Protective effects of C/EBPα on podocytes in diabetic nephropathy / 中华肾脏病杂志

Objective To explore the effects of C/EBPα knockout in podocyte on diabetic nephropathy and its mechanisms.Methods C/EBPαloxp/loxp mice were crossed with podocin-cre mice to obtain F1 hybrids and then propagated until homozygous mice (C/EBPαf/f) were obtained.Diabetic nephropathy (DN) models were established by low-dose streptozotocin (STZ,100 mg/kg) administration after 25 weeks of normal diet or 45％ high-fat diet treatment,and biochemical indicators of blood and urea were measured.The morphological characteristics and the proteins regulating oxidative stress and mitochondrial function were detected.Results The type 2 DN models were successfully constructed based on transgenic mice.The kidneys of 8-month-old C/EBPαf/f mice did not show obvious morphological changes,but after constructing DN models,they showed obvious renal impairment,inflammation and oxidative stress.Compared with wild-type DN mice,the protein levels of nephrin and E-cadherin in DN C/EBPαf/f mice with DN were significantly decreased (P ＜ 0.01);fibronectin and Nrf2 protein levels were all increased (all P ＜ 0.05).Keap1,phospho-AMPK and mitochondrial function related genes Pgc-1α protein levels were all decreased (all P ＜ 0.05).Conclusion Podocyte C/EBPα knockout exacerbates diabetic nephropathy by promoting fibrosis and inhibiting Pgc-1α-mediated mitochondrial antioxidant function.

Loss of Ranbp2 in motoneurons causes disruption of nucleocytoplasmic and chemokine signaling, proteostasis of hnRNPH3 and Mmp28, and development of amyotrophic lateral sclerosis-like syndromes.

The pathogenic drivers of sporadic and familial motor neuron disease (MND), such amyotrophic lateral sclerosis (ALS), are unknown. MND impairs the Ran GTPase cycle, which controls nucleocytoplasmic transport, ribostasis and proteostasis; however, cause-effect mechanisms of Ran GTPase modulators in motoneuron pathobiology have remained elusive. The cytosolic and peripheral nucleoporin Ranbp2 is a crucial regulator of the Ran GTPase cycle and of the proteostasis of neurological disease-prone substrates, but the roles of Ranbp2 in motoneuron biology and disease remain unknown. This study shows that conditional ablation of Ranbp2 in mouse Thy1 motoneurons causes ALS syndromes with hypoactivity followed by hindlimb paralysis, respiratory distress and, ultimately, death. These phenotypes are accompanied by: a decline in the nerve conduction velocity, free fatty acids and phophatidylcholine of the sciatic nerve; a reduction in the g-ratios of sciatic and phrenic nerves; and hypertrophy of motoneurons. Furthermore, Ranbp2 loss disrupts the nucleocytoplasmic partitioning of the import and export nuclear receptors importin ß and exportin 1, respectively, Ran GTPase and histone deacetylase 4. Whole-transcriptome, proteomic and cellular analyses uncovered that the chemokine receptor Cxcr4, its antagonizing ligands Cxcl12 and Cxcl14, and effector, latent and activated Stat3 all undergo early autocrine and proteostatic deregulation, and intracellular sequestration and aggregation as a result of Ranbp2 loss in motoneurons. These effects were accompanied by paracrine and autocrine neuroglial deregulation of hnRNPH3 proteostasis in sciatic nerve and motoneurons, respectively, and post-transcriptional downregulation of metalloproteinase 28 in the sciatic nerve. Mechanistically, our results demonstrate that Ranbp2 controls nucleocytoplasmic, chemokine and metalloproteinase 28 signaling, and proteostasis of substrates that are crucial to motoneuronal homeostasis and whose impairments by loss of Ranbp2 drive ALS-like syndromes.

Differential mouse-strain specific expression of Junctional Adhesion Molecule (JAM)-B in placental structures.

The junctional adhesion molecule (JAM)-B, a member of the immunoglobulin superfamily, is involved in stabilization of interendothelial cell-cell contacts, formation of vascular tubes, homeostasis of stem cell niches and promotion of leukocyte adhesion and transmigration. In the human placenta, JAM-B protein is abundant and mRNA transcripts are enriched in first-trimester extravillous trophoblast in comparison to the villous trophoblast. We here aimed to elucidate the yet unexplored spatio-temporal expression of JAM-B in the mouse placenta. We investigated and semi-quantified JAM-B protein expression by immunohistochemistry in early post-implantation si tes and in mid- to late gestation placentae of various murine mating combinations. Surprisingly, the endothelium of the placental labyrinth was devoid of JAM-B expression. JAM-B was mainly present in spongiotrophoblast cells of the junctional zone, as well as in the fetal vessels of the chorionic plate, the umbilical cord and in maternal myometrial smooth muscle. We observed a strain-specific placental increase of JAM-B protein expression from mid- to late gestation in Balb/c-mated C57BL/6 females, which was absent in DBA/2J-mated Balb/c females. Due to the essential role of progesterone during gestation, we further assessed a possible modulation of JAM-B in mid-gestational placentae deficient in the progesterone receptor (Pgr(-/-)) and observed an increased expression of JAM-B in Pgr(-/-) placentae, compared to Pgr(+/+) tissue samples. We propose that JAM-B is an as yet underappreciated trophoblast lineage-specific protein, which is modulated via the progesterone receptor and shows unique strain-specific kinetics. Future work is needed to elucidate its possible contribution to placental processes necessary to ensuring its integrity, ultimately facilitating placental development and fetal growth.

The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development.

Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.

Loss of glycogen synthase kinase 3 isoforms during murine oocyte growth induces offspring cardiac dysfunction.

Glycogen synthase kinase-3 (GSK3) is a constitutively active serine threonine kinase with 1) two isoforms (GSK3A and GSK3B) that have unique and overlapping functions, 2) multiple molecular intracellular mechanisms that involve phosphorylation of diverse substrates, and 3) implications in pathogenesis of many diseases. Insulin causes phosphorylation and inactivation of GSK3 and mammalian oocytes have a functional insulin-signaling pathway whereby prolonged elevated insulin during follicle/oocyte development causes GSK3 hyperphosphorylation, reduced GSK3 activity, and altered oocyte chromatin remodeling. Periconceptional diabetes and chronic hyperinsulinemia are associated with congenital malformations and onset of adult diseases of cardiovascular origin. Objectives were to produce transgenic mice with individual or concomitant loss of GSK3A and/or GSK3B and investigate the in vivo role of oocyte GSK3 on fertility, fetal development, and offspring health. Wild-type males bred to females with individual or concomitant loss of oocyte GSK3 isoforms did not have reduced fertility. However, concomitant loss of GSK3A and GSK3B in the oocyte significantly increased neonatal death rate due to congestive heart failure secondary to ventricular hyperplasia. Individual loss of oocyte GSK3A or GSK3B did not induce this lethal phenotype. In conclusion, absence of oocyte GSK3 in the periconceptional period does not alter fertility yet causes offspring cardiac hyperplasia, cardiovascular defects, and significant neonatal death. These results support a developmental mechanism by which periconceptional hyperinsulinemia associated with maternal metabolic syndrome, obesity, and/or diabetes can act on the oocyte and affect offspring cardiovascular development, function, and congenital heart malformation.

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity.

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.