Research and progression of TRPC6 and proteinuria induced by obesity / 中华内分泌外科杂志

The obesity related glomerulopathology attracts more and more attention due to the proteinuria induced by podocyte injury. TRPC6,which on the slit diaphragm of podocyte, and other factors contain together of the signal translation, cell polarization and cell skeleton structure of podocyte. Therefore the ion channels become the research front filed of kidney disease from the view on electrophysiology. Under this background, we introduce the biological function of TRPC6 and review its role on proteinuria induced by obesity. At last we sum up and outlook the application of TRPC6 as a treated target in proteinuria related obesity in the future.

MicroRNA-26a-5p protects against podocyte injury via inhibiting TRPC6 expression in diabetic kidney disease / 中华肾脏病杂志

Objective:To investigate the protective effect and potential mechanisms of microRNA-26a-5p (miR-26a-5p) on podocyte injury in diabetic kidney disease (DKD).Methods:(1) In vivo experiment: Four-week-old db/db mice were divided into db/db group, db/db+agomir-NC group and db/db+miR-26a-5p agomir group according to random number table method, with 10 mice in each group, and 10 db/m mice of the same week-old were set as normal control group. At the age of 10 weeks, pathological changes were observed through light and electron microscopy. Kidney weight/body weight (KW/BW), urinary albumin to creatinine ratio (ACR), fasting blood glucose (FBG) and other biochemical indicators were also detected. The position and expression of miR-26a-5p in kidney tissue were determined through fluorescence in situ hybridization and quantitative real-time PCR, while the expressions of transient receptor potential cation channel-6 (TRPC6) and Nephrin in kidney tissue were determined by Western blotting and immunohistochemistry. (2) In vitro experiment: The immortalized mouse podocytes (MPC5) were divided into 5 groups: normal glucose group, high mannitol group, high glucose group, high glucose+miR-26a-5p mimic group, and high glucose+mimic-NC group. The expressions of miR-26a-5p, TRPC6 and Nephrin were detected. Luciferase reporter assay was conducted to research the relationship of miR-26a-5p and TRPC6. Results:(1) In vivo experiment: Compared with db/m group, db/db mice exhibited lower KW/BW and disrupted conditions of ACR, FBG, total cholesterol, triglycerides and low density lipoprotein cholesterol (all P<0.01). Increased glomeruli volume, more extracellular matrix deposition, thicker basement membrane and more foot process fusion were observed by light and electron microscope. Increased expression of TRPC6 protein as well as decreased expression of Nephrin protein and miR-26a-5p were detected in kidney tissues of db/db mice ( P<0.05). Compared with db/db+agomir-NC group, db/db mice transfected by miR-26a-5p agomir exhibited less albuminuria, with less protein expression of TRPC6 and more Nephrin in kidney tissue (all P<0.05). (2) In vitro experiment: Compared with normal glucose group, high glucose-treated podocytes exhibited increased expression of TRPC6 ( P<0.05), as well as decreased expression of Nephrin ( P<0.05) and miR-26a-5p ( P<0.01). Compared with high glucose+mimic-NC group, lower expression of TRPC6 and higher expression of Nephrin were detected in podocytes transfected by miR-26a-5p mimic (both P<0.05). Luciferase reporter assay confirmed that miR-26a-5p could regulate the expression of TRPC6 precisely. Conclusions:The expression of miR-26a-5p in podocytes is down-regulated in the context of high glucose and miR-26a-5p protects podocytes from injury via inhibiting the expression of TRPC6 in DKD.

Research progress on the role of transient receptor potential canonical 6 in ischemia-reperfusion injury / 器官移植

Ischemia-reperfusion injury (IRI) refers to the reperfusion injury caused by the recovery of blood supply of ischemic tissues or organs, which commonly occurs in organ transplantation and other surgical procedures. IRI may cause a series of severe clinical issues, such as delayed graft function, acute kidney injury, myocardial infarction, ischemic stroke and circulatory arrest, etc. These events yield high incidence and fatality. At present, no effective solution has been available. Transient receptor potential canonical 6 (TRPC6), a member of Ca2+ channel family, is highly expressed in multiple types of cells. It may adjust many physiological functions by regulating intracellular Ca2+ concentration, which has become an important target for developing therapeutic drugs for multiple diseases. In this article, research progresses on the introduction and function of TRPC6, the association between TRPC6 and IRI and the therapeutic prospect of TRPC6 targeted drugs in IRI were reviewed, aiming to provide novel insights into the prevention and treatment of IRI during organ transplantation

Protective Mechanism of Danggui Shaoyaosan on Podocytes of Nephrotic Syndrome Rats Based on AngⅡ-TRPC6 Pathway / 中国实验方剂学杂志

Objective:To explore the protective effect and the mechanism of Danggui Shaoyaosan（DSS） on angiotensin Ⅱ （AngⅡ）/transient receptor potential cation channel 6 （TRPC6） pathway in nephrotic syndrome （NS） rats. Method:In animal experiments， doxorubicin （4 mg·kg^-1 for the 1^st week and 2 mg·kg^-1 for the 2^nd week） was injected twice to the tail vein of rats to induce NS model in 160 rats， which were then randomly divided into model group （normal saline）， losartan group （30 mg·kg^-1·d^-1）， and low-（4.3 g·kg^-1·d^-1）， medium-（8.6 g·kg^-1·d^-1）， and high-dose （17.2 g·kg^-1·d^-1） DSS groups. Besides， a normal group was also set. After intervention for four weeks， ultrastructure changes of the kidney were identified by transmission electron microscopy （TEM）. The 24-hour urine protein was detected by kits. Radioimmunoassay was used to detect the content of AngⅡ and Calcineurin （CaN） in plasma. Western blot was used to detect the protein expression of TRPC6， angiotensin Ⅱ type 1 receptor （AT1R）， podocyte slit diaphragm-specific protein （Nephrin）， and cysteine-aspartic acid protease-3 （Caspase-3） in the renal cortex. Immunohistochemistry was used to detect the expression of TRPC6 and AT1R in the slit diaphragm. In cell experiments， AngⅡ stimulated MPC5 podocytes. The cells were randomly divided into a normal group， an AngⅡ group， an AngⅡ+SAR7334 （TRPC6-specific inhibitor） group， an AngⅡ+5%DSS group， an AngⅡ+10%DSS group， and an AngⅡ+15%DSS group. Western blot was used to detect the protein expression of TRPC6， AT1R， Nephrin， and Caspase-3 in podocytes. Result:Compared with the normal group， the model group showed increased 24-hour urine protein content （<italic>P</italic><0.01） and AngⅡ and CaN in plasma （<italic>P</italic><0.01）， incomplete glomerular structure， the extensive fusion of podocyte process with elevated fusion rate （<italic>P</italic><0.01）， increased expression distribution of AT1R and TRPC6 in the renal cortex， and up-regulated protein expression of AT1R， TRPC6， and Caspase-3 in renal tissues （<italic>P</italic><0.01）， and reduced Nephrin protein expression （<italic>P</italic><0.01）. Compared with model group， the losartan group and the high-dose DSS group exhibited decreased 24-hour urine protein content （<italic>P</italic><0.01） and the content of AngⅡ and CaN in plasma （<italic>P</italic><0.01）， improved glomerular structure， reduced fusion rate of podocyte process （<italic>P</italic><0.01）， diminished expression distribution of TRPC6 and AT1R in the renal cortex， declining protein expression of AT1R， TRPC6 and Caspase-3 in renal tissues （<italic>P</italic><0.01）， and elevated Nephrin protein expression （<italic>P</italic><0.01）. Additionally， compared with the normal podocytes， AngⅡ-stimulated podocytes showed increased protein expression of AT1R， TRPC6 and Caspase-3 （<italic>P</italic><0.01）， and decreased expression of Nephrin （<italic>P</italic><0.01）. Compared with the AngⅡ group， the AngⅡ+SAR7334 group displayed reduced protein expression of AT1R， TRPC6， and Caspase-3 （<italic>P</italic><0.01） and increased protein expression of Nephrin （<italic>P</italic><0.01）. Conclusion:DSS can improve the pathological characteristics of NS presumedly by inhibiting the interaction between AngⅡ and TRPC6 in podocytes and improving the structural integrity of podocytes to repair the damage of glomerular molecular barrier and slow down the progression of NS-induced proteinuria.

Effect of TRPC6 knock-out on the mouse neuroinflammation induced by MPTP / 中国比较医学杂志

Objective To measure the level of microglia TRPC6 in mouse MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neuroinflammation model and investigate its role in cytokine production and dopaminergic neuron damages. Methods Microglia were sorted by magnetic beads labeled with CD11b antibody and the level of TRPC6 in MPTP-induced neuroinflammation models was measured by western blotting. The proliferation of microglia and damages of dopaminergic neurons induced by MPTP were analyzed by immunofluorescence in CD11b-TRPC6 -/ - mice. Meanwhile, the expression of cryαB and cytokines in microglia was measured by western blotting and real-time quantitative PCR, respectively. Results The level of microglia TRPC6 in MPTP-induced neuroinflammation model was up-regulated. The expression of cryαB was increased and the cytokine level was down-regulated in the microglia in MPTP-injected CD11b-TRPC6 -/ - mice. Moreover, the dopaminergic neuron survival was improved in the MPTP-induced neuroinflammation model after TRPC6 knock-out in the microglia. Conclusions The expression of TRPC6 in microglia is up-regulated after MPTP injection, while in CD11b-TRPC6 -/ - mice the MPTP-induced cytokine expression is reduced, contributing to the improvement of dopaminergic neuron survival.

Effects of astragaloside on TRPC6 expression on mouse podocyte induced by TGF-β1 / 中国免疫学杂志

Objective:To explore the possible mechanism of astragaloside involved in the mouse podocytes injury induced by TGF-β1 in vitro.Methods:Mouse podocytes were cultured in vitro and then all cell were divided into 5 groups:normal control group , TGF-β1 treatment group ,TGF-β1 treatment +astragaloside low dose group ,TGF-β1 treatment +astragaloside middle dose group and TGF-β1 treatment +astragaloside high dose group.The proliferation rate of each group was investigated by MTT assay ,the expression of TRPC6 protein and mRNA were measured by Western blot and RT-PCR respectively after 48 hours.Results:TGF-β1 can significantly inhibit the proliferation of podocytes ( P<0.05) ,fusions of foot processes or even effaced of podocytes were observed .TGF-β1 could also increase the expression of TRPC6.Astragaloside could reduce the inhibition of TGF-β1 to the proliferain of podocytes significantly ,make the cell shape tend to be normal,and reduce the expression of TRPC6 mRNA and protein with dose-effect relation.Conclusion:TRPC6 play an impor-tant role in the TGF-β1 induecd podocytes injury .Astragaloside can alleviate podocytes injury by reduce the expression of TRPC 6.

Characterization of a Trpc6 Transgenic Mouse Associated with Early Onset FSGS.

Rationale: Mutations in Transient Receptor Potential Channel 6 (TRPC6) gene are associated with autosomal dominant focal and segmental glomerulosclerosis (FSGS). The majority of the identified mutations affect the ion channel function. Since calcium channels are promising candidate drug targets, there is an an urgent need for a mouse model to assess new therapeutic drugs and to help delineate the pathogenic process leading to FSGS. We have previously reported the generation of three independent transgenic mouse lines carrying different Trpc6 mutations that display a glomerular disease comparable to the phenotype presented by individuals with FSGS. However, the utility of these models for drug testing is dampened by the late-onset of the presentation and the mild phenotypic manifestations. Methodology: In order to obtain a time-effective mouse model for Trpc6-associated FSGS we generated a new transgenic mutant Trpc6 mouse model emulating the amino acid change carried by the first pediatric patient of FSGS associated with a TRPC6 mutation: M132T. Results: Mice carrying the orthologous Trpc6 M131T transgene showed early onset proteinuria and early signs of FSGS. When exploring molecular consequences of the overexpression of this mutated form of Trpc6 in podocytes, differences in expression levels of Axin2 and β-catenin were found in glomeruli from transgenic Trpc6 M131T mice. These data supports the proposed molecular mechanisms related to the activation of calcineurin-NFAT/Wnt signaling, as outcome of the increased calcium influx caused by the mutated form of Trpc6. Conclusion: Given that the Trpc6 M131T mouse develops an early onset of FSGS-like phenotypes it represents a promising model for studying the pathogenesis of FSGS caused by TRPC6, facilitating the assessment of new drugs as treatments and allowing further studies to understand underlying molecular pathways involved in the development of the TRPC6 mediated disease.

Effects of TRPC6 on invasibility of low-differentiated prostate cancer cells

OBJECTIVE@#To study the expression of TRPC6 among prostate cancer cells, establish high expression cell lines of TRPC6, and to provide potential cell mode for prostate cancer oncogenesis and development.@*METHODS@#Occurrence and development of prostate cancer cells, PC3, PC-3 m DU145, 22 rv1, LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method. Calcium phosphate transfection method was used to package retrovirus pLEGFP-N1-TRPC6 and pLEGFP-N1-vector and infect the prostate cancer cells, a stable high expression of TRPC6 prostate cancer cells. Sable cell lines of TRPC6, matrix metalloproteinase (MMP) 2, MMP9 expression was detected by QPCR and Western blot. Change of cell invasion ability was detected by Transwell.@*RESULTS@#The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells. Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest, and high transfer cell line PC-3M express was the highest. Real-time fluorescent quantitative PCR and western blot results showed that after filter, the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously. Transwell experimental results showed that the overexpression of TRPC6 could promote the invasion ability of PC3 prostate cancer cells.@*CONCLUSIONS@#TRPC6 expressed in prostate cancer cells is in disorder, and its action may be associated with the invasion and metastasis of prostate cancer cells; successful establishment of stable high expression of TRPC6 prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer, and lay down the Foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer mechanism.

Effect of Down-regulation of TRPC6 on the Puromycin Aminonucleoside-induced Apoptosis of Mouse Podocytes / 华中科技大学学报（医学）（英德文版）

poptosis induced by PAN.

Methacholine and PDGF activate store-operated calcium entry in neuronal precursor cells via distinct calcium entry channels

Neurons are a diverse cell type exhibiting hugely different morphologies and neurotransmitter specifications. Their distinctive phenotypes are established during differentiation from pluripotent precursor cells. The signalling pathways that specify the lineage down which neuronal precursor cells differentiate remain to be fully elucidated. Among the many signáis that impinge on the differentiation of neuronal cells, cytosolic calcium (Ca2+) has an important role. However, little is known about the nature of the Ca2+ signáis involved in fate choice in neuronal precursor cells, or their sources. In this study, we show that activation of either muscarinic or platelet-derived growth factor (PDGF) receptors induces a biphasic increase in cytosolic Ca2+ that consists of reléase from intracellular stores followed by sustained entry across the plasma membrane. For both agonists, the prolonged Ca2+ entry occurred via a store-operated pathway that was pharmacologically indistinguishable from Ca2+ entry initiated by thapsigargin. However, muscarinic receptor-activated Ca2+ entry was inhibited by siRNA-mediated knockdown of TRPC6, whereas Ca2+ entry evoked by PDGF was not. These data provide evidence for agonist-specific activation of molecularly distinct store-operated Ca2+ entry pathways, and raise the possibility of privileged communication between these Ca2+ entry pathways and downstream processes.

Mutations of podocyte molecules in patients with late onset familial focal segmental glomerular sclerosis / 中华肾脏病杂志

Objective To investigate the mutations of pedocyte molecules in patients with late onset familial focal segmental glomerular sclerosis (FSGS). Methods Thirty-one pedigrees of late onset familial FSGS in Department of Nephrology, Shanghai Ruijin Hospital from Sep 1997 to Oct 2007 were enrolled in this study. The diagnosis standard of familial FSGS was as follows:(1) the age of presentation was more than 12 years old. (2) in one pedigree, two or more individuals were proven as FSGS by renal biopsy, or at least one was proven to be FSGS by renal biopsy, the others presented renal insufficiency or pmteinuria without precise causes. One hundred unrelated healthy people were screened as control group. Genomic DNA extracted from peripheral blood cells were amplified by PCR and then sequenced for mutations of NPHS2, ACTN4 and TRPC6. Results A novel missense heterozygotic mutation L316P of ACTN4 was identified inone pedigree. The mean onset age of the affected members of this pedigree was (38.7±7.4) years old and their kidney injury progress was slow. Proteinuria of the proband's brother was not improved by immunosuppressor. All 3 affected members of this family had such heterozygotic mutation. A novel missense heterozygotic mutation Q889K of TRPC6 was found in another pedigree. The mean onset age of the affected members in this pedigree was (38.0±4.2) years old. Three members presenting renal disease in this family all had such heterozygotic mutation but with different clinical manifestations. A quiescent mutation G467G of TRPC6 was also identified. Above variants were not found in healthy controls. No NPHS2 mutation was found to cause familial FSGS in these pedigrees. Conclusions A novel mutation L316P of ACTN4 and a new mutation Q889K of TRPC6 are identified in Chinese patients of late onset familial FSGS. No NPHS2 mutation is found to induce FSGS in these pedigrees.

Effects of overexpression of TRPC6 on angiotensin Ⅱ-induced apoptosis of mouse podocytes / 中华肾脏病杂志

Objective To study the effect of overexpression of TRPC6 on Ang Ⅱ-induced apoptosis of mouse podocytes in vitro and to explore the possible mechanisms. Methods Mouse TRPC6 cDNA eukaryotie expression vector pEGFP-NI-mTRPC6 was transfected to conditionally immortalized routine podocyte cell line by liposome. The fluorescent microscopy was used to examine the expression of EGFP after 24 hours. The change of TRPC6 protein expression was observed by Western-blot. Podocytes were treated by different concentrations of Ang Ⅱ. The podocyte intracellular calcium concentration was measured with laser-scanning con_focal microscope. The expression of Bax and Bcl-2 mRNA was assessed by RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot. The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst staining. Results About 35% of the cells expressed EGFP. An up-regulation of protein expression of TRPC6 was detected in podocytes when transfected with pEGFP-N1-mTRPC6 (P<0.01). The overexpression of TRPC6 promoted the Ang Ⅱ-induced influx of extracellular calcium and elevated the expression of Bax but decreased the expression of Bcl-2 (P<0.01, P<0.05). The apoptotic ratio of podocyte was (2.50±0.72)% when treated by low-dose Ang Ⅱ (10-10 mol/L), and it was increased to (4.33±0.45)% when transfected with pEGFP-N1-mTRPC6 (P <0.05 ). Transfection with pEGFP-NI-mTRPC6 increased apoptosis rate from (15.46± 1.40)% to (18.33±0.87)%(P<0.01) by high-dose Ang Ⅱ (10-6 mol/L). Conclusion TRPC6 plays an important role in the Ang Ⅱ-induced apoptosis of podocytes by promoting the influx of extraeellular calcium, which leads to the apoptosis cascade initiation.