Angiotensin II Type I Receptor Agonistic Autoantibody Induces Podocyte Injury via Activation of the TRPC6- Calcium/Calcineurin Pathway in Pre-Eclampsia.

Angiotensin II type I receptor agonistic autoantibody (AT1-AA) is closely related to pre-eclampsia, which is characterized by proteinuria and hypertension. AT1-AA has been shown to enhance the effect of AngII in pre-eclampsia, such as production of endothelin-1, activation of ROS, and vasoconstriction, which are considered to be associated with hypertension; however, whether or not AT1-AA participates in podocyte damage leading to the generation of proteinuria has not been reported. In this study we investigated the role of pre-eclamptic serum AT1-AA on podocytes and the mechanism underlying the generation of proteinuria. The levels of AT1-AA isolated from pre-eclamptic sera were determined by an enzyme-linked immunosorbent assay. Human podocytes were cultured in vitro and treated with various concentrations of AT1-AA. Whether or not an ERK1/2 inhibitor and TRPC6 siRNA inhibit the effect of AT1-AA on podocytes was determined. Western blot was used to detect the expression of podocyte-specific proteins (nephrin, synaptopodin, and podocin) and the phosphorylation of ERK1/2 and TRPC6. The arrangement of F-actin was observed by immunofluorescence. A Calcineurin Cellular Activity Assay Kit was used to detect calcineurin activity. Changes in the intracellular Ca2+ concentration was determined by confocal laser. AT1-AA induced a decrease in podocyte-specific protein expression and calcineurin activity and increased expression of p-ERK1/2 and TRPC6 protein and the intracellular Ca2+ concentration. Immunofluorescence revealed rearrangement of F-actin. PD98059, an inhibitor of ERK1/2, and TRPC6 siRNA attenuated the decreased expression of podocyte-specific proteins and decreased intracellular Ca2+ concentration. The expression of TRPC6 was reduced following the addition of ERK1/2 inhibitor. AT1-AA induced podocyte damage in a dose-dependent manner. The underlying mechanism might involve activation of the TRPC6 -calcium/calcineurin pathway. This study provides new details regarding podocyte injury and the mechanism underlying the generation of proteinuria in pre-eclampsia.

Yu Y ，Zhang L ，Xu G ，Wu Z ，Li Q ，Gu Y ，Niu J ... -  《-》

The Calcium-Dependent Protease Calpain-1 Links TRPC6 Activity to Podocyte Injury.

The hallmark of podocytopathies, such as FSGS, is podocyte injury resulting in proteinuria. Transient receptor potential channel C6 (TRPC6) is a calcium-conducting ion channel expressed at the slit diaphragm. TRPC6 gain-of-function mutations and glomerular TRPC6 overexpression are associated with proteinuria. However, the pathways linking TRPC6 to podocyte injury, which is characterized by loss of the slit diaphragm protein nephrin, activation of several intracellular pathways (including calcineurin-NFAT signaling), and cytoskeletal rearrangement, remain elusive. We tested whether the calcium-dependent protease calpain-1 mediates TRPC6-dependent podocyte injury in human and experimental FSGS and cultured podocytes. Compared with kidneys of healthy controls, kidneys of patients with FSGS had increased TRPC6 expression, increased calpain and calcineurin activity, and reduced expression of the calpain target Talin-1, which links the actin cytoskeleton to integrins and is critical for podocyte cytoskeletal stability. In a rat model of human FSGS, increased glomerular and urinary calpain activity associated with reduced Talin-1 abundance, enhanced calcineurin activity, and increased proteinuria. Treatment with the calpain inhibitor calpeptin prevented these effects. In cultured podocytes, pharmacologic stimulation of TRPC6-dependent calcium influx increased calpain-1 and calcineurin activity and reduced Talin-1 expression, and knockdown of TRPC6 or calpain-1 prevented these effects. We elucidated a novel mechanism that links TRPC6 activity to calpain-1 activation and through Talin-1 loss and possibly, calcineurin activation, the podocyte injury characterizing FSGS. Therefore, calpain-1 and/or TRPC6 inhibition could be future therapeutic options to treat patients with FSGS or other podocytopathies.

Verheijden KAT ，Sonneveld R ，Bakker-van Bebber M ，Wetzels JFM ，van der Vlag J ，Nijenhuis T ... -  《-》

Angiotensin II induces calcium/calcineurin signaling and podocyte injury by downregulating microRNA-30 family members.

Zhao Y ，Wu J ，Zhang M ，Zhou M ，Xu F ，Zhu X ，Zhou X ，Lang Y ，Yang F ，Yun S ，Shi S ，Liu Z ... -  《-》

Tetrandrine Suppresses Transient Receptor Potential Cation Channel Protein 6 Overexpression- Induced Podocyte Damage via Blockage of RhoA/ROCK1 Signaling.

Podocyte damage is common in many renal diseases characterized by proteinuria. Transient receptor potential cation channel protein 6 (TRPC6) plays an important role in renal function through its regulation of intracellular Ca2+ influx and RhoA/ROCK pathways. Chinese herb Stephania tetrandra, with the main active component being tetrandrine, has been used for the treatment of various kidney diseases for several years and has shown a positive effect. This study aimed at investigating the effect and mechanism of tetrandrine in podocyte damage induced by high expression of TRPC6. Immortalized, differentiated murine podocytes, MPC5 were treated with valsartan (0-800 μM) and tetrandrine (0-40 μM) for 48 h. The maximum safe concentrations of valsartan and tetrandrine were selected using a cell viability assay. MPC5 podocytes stably expressing TRPC6 were constructed using a lentivirus packaging system, followed by treatment with valsartan, tetrandrine, and Y-27632 for 48 h and U73122 (10 μM) for 10 min. The RhoA/ROCK pathway and podocyte-specific proteins (nephrin and synaptopodin) levels were quantified. Podocyte apoptosis and intracellular Ca2+ concentration were measured. Maximum safe concentrations of 100 μM valsartan and 10 μM tetrandrine showed no observable toxicity in podocytes. MPC5 podocytes stably expressing TRPC6 had higher intracellular Ca2+ influx, apoptotic percentages, and expression of RhoA/ROCK proteins, but lower expression of nephrin and synaptopodin proteins. U73122 treatment for 10 min did not inhibit TRPC6, but suppressed RhoA/ROCK protein. Y-27632 decreased ROCK1 expression, but did not influence the expression of TRPC6 protein. Both 100 μM valsartan and 10 μM tetrandrine for 48 h significantly inhibited intracellular Ca2+ influx, apoptosis, and RhoA/ROCK pathway, and increased nephrin and synaptopodin proteins in podocytes stably expressing TRPC6. Elevated TRPC6 expression can lead to podocyte injury by inducing intracellular Ca2+ influx and apoptosis of podocytes, and this effect may be mediated by activation of the RhoA/ROCK1 pathway. Tetrandrine can alleviate podocyte injury induced by TRPC6 expression through inhibition of the RhoA/ROCK pathway, suggesting a protective role in podocyte damage.

Yu J ，Zhu C ，Yin J ，Yu D ，Wan F ，Tang X ，Jiang X ... -  《Drug Design Development and Therapy》

Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway.

The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.

Nijenhuis T ，Sloan AJ ，Hoenderop JG ，Flesche J ，van Goor H ，Kistler AD ，Bakker M ，Bindels RJ ，de Boer RA ，Möller CC ，Hamming I ，Navis G ，Wetzels JF ，Berden JH ，Reiser J ，Faul C ，van der Vlag J ... -  《-》