| During the last decades, podocyte injury has been reported to be critical in the occurrence of proteinuria. It exists in many glomerular diseases such as minimal change nephropathy (MCN), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), diabetic nephropathy (DN) and lupus nephritis (LN). There are several mechanisms involved in podocyte injury including cytoskeleton damage, slit membrane abnormality, mitochondrial dysfunction, nuclear transcription factors change, intracellular calcium homeostasis unbalance, etc.Calcium, as the second messenger, plays an important role in many cellular functions, such as secretion, excitation/contraction coupling, motility, transcription, proliferation, division or apoptosis. In podocytes, Ca2+ regulates the actin dynamics. Altered Ca2+ signaling may cause the changes of podocyte slit diagram proteins and cytoskeleton proteins, which mediate remodeling of the actin cytoskeleton and foot process effacement. Strict handling of intracellular calcium is necessary to maintain these functions. Calcium homeostasis within the cell involves the action of Ca2+ entry channels in the plasma membrane (PM), Ca2+ pump and Ca2+ release channels in the intracellular endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) stores. There are three main Ca2+ entry channels in the PM:voltage-gated-Ca2+ channels (VGCC), receptor-operated channels (ROC) and store-operated Ca2+ channels (SOC).Previous studies have shown that transient receptor potential canonical 6(TRPC6) and TRPC5 channels are two important Ca2+ influx pathways in podocytes. A gain of function mutation in TRPC6 was found in families with hereditary FSGS, and inhibition of the TRPC5 channel has been reported to reduce proteinuria in an experimental model. It is known that TRPC6 and TRPC5 belong to ROC, while SOC is the main Ca2+ influx channel in non-excited cells. However, the association of SOC with the calcium signal in podocytes remains unknown.Stromal interaction molecule 1 (STIM1) is an important signal molecule of SOC. It is a singe-pass transmembrane protein primarily located in the ER membrane and acts as an ER Ca2+ sensor. Upon Ca2+ store depletion, STIM1 redistributes to ER-PM junctions, where it activates Orail and induces Ca2+ entry into the cytosol. Previous studies have shown that RNAi-mediated knockdown of STIM1 inhibited SOC current in HEK293 cells, while over-expression of STIM1 enhanced SOC current. The N-terminal domain mutation resulted in that STIM1 lost the function of Ca2+ sensor, and can not gather and activate SOC when Ca2+ store depletion, thus it may cause the imbalance of intracellular calcium homeostasis. STIM1 is involved in many diseases such as cancer related diseases, immunity related diseases, diabetes, etc. In 2009, Capucine Picard and colleagues reported on three siblings from one kindred with a clinical syndrome of immunodeficiency and autoimmunity. Two of these patients had a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca2+ Influx. Additionally, one child died" from severe nephrotic syndrome which was resistant to therapy. This indicates that STIM1 may play an important role in podocyte injury. Recent studies have demonstrated that high glucose and diabetes enhanced store-operated Ca2+entry (SOCE) and increased expression of STIM1/Orail in mesangial cells. However, the role of SOC and its signaling proteins in podocytes remains unknown.The previous studies of our group have shown that mitochondrial dysfunction was an early event in podocyte injury, and inhibiting mitochondrial function might protect against aldosteron-induced proteinuria and podocyte injury. STIM1 has been reported to be involved in the regulation of mitochondrial shape and bioenergetics, and play a role in oxidative stress in mouse embryonic fibroblasts (MEFs). In neurons, inhibition of SOC by STIM1 RNAi allevieated overload of intracellular Ca2+, restored mitochondrial membrane poteintial (MMP), reduced ER stress and mitochondrial dysfunction and eventually inhibited cell apoptosis. A growing body of evidence indicates that podocytes are similar to neurons, for instance, both of them have similar cellular phenotype and structure. However, whether STIM1 plays the same role in podocytes warrants further investigation.From the above, primary or secondary abnormalites of STIM1 may cause imbalance of intracellular Ca2+ homeostasis, oxidative stress and mitochondrial dysfunction, which may lead to podocyte injury and the occurrence of glomerular diseases. In addition, podocyte-associated proteins including slit diaphragm (SD) proteins and cytoskeleton proteins are very important in podocyte injury. Therefore, we hypothesized that abnormal expression of STIM1 mediates podocyte injury by inducing the imbalance of Ca2+ homeostasis, altering the expression of podocyte-associated molecules, inducing oxidative stress and mitochondrial dysfunction.The present study firstly examined the expression of STIMl in injured podocytes, experimental nephropathy animal models and FSGS patients. Then based on that, we upregulated or downregulated STIM1 expression in podocytes to explore the role of STIM1 in podocyte injury. This study provided experimental basis for further understanding of the relationship between calcium signal and podocyte injury, and it provided new insight to elucidate the pathogenesis and the therapy of kidney diseases.Part 1:STIM1 expression in injured podocytes, experimental nephropathy animal models and FSGS patientsObjective:To explore STIM1 expression in injured podocytes, experimental nephropathy animal models and FSGS patients.Methods:MPC5 mouse podocytes were cultured in vitro and treated with adriamycin (Adr) for 24 hours to build the cellular model of podocyte injury. Adriamycin nephropathy mouse model and puromycin aminonucleoside (PAN) nephropathy rat model were established. Normal mice or rats were used as the control group. Renal biopsy tissues of FSGS and MCN patients were collected. The paracarcinoma tissues from patients who received kidney tumor resection were used as the control group. We performed real-time RT-PCR and western blot to examine the mRNA and protein levels of STIM1 respectively. Immunohistochemistry was used to detect the expression of STIM1 in kidney tissues.Results:(1) The cellular level:the mRNA levels of STIM1 were increased in a dose dependent manner in podocytes treated with different concerntrations of Adr, and the differences were significant from the concerntration of 250ng/ml in which group the mRNA level of STIM1 was increased to 4.62 fold. The protein level of STIM1 was also significantly increased in Adr-treated podocytes. (2) The animal model level: compared with the control group, the expressions of STIM1 significantly increased in Adr nephropathy mouse model and PAN nephropathy rat model. The mRNA level was 2.68 fold as great as the control group on the 3rd day after the Adr injection, then the expression decreased a little, but still higher than the sham group, and it maintained a high level in the 5th week, about 2.36 fold of the sham group. The protein level of Adr-mice was increased to 2.02 fold. The mRNA and protein levels of PAN-rats were increased to 3.85 and 2.25 fold (p<0.05). Immunohistochemical staining showed that STIM1 mainly expressed in glomeruli and renal interstitial blood vessels in normal mouse and rat renal tissues, little in renal tubular cells, and significantly increased in Adr or PAN nephropathy animal models. (3) Expression of STIM1 in FSGS patients:STIM1 mainly expressed in the tubular cells and renal interstitial blood vessels, little in glomeruli. Difference between the MCN patients and the control group were not significant; however, STIM1 expression markedly increased in the tubular and glomerular cells in FSGS patients.Conclusion:(1) STIM1 mainly expressed in glomeruli and renal interstitial blood vessels, little in tubular cells in mouse and rat kidney. However, in human normal renal cortex, it mainly expressed in the tubular cells and interstitial blood vessels, little in glomeruli. (2) STIM1 expression was significantly increased in Adr-treated podocytes and in the renal tissues from Adr nephropathy mouse model, PAN nephropathy rat model and FSGS patients. These indicated STIM1 may play a role in podocyte injury.Part 2:Mechanisms of STIMl-overexpression in inducing podocyte injuryObjective:We explored the effects of STIM1 overexpression on podocyte associated proteins, podocyte apoptosis, intracellular Ca2+concerntration, ER stress related molecules and mitochondrial function.Methods:The recombinant plasmid of STIM1, identified by restriction enzyme digestion, PCR and sequencing was transfected into MPC5 cells via lipofectamine 2000. Then cells were treated with Adr (250ng/ml) for 24-48h. We devided podocytes into four groups:the empty vector control group (Vehi), the overexpressing STIM1 group (p-STIM1), the empty vector control+Adr group (Vehi+Adr) and the overexpressing STIM1+Adr group (p-STIM1+Adr). Real-time RT-PCR and western blot were used to examine the expression levels of STIM1, some podocyte-associated molecules (podocin, a-actinin-4, TRPC6) and ER stress related molecules (GRP78, GRP94, CHOP) in the transfected MPC5 cells. Apoptosis of podocytes was assessed by flow cytometry. The intracellular Ca2+ concentration was examined by Fluo-3/AM fluorescence. Mitochondrial morphology was observed with transmission electron microscope (TEM). The mitochondrial membrane potential (MMP) was detected by JC-1 staining. The production of reactive oxygen species (ROS) was explored by DCFH-DA fluorescence. Mitochondrial DNA (mtDNA) was measured by real-time PCR.Results:(1) Restriction enzyme digestion, PCR and sequencing results demonstrated the recombinant plasmid was constructed successfully. (2) The mRNA and protein levels of STIM1 were significantly increased to 3.50 fold and 1.92 fold respectively in the recombinant plasmid group compared with the cellular control group and the Vehi group (p<0.05), which confirmed the successful construction of STIM1 overexpressing cellular model. (3) Compared with the Vehi group, STIM1 overexpression increased the podocyte apoptosis rate to 1.23 fold over the control group (p<0.05), downregulated the expression of podocin by 49.8% for mRNA level and 60.0% for protein level, upregulated the expression of a-actinin-4 to 1.73 fold for mRNA level and 1.85 fold for protein level (p<0.05). However, it showed no significant effect on the expression of TRPC6 (p>0.05). Compared with the Vehi+Adr group, STIM1+Adr group showed higher apoptosis rate (1.44 fold over the control) (p<0.05). (4) Compared with the Vehi group, the intracellular Ca2+ concentration increased by 23% in STIM1 overexpressing group,46% in Vehi+Adr group, and 80% in p-STIM1+Adr group which was 1.23 fold as great as that in Vehi+Adr group (p<0.05). (5) Compared with the Vehi group, the expression of GRP78, GRP94, CHOP in p-STIMl group increased, however, the differences were not significant. Compared with the Vehi+Adr group, the mRNA expressions of GRP78, GRP94, CHOP in p-STIM1+Adr group were increased by 33%,26% and 55% (p<0.05), and the protein expressions were increased by 17%,18% and 23% (p<0.05). (6) With TEM, we observed swollen and tubular mitochondria with disorganized and fragmented cristae in p-STIM1 group, vacuolation of mitochondria in Adr group, and more severe changes of mitochondria in p-STIM1+Adr group. Compared with the Vehi group, MMP and mtDNA copies were decreased by.30% and 18% in p-STIM1 group, and the production of ROS increased to 2.16 fold over the control group. Compared with the Adr group, MMP and mtDNA copies decreased by 22% and 48% in p-STIM1+Adr group, and the production of ROS increased to 1.44 fold (p<0.05).Conclusion:Overexpression of STIM1 induced the podocyte injury by increasing the intracellular Ca2+ concerntration, affecting the expression of podocyte-associated molecules (podocin and a-actinin-4), inducing cell apoptosis, ER stress and mitochondrial dysfunction.Part 3:STIM1 knockdown protected against Adr-induced podocyte injuryObjective:The present study was conducted to explore the role of STIM1 knockdown in Adr-induced podocyte injury.Methods:MPC5 cells were cultured in vitro, transfected transiently with STIM1 siRNA, then treated with Adr (250ng/ml) for 24-48h. We divided cells into four groups:negative control group (NC), STIM1 siRNA group (si-STIM1), Adr group (NC+Adr) and STM1 siRNA+Adr group (si-STIMl+Adr). Real-time RT-PCR and western blot were used to examine the mRNA and protein expression levels of STIM1, podocyte-associated molecules (podocin, a-actinin-4, TRPC6) and ER stress related molecules (GRP78, GRP94, CHOP) in the transfected MPC5 cells. Apoptosis of podocytes was assessed by flow cytometry. The intracellular Ca2+ concentration was examined by Fluo-3/AM fluorescence. Mitochondrial morphology was observed with TEM. The MMP was detected by JC-1 staining. The production of ROS was measured by DCFH-DA fluorescence. MtDNA copy numbers were assessed by real-time PCR.Results:(1) The mRNA and protein level of STIM1 in the si-STIM1 group was significantly decreased to 49% and 63% of the NC group (p<0.05). (2) Compared with the NC group, the mRNA and protein levels of podocin were decreased by 20% and 30% in the si-STIM1 group (p<0.05), while there were no significant changes of a-actinin-4 and TRPC6; compared with the NC+Adr group, the mRNA level of podocin was decreased by 30% in the si-STIM1+Adr group(p<0.05), but the protein level did not change significantly. No significant changes of a-actinin-4 and TRPC6 expressions were detected. Compared with the NC+Adr group, STIM1 knockdown (KD) decreased cellular apoptosis by 33% which showed it attenuated Adr-induced podocyte injury. (3) Compared with the NC+Adr group, STIM1-KD decreased the concentration of cytoplasm Ca2+ by 24% (p<0.05). (4) Compared with the NC+Adr group, the mRNA expressions of GRP78, GRP94 and CHOP in si-STIM1+Adr group were decreased by 19.6%,20.1% and 27.0% (p<0.05). The protein levels of them were decreased by 32%,14.5% and 27.9% (p<0.05). (5) Using TEM, we observed the vacuolation of mitochondria in NC+Adr group. The mitochndria were swollen, and the cristae were irregular arranged or fragmented. However, STIM1-KD improved the morphological changes of mitochondria induced by Adr. Typical mitochondria with intact cristae were seen in si-STIM1+Adr group. Compared with the NC+Adr group, STIM1-KD improved the MMP and mtDNA copies to 1.26 and 1.77 fold, and decreased ROS production to 76.3% (p<0.05).Conclusion:STIM1-KD might protect against Adr-induced podocyte injury by balancing the intracellular Ca2+ concentration, attenuating ER stress and mitochondrial dysfunction, and decreasing the apoptosis rate of podocytes. However, downregulation of STIM1 in podocytes also affected the expression of podocin which need further investigation. |
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