Maternal age affects pronuclear and chromatin dynamics, morula compaction and cell polarity, and blastulation of human embryos.

STUDY QUESTION: Does maternal ageing impact early and late morphokinetic and cellular processes of human blastocyst formation? SUMMARY ANSWER: Maternal ageing significantly affects pronuclear size and intra- and extra-nuclear dynamics during fertilization, dysregulates cell polarity during compaction, and reduces blastocoel expansion. WHAT IS KNOWN ALREADY: In ART, advanced maternal age (AMA) affects oocyte yield, fertilization, and overall developmental competence. However, with the exception of chromosome segregation errors occurring during oocyte meiosis, the molecular and biochemical mechanisms responsible for AMA-related subfertility and reduced embryo developmental competence remain unclear. In particular, studies reporting morphokinetics and cellular alterations during the fertilization and pre-implantation period in women of AMA remain limited. STUDY DESIGN, SIZE, DURATION: A total of 2058 fertilized oocytes were stratified by maternal age according to the Society for Assisted Reproductive Technology classification (<35, 35-37, 38-40, 41-42, and >42 years) and retrospectively analysed. AMA effects were assessed in relation to: embryo morphokinetics and morphological alterations; and the presence and distribution of cell polarity markers-Yes-associated protein (YAP) and protein kinase C-ζ (PKC-ζ)-involved in blastocyst morphogenesis. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 1050 cycles from 1050 patients met the inclusion criteria and were analysed. Microinjected oocytes were assessed using a time-lapse culture system. Immature oocytes at oocyte retrieval and mature oocytes not suitable for time-lapse monitoring, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared among groups. Furthermore, 20 human embryos donated for research by consenting couples were used for immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: Static microscopic observation revealed that blastocyst formation and expansion were impaired in the 41-42 and >42-year groups (P < 0.0001). The morphological grades of the inner cell mass and trophectoderm were poorer in the >42-year group than those in the <35-year group (P = 0.0022 and P < 0.0001, respectively). Time-lapse microscopic observation revealed a reduction in nucleolus precursor body alignment in female pronuclei in the 41-42 and >42-year groups (P = 0.0010). Female pronuclear area decreased and asynchronous pronuclear breakdown increased in the >42-year group (P = 0.0027 and P < 0.0122, respectively). Developmental speed at cleavage stage, incidence of irregularity of first cleavage, type and duration of blastomere movement, and number of multinucleated cells were comparable among age groups. Delayed embryonic compaction and an increased number of extruded blastomeres were observed in the >42-year group (P = 0.0002 and P = 0.0047, respectively). Blastulation and blastocyst expansion were also delayed in the 41-42 and >42-year groups (P < 0.0001 for both). YAP positivity rate in the outer cells of morulae and embryo PKC-ζ immunoflourescence decreased in the >42-year group (P < 0.0001 for both). LIMITATIONS, REASONS FOR CAUTION: At the cellular level, the investigation was limited to cell polarity markers. Cell components of other developmental pathways should be studied in relation to AMA. WIDER IMPLICATIONS OF THE FINDINGS: The study indicates that maternal ageing affects the key functions of embryo morphogenesis, irrespective of the well-established influence on the fidelity of oocyte meiosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Association between a deep learning-based scoring system with morphokinetics and morphological alterations in human embryos.

RESEARCH QUESTION: What is the association between the deep learning-based scoring system, iDAScore, and biological events during the pre-implantation period? DESIGN: Retrospective observational study of patients (n = 925) who underwent oocyte retrieval in a clomiphene citrate-based minimal stimulation cycle and obtained expanded blastocysts between October 2019 and December 2020. The association between iDAScore with morphokinetics and morphological alteration during fertilization, cleavage stage, compaction and blastocyst stage was analysed. RESULTS: The duration of the cytoplasmic halo was significantly prolonged in low-scoring blastocysts (P < 0.0001). The timing of female and male pronuclei breakdown was significantly delayed in low-scoring blastocysts compared with high-scoring blastocysts (P < 0.0001 in both). Embryos with either trichotomous, multi-chotomous, rapid or reverse cleavage or asymmetric division had a lower score than embryos with normal cleavage (P < 0.0001-0.0098). The cell number and amount of blastomere fragmentation on days 2 and 3 were significantly associated with iDAScore (P < 0.0001-0.0008). Delayed compaction, blastulation and blastocyst expansion were observed in low-scoring embryos (P < 0.0001 in all). The incidence of blastomere exclusion and extrusion during embryonic compaction was significantly higher in low-scoring embryos than in high-scoring embryos (P ≤ 0.0001 in both). Blastocyst morphology was significantly associated with iDAScore (P < 0.0001). Multiple linear regression analysis revealed that, during the transformation to blastocyst stage, morphokinetic and morphological events were strongly associated with iDAScore (P < 0.0001-0.0116). CONCLUSIONS: iDAScore was significantly correlated with morphokinetics and morphological alterations of pre-implantation embryos, especially during the late pre-implantation period. Our findings contribute to research on deep learning model-based embryo selection, which may provide patients with a compelling explanation of blastocyst selection.

Human 1PN and 3PN zygotes recapitulate all morphokinetic events of normal fertilization but reveal novel developmental errors.

STUDY QUESTION: Does mono- (1PN) and tri-pronuclear (3PN) fertilization recapitulate the morphokinetic changes of normal bi-pronuclear (2PN) fertilization? SUMMARY ANSWER: Abnormal fertilization retraces the overall choreography of normal fertilization but reveals novel morphokinetic phenomena and raises scientifically and clinically relevant questions. WHAT IS KNOWN ALREADY: ART has allowed the extracorporeal observation of early human development. Time-lapse technology (TLT) has revealed the complexity of the morphokinetic changes underpinning fertilization and the importance of this process for the genetic and cellular integrity of the embryo. Abnormal fertilization has remained neglected, despite its relevance to the physiology and pathology of early human development. STUDY DESIGN, SIZE, DURATION: This retrospective study involved TLT observation of normally (2PN, N = 2517) and abnormally (1PN, N = 41; 3PN, N = 27) fertilized oocytes generated in ICSI cycles performed between October 2019 and December 2020. Oocyte retrieval was carried out after clomiphene citrate-based minimal ovarian stimulation. Oocytes of patients with different diagnoses of infertility were included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study included 1231 couples treated for diverse infertility causes. The fraction of male factor cases was substantial (36.1%). Microinjected oocytes were assessed by a combined TLT-culture system. Oocytes not suitable for TLT assessment, owing to an excess of residual corona cells or inadequate orientation for correct observation, were not analysed. Phenomena relevant to meiotic resumption, pronuclear dynamics, cytoplasmic/cortical modifications, cleavage patterns and embryo quality were annotated and compared between groups. MAIN RESULTS AND THE ROLE OF CHANCE: Extrusion of the second polar body (PBII) was observed in almost all 2PN/1PN (99.9% and 100.0%, respectively) and in a vast majority of 3PN zygotes (92.1%). Rates of PBII fusion with the ooplasm were much higher in 1PN and 3PN zygotes (P < 0.0001 versus 2PN). The cytoplasmic wave was observed not only in 2PN and 3PN but also in 1PN zygotes (positivity rates of 99.8% and 100% and 82.9%, respectively; P < 0.0001). More rarely, 2PN and 1PN zygotes emitted a third polar body (PBIII). The average times of this event were comparable. The presence and position of the cytoplasmic halo were comparable among the three classes of zygotes. In the 1PN group, the single PN was maternally or paternally derived in 17 and 24 zygotes, respectively, while in the vast majority of 3PN zygotes (121/127) the supernumerary PN was of maternal origin. Average times of maternal PN appearance were comparable, while average times of paternal PN appearance were delayed in 3PN zygotes (P = 0.0127). Compared with the control group, the area of the maternal PN was larger in 1PN zygotes, but smaller in 3PN zygotes (P < 0.0001). The paternal PNs displayed the same trend (P < 0.0001), although such values were consistently smaller than maternal PNs. The area of the third PN in the 3PN group was on average more than 50% smaller than those of maternal and paternal PNs. In maternal PNs of 3PN zygotes, nucleolus precursor bodies (NPBs) aligned along the area of PN juxtaposition at a lower rate compared with the 2PN group. The rate of NPB alignment was ∼50% smaller in 1PN zygotes (P = 0.0001). In paternal PNs, the rates of NPB alignment were not statistically different among the three groups. Asynchronous PN breakdown was increased in 3PN compared with 2PN zygotes (P = 0.0026). In 1PN zygotes, a developmental delay was observed starting from the disappearance of the cytoplasmic halo, reaching 9 h at the time of the first cleavage (P < 0.0001). Higher rates of abnormal cleavage patterns and blastomere fragmentation (P < 0.0001) were observed in 1PN compared to 2N and 3PN zygotes. Cleavage progression was increasingly affected after abnormal fertilization, especially 1PN, finally resulting in blastocyst formation rates of 70.2%, 12.2% and 53.5% in 2PN, 1PN and 3PN embryos, respectively (P < 0.0001). Both maternal and paternal ages were higher in cases involving 3PN fertilization. LIMITATIONS, REASONS FOR CAUTION: The study data were obtained from ICSI, but not standard IVF, treatments carried out in a single centre. The study findings therefore require independent verification. WIDER IMPLICATIONS OF THE FINDINGS: This study reports the first detailed morphokinetic map of human abnormal fertilization. Collectively, this evidence prompts new scientific hypotheses and raises clinical questions relevant to the aetiology and the treatment of abnormal fertilization. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the participating institutions. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Blastomere movement post first cell division correlates with embryonic compaction and subsequent blastocyst formation.

BACKGROUND: Blastomere movement (BMov) occurs after the first cell division in human embryos. This movement has been suggested as a prognostic parameter for pregnancy outcome prediction following cleavage-stage embryo transfer. However, the effect of BMov on preimplantation development and pregnancy outcome after blastocyst transfer remains unclear. Therefore, this study aimed to evaluate whether BMov after the first cell division is correlated with blastocyst formation rate and live birth rate after single vitrified-warmed blastocyst transfer (SVBT). METHODS: Nine hundred and sixty-six embryos cultured in the EmbryoScope+® time-lapse system were retrospectively analyzed. The BMov type was categorized into three groups; namely, bouncing, wobbling, and twist-and-crumble. The BMov duration (dBMov) between the first (t2) and second cell division (t3) was monitored, and the ratio of dBMov to the duration of the 2-cell stage was calculated [dBMov/(t3-t2)]. Developmental rates to the 4-cell, 8-cell, morula, blastocyst, and expanded blastocyst stages were assessed, as well as blastocyst morphological grade. The correlations between dBMov and clinical pregnancy, ongoing pregnancy, and live birth rates were evaluated. RESULTS: Increased dBMov/(t3-t2) was significantly correlated with decreased developmental rates to the 8-cell, morula, blastocyst, and expanded blastocyst stages, especially from the 4-cell stage to the morula stage. Analysis of different types of BMov revealed that embryos with bouncing movement exhibited significantly higher developmental rates to the 8-cell, morula, blastocyst, and expanded blastocyst stages compared with embryos with twist-and-crumble movement. The morphological quality of blastocyst-stage embryos with twist-and-crumble movement was significantly lower than that of embryos with bouncing and wobbling movements. The rates of clinical pregnancy, ongoing pregnancy, and live birth after SVBT were not correlated with BMov type or duration. CONCLUSIONS: Embryonic compaction and subsequent blastocyst formation are adversely affected by twist-and-crumble movement and prolonged movement after the first cell division. Our results indicate that the preimplantation developmental competence of human embryos could be predicted by assessing BMov after the first cell division on day 1.

Phylogenetic Analysis of the Synnema-Producing Genus Synnemapestaloides.

Synnemapestaloides rhododendri, the type species of the genus Synnemapestaloides, is a pathogen of Rhododendron brachycarpum. This fungus produces six-celled conidia with appendages at both end cells, and are generated by annellidic conidiogenous cells on the synnema. These conidial structures are similar to those of the genus Pestalotia. The monotypic genus Synnemapestaloides is currently classified in the family Amphisphaeriaceae solely based on conidial morphology. Here we demonstrate that Synnemapestaloides represents a distinct genus in the family Sporocadaceae (Amphisphaeriales) based on differences in the nucleotide sequences of the partial large subunit rDNA gene, the rDNA internal transcribed spacer, and the partial ß-tubulin. The genus most closely related to Synnemapestaloides is Seimatosporium and the species most similar to Synnemapestaloides rhododendri is Seim. foliicola which produces short synnema-like conidiomata (sporodochia). These results demonstrate that Seim. foliicola should be transferred to Synnemapestaloides, and also demonstrate that Sporocadaceae can have synnematal in addition to pycnidial and acervular conidiomata.

Identification of quantitative trait loci for diabetic nephropathy in KK-Ay/Ta mice.

BACKGROUND: The pathogenesis and development of human diabetic nephropathy involves genetic factors. Since human diabetic nephropathy is a heterogeneous disorder, identification of responsible gene loci is difficult. We studied candidate gene loci for diabetic nephropathy, using quantitative trait locus (QTL) analysis of a spontaneous animal model for diabetic nephropathy: KK-Ay/Ta × normal BALB/cA F2 intercross mice. METHODS: We examined 270 (KK-Ay/Ta × BALB/cA) F2 intercross mice for their urinary albumin to creatinine ratios (ACRs), HbA1c and fasting body weights (FBW) at 8, 12, 16 and 20 weeks. Genotypes were investigated using 86 microsatellite markers with QTL analysis. RESULTS: ACR in mice at 20 weeks and ACR gain showed a suggestive linkage to chromosome 9 (log of the odds [LOD] scores: 3.8 and 3.4, respectively; designated ACR-1). Gene loci contributing to HbA1c indicated a significant linkage to chromosome 7 (LOD: 5.8 and 8.9) in mice at 8 and 20 weeks (designated HbA1c-1), and FBW indicated a significant linkage to chromosome 1 (LOD: 5.5 and 5.2) in mice at 8 and 12 weeks (designated Fbw-1). At 20 weeks, glomerular to Bowman's capsule volume (G/B) ratio of F2 mice homozygous BB for D9Mit66 was significantly higher than that in homozygous KK and heterozygous KB F2 progeny. The sizes of pancreatic islets in F2 progeny homozygous KK and heterozygous KB for D7Mit100 were larger than those in homozygous BB F2 progeny. CONCLUSION: QTL analysis of KK-Ay/Ta mice revealed several new loci contributing to diabetic nephropathy and related phenotypes. Thus, it appears that type 2 diabetes and nephropathy of KK-Ay/Ta mice have different genetic factors.

Combination effects of enalapril and losartan on lipid peroxidation in the kidneys of KK-Ay/Ta mice.

BACKGROUND: Adenosine monophosphate activated protein kinase (AMPK) has a protective effect on lipid peroxidation. Adiponectin and AMPK might have a role in the pathogenesis of diabetic nephropathy. Blockade of the renin-angiotensin system (RAS) increases adiponectin levels and reduces oxidative stress. The objective of the present study was to examine lipid peroxidation via adiponectin and AMPK activation in the kidneys of KK-A(y)/Ta mice by RAS inhibitors, such as enalapril and/or losartan. METHODS: KK-A(y)/Ta mice were given enalapril (2.5 mg/kg/day) and/or losartan (25 mg/kg/day), or hydralazine (25 mg/kg/day) in the drinking water for 8 weeks starting at 8 weeks of age. They were divided into 5 groups as follows: enalapril 2.5 mg/kg/day treatment group (n = 5), losartan 25 mg/kg/day treatment group (n = 5), enalapril 2.5 mg/kg/day + losartan 25 mg/kg/day combination treatment group (n = 5), hydralazine 25 mg/kg/day treatment group (n = 5) and tap water group as the untreated group (n = 5). The urinary albumin/creatinine ratio (ACR), serum adiponectin and systemic blood pressure were measured as test parameters. Expressions of adiponectin, phospho-AMPKalpha (p-AMPKalpha) and phospho-acetyl CoA carboxylase(beta) (p-ACC(beta)) in the kidneys were evaluated by Western blot analyses. Pathological changes of glomeruli were evaluated by light microscopy. Accumulations of N(epsilon)-(carboxymethyl) lysine (CML), malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) in glomeruli were evaluated by immunohistochemical analyses. RESULTS: Enalapril and/or losartan improved levels of urinary ACR with activation of adiponectin, p-AMPKalpha and p-ACC(beta) in the kidneys. CML, MDA and 4-HNE expressions in glomeruli were significantly suppressed by enalapril and/or losartan, especially in the combination treatment group. CONCLUSIONS: It appears that enalapril and/or losartan, especially in combination, inhibited accumulation of CML/MDA/4-HNE in diabetic renal tissues. These effects might be related to lipid peroxidation via tissue-specific activation of adiponectin and AMPK.

Effect of pitavastatin on type 2 diabetes mellitus nephropathy in KK-Ay/Ta mice.

It is generally considered that 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) have renoprotective effects via a pathway independent of their cholesterol-lowering cascade. In the kidneys of diabetic nephropathy, monomeric endothelial nitric oxide synthase (eNOS) is thought to be overexpressed; and its dimerization is suppressed. In the present study, we investigated the expression of eNOS and oxidative stress in type 2 diabetes mellitus KK-Ay/Ta mice treated with pitavastatin, one of the statins. The KK-Ay/Ta mice were divided into 3 groups and given pitavastatin intraperitoneally starting at 8 weeks of age for 8 weeks: pitavastatin 3 mg/(kg d) (n=5), pitavastatin 10 mg/(kg d) (n=5), and a control group (n=10). The urinary albumin-creatinine ratio (ACR), urinary 8-hydroxy-2'-deoxyguanosine, body weight, fasting blood glucose, hemoglobin A1c, total cholesterol, and triglyceride were measured; and the intraperitoneal glucose tolerance test was performed. The eNOS, nitrotyrosine, and p47 phox were evaluated by immunohistochemical analyses and/or Western blot analyses. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression in the kidneys was evaluated using a real-time polymerase chain reaction assay. Pitavastatin improved the levels of urinary ACR and 8-hydroxy-2'-deoxyguanosine, intraperitoneal glucose tolerance test, and hemoglobin A1c. Protein levels of monomeric eNOS, nitrotyrosine, and p47 phox in the kidneys were decreased in the pitavastatin-treated groups. Guanosine triphosphate cyclohydrolase 1 messenger RNA expression was significantly increased in the pitavastatin groups. There were no significant changes in body weight, levels of fasting blood glucose, serum total cholesterol, triglyceride, and blood pressure among all groups. Pitavastatin improved urinary ACR apparently because of suppression of eNOS uncoupling and its antioxidant effect in the kidneys of KK-Ay/Ta mice.

Effect of pyridoxamine (K-163), an inhibitor of advanced glycation end products, on type 2 diabetic nephropathy in KK-A(y)/Ta mice.

Advanced glycation end products (AGEs) from the Maillard reaction contribute to the pathogenesis of diabetes-associated complications such as diabetic nephropathy. In therapeutic interventions for reducing AGEs, many compounds have been reported as AGE inhibitors. The objective of the present study was to examine the effect of pyridoxamine (K-163), an AGE inhibitor, in type 2 diabetic KK-A(y)/Ta mice. KK-A(y)/Ta mice were given pyridoxamine (200 or 400 mg/kg per day) starting at 8 weeks of age for 12 weeks. They were divided into 3 groups as follows: pyridoxamine 200 mg/kg per day treatment group (n = 10), pyridoxamine 400 mg/kg per day treatment group (n = 10), and a tap water group as the control group (n = 20). The urinary albumin/creatinine ratio (ACR), body weight (BW), levels of fasting and casual blood glucose, blood glycated hemoglobin (HbA(1c)), fasting serum insulin, triglyceride (TG), total cholesterol (T-Cho), and 3-deoxyglucosone (3DG), and systemic blood pressure were measured as biochemical parameters. N(epsilon)-(Carboxymethyl)lysine (CML) and nitrotyrosine accumulations in glomeruli were evaluated by immunohistochemical analyses. Transforming growth factor beta1 (TGF-beta1) and laminin-beta1 messenger RNA expressions in the kidneys were evaluated by real-time polymerase chain reaction. Pyridoxamine, especially at 400 mg/kg per day, improved the levels of urinary ACR, fasting serum TG, and 3DG. CML and nitrotyrosine accumulations in glomeruli were decreased. Furthermore, large doses of pyridoxamine prevented not only urinary ACR but also increases of BW, casual blood glucose, and HbA(1c). TGF-beta1 and laminin-beta1 messenger RNA expressions in kidneys were significantly lower than those in the controls. There were no significant changes in the levels of fasting blood glucose, serum T-Cho, and systemic blood pressure among all groups. It appears that pyridoxamine improved urinary ACR by its anti-AGE and anti-oxidant effects in the kidneys of KK-A(y)/Ta mice.

Effects of eicosapentaenoic acid on the early stage of type 2 diabetic nephropathy in KKA(y)/Ta mice: involvement of anti-inflammation and antioxidative stress.

Eicosapentaenoic acid (EPA) has been reported to have beneficial effects on the progression of various renal diseases including diabetic nephropathy; however, the precise mechanisms are not completely understood. We examined the effects of EPA on the early stage of type 2 diabetic nephropathy in KKA(y)/Ta mice and the possible role of inflammation, oxidative stress, and growth factor in this process. KKA(y)/Ta mice were divided into 2 groups. The treatment group was injected with EPA ethyl ester at 1 g/kg per day intraperitoneally from 12 to 20 weeks of age and the control group was injected with saline. Renal morphologic examinations were performed after 8 weeks of treatment. Glomerular macrophage infiltration and expression of monocyte chemoattractant protein 1, malondialdehyde (MDA), nitrotyrosine, transforming growth factor beta1 (TGF-beta1), and type I collagen were evaluated. Eicosapentaenoic acid decreased the levels of urinary albumin, serum triglyceride and MDA, and improved glucose intolerance in KKA(y)/Ta mice. Morphometric analysis showed that accumulation of extracellular matrix and the tubulointerstitial fibrosis area were significantly decreased after treatment. Immunohistochemistry revealed that glomerular macrophage infiltration and the expression of MDA and nitrotyrosine in KKA(y)/Ta mice were increased and were inhibited by EPA treatment. Protein and gene expression levels of monocyte chemoattractant protein 1, TGF-beta1, and type I collagen, which were evaluated by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction, were down-regulated in the EPA treatment group. In conclusion, EPA improves type 2 diabetic nephropathy in KKA(y)/Ta mice. This beneficial effect might be mediated by attenuation of metabolic abnormalities and inhibition of renal inflammation, oxidative stress, and TGF-beta expression.

Glomerular changes in the KK-Ay/Ta mouse: a possible model for human type 2 diabetic nephropathy.

BACKGROUND: In type 2 diabetic nephropathy, there is no animal model which has been completely matched with humans. Advanced glycation end products (AGE) and transforming growth factor-beta (TGF-beta) are closely related to hyperglycaemia and their pathobiochemistry could explain diabetic nephropathy. The objective of the present study was to evaluate the KK-A(y)/Ta mouse as a suitable model for type 2 diabetic nephropathy including pathological changes and immunohistochemical analyses of AGE and TGF-beta, compared with the non-diabetic BALB/cA mouse. METHODS: The urinary albumin/creatinine ratio (ACR), body weight (BW), fasting and casual blood glucose, blood haemoglobin A(1c) (HbA(1c)), creatinine clearance (Ccr) and blood pressure were measured for phenotypic characterisation. The pathological changes of glomeruli were evaluated by light microscopy, immunofluorescence and electron microscopy. AGE and TGF-beta accumulation were evaluated by immunoperoxidase staining. RESULTS: The mean levels of ACR, casual blood glucose, blood HbA(1c) and Ccr in KK-A(y)/Ta mice were higher than those in age-matched non-diabetic BALB/cA mice after 12 weeks of age. There were no significant changes in the levels of systemic blood pressure among all groups. The pathological changes of glomeruli in KK-A(y)/Ta mice were consistent with those in the early stage of human diabetic nephropathy. AGE and TGF-beta protein appeared to be localised in the glomerular mesangial matrices. CONCLUSION: It appears that KK-A(y)/Ta mice, especially in terms of histopathological findings, are a suitable animal model for the early stage of type 2 diabetic nephropathy.

Eicosapentaenoic acid ameliorates diabetic nephropathy of type 2 diabetic KKAy/Ta mice: involvement of MCP-1 suppression and decreased ERK1/2 and p38 phosphorylation.

BACKGROUND: Previous studies reported that eicosapentaenoic acid (EPA) was effective against any renal diseases including diabetic nephropathy. Monocyte chemoattractant protein-1 (MCP-1) is a regulating macrophage recruitment protein, which is up-regulated in patients with diabetic nephropathy. The objectives of the present study were to evaluate the effects of EPA including renal MCP-1 expression in diabetic KKAy/Ta mice, MCP-1 production and signal transduction in mouse mesangial cells (MMCs). METHODS: KKAy/Ta mice were injected with EPA ethyl ester (1 g/kg/day) intraperitoneally. Immunohistochemical staining of MCP-1, F4/80, phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phospho-p38 in the renal sections were performed. EPA or specific inhibitors were incorporated in MMCs, and the levels of supernatant MCP-1 were measured. The effect of EPA on ERK1/2, c-jun NH2-terminal kinase (JNK), p38 or phosphoinositide 3-kinase (PI3K) activity in MMCs was examined using Western blot. RESULTS: EPA decreased the levels of serum triglycerides, leptin, urinary albumin and MCP-1, and improved glucose intolerance, mesangial matrix accumulation and tubulointerstitial fibrosis in KKAy/Ta mice. Immunohistochemical staining of MCP-1 and F4/80 in the glomeruli and tubulointerstitial regions was decreased in the EPA-treated group. EPA and specific inhibitors of ERK1/2, JNK and PI3K decreased levels of MCP-1 in MMCs. EPA suppressed phosphorylation of ERK1/2 and p38 in MMCs, and decreased p-ERK positive cells in glomeruli of KKAy/Ta mice. CONCLUSIONS: EPA ameliorates diabetic nephropathy of type 2 diabetic KKAy/Ta mice. We propose that the observed down-regulation of MCP-1 is critically involved in the beneficial effect of EPA, probably in concert with improvement of other clinical parameters.

Genetic susceptibility to type 2 diabetic nephropathy in human and animal models.

Diabetic nephropathy is the most common cause of end-stage renal disease (ESRD) in Japan, Western Europe, and the United States. Mega studies such as Diabetes Control and Complication Trial (DCCT), Epidemiology of Diabetes Interventions and Complications (EDIC), and the United Kingdom Prospective Diabetes Study (UKPDS) clarified that poor glycemic and blood pressure control are undoubtedly involved in the development of nephropathy. However, these factors are not sufficient to predict which diabetic patients will develop renal disease, because not all patients with poor glycemic and blood pressure control develop renal disease. Since ethnic variations and familial clustering of diabetic nephropathy have been observed, genetic factors might contribute to susceptibility to this disease. Several methods such as (genome wide) association studies, sib-pair analysis, and quantitative trait loci (QTLs) analysis are available to examine polygenic diseases. However, no mutations that could explain the majority of nephropathy cases have been identified so far. The development of most diabetic nephropathy might be explained by the polygenic effect (i.e. many minor gene-gene interactions might be very important in the development of nephropathy). Identification of candidate genes of nephropathy enables targeting of therapy in patients at risk and development of novel therapeutic agents.

Altered mouse cholinephosphotransferase gene expression in kidneys of type 2 diabetic KK/TA mouse.

It is generally considered that genetic factors may contribute to the susceptibility of type 2 diabetic nephropathy. The purpose of the present study is to identify molecules that contribute to the development and/or progression of this disease. Differential display was performed to isolate genes in the kidney using the KK/Ta mouse model of type 2 diabetes. The differential expression of 8 randomly chosen candidate genes (DN1-8) were verified by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis. DN1-3 (Zn-alpha2-glycoprotein, vascular endothelial growth factor receptor [VEGFR]-2, and lactate dehydrogenase [LDH]) were overexpressed and DN7-8 (peroxisome proliferator-activated receptor [PPAR]-interacting protein [PRIP], unknown) were underexpressed in the KK/Ta mouse kidney. DN4-6 (Ezrin, transcobalamin 2, aldo-ketoreductase) did not differ between KK/Ta and control (BALB/c) mice. DN8 only showed no significant sequence similarity to previously reported genes. Molecular cloning revealed that full-length DN8 shares 89% identity with human cholinephosphotransferase 1 (hCHPT1), and we designated it as "putative" mouse cholinephosphotransferase 1 (mCHPT1). The putative mCHPT1 gene was most closely mapped to the D10Mit94 locus with the highest logarithm of odds (lod) score. In situ hybridization revealed the levels of glomerular putative mCHPT1 in BALB/c mice tended to be slightly higher than those in KK/Ta mice. The altered renal mRNA expression of these genes may be involved in the development and/or progression of diabetic nephropathy.