14-3-3 Proteins stabilize actin and vimentin filaments to maintain processes in renal glomerular podocyte.

14-3-3 proteins are a ubiquitously expressed family of adaptor proteins. Despite exhibiting high sequence homology, several 14-3-3 isoforms have isoform-specific binding partners and roles. We reported that 14-3-3ß interacts with FKBP12 and synaptopodin to maintain the structure of actin fibers in podocytes. However, the precise localization and differential role of 14-3-3 isoforms in kidneys are unclear. Herein, we showed that 14-3-3ß in glomeruli was restricted in podocytes, and 14-3-3σ in glomeruli was expressed in podocytes and mesangial cells. Although 14-3-3ß was dominantly co-localized with FKBP12 in the foot processes, a part of 14-3-3ß was co-localized with Par3 at the slit diaphragm. 14-3-3ß interacted with Par3, and FKBP12 bound to 14-3-3ß competitively with Par3. Deletion of 14-3-3ß enhanced the interaction of Par3 with Par6 in podocytes. Gene silencing for 14-3-3ß altered the structure of actin fibers and process formation. 14-3-3ß and synaptopodin expression was decreased in podocyte injury models. In contrast, 14-3-3σ in podocytes was expressed in the primary processes. 14-3-3σ interacted with vimentin but not with the actin-associated proteins FKBP12 and synaptopodin. Gene silencing for 14-3-3σ altered the structure of vimentin fibers and process formation. 14-3-3σ and vimentin expression was increased in the early phase of podocyte injury models but was decreased in the late stage. Together, the localization of 14-3-3ß at actin cytoskeleton plays a role in maintaining the foot processes and the Par complex in podocytes. In contrast, 14-3-3σ at vimentin cytoskeleton is essential for maintaining primary processes.

Th17 Cells Participate in Thy1.1 Glomerulonephritis Which Is Ameliorated by Tacrolimus.

INTRODUCTION: Thy1.1 glomerulonephritis (Thy1.1 GN) in rats is widely used as an experimental model of mesangial proliferative glomerulonephritis (GN). We previously reported that T-helper (Th) cells were accumulated in glomeruli from the early phase of this model and that not Th2 cells but Th1 cells play an important role in the development of glomerular alterations. Although Th17 is reported to be involved in the pathogenesis of several autoimmune diseases, the role of Th17 cells in the pathogenesis of mesangial alterations in Thy1.1 GN remains unclear. METHODS: The kinetics of the infiltration of subsets of Th cells and the expression of IL-17 in Thy1.1 GN were analyzed. Next, the localization and the cell types of IL-17 receptor (IL-17R)-positive cells and IL-6-positive cells were analyzed. Then, the effect of tacrolimus on the expressions of Th17-related cytokines in Thy1.1 GN was analyzed. RESULTS: Not only Th1 cells but also Th17 cells were recruited into glomeruli from the early phase of the disease. mRNA expression of IL-17 in glomeruli was elevated. The increased positive expression of IL-17R was detected in the mesangial area, and some of IL-17R-positive cells were co-stained with IL-6. Tacrolimus treatment ameliorated mesangial alterations by suppressing the expressions of Th17-related cytokines such as IL-17 and IL-6. CONCLUSION: Th17 cells participate in the development of Thy1.1 GN, a mimic of mesangial proliferative GN, and Th17 cells and their related cytokines are pertinent therapeutic targets.

Tacrolimus ameliorates podocyte injury by restoring FK506 binding protein 12 (FKBP12) at actin cytoskeleton.

FKBP12 was identified as a binding protein of tacrolimus (Tac). Tac binds to FKBP12 and exhibits immunosuppressive effects in T cells. Although it is reported that Tac treatment directly ameliorates the dysfunction of the podocyte in nephrotic syndrome, the precise pharmacological mechanism of Tac is not well understood yet. It is also known that FKBP12 functions independently of Tac. However, the localization and the physiological function of FKBP12 are not well elucidated. In this study, we observed that FKBP12 is highly expressed in glomeruli, and the FKBP12 in glomeruli is restricted in podocytes. FKBP12 in cultured podocytes was expressed along the actin cytoskeleton and associated with filamentous actin (F-actin). FKBP12 interacted with the actin-associated proteins 14-3-3 and synaptopodin. RNA silencing for FKBP12 reduced 14-3-3 expression, F-actin staining, and process formation in cultured podocytes. FKBP12 expression was decreased in the nephrotic model caused by adriamycin (ADR) and the cultured podocyte treated with ADR. The process formation was deteriorated in the podocytes treated with ADR. Tac treatment ameliorated these decreases. Tac treatment to the normal cells increased the expression of FKBP12 at F-actin in processes and enhanced process formation. Tac enhanced the interaction of FKBP12 with synaptopodin. These observations suggested that FKBP12 at actin cytoskeleton participates in the maintenance of processes, and Tac treatment ameliorates podocyte injury by restoring FKBP12 at actin cytoskeleton.

Synbindin Downregulation Participates in Slit Diaphragm Dysfunction.

INTRODUCTION: Synbindin, originally identified as a neuronal cytoplasmic molecule, was found in glomeruli. The cDNA subtractive hybridization technique showed the mRNA expression of synbindin in glomeruli was downregulated in puromycin aminonucleoside (PAN) nephropathy, a mimic of minimal-change nephrotic syndrome. METHODS: The expression of synbindin in podocytes was analyzed in normal rats and 2 types of rat nephrotic models, anti-nephrin antibody-induced nephropathy, a pure slit diaphragm injury model, and PAN nephropathy, by immunohistochemical analysis and RT-PCR techniques. To elucidate the function of synbindin, a gene silencing study with human cultured podocytes was performed. RESULTS: Synbindin was mainly expressed at the slit diaphragm area of glomerular epithelial cells (podocytes). In both nephrotic models, decreased mRNA expression and the altered staining of synbindin were already detected at the early phase when proteinuria and the altered staining of nephrin, a key molecule of slit diaphragm, were not detected yet. Synbindin staining was clearly reduced when severe proteinuria was observed. When the cultured podocytes were treated with siRNA for synbindin, the cell changed to a round shape, and filamentous actin structure was clearly altered. The expression of ephrin-B1, a transmembrane protein at slit diaphragm, was clearly lowered, and synaptic vesicle-associated protein 2B (SV2B) was upregulated in the synbindin knockdown cells. CONCLUSION: Synbindin participates in maintaining foot processes and slit diaphragm as a downstream molecule of SV2B-mediated vesicle transport. Synbindin downregulation participates in slit diaphragm dysfunction. Synbindin can be an early marker to detect podocyte injury.

Nephrin-Ephrin-B1-Na+/H+ Exchanger Regulatory Factor 2-Ezrin-Actin Axis Is Critical in Podocyte Injury.

Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1-associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the nephrin-ephrin-B1 complex at the slit diaphragm. The nephrin-ephrin-B1-NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1-NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of nephrin-ephrin-B1-NHERF2-ezrin-actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.

Xanthine oxidoreductase inhibitor topiroxostat ameliorates podocyte injury by inhibiting the reduction of nephrin and podoplanin.

BACKGROUND: Topiroxostat, an inhibitor of xanthine oxidoreductase (XOR) was shown to reduce urinary albumin excretion of hyperuricemic patients with chronic kidney disease. However, its pharmacological mechanism is not well understood. In this study, we examined the effects of topiroxostat on glomerular podocytes. Podocyte is characterized by foot process and a unique cell-cell junction slit diaphragm functioning as a final barrier to prevent proteinuria. METHODS: The effects of topiroxostat on the expressions of podocyte functional molecules were analysed in db/db mice, a diabetic nephropathy model, anti-nephrin antibody-induced rat podocyte injury model and cultured podocytes treated with adriamycin. RESULTS: Topiroxostat treatment ameliorated albuminuria in db/db mice. The expression of desmin, a podocyte injury marker was increased, and nephrin and podocin, key molecules of slit diaphragm, and podoplanin, an essential molecule in maintaining foot process were downregulated in db/db mice. Topiroxostat treatment prevented the alterations in the expressions of these molecules in db/db mice. XOR activity in kidney was increased in rats with anti-nephrin antibody-induced podocyte injury. Topiroxostat treatment reduced XOR activity and restored the decreased expression of nephrin, podocin and podoplanin in the podocyte injury. Furthermore, topiroxostat enhanced the expression of podoplanin in injured human cultured podocytes. CONCLUSIONS: Podocyte injury was evident in db/db mice. Topiroxostat ameliorated albuminuria in diabetic nephropathy model by preventing podocyte injury. Increase of XOR activity in kidney contributes to development of podocyte injury caused by stimulation to slit diaphragm. Topiroxostat has an effect to stabilize slit diaphragm and foot processes by inhibiting the reduction of nephrin, podocin and podoplanin.

Synaptic Vesicle Protein 2B Negatively Regulates the Amyloidogenic Processing of AßPP as a Novel Interaction Partner of BACE1.

BACKGROUND: Given that amyloid-ß (Aß) peptide is produced and released at synapses, synaptic Aß is one of the promising therapeutic targets to prevent synaptic dysfunction in Alzheimer's disease (AD). Although Aß production begins with the cleavage of the amyloid-ß protein precursor (AßPP) by ß-site AßPP cleaving enzyme 1 (BACE1), the mechanism on how BACE1 is involved in AßPP processing at synapses remains unclear. OBJECTIVE: This study aimed to identify novel BACE1 interacting proteins regulating Aß production at the synapse. METHODS: BACE1 interacting proteins were pulled down using a mass spectrometry-based proteomics of wild-type (WT) rat brain synaptoneurosome lysates utilizing anti-BACE1 antibody. Then, a novel BACE1 interactor was identified and characterized using experimental systems that utilized transfected cells and knockout (KO) mice. RESULTS: Synaptic vesicle protein 2B (SV2B) was identified as a novel presynaptic interaction partner of BACE1. In HEK293 cells, co-overexpression of SV2B with BACE1 significantly reduced the sAßPPß and Aß levels released in the media; thus, SV2B overexpression negatively affected the AßPP cleavage by BACE1. Compared with those of WT mice, the hippocampal lysates of SV2B knockout mice had significantly elevated Aß levels, whereas the ß-secretase activity and the AßPP and BACE1 protein levels remained unchanged. Finally, a fractionation assay revealed that BACE1 was mislocalized in SV2B KO mice; hence, SV2B may be involved in BACE1 trafficking downregulating the amyloidogenic pathway of AßPP. CONCLUSION: SV2B has a novel role of negatively regulating the amyloidogenic processing of AßPP at the presynapses.

New insight into podocyte slit diaphragm, a therapeutic target of proteinuria.

Dysfunction of slit diaphragm, a cell-cell junction of glomerular podocytes, is involved in the development of proteinuria in several glomerular diseases. Slit diaphragm should be a target of a novel therapy for proteinuria. Nephrin, NEPH1, P-cadherin, FAT, and ephrin-B1 were reported to be extracellular components forming a molecular sieve of the slit diaphragm. Several cytoplasmic proteins such as ZO-1, podocin, CD2AP, MAGI proteins and Par-complex molecules were identified as scaffold proteins linking the slit diaphragm to the cytoskeleton. In this article, new insights into these molecules and the pathogenic roles of the dysfunction of these molecules were introduced. The slit diaphragm functions not only as a barrier but also as a signaling platform transfer the signal to the inside of the cell. For maintaining the slit diaphragm function properly, the phosphorylation level of nephrin is strictly regulated. The recent studies on the signaling pathway from nephrin, NEPH1, and ephrin-B1 were reviewed. Although the mechanism regulating the function of the slit diaphragm had remained unclear, recent studies revealed TRPC6 and angiotensin II-regulating mechanisms play a critical role in regulating the barrier function of the slit diaphragm. In this review, recent investigations on the regulation of the slit diaphragm function were reviewed, and a strategy for the establishment of a novel therapy for proteinuria was proposed.

Partitioning-Defective-6-Ephrin-B1 Interaction Is Regulated by Nephrin-Mediated Signal and Is Crucial in Maintaining Slit Diaphragm of Podocyte.

Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with nephrin, and Par-3 interacted with nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-nephrin were linked via extracellular sites of ephrin-B1 and nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/nephrin. Stimulation to nephrin phosphorylated not only nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

Nephrin-Binding Ephrin-B1 at the Slit Diaphragm Controls Podocyte Function through the JNK Pathway.

Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-nephrin antibody promoted the phosphorylation of nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and nephrin occurred in the early phase (24 hours) of anti-nephrin antibody-induced nephropathy.Conclusions Through interactions with nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

Possible role for glomerular-derived angiotensinogen in nephrotic syndrome.

BACKGROUND AND OBJECTIVE: Renin-angiotensin system (RAS) inhibitors reduce glomerular injury and proteinuria, indicating that angiotensin II (Ang II) is involved in glomerular diseases. Although the local RAS is reported to play an essential role in maintaining local tissue functions, the role of the local RAS in regulating glomerular function is not well evaluated. In this study, we analyzed the glomerular expression of RAS components in nephrotic models and the effect of Ang II receptor blockers (ARB) on the expression of angiotensinogen (AGT). METHODS: The levels of glomerular expression of RAS components were analyzed in two nephrotic models: anti-nephrin antibody-induced nephropathy and PAN nephropathy, a mimic of human minimal change nephrotic syndrome. The effect of the ARB irbesartan on the expression of AGT in the nephrotic model was analyzed. RESULTS: Glomerular expression of AGT and the receptors for Ang II was clearly increased in the nephrotic models, while the expression levels of renin, ACE and ACE2 were decreased. ARB treatment suppressed the increase of glomerular expression of AGT in the nephrotic model. CONCLUSION: It is conceivable that the promoted local RAS action participated in the glomerular dysfunction, and that ARB treatment ameliorated slit diaphragm injury by inhibiting the positive feedback loop of the activated local Ang II action.

Role of calcineurin (CN) in kidney glomerular podocyte: CN inhibitor ameliorated proteinuria by inhibiting the redistribution of CN at the slit diaphragm.

Although calcineurin (CN) is distributed in many cell types and functions in regulating cell functions, the precise roles ofCNremained in each type of the cells are not well understood yet. ACNinhibitor (CNI) has been used for steroid-resistant nephrotic syndrome. ACNIis assumed to ameliorate proteinuria by preventing the overproduction of T-cell cytokines. However, recent reports suggest thatCNIhas a direct effect on podocyte. It is accepted that a slit diaphragm (SD), a unique cell-cell junction of podocytes, is a critical barrier preventing a leak of plasma protein into urine. Therefore, we hypothesized thatCNIhas an effect on theSD In this study, we analyzed the expression ofCNin physiological and in the nephrotic model caused by the antibody against nephrin, a critical component of theSD We observed thatCNis expressed at theSDin normal rat and human kidney sections and has an interaction with nephrin. The staining ofCNat theSDwas reduced in the nephrotic model, whileCNactivity in glomeruli was increased. We also observed that the treatment with tacrolimus, aCNI, in this nephrotic model suppressed the redistribution ofCN, nephrin, and otherSDcomponents and ameliorated proteinuria. These observations suggested that the redistribution and the activation ofCNmay participate in the development of theSDinjury.

Dickkopf 3 Promotes the Differentiation of a Rostrolateral Midbrain Dopaminergic Neuronal Subset In Vivo and from Pluripotent Stem Cells In Vitro in the Mouse.

Wingless-related MMTV integration site 1 (WNT1)/ß-catenin signaling plays a crucial role in the generation of mesodiencephalic dopaminergic (mdDA) neurons, including the substantia nigra pars compacta (SNc) subpopulation that preferentially degenerates in Parkinson's disease (PD). However, the precise functions of WNT1/ß-catenin signaling in this context remain unknown. Stem cell-based regenerative (transplantation) therapies for PD have not been implemented widely in the clinical context, among other reasons because of the heterogeneity and incomplete differentiation of the transplanted cells. This might result in tumor formation and poor integration of the transplanted cells into the dopaminergic circuitry of the brain. Dickkopf 3 (DKK3) is a secreted glycoprotein implicated in the modulation of WNT/ß-catenin signaling. Using mutant mice, primary ventral midbrain cells, and pluripotent stem cells, we show that DKK3 is necessary and sufficient for the correct differentiation of a rostrolateral mdDA neuron subset. Dkk3 transcription in the murine ventral midbrain coincides with the onset of mdDA neurogenesis and is required for the activation and/or maintenance of LMX1A (LIM homeobox transcription factor 1α) and PITX3 (paired-like homeodomain transcription factor 3) expression in the corresponding mdDA precursor subset, without affecting the proliferation or specification of their progenitors. Notably, the treatment of differentiating pluripotent stem cells with recombinant DKK3 and WNT1 proteins also increases the proportion of mdDA neurons with molecular SNc DA cell characteristics in these cultures. The specific effects of DKK3 on the differentiation of rostrolateral mdDA neurons in the murine ventral midbrain, together with its known prosurvival and anti-tumorigenic properties, make it a good candidate for the improvement of regenerative and neuroprotective strategies in the treatment of PD. Significance statement: We show here that Dickkopf 3 (DKK3), a secreted modulator of WNT (Wingless-related MMTV integration site)/ß-catenin signaling, is both necessary and sufficient for the proper differentiation and survival of a rostrolateral (parabrachial pigmented nucleus and dorsomedial substantia nigra pars compacta) mesodiencephalic dopaminergic neuron subset, using Dkk3 mutant mice and murine primary ventral midbrain and pluripotent stem cells. The progressive loss of these dopamine-producing mesodiencephalic neurons is a hallmark of human Parkinson's disease, which can up to now not be halted by clinical treatments of this disease. Thus, the soluble DKK3 protein might be a promising new agent for the improvement of current protocols for the directed differentiation of pluripotent and multipotent stem cells into mesodiencephalic dopaminergic neurons and for the promotion of their survival in situ.

Alteration in the podoplanin-ezrin-cytoskeleton linkage is an important initiation event of the podocyte injury in puromycin aminonucleoside nephropathy, a mimic of minimal change nephrotic syndrome.

Podoplanin was identified as a protein associated with the transformation of arborized foot processes of glomerular epithelial cells (podocytes) to flat feet. However, the function of podoplanin in the podocyte is not yet fully clarified. In this study, we analyzed the molecular nature of podoplanin, and its expression in rat nephrotic models and patients with minimal change nephrotic syndrome (MCNS). We demonstrated here that podoplanin has two forms: one contains abundant sialic acid and the other a lesser amount of sialic acid. Podoplanin bound ezrin to interact with the cytoskeleton. The silencing of podoplanin in cultured podocytes caused a change in the cell shape and the distribution of ezrin and actin. The expression of podoplanin was clearly reduced before the onset of proteinuria in puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, and the decrease in the expression of podoplanin became more evident at the proteinuric stage. Podoplanin was detected in normal urine samples, and the amount of urinary podoplanin markedly increased on day 1 of PAN nephropathy. Urinary ezrin was also detected. The amount of the phosphorylated ezrin was reduced, while the amount of the podoplanin-interacting ezrin increased. The podoplanin expression was reduced in a patient with active-phase MCNS. It is conceivable that the alteration of the podoplanin-ezrin-cytoskeleton linkage is an important event of the podocyte injury in MCNS.

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.

Angiotensin II type 1 receptor blockade ameliorates proteinuria in puromycin aminonucleoside nephropathy by inhibiting the reduction of NEPH1 and nephrin.

BACKGROUND: The precise pathogenic mechanism and role of angiotensin II (Ang II) action in the development of proteinuria in minimal change nephrotic syndrome (MCNS) is uncertain. METHODS: The glomerular expressions of the slit diaphragm (SD) molecules nephrin, podocin and NEPH1 in rat puromycin aminonucleoside (PAN) nephropathy, a mimic of MCNS, were analyzed. The effects of Ang II receptor blockade (ARB) (irbesartan 15 mg/kg body weight/day) on proteinuria and on the expression of the SD molecules were analyzed. RESULTS: mRNA expressions of nephrin, podocin and NEPH1 were decreased to an undetectable level at 1 h. The staining of these SD molecules shifted to a discontinuous pattern, and their intensity was reduced. NEPH1 staining was reduced to an undetectable level on day 10. ARB treatment ameliorated the peak value of proteinuria (237.6 ± 97.0 vs. 359.0 ± 63.3 mg/day, p < 0.05), and prevented the decrease in the mRNA expression of the SD molecules (nephrin 66.96 %, podocin 60.40 %, NEPH1 77.87 % of normal level). The immunofluorescence staining of NEPH1 was restored by ARB. ARB treatment enhanced the expression of NEPH1 of normal rats. CONCLUSIONS: Dysfunction of the SD molecules including NEPH1 is a crucial initiation event of PAN nephropathy. ARB treatment ameliorates proteinuria in PAN nephropathy by inhibiting the reduction of NEPH1 and nephrin. Ang II action regulates the expression of NEPH1 and nephrin in not only the pathological but also physiological state.

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.

Beta4-galactosyltransferase-5 is a lactosylceramide synthase essential for mouse extra-embryonic development.

Glycosphingolipids (GSLs) are important for various biological functions in the nervous system, the immune system, embryogenesis and in other tissues and processes. Lactosylceramide (LacCer), which is synthesized from glucosylceramide (GlcCer) by LacCer synthase, is a core structure of GSLs, including gangliosides. LacCer synthase was reported to be synthesized by the beta4-galactosyltransferase-6 (beta4GalT-6) gene in the rat brain. However, the existence of another LacCer synthase gene was shown in cultured cells lacking beta4GalT-6. Here, we report that LacCer synthase is mainly synthesized by the beta4GalT-5 gene during early mouse embryogenesis, and its disruption is embryonic lethal. beta4GalT-5-deficient embryos showed developmental retardation from E7.5 and died by E10.5 as reported previously. LacCer synthase activity was significantly reduced in beta4GalT-5-deficient embryos and extra-embryonic endoderm (XEN) cells derived from blastocysts, and it was recovered when beta4GalT-5 cDNA was introduced into beta4GalT-5-deficient XEN cells. The amounts of LacCer and GM3 ganglioside were drastically reduced, while GlcCer accumulated in the beta4GalT-5-deficient XEN cells. Hematoma and ectopically accumulated trophoblast giant cells were observed in the anti-mesometrial pole of the extra-embryonic tissues, although all three embryonic layers formed. beta4GalT-5-deficient embryos developed until E12.5 as chimeras with wild-type tetraploid cells, which formed the extra-embryonic membranes, indicating that extra-embryonic defects caused the early embryonic lethality. Our results suggest that beta4GalT-5 is essential for extra-embryonic development during early mouse embryogenesis.

Wtap is required for differentiation of endoderm and mesoderm in the mouse embryo.

Wilms' tumor 1-associating protein (WTAP) was previously identified as a protein associated with Wilms' tumor-1 (WT-1) protein that is essential for the development of the genitourinary system. Although WTAP has been suggested to function in alternative splicing, stabilization of mRNA, and cell growth, its in vivo function is still unclear. We generated Wtap mutant mice using a novel gene-trap approach and showed that Wtap mutant embryos exhibited defective egg-cylinder formation at the gastrulation stage and died by embryonic day 10.5. Although they could form extraembryonic tissues and anterior visceral endoderm, Wtap mutant embryos and embryonic stem cells failed to differentiate into endoderm and mesoderm. The chimera analysis showed that Wtap in extraembryonic tissues was required for the formation of mesoderm and endoderm in embryonic tissues. Taken together, our findings indicate that Wtap is indispensable for differentiation of mesoderm and endoderm in the mouse embryo.

A novel gene trapping for identifying genes expressed under the control of specific transcription factors.

Gene trapping is a powerful method for identifying novel genes and for analyzing their functions. It is, however, difficult to select trapped genes on the basis of their function. To identify genes regulated by transcription factors that are important in the mesodermal formation, we selected trapped ES clones by infection of adenoviral vectors expressing Pax1, Brachyury, and Foxa2. Among 366 trapped genes, seven seemed to be controlled by these transcription factors in the first screening. The trapped genes were identified by 5' RACE, and a Northern blotting revealed that expressions of three trapped genes were regulated by these transcription factors. Expression patterns of Cx43 and HP1gamma implicated their functional relationships to Foxa2 in the formation of the notochord and the neural tube. Furthermore, Wtap mutant mice derived from the trapped clone showed defects in the mesendoderm formation. Our results indicate that trapped ES clones could be selected effectively using transcription factors.