Jundong Jiao

Circulating microRNA-1 as a potential novel biomarker for acute myocardial infarction

Recent studies have revealed the role of microRNAs (miRNAs) in a variety of basic biological and pathological processes and the association of miRNA signatures with human diseases. Circulating miRNAs have been proposed as sensitive and informative biomarkers for multiple cancers diagnosis. We have previously documented aberrant up-regulation of miR-1 expression in ischemic myocardium and the consequent slowing of cardiac conduction. However, whether miR-1 could be a biomarker for predicting acute myocardial infarction (AMI) is unclear. In the present study, we recruited 159 patients with or without AMI for quantification of miR-1 level in plasma using real-time RT-PCR method. We performed Wilcoxon rank sum and signed rank tests for comparison. Univariable linear regression and logistics regression analyses were performed to assess the potential correlation between miR-1 and known AMI markers. We also conducted receiver-operator characteristic curve (ROC) analysis to evaluate the diagnostic ability of miR-1. We found that: miR-1 level was significantly higher in plasma from AMI patients compared with non-AMI subjects and the level was dropped to normal on discharge following medication. Increased circulating miR-1 was not associated with age, gender, blood pressure, diabetes mellitus or the established biomarkers for AMI. However, miR-1 level was well correlated with QRS by both univariable linear and logistics regression analyses. The area under ROC curve (AUC) was 0.7740 for separation between non-AMI and AMI patients and 0.8522 for separation AMI patients under hospitalization and discharge. Collectively, our results revealed that circulating miR-1 may be a novel, independent biomarker for diagnosis of AMI.

High glucose-induced apoptosis in cultured podocytes involves TRPC6-dependent calcium entry via the RhoA/ROCK pathway.

Increasing evidence indicates that podocyte apoptosis is a key event in the development of diabetic nephrology. However, the underlying mechanism of this apoptosis remains poorly understood. In this study, we report that high levels of glucose enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) influx, but glucose levels did not affect TRPC1 and TRPC5 expression. TRPC6 knockdown by siRNA interference attenuated the observed increase in glucose-induced podocyte apoptosis. High glucose levels also increased the generation of ROS; inhibition of ROS activity by N-acetyl-l-cysteine attenuated the high glucose-induced increase in TRPC6 expression and Ca(2+) influx. Exogenous treatment with H2O2 mimicked the high glucose response, resulting in an increase in TRPC6 expression and Ca(2+) influx. Taken together, these data suggest that high glucose levels induce ROS, thereby mediating TRPC6 expression and Ca(2+) influx. Because RhoA activity is increased following TRPC6 activation, we investigated whether TRPC6 is involved in high glucose-induced apoptosis via the RhoA/ROCK pathway. We report that high glucose levels produced an increase in RhoA activity, and this effect was abolished by the knockdown of TRPC6. Moreover, inhibition of the RhoA/ROCK pathway by a ROCK inhibitor, Y27632, also attenuated high glucose-induced apoptosis. We conclude that TRPC6 is involved in high glucose-induced podocyte apoptosis through the RhoA/ROCK pathway.

Calcium Sensing Receptor Modulates Extracellular Calcium Entry and Proliferation via TRPC3/6 Channels in Cultured Human Mesangial Cells

Calcium-sensing receptor (CaSR) has been demonstrated to be present in several tissues and cells unrelated to systemic calcium homeostasis, where it regulates a series of diverse cellular functions. A previous study indicated that CaSR is expressed in mouse glomerular mesangial cells (MCs), and stimulation of CaSR induces cell proliferation. However, the signaling cascades initiated by CaSR activation in MCs are currently unknown. In this study, our data demonstrate that CaSR mRNA and protein are expressed in a human mesangial cell line. Activating CaSR with high extracellular Ca2+ concentration ([Ca2+]o) or spermine induces a phospholipase C (PLC)-dependent increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, the CaSR activation-induced increase in [Ca2+]i results not only from intracellular Ca2+ release from internal stores but also from canonical transient receptor potential (TRPC)-dependent Ca2+ influx. This increase in Ca2+ was attenuated by treatment with a nonselective TRPC channel blocker but not by treatment with a voltage-gated calcium blocker or Na+/Ca2+ exchanger inhibitor. Furthermore, stimulation of CaSR by high [Ca2+]o enhanced the expression of TRPC3 and TRPC6 but not TRPC1 and TRPC4, and siRNA targeting TRPC3 and TRPC6 attenuated the CaSR activation-induced [Ca2+]i increase. Further experiments indicate that 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of receptor-operated calcium channels, significantly enhances the CaSR activation-induced [Ca2+]i increase. Moreover, under conditions in which intracellular stores were already depleted with thapsigargin (TG), CaSR agonists also induced an increase in [Ca2+]i, suggesting that calcium influx stimulated by CaSR agonists does not require the release of calcium stores. Finally, our data indicate that pharmacological inhibition and knock down of TRPC3 and TRPC6 attenuates the CaSR activation-induced cell proliferation in human MCs. With these data, we conclude that CaSR activation mediates Ca2+ influx and cell proliferation via TRPC3 and TRPC6 in human MCs.

Alpha1-Adrenergic Receptor Activation Stimulates Calcium Entry and Proliferation via TRPC6 Channels in Cultured Human Mesangial Cells

Background and Aims: There is accumulating evidence that sympathetic nervous hyperactivity contributes to the pathogenesis of glomerular sclerosis independent of blood pressure effects. A previous study showed that α₁-adrenoceptor (α₁-AR) antagonists inhibit mesangial cell (MC) proliferation. However, the underlying mechanism remains unclear. Methods and Results: We found that α₁-AR is expressed in a human mesangial cell line. The α₁-AR agonist phenylephrine (PE) induced Ca²⁺ influx as well as release from intracellular Ca²⁺ stores. Blockade of TRPC6 with siRNA, anti-TRPC6 antibodies and a TRPC blocker attenuated the PE-induced [Ca²⁺]_i increase. Additionally, the PE-induced [Ca²⁺]_i increase was phospholipase C dependent. Furthermore, PE induced a [Ca²⁺]_i increase even when the intracellular Ca²⁺ stores were already depleted. This effect was mimicked by an analog of diacylglycerol. These results suggested that, upon α₁-AR stimulation, TRPC6 mediates Ca²⁺ influx via a receptor-operated Ca²⁺ entry mechanism. Finally, TRPC6 contributes to the PE-induced MC proliferation. The mechanisms are associated with the extracellular signal-regulated kinase (ERK) signaling pathway because blockade of TRPC6 and chelation of extracellular Ca²⁺ abrogated PE-induced ERK1/2 abrogated PE-induced ERK1/2 phosphorylation. Conclusion: TRPC6 channels are involved in α₁-AR activation-induced Ca²⁺ entry, which mediates proliferation via ERK signaling in human MCs

Chronic hypoxia in cultured human podocytes inhibits BKCa channels by upregulating its β4-subunit.

Accumulating evidence suggests that podocyte hypoxia is an alternative mechanism for the pathogenesis of renal diseases. Functional, large-conductance, calcium-activated potassium channels (BK(Ca) channels) are expressed in podocytes as mechanosensitive channels; however, whether BK(Ca) channels are involved in the podocyte response to chronic hypoxia and the possible underlying mechanisms remain unclear. Here, we use the patch clamp technique to show that the exposure of human podocytes to 2% O(2) for 24 h causes a significant reduction in BK(Ca) channel currents. Molecular biology experiments showed that chronic hypoxia increased BK(Ca) channel β4-subunit mRNA and protein expression, but not the expression of the BK(Ca) pore-forming α- or β3-subunits. Furthermore, chronic hypoxia shifted the channel activation range toward more depolarized voltages and slowed its activation kinetics, which are similar to the properties conferred by the β4-subunit. We conclude that BK(Ca) channels are involved in the response of podocytes to chronic hypoxia via the upregulation of the β4-subunit. These findings provide new insight into the mechanism underlying the cellular responses of podocytes to hypoxia.

The upregulation of TRPC6 contributes to Ca2+ signaling and actin assembly in human mesangial cells after chronic hypoxia

There is increasing evidence that mesangial cells are important targets of chronic hypoxia injury. Impaired Ca(2+) signaling has been found in mesangial cells (MCs) subjected to chronic hypoxia. However, the mechanisms underlying this phenomenon have not yet been defined. In the present study, we found that chronic hypoxia enhanced the expression of TRPC6 and TRPC6-dependent Ca(2+) entry, and TRPC6 knockdown inhibited the chronic hypoxia-induced increase in [Ca(2+)]i, suggesting that TRPC6-mediated Ca(2+) entry is responsible for the elevated [Ca(2+)]i induced by chronic hypoxia in MCs. In addition, TRPC6 knockdown attenuated chronic hypoxia-induced actin assembly and actin reorganization. We concluded that the upregulation of TRPC6 is involved in the Ca(2+) signaling and actin assembly in human MCs after chronic hypoxia. These findings provide new insight into the mechanisms underlying the cellular response of MCs to hypoxia.

The use of functional chemical-protein associations to identify multi-pathway renoprotectants.

Typically, most nephropathies can be categorized as complex human diseases in which the cumulative effect of multiple minor genes, combined with environmental and lifestyle factors, determines the disease phenotype. Thus, multi-target drugs would be more likely to facilitate comprehensive renoprotection than single-target agents. In this study, functional chemical-protein association analysis was performed to retrieve multi-target drugs of high pathway wideness from the STITCH 3.1 database. Pathway wideness of a drug evaluated the efficiency of regulation of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in quantity. We identified nine experimentally validated renoprotectants that exerted remarkable impact on KEGG pathways by targeting a limited number of proteins. We selected curcumin as an illustrative compound to display the advantage of multi-pathway drugs on renoprotection. We compared curcumin with hemin, an agonist of heme oxygenase-1 (HO-1), which significantly affects only one KEGG pathway, porphyrin and chlorophyll metabolism (adjusted p = 1.5×10−5). At the same concentration (10 µM), both curcumin and hemin equivalently mitigated oxidative stress in H2O2-treated glomerular mesangial cells. The benefit of using hemin was derived from its agonistic effect on HO-1, providing relief from oxidative stress. Selective inhibition of HO-1 completely blocked the action of hemin but not that of curcumin, suggesting simultaneous multi-pathway intervention by curcumin. Curcumin also increased cellular autophagy levels, enhancing its protective effect; however, hemin had no effects. Based on the fact that the dysregulation of multiple pathways is implicated in the etiology of complex diseases, we proposed a feasible method for identifying multi-pathway drugs from compounds with validated targets. Our efforts will help identify multi-pathway agents capable of providing comprehensive protection against renal injuries.

Calcium-Sensing Receptor Stimulation in Cultured Glomerular Podocytes Induces TRPC6-Dependent Calcium Entry and RhoA Activation

Background/Aims: Recent studies provided compelling evidence that stimulation of the calcium sensing receptor (CaSR) exerts direct renoprotective action at the glomerular podocyte level. This protective action may be attributed to the RhoA-dependent stabilization of the actin cytoskeleton. However, the underlying mechanisms remain unclear. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure canonical transient receptor potential 6 (TRPC6) protein expression and RhoA activity. Stress fibers were detected by FITC-phalloidin. Results: Activating CaSR with a high extracellular Ca2+ concentration ([Ca2+]o) or R-568 (a type II CaSR agonist) induces an increase in the [Ca2+]i in a dose-dependent manner. This increase in [Ca2+]i is phospholipase C (PLC)-dependent and is smaller in the absence of extracellular Ca2+ than in the presence of 0.5 mM [Ca2+]o. The CaSR activation-induced [Ca2+]i increase is attenuated by the pharmacological blockage of TRPC6 channels or siRNA targeting TRPC6. These data suggest that TRPC6 is involved in CaSR activation-induced Ca2+ influx. Consistent with a previous study, CaSR stimulation results in an increase in RhoA activity. However, the knockdown of TRPC6 significantly abolished the RhoA activity increase induced by CaSR stimulation, suggesting that TRPC6-dependent Ca2+ entry is required for RhoA activation. The activated RhoA is involved in the formation of stress fibers and focal adhesions in response to CaSR stimulation because siRNA targeting RhoA attenuated the increase in the stress fiber mediated by CaSR stimulation. Moreover, this effect of CaSR activation on the formation of stress fibers is also abolished by the knockdown of TRPC6. Conclusion: TRPC6 is involved in the regulation of stress fiber formation and focal adhesions via the RhoA pathway in response to CaSR activation. This may explain the direct protective action of CaSR agonists.

TRPC6-Mediated Ca 2+ Signaling is Required for Hypoxia-Induced Autophagy in Human Podocytes

Background/Aims: Intracellular Ca2+ signaling plays an important role in the regulation of autophagy. However, very little is known about the role of Ca2+ influx, which is induced by plasma membrane Ca2+ channels. Our previous study showed that transient receptor potential canonical channel-6 (TRPC6), a major Ca2+ influx pathway in podocytes, was activated by hypoxia. Here, we investigated whether TRPC6 is involved in hypoxia-induced autophagy in cultured human podocytes. Methods: In the present study, an immortalized human podocyte cell line was used. Fluo-3 fluorescence was utilized to determine intracellular Ca2+ concentration ([Ca2+]i), and western blotting was used to measure autophagy and protein expression. Results: We found that blockade TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker attenuated hypoxia-induced autophagy, while enhancement of TRPC6 activity with a TRPC6 activator enhanced hypoxia-induced autophagy. Furthermore, TRPC6-dependent Ca2+ signaling is responsible for hypoxia-induced autophagy since both an intracellular and extracellular Ca2+ chelator abolished hypoxia-induced autophagy. Moreover, we found that blockade of TRPC6 by using either TRPC6 siRNA or a TRPC6 blocker decreased the expression of adenosine monophosphate-activated protein kinase (AMPK), an important signaling molecule in Ca2+-dependent autophagy activation, which is activated under hypoxic conditions. These data suggest that the involvement of TRPC6 in hypoxia-induced autophagy is associated with AMPK signaling. Conclusion: TRPC6 is essential for hypoxia-induced autophagy in podocytes.