Assisted reproductive technologies are slightly associated with maternal lack of affection toward the newborn: The Japan Environment and Children's Study.

AIM: To evaluate the association between use of assisted reproductive technology (ART) and mother-to-infant bonding. METHODS: Using nationwide birth cohort study with periodical follow-ups for mothers and children during pregnancy and at 1 year after delivery, mothers were classified following three groups; infertility group with ART: 2792 mothers; infertility group with non-ART treatment (ovulation induction and intrauterine insemination): 3835 mothers and unaided pregnancy group: 78 726 mothers. Data on maternal and child health as well as basic characteristics were collected via medical records and self-administered questionnaires. The Japanese version of Mother-to-Infant Bonding Scale was used to evaluate maternal bonding style. To evaluate the association between ART and maternal bonding toward babies, multivariate analysis was used with adjustment for potential confounders such as babies' sex, socio-economic status and history of maternal mental disorders. RESULTS: Multivariate logistic regression analysis indicated that ART was slightly but significantly associated with an increased risk of maternal lack of affection toward their newborn (adjusted odds ratio 1.10, 95% confidence interval 1.02-1.20), while non-ART was significantly associated with both maternal lack of affection and anger/rejection toward the newborn. No substantial association was observed between ART and overall Mother-to-Infant Bonding Scale score (adjusted odds ratio 1.03, 95% confidence interval 0.94-1.12). Very slight negative correlations were observed between poor mother-to-infant bonding and good infant mental and physical development at both 6 months and 1 year. CONCLUSION: ART may exert a negative influence on maternal emotion after delivery, but this does not have strong correlation with child development evaluated by mothers.

The risk of secondary sex ratio imbalance and increased monozygotic twinning after blastocyst transfer: data from the Japan Environment and Children's Study.

BACKGROUND: Some studies have suggested that blastocyst transfer is associated with i) imbalance in the secondary sex ratio (SSR) (which favors male offspring), ii) increased incidence of monozygotic twins (MZT). In contrast, others have not found these changes. In this study, we evaluated the association between blastocyst transfer and SSR and MZT, considering potential parental confounders. METHODS: The Japan Environment and Children's Study is a large, nationwide longitudinal birth cohort study funded by the Ministry of the Environment of Japan. We used this large dataset, including 103,099 pregnancies, to further investigate the association between blastocyst transfer, SSR and MZT, using spontaneously conceived pregnancies, non-assisted reproductive technology (non-ART) treatment (intrauterine insemination and ovulation induction with timed intercourse) and cleavage stage embryo transfer for comparison. We evaluated the association with each group, the SSR, and the frequency of MZT, calculating the adjusted odds ratio (AOR) using multivariable logistic regression analyses, adjusting for potential parental confounders such as basic health and socioeconomic status. RESULTS: For each group (spontaneous conception vs. non-ART treatment vs. cleavage stage embryo transfer vs. blastocyst transfer), the percentages of males were 51.3% vs 50.7% vs 48.9% vs 53.4% and the monozygotic twinning rates per pregnancy were 0.27% vs 0.11% vs 0.27% vs 0.99% respectively. Multivariate logistic regression analyses indicated that blastocyst transfer was significantly associated with a higher SSR and higher incidence of MZT than the other three groups (SSR: AOR 1.095, 95% CI1.001-1.198; MZT: AOR 4.229, 95% CI 2.614-6.684). CONCLUSIONS: There are significant relationships between blastocyst transfer and SSR imbalance and a higher occurrence of MZT.

Association of four imprinting disorders and ART.

BACKGROUND: Human-assisted reproductive technologies (ART) are a widely accepted treatment for infertile couples. At the same time, many studies have suggested the correlation between ART and increased incidences of normally rare imprinting disorders such as Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS), Prader-Willi syndrome (PWS), and Silver-Russell syndrome (SRS). Major methylation dynamics take place during cell development and the preimplantation stages of embryonic development. ART may prevent the proper erasure, establishment, and maintenance of DNA methylation. However, the causes and ART risk factors for these disorders are not well understood. RESULTS: A nationwide epidemiological study in Japan in 2015 in which 2777 pediatrics departments were contacted and a total of 931 patients with imprinting disorders including 117 BWS, 227 AS, 520 PWS, and 67 SRS patients, were recruited. We found 4.46- and 8.91-fold increased frequencies of BWS and SRS associated with ART, respectively. Most of these patients were conceived via in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), and showed aberrant imprinted DNA methylation. We also found that ART-conceived SRS (ART-SRS) patients had incomplete and more widespread DNA methylation variations than spontaneously conceived SRS patients, especially in sperm-specific methylated regions using reduced representation bisulfite sequencing to compare DNA methylomes. In addition, we found that the ART patients with one of three imprinting disorders, PWS, AS, and SRS, displayed additional minor phenotypes and lack of the phenotypes. The frequency of ART-conceived Prader-Willi syndrome (ART-PWS) was 3.44-fold higher than anticipated. When maternal age was 37 years or less, the rate of DNA methylation errors in ART-PWS patients was significantly increased compared with spontaneously conceived PWS patients. CONCLUSIONS: We reconfirmed the association between ART and imprinting disorders. In addition, we found unique methylation patterns in ART-SRS patients, therefore, concluded that the imprinting disorders related to ART might tend to take place just after fertilization at a time when the epigenome is most vulnerable and might be affected by the techniques of manipulation used for IVF or ICSI and the culture medium of the fertilized egg.

Lack of association between receiving ART treatment and parental psychological distress during pregnancy: Preliminary findings of the Japan Environment and Children's Study.

In a nationwide population-based birth cohort study in Japan, pregnant women and their partners were evaluated for psychological distress as part of the first and second/third trimester health checks. Participants were divided into three groups: an infertility group receiving assisted reproductive technology (ART) treatment (239 mothers and 151 fathers); an infertility group receiving non-ART treatment (350 mothers and 215 fathers); and a spontaneous pregnancy group (8514 mothers and 5110 fathers). Data on maternal and child health as well as basic characteristics were collected via medical records and self-administered questionnaires. The Kessler Six-item Psychological Distress Scale was employed for eligible women and their partners. Multivariate logistic regression analysis was used to evaluate the association between psychological distress experienced during pregnancy and ART treatment, with adjustment for potential confounders such as basic health status and socio-economic status. The mothers who received ART treatment suffered less psychological distress than the mothers in the other two groups. In multivariate analysis adjusted for background characteristics, no significant association was observed between persistent maternal distress and ART treatment (adjusted odds ratio 0.79, 95% confidence interval 0.49-1.26). Higher socio-economic status among couples receiving ART treatment may explain, in part, the lack of association between ART treatment and parental distress during pregnancy.

Genome-wide microRNA expression profiling in placentae from frozen-thawed blastocyst transfer.

BACKGROUND: Frozen-thawed embryo transfer (FET) is increasingly available for the improvement of the success rate of assisted reproductive technologies other than fresh embryo transfer (ET). There have been numerous findings that FET provides better obstetric and perinatal outcomes. However, the birth weight of infants conceived using FET is heavier than that of those conceived via ET. In addition, some reports have suggested that FET is associated with perinatal diseases such as placenta accreta and pregnancy-induced hypertension (PIH). RESULTS: In this study, we compared the microRNA (miRNA) expression profiles in term placentae derived from FET, ET, and spontaneous pregnancy (SP). We identified four miRNAs, miR-130a-3p, miR-149-5p, miR-423-5p, and miR-487b-3p, that were significantly downregulated in FET placentae compared with those from SP and ET. We found that DNA methylation of MEG3-DMR, not but IG-DMR, was associated with miRNA expression of the DLK1-DIO3 imprinted domain in the human placenta. In functional analyses, GO terms and signaling pathways related to positive regulation of gene expression, growth, development, cell migration, and type II diabetes mellitus (T2DM) were enriched. CONCLUSIONS: This study supports the hypothesis that the process of FET may increase exposure of epigenome to external influences.

Factors associated with aberrant imprint methylation and oligozoospermia.

Disturbingly, the number of patients with oligozoospermia (low sperm count) has been gradually increasing in industrialized countries. Epigenetic alterations are believed to be involved in this condition. Recent studies have clarified that intrinsic and extrinsic factors can induce epigenetic transgenerational phenotypes through apparent reprogramming of the male germ line. Here we examined DNA methylation levels of 22 human imprinted loci in a total of 221 purified sperm samples from infertile couples and found methylation alterations in 24.8% of the patients. Structural equation model suggested that the cause of imprint methylation errors in sperm might have been environmental factors. More specifically, aberrant methylation and a particular lifestyle (current smoking, excess consumption of carbonated drinks) were associated with severe oligozoospermia, while aging probably affected this pathology indirectly through the accumulation of PCB in the patients. Next we examined the pregnancy outcomes for patients when the sperm had abnormal imprint methylation. The live-birth rate decreased and the miscarriage rate increased with the methylation errors. Our research will be useful for the prevention of methylation errors in sperm from infertile men, and sperm with normal imprint methylation might increase the safety of assisted reproduction technology (ART) by reducing methylation-induced diseases of children conceived via ART.

Epigenetic alterations in sperm associated with male infertility.

The most common form of male infertility is a low sperm count, known as oligozoospermia. Studies suggest that oligozoospermia is associated with epigenetic alterations. Epigenetic alterations in sperm, which may arise due to the exposure of gametes to environmental factors or those that pre-exist in the sperm of infertile individuals, may contribute to the increased incidence of normally rare imprinting disorders in babies conceived after assisted reproductive technology using the sperm of infertile men. Genomic imprinting is an important developmental process whereby the allelic activity of certain genes is regulated by DNA methylation established during gametogenesis. The aberrant expression of several imprinted genes has been linked to various diseases, malignant tumors, lifestyle and mental disorders in humans. Understanding how infertility and environmental factors such as reproductive toxicants, certain foods, and drug exposures during gametogenesis contribute to the origins of these disorders via defects in sperm is of paramount importance. In this review, we discuss the association of epigenetic alterations with abnormal spermatogenesis and the evidence that epigenetic processes, including those required for genomic imprinting, may be sensitive to environmental exposures during gametogenesis, fertilization and early embryonic development. In addition, we review imprinting diseases and their relationships with environmental factors. While the plasticity of epigenetic marks may make these more susceptible to modification by the environment, this also suggests that aberrant epigenetic marks may be reversible. A greater understanding of this process and the function of epidrugs may lead to the development of new treatment methods for many adult diseases in the future.

SV2B is essential for the integrity of the glomerular filtration barrier.

The glomerular visceral epithelial cell (podocyte) is characterized as a specialized structure of the interdigitating foot processes, covering the outer side of the glomerular basement membrane (GBM). The neighboring foot processes are connected by a slit diaphragm, which is a key structure regulating the barrier function of the glomerular capillary wall to prevent proteinuria. We have previously reported that synaptic vesicle protein 2 B (SV2B) is expressed in the podocyte and that the expression is clearly decreased in nephrotic models. However, the precise function of SV2B in the podocyte is unclear. To investigate the role of SV2B in maintaining the podocyte function and to better understand the function of the neuron-like vesicle expressing SV2B in the podocyte, we analyzed them with SV2B knockout (KO) mice. An increase in the amount of proteinuria, effacement of the foot process of the podocyte, and alterations of the GBM were detected in SV2B KO mice. It was also found that the expression of CD2AP, nephrin, and NEPH1, the functional molecules of the slit diaphragm, and laminin, a critical component of the GBM, is clearly altered in SV2B KO mice. Synaptotagmin and neurexin, which have a role in the synaptic vesicle docking in neurons, are downregulated in the kidney cortex of SV2B KO mice. We have previously reported that neurexin interacts with CD2AP, and the present study shows that SV2B interacts with CD2AP. These findings suggest that the SV2B-neurexin complex is involved in the formation and maintenance of the slit diaphragm. In addition, SV2B is densely expressed close to the cell surface in the presumptive podocyte in the early stage of glomerulogenesis. These results suggest that SV2B has an essential role in the formation and maintenance of the glomerular capillary wall.

Imprinting methylation errors in ART.

There has been an increase in incidence reports of rare imprinting disorders associated with assisted reproductive technology (ART). ART, including in vitro fertilization and intracytoplasmic sperm injections, is an important treatment for infertile people of reproductive age and increasingly produces children. The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that ART techniques themselves may predispose embryos to acquire imprinting errors and diseases. In this review, we note that the particular steps of ART may be prone to induction of imprinting methylation errors during gametogenesis, fertilization and early embryonic development. In addition, we explain imprint-associated diseases and their causes. Moreover, from a Japanese nationwide epidemiological study of imprint-associated diseases, we determine their associations with ART. Epigenetic studies will be required to understand the pathogenesis, ART-related risk factor(s) and what precautions can be taken to prevent the occurrence of input methylation errors. We hope that the constitution of children born after each ART procedure will reveal the safest and most ethical approach to use, which will be invaluable for the future development of standard ART.

Therapeutic target for nephrotic syndrome: Identification of novel slit diaphragm associated molecules.

The slit diaphragm bridging the neighboring foot processes functions as a final barrier of glomerular capillary wall for preventing the leak of plasma proteins into primary urine. It is now accepted that the dysfunction of the sit diaphragm contributes to the development of proteinuria in several glomerular diseases. Nephrin, a gene product of NPHS1, a gene for a congenital nephrotic syndrome of Finnish type, constitutes an extracellular domain of the slit diaphragm. Podocin was identified as a gene product of NPHS2, a gene for a familial steroid-resistant nephrotic syndrome of French. Podocin binds the cytoplasmic domain of nephrin. After then, CD2 associated protein, NEPH1 and transient receptor potential-6 were also found as crucial molecules of the slit diaphragm. In order to explore other novel molecules contributing to the development of proteinuria, we performed a subtraction hybridization assay with a normal rat glomerular RNA and a glomerular RNA of rats with a puromycin aminonucleoside nephropathy, a mimic of a human minimal change type nephrotic syndrome. Then we have found that synaptic vesicle protein 2B, ephrin-B1 and neurexin were already downregulated at the early stage of puromycin aminonucleoside nephropathy, and that these molecules were localized close to nephrin. It is conceivable that these molecules are the slit diaphragm associated molecules, which participate in the regulation of the barrier function. These molecules could be targets to establish a novel therapy for nephrotic syndrome.

DNA methylation errors in imprinting disorders and assisted reproductive technology.

There have been increased incident reports of rare imprinting disorders associated with assisted reproductive technology (ART). ART is an important treatment for infertile people of reproductive age and is increasingly common. The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquisition of imprinting errors and disease. It is still unknown, however, at what point(s) these imprinting errors arise, or the risk factors. In this review it was hypothesized that the particular steps of the ART process may be prone to induction of imprinting methylation errors during gametogenesis, fertilization and early embryonic development. In addition, imprinting diseases and their causes are explained. Moreover, using a Japanese nationwide epidemiological study of imprinting diseases, their association with ART is determined. Epigenetic studies are required to understand the pathogenesis of this association; the ART-related risk factor(s); and the precautions that can be taken to prevent the occurrence of these syndromes. It is hoped that the constitution of children born after ART will indicate the safest and most ethical approach to use, which will be invaluable for the future development of standard ART treatment.

Stability of genomic imprinting in human induced pluripotent stem cells.

BACKGROUND: hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs. RESULTS: We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture. CONCLUSIONS: Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.

Characterization of DNA methylation errors in patients with imprinting disorders conceived by assisted reproduction technologies.

BACKGROUND: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquire imprinting errors and diseases. However, it is still unknown at what point(s) these imprinting errors arise, or the risk factors. METHODS: In 2009 we conducted a Japanese nationwide epidemiological study of four well-known imprinting diseases to determine any association with ART. Using bisulfite sequencing, we examine the DNA methylation status of 22 gametic differentially methylated regions (gDMRs) located within the known imprinted loci in patients with Beckwith-Wiedemann syndrome (BWS, n=1) and also Silver-Russell syndrome (SRS, n= 5) born after ART, and compared these with patients conceived naturally. RESULTS: We found a 10-fold increased frequency of BWS and SRS associated with ART. The majority of ART cases showed aberrant DNA methylation patterns at multiple imprinted loci both maternal and paternal gDMRs (5/6), with both hyper- and hypomethylation events (5/6) and also mosaic methylation errors (5/6). Although our study may have been limited by a small sample number, the fact that many of the changes were mosaic suggested that they occurred after fertilization. In contrast, few of the patients who were conceived naturally exhibited a similar pattern of mosaic alterations. The differences in methylation patterns between the patients who were conceived naturally or after ART did not manifest due to the differences in the disease phenotypes in these imprinting disorders. CONCLUSION: A possible association between ART and BWS/SRS was found, and we observed a more widespread disruption of genomic imprints after ART. The increased frequency of imprinting disorders after ART is perhaps not surprising given the major epigenetic events that take place during early development at a time when the epigenome is most vulnerable.

High-throughput detection of aberrant imprint methylation in the ovarian cancer by the bisulphite PCR-Luminex method.

BACKGROUND: Aberrant DNA methylation leads to loss of heterozygosity (LOH) or loss of imprinting (LOI) as the first hit during human carcinogenesis. Recently we developed a new high-throughput, high-resolution DNA methylation analysis method, bisulphite PCR-Luminex (BPL), using sperm DNA and demonstrated the effectiveness of this novel approach in rapidly identifying methylation errors. RESULTS: In the current study, we applied the BPL method to the analysis of DNA methylation for identification of prognostic panels of DNA methylation cancer biomarkers of imprinted genes. We found that the BPL method precisely quantified the methylation status of specific DNA regions in somatic cells. We found a higher frequency of LOI than LOH. LOI at IGF2, PEG1 and H19 were frequent alterations, with a tendency to show a more hypermethylated state. We detected changes in DNA methylation as an early event in ovarian cancer. The degree of LOI (LOH) was associated with altered DNA methylation at IGF2/H19 and PEG1. CONCLUSIONS: The relative ease of BPL method provides a practical method for use within a clinical setting. We suggest that DNA methylation of H19 and PEG1 differentially methylated regions (DMRs) may provide novel biomarkers useful for screening, diagnosis and, potentially, for improving the clinical management of women with human ovarian cancer.

[Genomic imprinting and carcinogenesis].

Genomic imprinting is an epigenetic marking and a stable transmission of monoallelic gene expression patterns in a parent of- origin-specific manner. Aberrant imprinting has been linked to a number of human genetic disorders, including congenital abnormalities, childhood cancer, behavior disorders, and cancer in adults. Imprinted genes play roles in carcinogenesis. Recently, progress in researched on epigenetic mechanisms of imprinted genes, in edition to analysis of the pathology of the oncogenetic mechanisms, has begun to be clinically applied to diagnostic methods, prevention, and cancer drug development.

Neurexin-1, a presynaptic adhesion molecule, localizes at the slit diaphragm of the glomerular podocytes in kidneys.

The slit diaphragm connecting the adjacent foot processes of glomerular epithelial cells (podocytes) is the final barrier of the glomerular capillary wall and serves to prevent proteinuria. Podocytes are understood to be terminally differentiated cells and share some common features with neurons. Neurexin is a presynaptic adhesion molecule that plays a role in synaptic differentiation. Although neurexin has been understood to be specifically expressed in neuronal tissues, we found that neurexin was expressed in several organs. Several forms of splice variants of neurexin-1α were detected in the cerebrum, but only one form of neurexin-1α was detected in glomeruli. Immunohistochemical study showed that neurexin restrictedly expressed in the podocytes in kidneys. Dual-labeling analyses showed that neurexin was colocalized with CD2AP, an intracellular component of the slit diaphragm. Immunoprecipitation assay using glomerular lysate showed that neurexin interacted with CD2AP and CASK. These observations indicated that neurexin localized at the slit diaphragm area. The staining intensity of neurexin in podocytes was clearly lowered, and their staining pattern shifted to a more discontinuous patchy pattern in the disease models showing severe proteinuria. The expression and localization of neurexin in these models altered more clearly and rapidly than that of other slit diaphragm components. We propose that neurexin is available as an early diagnostic marker to detect podocyte injury. Neurexin coincided with nephrin, a key molecule of the slit diaphragm detected in a presumptive podocyte of the developing glomeruli and in the glomeruli for which the slit diaphragm is repairing injury. These observations suggest that neurexin is involved in the formation of the slit diaphragm and the maintenance of its function.

Assessing loss of imprint methylation in sperm from subfertile men using novel methylation polymerase chain reaction Luminex analysis.

OBJECTIVE: To assess the clinical value of bisulfite polymerase chain reaction Luminex (BPL), an automated, high-throughput procedure for the detection of alterations in DNA methylation. DESIGN: Experimental prospective study. SETTING: University research laboratory and private in vitro fertilization (IVF) clinic. PATIENT(S): A total of 337 men, 61 with severe oligozoospermia, 67 with moderate oligozoospermia, and 209 with microscopically normozoospermia. INTERVENTION(S): The ejaculated sperm samples after the routine semen analysis with patients' consent. MAIN OUTCOME MEASURE(S): Examination of the methylation patterns of eight imprinted loci in sperm DNA, and confirmation with combined bisulfite PCR restriction analysis (COBRA). RESULT(S): A total of 47 cases (13.9%) showed abnormal methylation at one or more imprinted loci (18 paternal, 18 maternal, and 11 cases with alterations of both maternal and paternal imprints). CONCLUSION(S): The relative ease of the BPL method provides a practical method within a clinical setting to reduce the likelihood of abnormal samples being used in assisted reproduction treatments.

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip.

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.

Therapeutic targets in the podocyte: findings in anti-slit diaphragm antibody-induced nephropathy.

Recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the barrier in the glomerular capillary wall. Murine monoclonal antibody against nephrin, a molecule constituting the extracellular site of the slit diaphragm, caused severe proteinuria if injected into rats, in a complement- or inflammatory cell-independent manner. In this proteinuric state, not only nephrin but also other slit diaphragm-associated molecules are down-regulated. These observations suggest that the antibody alters the molecular composition of the slit diaphragm and, thereby, affects the glomerular permeability barrier. Recently, it was found that IP-10, SV2B, ephrin B1 and the receptors of angiotensin II were expressed in the podocyte, and that their expressions were clearly altered in anti-nephrin antibody-induced nephropathy. It is conceivable that these molecules are involved in the development of proteinuria in this model. IP-10 is assumed to play a role in maintaining the slit diaphragm function by regulating the cell cycle balance of the podocyte. SV2B and ephrin B1 play pivotal roles in the proper localization of the slit diaphragm component. In vivo and in vitro studies demonstrated that angiotensin II type 2 receptor-mediated action enhanced the expression of nephrin. We propose that these molecules could be novel therapeutic targets for proteinuria.

Slit diaphragm dysfunction in proteinuric states: identification of novel therapeutic targets for nephrotic syndrome.

Several recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the principal barrier in the glomerular capillary wall. Nephrin identified as a gene product mutated in congenital nephrotic syndrome located at the outer leaflet of plasma membranes of the slit diaphragm. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. Expression of nephrin was reduced in glomeruli of minimal change nephrotic syndrome. Some recent studies demonstrated that podocin, CD2-associated protein and NEPH1 are also functional molecules in the slit diaphragm, and their expressions are altered in membranous nephropathy and also in focal glomerulosclerosis. These observations suggested that the alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases. Recent studies of our group have demonstrated that type 1 receptor-mediated angiotensin II action reduced the expression of the slit diaphragm-associated molecules and that type 1 receptor blockade ameliorated proteinuria by preventing the function of angiotensin II on the slit diaphragm. By the subtraction hybridization techniques using glomerular cDNA of normal and proteinuric rats, we detected that synaptic vesicle protein 2B and ephrin B1 are involved in the maintenance of the barrier function of the slit diaphragm.