed that only CDH1-positive cells had colonizing activity in the recipient testis. Our data clearly demonstrated that spermatogenic stem cells reside among undifferentiated type A spermatogonia, which express CDH1.

Evolutionarily conserved low copy repeats (LCRs) in 22q11 mediate deletions, duplications, translocations, and genomic instability: an update and literature review.

Several constitutional rearrangements, including deletions, duplications, and translocations, are associated with 22q11.2. These rearrangements give rise to a variety of genomic disorders, including DiGeorge, velocardiofacial, and conotruncal anomaly face syndromes (DGS/VCFS/CAFS), cat eye syndrome (CES), and the supernumerary der(22)t(11;22) syndrome associated with the recurrent t(11;22). Chromosome 22-specific duplications or low copy repeats (LCRs) have been directly implicated in the chromosomal rearrangements associated with 22q11.2. Extensive sequence analysis of the different copies of 22q11 LCRs suggests a complex organization. Examination of their evolutionary origin suggests that the duplications in 22q11.2 may predate the divergence of New World monkeys 40 million years ago. Based on the current data, a number of models are proposed to explain the LCR-mediated constitutional rearrangements of 22q11.2.

Identification of the gene responsible for gelatinous drop-like corneal dystrophy

Gelatinous drop-like corneal dystrophy (GDLD; OMIM 204870) is an autosomal recessive disorder characterized by severe corneal amyloidosis leading to blindness, with an incidence of 1 in 300,000 in Japan. Our previous genetic linkage study localized the gene responsible to a 2.6-cM interval on chromosome 1p (ref. 6). Clinical manifestations, which appear in the first decade of life, include blurred vision, photophobia and foreign-body sensation. By the third decade, raised, yellowish-grey, gelatinous masses severely impair visual acuity, and lamellar keratoplasty is required for most patients. Here we report DNA sequencing, cDNA cloning and mutational analyses of four deleterious mutations (Q118X, 632delA, Q207X and S170X) in M1S1 (formerly TROP2 and GA733-1), encoding a gastrointestinal tumour-associated antigen. The Q118X mutation was the most common alteration in the GDLD patients examined, accounting for 33 of 40 (82.5%) disease alleles in our panel of families. Protein expression anaysis revealed aggregation of the mutated, truncated protein in the perinuclear region, whereas the normal protein was distributed diffusely in the cytoplasm with a homogenous or fine granular pattern. Our successful identification of the gene that is defective in GDLD should facilitate genetic diagnosis and potentially treatment of the disease, and enhance general understanding of the mechanisms of amyloidosis.

Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3′ untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3′ end of the fukutin coding region, a proximal part of the 3′ UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.

Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations

There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.

Another critical region for deletion of 22q11 : A study of 100 patients

Deletions at 22q11.1-q11.2 present with variable manifestations usually referred to as DiGeorge or velo-cardio-facial syndrome. We previously reported that deletions observed in patients with the syndrome can be subgrouped into three types (common large deletion, proximal deletion, and distal deletion) and demonstrated the presence of a second critical region for the syndrome. In order to characterize further the second critical region, a 22q11 deletion map was constructed from the data of 100 patients, using 12 DNA markers scattered in the common large deletion, and then a phenotype-genotype correlation was analyzed. The second critical region was found to correspond to the distal deletion encompassing the HCF2, cHKAD26, and D22S935 loci, and the proximal and distal deletions do not overlap each other. Although it seems that this condition is a contiguous gene syndrome, the phenotype of patients with these two types of deletion was indistinguishable from that of patients with the common large deletion. Thus, it is plausible that several genes located in the two segments corresponding to the two deleted regions are involved in the same developmental pathway or in an extremely long-range position effect.

Serum neutrophil gelatinase-associated lipocalin as a predictor of organ recovery from delayed graft function after kidney transplantation from donors after cardiac death.

Because of a worldwide shortage of renal grafts, kidneys procured from donors after cardiac death (DCD) have recently become an important source of renal transplants. However, DCD kidneys often have complications with delayed graft function (DGF) and recipients require hemodialysis (HD) in the early period after kidney transplantation (KTx). This study evaluated serum NGAL as a potential specific parameter to predict early functional recovery of transplanted DCD kidneys. The average serum neutrophil gelatinase-associated lipocalin (NGAL) level in normal samples was 53 ± 30 ng/ml, while that in patients with chronic renal failure requiring HD was markedly raised at 963 ± 33 ng/ml. In patients undergoing a living-related KTx from a living donor (n = 11), serum NGAL level decreased rapidly after KTx, and only in two cases, with serum NGAL levels over 400 ng/ml on postoperative day 1 (POD1), was HD required due to DGF. In contrast, all patients undergoing a KTx from a DCD (n = 5) required HD due to DGF. Even in these cases, serum NGAL levels decreased rapidly several days after a KTx prior to the recovery of urine output and preceding the decrease in serum creatinine level. The pattern of decline in serum NGAL was biphasic, the decrease after the second peak indicating a functional recovery within the next several days. These data suggest that monitoring of serum NGAL levels may allow us to predict graft recovery and the need for HD after a KTx from a DCD.

Comparative gene expression profiling of placentas from patients with severe pre-eclampsia and unexplained fetal growth restriction

BackgroundIt has been well documented that pre-eclampsia and unexplained fetal growth restriction (FGR) have a common etiological background, but little is known about their linkage at the molecular level. The aim of this study was to further investigate the mechanisms underlying pre-eclampsia and unexplained FGR.MethodsWe analyzed differentially expressed genes in placental tissue from severe pre-eclamptic pregnancies (n = 8) and normotensive pregnancies with or (n = 8) without FGR (n = 8) using a microarray method.ResultsA subset of the FGR samples showed a high correlation coefficient overall in the microarray data from the pre-eclampsia samples. Many genes that are known to be up-regulated in pre-eclampsia are also up-regulated in FGR, including the anti-angiogenic factors, FLT1 and ENG, believed to be associated with the onset of maternal symptoms of pre-eclampsia. A total of 62 genes were found to be differentially expressed in both disorders. However, gene set enrichment analysis for these differentially expressed genes further revealed higher expression of TP53-downstream genes in pre-eclampsia compared with FGR. TP53-downstream apoptosis-related genes, such as BCL6 and BAX, were found to be significantly more up-regulated in pre-eclampsia than in FGR, although the caspases are expressed at equivalent levels.ConclusionsOur current data indicate a common pathophysiology for FGR and pre-eclampsia, leading to an up-regulation of placental anti-angiogenic factors. However, our findings also suggest that it may possibly be the excretion of these factors into the maternal circulation through the TP53-mediated early-stage apoptosis of trophoblasts that leads to the maternal symptoms of pre-eclampsia.

Basement membrane fragility underlies embryonic lethality in fukutin-null mice

Fukuyama-type congenital muscular dystrophy (FCMD), associated with brain malformation due to defects in neuronal migration, is caused by mutations in fukutin. Several lines of evidence suggest that the fukutin protein plays a pivotal role in synthesis of O-mannosyl sugar moieties of alpha-dystroglycan, a cell surface laminin receptor. Here, through targeted disruption of the orthologous mouse fukutin gene, we show that the fukutin protein is essential, as homozygous-null embryos die by E9.5 of gestation. Fukutin-null embryos show phenotypic diversity, features of which include growth retardation, folding of the egg cylinder, leakage of maternal red blood cells into the yolk sac cavity, and an increased number of apoptotic cells in the ectoderm. Loss of immunoreactivity against sugar moieties in alpha-dystroglycan suggests a reduced laminin-binding capacity. Ultrastructural analysis shows thin and breached basement membranes (BMs). BM fragility may underlie all of these abnormal phenotypes, and maintenance of BM function may require fukutin-mediated glycosylation of alpha-dystroglycan early in embryonic development.