Abnormal podocyte TRPML1 channel activity and exosome release in mice with podocyte-specific Asah1 gene deletion

Podocytopathy and associated nephrotic syndrome (NS) have been reported in a knockout mouse strain (Asah1^fl/fl/Podo^Cre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy of these mice remains unknown. The present study tested whether exosome release from podocytes is enhanced due to Asah1 gene knockout, which may serve as a pathogenic mechanism switching on podocytopathy and associated NS in Asah1^fl/fl/Podo^Cre mice. We first demonstrated the remarkable elevation of urinary exosome excretion in Asah1^fl/fl/Podo^Cre mice compared with WT/WT mice, which was accompanied by significant Annexin-II (an exosome marker) accumulation in glomeruli of Asah1^fl/fl/Podo^Cre mice, as detected by immunohistochemistry. In cell studies, we also confirmed that Asah1 gene knockout enhanced exosome release in the primary cultures of podocyte isolated from Asah1^fl/fl/Podo^Cre mice compared to WT/WT mice. In the podocytes from Asah1^fl/fl/Podo^Cre mice, the interactions of lysosome and multivesicular body (MVB) were demonstrated to be decreased in comparison with those from their control littermates, suggesting reduced MVB degradation that may lead to increase in exosome release. Given the critical role of transient receptor potential mucolipin 1 (TRPML1) channel in Ca²⁺-dependent lysosome trafficking and consequent lysosome-MVB interaction, we tested whether lysosomal Ca²⁺ release through TRPML1 channels is inhibited in the podocytes of Asah1^fl/fl/Podo^Cre mice. By GCaMP3 Ca²⁺ imaging, it was found that lysosomal Ca²⁺ release through TRPML1 channels was substantially suppressed in podocytes with Asah1 gene deletion. As an Ac product, sphingosine was found to rescue TRPML1 channel activity and thereby recover lysosome-MVB interaction and reduce exosome release of podocytes from Asah1^fl/fl/Podo^Cre mice. Combination of N, N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, and sphingosine significantly inhibited urinary exosome excretion of Asah1^fl/fl/Podo^Cre mice. Moreover, rescue of Aash1 gene expression in podocytes of Asah1^fl/fl/Podo^Cre mice showed normal ceramide metabolism and exosome secretion. Based on these results, we conclude that the normal expression of Ac importantly contributes to the control of TRPML1 channel activity, lysosome-MVB interaction, and consequent exosome release from podocytes. Asah1 gene defect inhibits TRPML1 channel activity and thereby enhances exosome release, which may contribute to the development of podocytopathy and associated NS.     

Deletion of pancreatic β‐cell adenosine kinase improves glucose homeostasis in young mice and ameliorates streptozotocin‐induced hyperglycaemia

Severe reduction in the β‐cell number (collectively known as the β‐cell mass) contributes to the development of both type 1 and type 2 diabetes. Recent pharmacological studies have suggested that increased pancreatic β‐cell proliferation could be due to specific inhibition of adenosine kinase (ADK). However, genetic evidence for the function of pancreatic β‐cell ADK under physiological conditions or in a pathological context is still lacking. In this study, we crossed mice carrying LoxP‐flanked Adk gene with Ins2‐Cre mice to acquire pancreatic β ‐cell ADK deficiency (Ins2‐Cre^±Adk^fl/fl) mice. Our results revealed that Ins2‐Cre^+/‐Adk^fl/fl mice showed improved glucose metabolism and β‐cell mass in younger mice, but showed normal activity in adult mice. Moreover, Ins2‐Cre^±Adk^fl/fl mice were more resistant to streptozotocin (STZ) induced hyperglycaemia and pancreatic β‐cell damage in adult mice. In conclusion, we found that ADK negatively regulates β‐cell replication in young mice as well as under pathological conditions, such as STZ induced pancreatic β‐cell damage. Our study provided genetic evidence that specific inhibition of pancreatic β‐cell ADK has potential for anti‐diabetic therapy.     

Functional defect of peripheral neutrophils in mice with induced deletion of CXCR2

Type 2 CXC chemokine receptor CXCR2 plays roles in development, tumorigenesis, and inflammation. CXCR2 also promotes demyelination and decreases remyelination by actions toward hematopoietic cells and nonhematopoietic cells. Germline CXCR2 deficient (Cxcr2^‐/‐) mice reported in 1994 revealed the complexity of CXCR2 function and its differential expression in varied cell‐types. Here, we describe Cxcr2^fl/fl mice for which the targeting construct was generated by recombineering based on homologous recombination in E. coli. Without recombination Cxcr2^fl/fl mice have CXCR2 expression on neutrophils in peripheral blood, bone marrow and spleen. Cxcr2^fl/fl mice were crossed to Mx‐Cre mice in which Cre recombinase is induced by Type I interferons, elicited by injection with polyinosinic‐polycytidylic acid (poly(I:C)). CXCR2‐deficient neutrophils were observed in poly(I:C) treated Cxcr2^fl/fl::Mx‐Cre⁺ (Cxcr2‐CKO) mice, but not in poly(I:C) treated Cxcr2^f//+::Mx‐Cre⁺ mice. CXCR2 deletion was mainly observed peripherally but not in the CNS. Cxcr2‐CKO mice showed impaired neutrophil migration in sterile peritonitis. Cxcr2‐CKO mice reported here will provide a genetic reagent to dissect roles of CXCR2 in the neutrophil granulocyte lineage. Furthermore Cxcr2^fl/fl mice will provide useful genetic models to evaluate CXCR2 function in varied cell populations. genesis 51:587–595. © 2013 Wiley Periodicals, Inc.     

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1^Cre) mouse. Embryonic Wt1^Cre;Alk3^fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1^Cre;Alk3^fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1^Cre;Alk3^fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.     

PDK1对生发中心的生成、发育及功能的影响

目的:研究PDK1对生发中心(GC)的生成、发育及其功能的影响。方法:通过配种小鼠得到在GC B细胞中特异性敲除PDK1的小鼠,然后采用共聚焦显微镜观察小鼠脾脏GC的大小,多色流式细胞分析方法观测PDK1的敲除是否会影响小鼠B细胞的发育,ELISA技术检测PDK1敲除的小鼠经免疫后其体内产生抗体的能力是否受影响,结合平面脂双层抗原呈递系统及全内反射荧光显微镜成像系统(TIRFM)观测PDK1的敲除是否会影响小鼠脾脏IgG细胞P85的磷酸化水平。结果:PDK1的缺失并不会影响小鼠B细胞的发育,细胞群中成熟与不成熟B细胞所占比例均无显著性变化,但在GC B细胞中条件性敲除PDK1会影响小鼠脾脏GC的生成以及T细胞依赖性抗原免疫反应,而且同等条件活化后,GC B细胞中条件性敲除PDK1的小鼠脾脏IgG细胞其胞内分子P85的磷酸化水平显著降低。结论:PDK1对GC的生成、发育及功能都具有重要作用。     

Endothelial Arginine Resynthesis Contributes to the Maintenance of Vasomotor Function in Male Diabetic Mice

Aim

Argininosuccinate synthetase (ASS) is essential for recycling L-citrulline, the by-product of NO synthase (NOS), to the NOS substrate L-arginine. Here, we assessed whether disturbed arginine resynthesis modulates endothelium-dependent vasodilatation in normal and diabetic male mice.

Methods and Results

Endothelium-selective Ass-deficient mice (Ass^fl/fl/Tie2Cre^tg/− = Ass-KO^Tie2) were generated by crossing Ass^fl/fl mice ( = control) with Tie2Cre mice. Gene ablation in endothelial cells was confirmed by immunohistochemistry. Blood pressure (MAP) was recorded in 34-week-old male mice. Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Ass-KO^Tie2 and control animals. At the age of 10 weeks, diabetes was induced in control and Ass-KO^Tie2 mice by streptozotocin injections. Vasomotor responses of diabetic animals were studied 10 weeks later. MAP was similar in control and Ass-KO^Tie2 mice. Depletion of circulating L-arginine by arginase 1 infusion or inhibition of NOS activity with L-NAME resulted in an increased MAP (10 and 30 mmHg, respectively) in control and Ass-KO^Tie2 mice. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in healthy control and Ass-KO^Tie2 mice. However, in diabetic Ass-KO^Tie2 mice, relaxation responses to acetylcholine and endothelium-derived NO (EDNO) were significantly reduced when compared to diabetic control mice.

Conclusions

Absence of endothelial citrulline recycling to arginine did not affect blood pressure and systemic arterial vasomotor responses in healthy mice. EDNO-mediated vasodilatation was significantly more impaired in diabetic Ass-KO^Tie2 than in control mice demonstrating that endothelial arginine recycling becomes a limiting endothelial function in diabetes.     

Deletion of Fgfr1 in Osteoblasts Enhances Mobilization of EPCs into Peripheral Blood in a Mouse Endotoxemia Model

Endothelial progenitor cells (EPCs) contribute to neovascularization and vascular repair, and may exert a beneficial effect on the clinical outcome of sepsis. Osteoblasts act as a component of “niche” in bone marrow, which provides a nest for stem/progenitor cells and are involved in the formation and maintenance of stem/progenitor cells. Fibroblast growth factor receptor 1 (FGFR1) can regulate osteoblast activity and influence bone mass. So we explored the role of FGFR1 in EPC mobilization. Male mice with osteoblast-specific knockout of Fgfr1 (Fgfr1^fl/fl;OC-Cre) and its wild-type littermates (Fgfr1^fl/fl) were used in this study. Mice intraperitoneally injected with lipopolysaccharide (LPS) were used to measure the number of circulating EPCs in peripheral blood and serum stromal cell-derived factor 1α (SDF-1α). The circulating EPC number and the serum level of SDF-1α were significantly higher in Fgfr1^fl/fl;OC-Cre mice than those in Fgfr1^fl/fl mice after LPS injection. In cell culture system, SDF-1α level was also significantly higher in Fgfr1^fl/fl;OC-Cre osteoblasts compared with that in Fgfr1^fl/fl osteoblasts after LPS treatment. TRAP staining showed that there was no significant difference between the osteoclast activity of septic Fgfr1^fl/fland Fgfr1^fl/fl;OC-Cre mice. This study suggests that targeted deletion of Fgfr1 in osteoblasts enhances mobilization of EPCs into peripheral blood through up-regulating SDF-1α secretion from osteoblasts.     

RANKL from bone marrow adipose lineage cells promotes osteoclast formation and bone loss

Receptor activator of NF‐κB ligand (RANKL) is essential for osteoclast formation and bone remodeling. Nevertheless, the cellular source of RANKL for osteoclastogenesis has not been fully uncovered. Different from peripheral adipose tissue, bone marrow (BM) adipose lineage cells originate from bone marrow mesenchymal stromal cells (BMSCs). Here, we demonstrate that adiponectin promoter‐driven Cre expression (Adipoq^Cre ) can target bone marrow adipose lineage cells. We cross the Adipoq^Cre mice with rankl^fl/fl mice to conditionally delete RANKL from BM adipose lineage cells. Conditional deletion of RANKL increases cancellous bone mass of long bones in mice by reducing the formation of trabecular osteoclasts and inhibiting bone resorption but does not affect cortical bone thickness or resorption of calcified cartilage. Adipoq^Cre; rankl^fl/fl mice exhibit resistance to estrogen deficiency and rosiglitazone (ROS)‐induced trabecular bone loss but show bone loss induced by unloading. BM adipose lineage cells therefore represent an essential source of RANKL for the formation of trabecula osteoclasts and resorption of cancellous bone during remodeling under physiological and pathological conditions. Targeting bone marrow adiposity is a promising way of preventing pathological bone loss.     

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1^fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1^fl/flTie2cre). Macrophages from Gch1^fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1^fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1^fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1^fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1^fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1^fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.     

Hepatic Deletion of X-Box Binding Protein 1 in FXR Null Mice Leads to Enhanced Liver Injury

FXR regulates bile acid metabolism, and FXR null (Fxr^?/?) mice have elevated bile acid levels and progressive liver injury. The inositol-requiring enzyme 1α/X-box binding protein 1 (XBP1) pathway is a protective unfolded protein response pathway activated in response to endoplasmic reticulum stress. Here, we sought to determine the role of the inositol-requiring enzyme 1α/XBP1 pathway in hepatic bile acid toxicity using the Fxr^?/? mouse model. Western blotting and quantitative PCR analysis demonstrated that hepatic XBP1 and other unfolded protein response pathways were activated in 24-week-old Fxr^?/? compared with 10-week-old Fxr^?/? mice but not in WT mice. To further determine the role of the liver XBP1 activation in older Fxr^?/? mice, we generated mice with whole-body FXR and liver-specific XBP1 double KO (DKO, Fxr^?/?Xbp1^LKO) and Fxr^?/?Xbp1^fl/fl single KO (SKO) mice and characterized the role of hepatic XBP1 in cholestatic liver injury. Histologic staining demonstrated increased liver injury and fibrosis in DKO compared with SKO mice. RNA sequencing revealed increased gene expression in apoptosis, inflammation, and cell proliferation pathways in DKO mice. The proapoptotic C/EBP-homologous protein pathway and cell cycle marker cyclin D1 were also activated in DKO mice. Furthermore, we found that total hepatic bile acid levels were similar between the two genotypes. At age 60 weeks, all DKO mice and no SKO mice spontaneously developed liver tumors. In conclusion, the hepatic XBP1 pathway is activated in older Fxr^?/? mice and has a protective role. The potential interaction between XBP1 and FXR signaling may be important in modulating the hepatocellular cholestatic stress responses.     

Novel Endothelial Cell-Specific AQP1 Knockout Mice Confirm the Crucial Role of Endothelial AQP1 in Ultrafiltration during Peritoneal Dialysis

The water channel aquaporin-1 (AQP1) mediates about 50% ultrafiltration during a 2-hour hypertonic dwell in global AQP1 knockout (AQP1^-/-) mice. Although AQP1 is widely expressed in various cell types including mesothelial cells, the ultrafiltration has been assumed to be mediated via endothelial AQP1 of the peritoneum. The partial embryonic lethality and reduced body weight in AQP1^-/- mice may reflect potential confounding phenotypic effects evoked by ubiquitous AQP1 deletion, which may interfere with functional analysis of endothelial AQP1. Using a Cre/loxP approach, we generated and characterised endothelial cell- and time-specific AQP1 knockout (AQP1^fl/fl; Cdh5-Cre⁺) mice. Compared to controls, AQP1^fl/fl; Cdh5-Cre⁺ mice showed no difference in an initial clinical and biological analysis at baseline, including body weight and survival. During a 1-hour 3.86% mini-peritoneal equilibration test (mini-PET), AQP1^fl/fl; Cdh5-Cre⁺ mice exhibited strongly decreased indices for AQP1-related transcellular water transport (43.0% in net ultrafiltration, 93.0% in sodium sieving and 57.9% in free water transport) compared to controls. The transport rates for small solutes of urea and glucose were not significantly altered. Our data provide the first direct experimental evidence for the functional relevance of endothelial AQP1 to the fluid transport in peritoneal dialysis and thereby further validate essential predictions of the three-pore model of peritoneal transport.     

Impaired Clearance of Influenza A Virus in Obese,Leptin Receptor Deficient Mice Is Independent of Leptin Signaling in the Lung Epithelium and Macrophages

Rationale

During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.

Methods

We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR^fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr^fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.

Results

The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre^+/+/LepR^fl/fl and LysM-Cre^+/+/LepR^{fl /fl} mice exhibited improved survival.

Conclusions

Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.     

Myeloid‐specific GPCR kinase‐2 negatively regulates NF‐κB1p105‐ERK pathway and limits endotoxemic shock in mice

G‐protein‐coupled receptor kinase 2 (GRK2) is a member of a kinase family originally discovered for its role in the phosphorylation and desensitization of G‐protein‐coupled receptors. It is expressed in high levels in myeloid cells and its levels are altered in many inflammatory disorders including sepsis. To address the physiological role of myeloid cell‐specific GRK2 in inflammation, we generated mice bearing GRK2 deletion in myeloid cells (GRK2^?mye). GRK2^?mye mice exhibited exaggerated inflammatory cytokine/chemokine production, and organ injury in response to lipopolysaccharide (LPS, a TLR4 ligand) when compared to wild‐type littermates (GRK2^fl/fl). Consistent with this, peritoneal macrophages from GRK2^?mye mice showed enhanced inflammatory cytokine levels when stimulated with LPS. Our results further identify TLR4‐induced NF‐κB1p105‐ERK pathway to be selectively regulated by GRK2. LPS‐induced activation of NF‐κB1p105‐MEK‐ERK pathway is significantly enhanced in the GRK2^?mye macrophages compared to GRK2^fl/fl cells and importantly, inhibition of the p105 and ERK pathways in the GRK2^?mye macrophages, limits the enhanced production of LPS‐induced cytokines/chemokines. Taken together, our studies reveal previously undescribed negative regulatory role for GRK2 in TLR4‐induced p105‐ERK pathway as well as in the consequent inflammatory cytokine/chemokine production and endotoxemia in mice. J. Cell. Physiol. 226: 627–637, 2011. © 2010 Wiley‐Liss, Inc.     

Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7 ^fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7 ^fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7 ^fl/flK14-Cre-ER^T2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.     

Impaired autophagic flux and dedifferentiation in podocytes lacking Asah1 gene: Role of lysosomal TRPML1 channel

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1^fl/fl/Podo^cre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (Ac). However, the pathogenesis of podocytopathy in these mice remains unclear. The present study tested whether Ac deficiency impairs autophagic flux in podocytes through blockade of transient receptor potential mucolipin 1 (TRPML1) channel as a potential pathogenic mechanism of podocytopathy in Asah1^fl/fl/Podo^cre mice. We first demonstrated that impairment of autophagic flux occurred in podocytes lacking Asah1 gene, which was evidenced by autophagosome accumulation and reduced lysosome-autophagosome interaction. TRPML1 channel agonists recovered lysosome-autophagosome interaction and attenuated autophagosome accumulation in podocytes from Asah1^fl/fl/Podo^cre mice, while TRPML1 channel inhibitors impaired autophagic flux in WT/WT podocytes and worsened autophagic deficiency in podocytes lacking Asah1 gene. The effects of TRPML1 channel agonist were blocked by dynein inhibitors, indicating a critical role of dynein activity in the control of lysosome movement due to TRPML1 channel-mediated Ca²⁺ release. It was also found that there is an enhanced phenotypic transition to dedifferentiation status in podocytes lacking Asah1 gene in vitro and in vivo. Such podocyte phenotypic transition was inhibited by TRPML1 channel agonists but enhanced by TRPML1 channel inhibitors. Moreover, we found that TRPML1 gene silencing induced autophagosome accumulation and dedifferentiation in podocytes. Based on these results, we conclude that Ac activity is essential for autophagic flux and maintenance of differentiated status of podocytes. Dysfunction or deficiency of Ac may impair autophagic flux and induce podocyte dedifferentiation, which may be an important pathogenic mechanism of podocytopathy and associated nephrotic syndrome.     

Hepatic Xbp1 Gene Deletion Promotes Endoplasmic Reticulum Stress-induced Liver Injury and Apoptosis

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, then the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1^LKO) and Xbp1^fl/fl control mice were subjected to varying levels and durations of pharmacologic ER stress. Xbp1^LKO and Xbp1^fl/fl mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 h, Xbp1^fl/fl controls showed complete resolution of UPR activation and no liver injury, indicating successful adaptation to the stress. Conversely, Xbp1^LKO mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and, ultimately, fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.