Canonical Transient Receptor Potential 6 Channel: A New Target of Reactive Oxygen Species in Renal Physiology and Pathology

Abstract

Significance: Regulation of Ca²⁺ signaling cascade by reactive oxygen species (ROS) is becoming increasingly evident and this regulation represents a key mechanism for control of many fundamental cellular functions. Canonical transient receptor potential (TRPC) 6, a member of Ca²⁺-conductive channel in the TRPC family, is widely expressed in kidney cells, including glomerular mesangial cells, podocytes, tubular epithelial cells, and vascular myocytes in renal microvasculature. Both overproduction of ROS and dysfunction of TRPC6 channel are involved in renal injury in animal models and human subjects. Although regulation of TRPC channel function by ROS has been well described in other tissues and cell types, such as vascular smooth muscle, this important cell regulatory mechanism has not been fully reviewed in kidney cells. Recent Advances: Accumulating evidence has shown that TRPC6 is a redox-sensitive channel, and modulation of TRPC6 Ca²⁺ signaling by altering TRPC6 protein expression or TRPC6 channel activity in kidney cells is a downstream mechanism by which ROS induce renal damage. Critical Issues: This review highlights how recent studies analyzing function and expression of TRPC6 channels in the kidney and their response to ROS improve our mechanistic understanding of oxidative stress-related kidney diseases. Future Directions: Although it is evident that ROS regulate TRPC6-mediated Ca²⁺ signaling in several types of kidney cells, further study is needed to identify the underlying molecular mechanism. We hope that the newly identified ROS/TRPC6 pathway will pave the way to new, promising therapeutic strategies to target kidney diseases such as diabetic nephropathy. Antioxid. Redox Signal. 25, 732–748.

Introduction

Canonical transient receptor potential (TRPC) proteins constitute one of the major subfamilies of the TRP superfamily first identified in Drosophila melanogaster (138). There are seven subtypes, designated TRPC1-7, of which TRPC2 is a pseudogene in humans. The remaining six proteins can be divided phylogenetically based on their structure, with TRPC1 most closely related to TRPC4 and TRPC5, and TRPC3 grouped with TRPC6 and TRPC7 (153, 161).

Over the past 20 years, the seven TRPC members have been the focus of intensive investigations in many cell types, including kidney cells. TRPCs may function as store-operated channels activated by depletion of intracellular Ca²⁺ stores (151) or as receptor-operated channels activated by G protein-coupled or receptor tyrosine kinase signaling pathways (79). In both cases, TRPCs act as nonselective cation channels, allowing Ca²⁺ influx. No matter whether their activation occurs via store-operated or receptor-activated mechanisms, increasing evidence demonstrates that TRPCs have functional significance in cellular Ca²⁺ signaling. This gives them the potential to contribute to the regulation of numerous Ca²⁺-dependent cellular functions from cell growth to myocyte contraction.

Like other TRPC family members, TRPC6 comprises six transmembrane spanning regions with intracellular N- and C-termini and a presumed pore-forming loop between transmembrane spanning regions 5 and 6. Other conserved regions include three ankyrin repeats, a coiled-coil domain, and caveolin binding region in the N-terminus. The C-terminus is characterized by a TRP motif (EWKFAR), another coiled-coil domain, a proline-rich motif, and a calmodulin/IP₃R binding region (38, 205) (Fig. 1). TRPC6 is widely expressed in a variety of tissues and organs, including the kidney (2). Particularly, a link between an altered expression level or dysfunction of TRPC6 channel and renal diseases has been firmly established (38).

FIG. 1.

TRPC6 channel structure. TRPC6 is characterized by six transmembrane domains with a pore-forming region found between transmembrane domains 5 and 6. The N-terminus of TRPC6 contains three Ank, one CC, and one CBD, and the C-terminus contains one CIRB and highly conserved TRP box. Several disease-related point mutations (indicated by arrows) have been discovered in TRPC6. Amino acid changes are indicated by the single-letter code and a number for the exact position in the protein. *A stop codon (truncation mutant). CBD, caveolin-1 binding domain; CC, coiled-coiled domain; CIRB, calmodulin/IP₃R binding domain; TRPC, canonical transient receptor potential.

Although TRPC6 is acknowledged as a lipid (such as dicylglycerol) and mechanosensitive channel (6, 7, 85, 91, 95, 113, 116, 188), evidence has accumulated that it is also a redox-sensitive channel (70). Recent studies have called attention to the modulation of TRPC6 channel by the cellular redox in kidney cells, which may underlie a new mechanism for development of some kidney diseases. This review summarizes recent studies on the importance of the ROS/TRPC6 signaling pathway in regulation of renal function and, importantly, the contribution of this intracellular pathway to kidney diseases. We hope that this review can provide new information for people in the field of both TRPC6 biology/pathology and renal physiology/pathology.

Distribution of TRPC6 in Renal Microvasculature

TRPC6 protein is widely expressed in blood vessels of varying sizes and plays a role in the regulation of vascular tone by contributing to smooth muscle contraction as well as endothelium-dependent vasorelaxation (6, 7, 39, 226). For instance, TRPC6 is expressed in myocytes of portal vein (6, 96), pulmonary artery (120, 207), cerebellar arteries (216), mesenteric arteries (7, 42), and aorta (42) and in the endothelial cells of pulmonary artery (108, 184) and aorta (32). TRPC6 in these vascular cells can regulate basal vascular tone, agonist-induced vasoconstriction and vasodilatation, vascular endothelial permeability, vascular endothelial cell migration, and vascular myocyte proliferation (2, 40, 63, 108, 213).

The findings in the extrarenal vasculature raise an interest in studying expression and function of TRPC6 channel in the renal microvasculature (Fig. 2). Different from microcirculation in other tissues/organs, renal microcirculation contains two sets of capillaries, termed glomerular and peritubular capillaries, which are arranged in series and separated by efferent arterioles. The afferent and efferent arterioles are major resistance vessels in the kidney and their contractile state controls the renal blood flow. Furthermore, the microcirculation of the kidney is regionally specialized. In the cortex, afferent and efferent arterioles govern the driving forces that promote glomerular filtration. A dense peritubular capillary plexus surrounds the proximal and distal convoluted tubules to accommodate enormous reabsorption of glomerular filtrates.

FIG. 2.

Distribution of TRPC6 in nephron and renal microvasculature.

By adjusting the resistance of the afferent and efferent arterioles, the kidneys can regulate the hydrostatic pressures in both the glomerular and the peritubular capillaries, thereby changing the rate of glomerular filtration and/or tubular reabsorption in response to body homeostatic demands. TRPC channels in the renal microvessels may help to regulate renal function by contributing to the regulation of driving force for glomerular filtration and tubular reabsorption, making their potential role in the renal microcirculatory system of particular interest.

Earlier studies have demonstrated that afferent and efferent arterioles expressed different isoforms of TRPC channels. TRPC1 is present in the efferent arterioles (198), but TRPC6 is expressed in the afferent arterioles. In the freshly isolated vascular smooth muscle cells derived from rat preglomerular vessels, Fellner and Arendshorst found that nifedipine or verapamil only partially inhibited vasopressin-induced Ca²⁺ entry (52). In the presence of the voltage-gated Ca²⁺ channel blockers, depletion of internal Ca²⁺ stores with cyclopiazonic acid or thapsigargin significantly raised intracellular Ca²⁺ concentration in response to Ca²⁺ readmission. They further found that TRPC blockade with 2-aminoethoxydiphenyl borane inhibited angiotensin II (Ang II)-induced Ca²⁺ influx, while flufenamic acid, which has been shown to activate TRPC6, resulted in an increased Ca²⁺ influx (51). The authors concluded that TRPC6 participated in agonist-evoked Ca²⁺ signaling in afferent arterioles.

It should be noted that several other subtypes of TRPCs (TRPC1, 3, 4, 5, and 7) were also detected at both mRNA and protein levels in the preglomerular resistance vessels, including afferent arterioles (47, 198). However, their role in contractile function of the afferent arterioles has not been studied yet.

Distribution of TRPC6 in Glomerular Cells

The glomerulus is the filtering unit of kidney. The mature glomerulus contains four cell types: parietal epithelial cells that form Bowman's capsule, podocytes that cover the outmost layer of the glomerular filtration membrane, endothelial cells that are lined along the luminal side of the wall of glomerular capillaries, and mesangial cells (MCs) that sit between the capillary loops (135, 190). Over the past 10 years, studies have been conducted to examine expression of TRPC6 in specific type of glomerular cells and the implications of TRPC6 in renal function. Although the results from different groups are not completely consistent, and some are even controversial, a consensus among the studies is that TRPC6 is expressed in glomerular cells and plays important roles in glomerular physiology and pathology (Fig. 2).

Expression of TRPC6 in glomerular MCs

Glomerular MCs and their matrix form the central stalk of the glomerulus. MCs comprise approximately one-third of the decapsulated glomerular cell population. In the glomerulus, MCs have many beneficial roles, which include secretion of growth factors that allow normal cell turnover, production of mesangial matrix to provide structural support for glomerular capillaries, modulation of glomerular hemodynamics through their contractile properties, and phagocytosis of apoptotic cells or immune complexes formed at or delivered to the glomerular capillaries (1, 130, 189). As in most cell types, Ca²⁺ signaling plays a central role in mediating and regulating those cellular processes in MCs. Ca²⁺ influx across the plasma membrane is a major component of MC Ca²⁺ response to a variety of stimuli (123). Among several types of Ca²⁺-conductive channels in the plasma membrane, TRPC channels have recently caught intensive attention. Over the past 10 years, we and others have extensively studied the function and regulatory mechanisms of TRPC channels, particularly TRPC6, in MCs.

In an earlier study, we identified the presence of TRPC6 protein in cultured human MCs (187). Expression of TRPC6 protein in human MCs was also reported by several other groups (87, 110, 119, 131, 158, 233). However, Goel et al. reported that rat MCs only expressed TRPC1 protein (61). The distinct results from different groups may be due to different antibodies used in those studies. It is also possible that the TRPC protein distribution in MCs differs among species.

MCs have a contractile phenotype similar to vascular smooth muscle cells. The contractile properties of MCs enable them to alter intraglomerular capillary flow and glomerular ultrafiltration surface area and thereby the glomerular filtration rate (41, 130, 189). MC contractile function is controlled by intracellular Ca²⁺ concentration, which is, to a large extent, attributed to Ca²⁺ influx through Ca²⁺ channels in the plasma membrane. TRPC6 is a Ca²⁺-conductive channel and can be activated by chemical (such as dicylglycerol) and physical factors (such as stretch) (85, 91, 95, 116, 188), both of which are physiological stimuli to MCs. Therefore, TRPC6 may regulate MC contractility. This speculation was confirmed by our recent study in which TRPC6 mediated Ca²⁺ entry and contraction of cultured human MCs in response to Ang II (72).

Diabetic nephropathy is a major and devastating complication of diabetes. At the very onset of diabetes, a dominant pathophysiological characteristic is the development of renal glomerular hyperfiltration (13, 214). This early hemodynamic phenotype provokes the subsequent demise of a diabetic kidney. The diabetic hyperfiltration is derived from a combined decreased responsiveness of both the renal afferent arterioles and glomerular MCs to vasoconstrictors (13, 27, 104, 144, 218). In diabetes, mesangial contractile function is impaired, and reduced Ca²⁺ influx is believed to be a major contributing factor to the hypocontractility (144, 218).

Since TRPC6 channel mediates Ca²⁺ response in MCs, it is conceivable that dysfunction of the channel may play a role in diabetic hyperfiltration. We recently found that expression of TRPC6, but not TRPC1 and TRPC3, was downregulated in glomeruli of streptozotocin (STZ) diabetic rats as well as in MCs treated with high glucose (71). Both high-glucose treatment and knockdown of TRPC6 attenuated Ang II-stimulated membrane currents and Ca²⁺ influx in MCs (71). This high-glucose/diabetes-induced decrease in TRPC6 protein expression was MC specific because this response was not observed in the aorta and the heart tissue isolated from those diabetic rats (72) and in high-glucose-treated podocytes (71).

Since our study was conducted in rats only 2 weeks after STZ injection, the decrease in abundance of TRPC6 protein might indicate an early change in the development of diabetic nephropathy. However, it is not clear whether the decrease of TRPC6 protein in MCs is the cause or consequence of diabetic kidney disease. Elucidation of this cause–effect relationship needs further studies, for instance, studies to investigate whether MC-specific knockout/knockdown of TRPC6 can initiate diabetic kidney disease and whether hyperfiltration in diabetic animals can be normalized by rescuing TRPC6 protein in MCs.

In addition to regulation of MC contraction, TRPC6 may also play a role in MC proliferation. Ca²⁺-sensing receptor is expressed in MCs and stimulation of the receptor promotes proliferation (112). Meng et al. found that activation of Ca²⁺-sensing receptor increased expression of TRPC6 in MCs. Knocking down of TRPC6 attenuated Ca²⁺-sensing receptor-mediated elevation of intracellular Ca²⁺ and MC proliferation (131), suggesting contributions of TRPC6 to the MC responses.

Ang II is a known proliferative factor in many cell types, including MCs (98, 171). Studies showed that Ang II stimulated MC proliferation by increasing the expression level of TRPC6 protein (158, 233). In addition, TRPC6 also contributed to phenylephrine (α1-adrenergic receptor agonist)-induced MC proliferation (110). Interestingly, the ERK signaling pathway can work as a mechanism both upstream (in Ang II signaling pathway) (233) and downstream (in α1-adrenergic receptor signaling pathway) (110) TRPC6 expression. Excessive proliferation of MCs is a characteristic pathological feature of glomerulosclerosis. Both an overactive Ang II system and sympathetic nervous hyperactivity (overactive α1-adrenergic receptor signaling pathway) in the kidney contribute to the pathogenesis of glomerulosclerosis in chronic kidney disease (22, 103, 133, 147, 206, 220). Therefore, TRPC6 may be used as a therapeutic target for treating renal fibrosis in chronic kidney disease.

Expression of TRPC6 in podocytes

Podocytes are pericyte-like cells with a complex cellular organization consisting of a cell body, major processes, and foot processes. The foot processes of neighboring podocytes interdigitate each other, forming filtration slits that are bridged by the glomerular slit diaphragm (75). Podocytes are highly differentiated cells with limited capability to undergo cell division in the adult, and the loss of podocytes is a hallmark of progressive kidney disease. Podocytes form the glomerular filtration barrier along with the fenestrated glomerular capillary endothelium and glomerular basement membrane and therefore play a significant role in the regulation of glomerular filtration (204). Disruption of the podocyte slit diaphragm is associated with severe proteinuria, including that associated with focal and segmental glomerulosclerosis (FSGS).

Like many other cell types, podocytes also express multiple subtypes of TRPC proteins. In an earlier study, TRPC3 and TRPC6 proteins were detected in rat podocytes (61). This was verified by a later study from Kim et al. who showed colocalizations of both TRPC3 and TRPC6 with large-conductance Ca²⁺-activated K⁺ channels (105). TRPC6 protein was also found in the podocytes of the freshly isolated rat glomeruli (93).

An enormous impetus of studying the importance of TRPC6 in podocyte biology, physiology, and pathology is derived from a breakthrough finding that a gain-of-function mutation of TRPC6 in podocytes was associated with familial FSGS, a pathologic lesion that frequently causes the nephritic syndrome and ensuing renal failure (136, 219). Through whole-genome linkage analysis, fine mapping, and candidate gene screening, Winn et al. identified a mutated trpc6 gene in a large family, most members of which progressed to end-stage renal disease (219). Subsequently, Reiser et al. identified TRPC6 mutations in five other unrelated families with autosomal dominant FSGS (163). More TRPC6 mutations were recently found in late-onset FSGS as well as childhood FSGS patients (57, 88, 235). So far, 14 different mutations in the trpc6 gene have been identified in nine families from different ethnic backgrounds and in five sporadic patients (57, 88, 163, 174, 219, 235). Most of these mutations were gain-of-function mutations that resulted in increased Ca²⁺ entry.

Although the associations of TRPC6 mutations in podocytes with FSGS have been firmly established, the underlying mechanisms are still unclear. Multiple mechanisms may be involved. For instance, increased intracellular Ca²⁺ derived from overactive TRPC6 channel may modify the contractile structure or actin cytoskeleton arrangement of podocyte foot processes, resulting in an alteration of the ultrafiltration coefficient (99, 137, 202). Another possible mechanism is that increased intracellular Ca²⁺ may cause loss of podocytes through apoptosis, detachment, or lack of proliferation, resulting in glomerulosclerosis (152). It is also possible that abnormally high intracellular Ca²⁺ limits normal podocyte proliferation (182).

Consistent with the role of hyperactive TRPC6 mutations in hereditary FSGS, an increased expression of TRPC6 was found in acquired forms of proteinuric kidney diseases, including minimal change disease and membranous glomerulonephritis (137). Overexpression of TRPC6 in podocytes disrupts actin cytoskeleton, which may result in slit diaphragm dysfunction and subsequent proteinuria in mice transiently overexpressing TRPC6 (137).

Because Ang II plays a critical role in the generation of proteinuria and progression of kidney injury (22, 103, 133, 147, 220), a role of TRPC6 channel in the Ang II signaling pathway in podocytes has been intensively studied. An earlier study by Zhang et al. demonstrated that Ang II-induced podocyte apoptosis resulted from augmentation of TRPC6-mediated Ca²⁺ influx (231). Ang II treatment increased TRPC6 protein expression through the ERK/NF-κB pathway (231), and the ERK1/2 pathway was activated by gain-of-function TRPC6 mutants in cultured podocytes (34). Therefore, there may be a positive feedback loop between ERK1/2 and TRPC6, which can accelerate Ang II-induced podocyte injury.

Nijenhuis et al. also reported that Ang II increased TRPC6 mRNA and protein expression levels in cultured mouse podocytes (142). They further showed that Ang II infusion enhanced glomerular and podocyte TRPC6 expression in rats. In rat models for human FSGS and increased renin–angiotensin system activity, glomerular and podocyte TRPC6 expression was increased in an Ang II-dependent manner and the increase in TRPC6 expression correlated with podocyte injury and glomerular damage (142). Using in vitro and in vivo settings, they demonstrated that Ang II induced podocyte injury by enhancing TRPC6 expression via a calcineurin/NFAT-positive feedback signaling pathway (142).

Indeed, several TRPC6 mutations previously shown to enhance channel activity caused constitutive activation of the calcineurin/NFAT pathway in podocytes (175), and activation of calcineurin increased TRPC6 expression in podocytes in a murine model of podocyte-specific constitutively active Gq α subunit (208). In another study, Eckel et al. infused TRPC6-deficient and wild-type control mice with Ang II for 28 days. Although both groups developed similar levels of hypertension, TRPC6-deficient mice had significantly less albuminuria (44), presumably due to lack of TRPC6-dependent Ca²⁺ influx upon Ang II stimulation. Therefore, TRPC6 adversely influences the glomerular filter and enhances Ang II-induced kidney injury.

In addition to increasing number of TRPC6 channels, Ang II may also increase activity of existing TRPC6 channels in podocytes. This has been demonstrated in podocytes of freshly isolated rat glomeruli (94). Therefore, Ang II has both chronic and acute effects on TRPC6 channels, the former is to increase abundance of TRPC6 proteins and the latter is to increase open probability of the channel.

Effects of TRPC6 on podocyte function may be context dependent. In contrast to those studies described above, Kistler et al. reported that overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure, but protected podocytes from complement-mediated injury (109). Consistently, TRPC6 inactivation increased podocyte susceptibility to complement. They further showed that the TRPC6 effect was mediated by Ca²⁺/calmodulin-dependent protein kinase II mechanism (109). Their study suggests a dual and context-dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to FSGS.

Recently, TRPC5 was found to promote podocyte motility by antagonizing the TRPC6 signaling pathway. Tian et al. demonstrated that podocytes contained two distinct molecular complexes of TRPC5-Rac1 and TRPC6-RhoA, which are spatially, biochemically, and functionally segregated (202). Both Rac1 and RhoA are the members of the Rho family of small guanosine triphosphatases, with Rac1 promoting cell motility and RhoA enhancing contractility (46). In accordance with the distinct functions of Rac1 and RhoA, TRPC5 promoted a motile phenotype, whereas TRPC6 maintained a contractile phenotype of podocytes (202). The antagonistic and mutually inhibitory TRPC5 and TRPC6 signaling is well coordinated and maintains the balance between contractility and motility of podocytes.

The findings from the Tian et al. study are consistent with the previous reports of increasing neuronal cell motility and vascular smooth muscle cell migration by TRPC5 (76, 222) and inhibiting endothelial cell migration by TRPC6 (184), thus highlighting complicated TRPC channel signaling in regulation of podocyte structure and function (74).

Although several other isoforms of TRPCs were also detected in podocytes either at the mRNA level or protein level, their physiological and pathological relevance in podocytes has not been studied, probably due to their low expression levels and/or no contribution to agonist-stimulated Ca²⁺ response in podocytes (93, 94, 163, 202).

Expression of TRPC6 in endothelial cells

Glomerular endothelial cells line the inner surface of the glomerular capillaries. Compared with the endothelial cells in most vascular beds, glomerular endothelial cells are extremely flat and perforated by dense arrays of transcellular pores, the fenestrae. This phenotype is critical for the high glomerular water and electrolyte permeability (145).

Many TRPC isoforms have been found in endothelial cells of extrarenal vasculature, as described previously (11, 32, 48, 55, 154, 184, 226, 227). However, studies on TRPC channels in the glomerular endothelial cells are scarce. Using immunofluorescent staining of rat kidney sections, Reiser et al. were able to detect TRPC6 protein expression in glomerular endothelial cells (163). However, the function of TRPC6 in the endothelial cells was not examined in that study. Since TRPC6 in endothelial cells plays an important role in vascular endothelial permeability (2, 40, 108), it may be worth studying the TRPC6 function in glomerular endothelial cells, which may provide new information on regulation of glomerular filtration.

Distribution of TRPC6 in Tubular Epithelial Cells

Renal tubules comprise four major segments, that is, the proximal tubule, Henle's loop, distal tubule, and collecting duct. Each segment is made up of a layer of epithelial cells that are uniquely suited to perform specific transport functions. By the processes of selective reabsorption of solutes and water and secretion of solutes, the renal tubules modulate the volume and composition of urine, which in turn allow the tubules to precisely control the volume, osmolality, composition, and pH of the extracellular and intracellular fluids. Accumulating evidence shows that TRPC6 is expressed in the epithelial cells of different segments and regulates water and solute transport in the tubules (Fig. 2).

Expression of TRPC6 in proximal tubule

TRPC6 (160) was found in cultured human-derived renal proximal tubular cell lines (HK-2) (172). A study further suggested that TRPC6 was involved in hepatocyte growth factor-induced growth, migration, cytoskeletal reorganization, and tubulogenesis of proximal tubular epithelial cells (160).

Expression of TRPC6 in distal tubules

TRPC6 expression in the distal tubules has been extensively studied. In cultured polarized MDCK cells, which resemble the intercalated cells of the renal cortical collecting duct (146), Bandyopadhyay et al. detected TRPC6, which was localized both apically and basolaterally (12). Goel et al. performed a series of studies in vivo (rats) and in the cultured mouse collecting duct cell line and comprehensively examined distributions and functions of TRPC6 in distal nephron. TRPC6 was detected in specific tubular cells of the cortex and both the outer and inner medullas (61). Using specific markers for different tubular segments, they further revealed that TRPC6 was expressed in principal cells of the collecting duct (61). Consistent with Bandyopadhyay's findings (12), Goel et al. also found that TRPC6 was expressed in both the basolateral and apical membranes in polarized cultures of M1 and IMCD-3 collecting duct cells (61).

Although both TRPC3 and 6 exist in principal cells of the collecting duct, only TRPC3-containing intracellular vesicles translocated to the apical membrane along with the water channel, aquaporin-2, upon stimulation with the antidiuretic hormone, arginine vasopressin (60, 62). TRPC3 channels were also responsible for transepithelial apical-to-basolateral Ca²⁺ flux stimulated by apical ATP in principal cells of the inner medullary collecting duct (59, 60). However, the function of TRPC6 channel in this tubule has not been studied.

Distribution of TRPC6 in Other Renal Cells

Juxtaglomerular (JG) cells of the kidneys are a group of specialized cells in renal microcirculation. They are modified smooth muscle cells located in the walls of the afferent arterioles immediately proximal to the glomeruli. The major function of the JG cells is to synthesize, store, and release renin into the blood flow for initiating the renin–angiotensin–aldosterone system. Ca²⁺ plays an unusual role in the control of renin secretion from the renal JG cells in the way that an increase in the Ca²⁺ concentration inhibits the exocytosis of renin. The transcript of TRPC6 was detected in mouse JG cells (149). TRPC6 might be responsible for adenosine-evoked inhibition of renin release (149).

Fibroblasts, when activated, undergo a phenotypic change to myofibroblasts, which is a key process of fibrosis. TRPC6 is expressed in renal fibroblasts and plays a role in growth and proliferation of the cells. In cultured rat renal fibroblasts, TRPC6 protein was found by forming a macromolecular complex with TRPC3 (173). However, functional studies showed that specific knockdown of TRPC6 did not inhibit agonist-stimulated Ca²⁺ entry in cultured fibroblasts (173). Therefore, the function of TRPC6 in renal fibroblasts needs to be further studied.

Reactive Oxygen Species and Ca²⁺ Signaling

Reactive oxygen species (ROS) are ubiquitous, highly reactive short-lived derivatives of oxygen metabolism produced in all biological systems that react with surrounding molecules at the site of formation. These species include free radicals, such as the superoxide anion (O₂^.−) and the hydroxyl radical (.OH), and nonradicals, such as H₂O₂. Compared with radicals, H₂O₂ has longer half-life and diffusion distance and is more stable. Different from O₂^.−, which remains near the site of its production, H₂O₂ can diffuse across membranes with the help of aquaporin proteins (15–17, 89, 132) and through the cytosol (10). Therefore, H₂O₂ has been focused on in most studies to investigate the roles of ROS in regulation of biological, physiological, and pathological processes.

It has been acknowledged that ROS, at a low level, play an essential role in multiple cellular signal transduction pathways. However, under conditions of oxidative stress, they contribute to a series of cellular dysfunctions. This is particularly true for H₂O₂. H₂O₂ has been identified as a second messenger molecule (90, 165) and initiates/regulates a diversity of cellular effects, such as cell shape changes, the formation of functional actomyosin structures, and vascular tone (42, 221). However, overproduction of H₂O₂ has been linked to metabolic syndrome (167), cardiovascular diseases (29, 77, 176), atherosclerosis (126, 185), hypertension (114, 180), and diabetic complications (65, 80, 102, 168). Like in all other organs/tissues, ROS not only play important roles in renal biology and physiology (41, 148, 177, 179) but also contribute to acute and chronic kidney injury (14, 64–67, 80, 97, 176, 199).

ROS are constitutively produced from the mitochondrial electron transport chain (30) and oxidases distributed in subcellular compartments (3, 25, 86, 101, 127), for example, NADPH oxidases (cytoplasm, plasma membrane, nuclei), xanthine oxidase (cytoplasm, extracellular), endoplasmic reticulum oxidase, sulfhydryl oxidases (nuclei, extracellular), thiol oxidases (plasma membrane, plasma), and monoamine oxidases (mitochondria outer membrane). Currently, there is substantial evidence that the phagocyte-like Nox family enzymes are a major source of ROS in the kidney (58, 64–68, 177). Particularly, associations of overproduction of ROS from activated NADPH oxidases with structural and functional deteriorations in diabetic nephropathy have recently been firmly established (19, 65–67, 97, 164, 178, 199, 228).

Multiple downstream pathways mediate ROS effects on cellular processes. These include direct and indirect oxidation of macromolecules (lipid, DNA, and proteins) (101, 183, 225), modulation of protein kinases (33, 56, 72, 83, 84, 191, 195), protein tyrosine phosphatases (37, 121, 165, 186, 197, 215), and transcription factors (41, 82, 100, 115, 166, 211). This is particularly true in the kidney. Alterations of activities of PKC, Akt, ERK1/2, and signaling pathways of TGF-β1, NF-κB, and Nrf-2 by ROS have been reported in glomerular cells and tubular epithelial cells (20, 41, 68, 69, 72, 82–84, 100, 115, 140, 166, 191, 211).

Among those ROS-associated pathways, the Ca²⁺ signaling pathway is an important target for modulation of cellular processes by ROS under physiological and pathological conditions. Intracellular Ca²⁺ represents a convergent point of many signal transduction pathways and modulates a diverse array of cellular activities ranging from contraction to cell growth. Cells generate Ca²⁺ signals through both internal and external Ca²⁺ sources. A major internal Ca²⁺ source is the ER/SR, which releases the stored Ca²⁺ in the lumen into the cytoplasm through IP₃R or RyR. In terms of external source, several types of Ca²⁺-conductive ion channels in the plasma membrane are involved in controlling Ca²⁺ entry in response to a variety of stimuli. Elevated cytosolic Ca²⁺ is rapidly reduced back toward baseline by NCX (18) and PMCA in the plasma membrane and SERCA (26, 134, 150). The Ca²⁺ influx from the extracellular compartment and Ca²⁺ release from ER/SR, in concert with Ca²⁺ extrusion to outside the cell and Ca²⁺ sequestration into the ER/SR, maintain cytoplasmic Ca²⁺ homeostasis (Fig. 3).

FIG. 3.

Cellular components involved in Ca²⁺ homeostasis. All of those components are the targets of ROS. For simplicity, several other pathways, which also regulate the cytosolic Ca²⁺ level, are intentionally omitted, such as the nucleus and the mitochondria that contribute to remove Ca²⁺ from the cytoplasm. ROS, reactive oxygen species.

Enormous studies have demonstrated that all those Ca²⁺ homeostatic proteins are redox sensitive and thus are the targets of ROS (50, 78, 90, 196, 203) (Fig. 3). TRPC6, a Ca²⁺-permeable channel and widely expressed in a variety of tissues/organs, has been revealed to sense ROS and is involved in ROS-induced diverse physiological and pathological responses (111, 203). In the kidney, accumulating evidence has indicated that regulation of TRPC6 protein abundance or channel activity in different parts of the nephron may underlie ROS-induced changes in renal function.

Regulation of TRPC6 Channel by ROS in Kidney Cells

Regulation of TRPC6 by ROS in glomerular MCs

Our earlier study demonstrated that TRPC6 regulated contractile function of MCs (72) and a reduced abundance of TRPC6 protein in MCs might contribute to glomerular hyperfiltration in the early stage of diabetes (71). We recently carried out a series of studies to investigate the mechanism underlying the decrease in TRPC6 protein expression by diabetes. We found that the diabetes/high-glucose-induced downregulation of TRPC6 protein level was mediated by the elevated intracellular level of ROS (72). In cultured MCs, H₂O₂ or overexpression of Nox4 recapitulated the diabetes/high-glucose effect on TRPC6 expression in MCs, and antioxidant treatment or knockdown of Nox4 significantly suppressed high-glucose-induced decrease in TRPC6 expression (72). Importantly, in vivo antioxidant treatment significantly restored TRPC6 expression in glomerulus in diabetic rats (72, 122). We further showed that the ROS effect was mediated by activation of PKC, which phosphorylated NF-κB, resulting in repression of TRPC6 transcription (72, 211).

These findings were supported by Sun et al. who recently reported that astragaloside IV, a potent antioxidant, prevented high-glucose-induced decrease in TRPC6 protein expression through inhibition of the Nox4/Akt/NF-κB pathway (193). Therefore, there is a signaling cascade of high glucose/Nox4/ROS/PKC or Akt/NF-κB/TRPC6 in diabetic MCs, which leads to reduction of TRPC6 protein expression (Fig. 4).

FIG. 4.

Diagram depicting the pathways involved in TRPC6 protein downregulation in glomerular mesangial cells in diabetes.

However, a recent study by Liao et al. showed that chronic hypoxia increased TRPC6 protein expression in MCs (119). Because chronic hypoxia is usually accompanied with oxidative stress (124, 143, 155, 157), the results from the Liao et al. study seem to contradict with ours. However, multiple signaling pathways, such as Ang II signaling, are activated by chronic hypoxia (35, 54, 128, 129, 159). Thus, upregulation of TRPC6 expression might be attributed to an ROS independent mechanism. Indeed, Ang II has been shown to increase the TRPC6 protein expression level in MCs (158, 233).

It should be noted that all aforementioned studies are chronic effects of ROS, that is, altering abundance of TRPC protein. However, ROS can also acutely regulate TRPC6 channel function. In TRPC6-expressing HEK293 cells and vascular smooth muscle cells, H₂O₂ evoked TRPC6-mediated membrane currents in a few minutes by stimulating translocation of cytosolic TRPC6 protein to the cell surface (70). The ROS-induced acute activation of TRPC6 channels in vascular myocytes played an important role in controlling vasoconstrictor-regulated vascular tone (42). Therefore, when considering the overall effect of ROS on TRPC6 channels in MCs, both chronic and acute effects should be taken into account.

Regulation of TRPC6 by ROS in podocytes

As described previously, TRPC6 channels play a critical role in maintaining podocyte structural and functional integrity, and dysfunction of the channels can cause podocyte injury, leading to advanced kidney diseases such as FSGS and diabetic nephropathy (45, 57, 88, 92, 136, 137, 139, 162, 163, 194, 219, 232, 234, 235). Accumulating evidence indicates that TRPC6 expression and function in podocytes are under control of ROS.

Wang et al. were the first group to report increased TRPC6 protein expression by ROS in podocytes (212). In the puromycin aminonucleoside (PAN) rat model of FSGS, they found significant podocyte injury, which was accompanied by increased Nox4 expression and activity, and TRPC6 protein expression in glomeruli from PAN nephrosis rats. Apocynin, an inhibitor of NADPH oxidases, ameliorated the podocyte injury and reduced the PAN-induced TRPC6 expression (212). These in vivo findings were nicely recapitulated in cultured podocytes (212). Therefore, NADPH oxidase-derived ROS contributed to PAN-induced upregulation of TRPC6 expression and podocyte injury.

Diabetic nephropathy is characterized with podocyte injury (45, 92, 139, 162, 194, 232, 234). Increased TRPC6 channel expression and activity have been associated with impaired podocyte structure and function (136, 137, 163, 219, 235). High glucose or hyperglycemia is the major trigger of development of diabetic kidney disease (81, 200). In cultured human podocytes, Thilo et al. recently found that high-glucose treatment significantly increased TRPC6 expression at both mRNA and protein levels. These responses were blocked by tempol, a mimetic of superoxide dismutase, and mimicked by application of oxidative stress (201). Therefore, high glucose increased abundance of TRPC6 protein through ROS in podocytes.

Recent studies showed that insulin signaling to podocytes is crucial for the normal function of the entire glomerulus (23, 217). In cultured mouse podocytes, Kim et al. found that insulin evoked a rapid increase in the cell surface expression of functional TRPC6 (106). This response was mimicked by treating podocytes with H₂O₂. Furthermore, insulin treatment increased H₂O₂ production by mobilizing Nox4 to the cell surface. In addition, the insulin-stimulated surface expression of TRPC6 was reduced by pretreatment with diphenylene iodonium, an inhibitor of NADPH oxidases, by siRNA knockdown of Nox4, and by several antioxidants (106). This study suggests that insulin increases the surface expression of TRPC6 in podocytes through Nox4-derived ROS.

However, the conclusion from the in vitro studies may need to be verified in intact animals. In addition, the pathological relevance of the insulin-promoted cell surface translocation of TRPC6 in podocytes may need to be further elucidated. For instance, how this pathway contributes to podocyte injury in diabetic nephropathy, which is usually associated with insulin deficiency or impairment of insulin signaling. Nevertheless, this study at least revealed that ROS stimulate membrane trafficking of TRPC6 in podocytes, which is consistent with our findings in HEK293 cells (70) and vascular smooth muscle cells (42).

In addition to growth factor signaling (such as insulin signaling), G protein-coupled receptor signaling may also regulate TRPC6 expression and function through generation of ROS in podocytes. Studies demonstrated that Ang II and ATP activated TRPC6 channels through Gq-coupled AT1 and P2Y receptors, respectively, in cultured mouse podocyte cell lines and in primary rat podocytes in ex vivo glomeruli (9, 169). Both Ang II and ATP effects on TRPC6 relied on generation of ROS because pretreating podocytes with ROS quencher or NADPH oxidase inhibitors eliminated or reduced the Ang II and ATP activation of TRPC6 (9, 169). Since Ang II plays a major role in the progression of chronic kidney disease (22, 103, 133, 147, 220) and ATP is an important mediator of tubuloglomerular feedback (5, 24, 28, 49, 192), the findings that ROS mediated Ang II- and ATP-induced TRPC6 activation in podocytes may have important physiological and pathological relevance.

It has been widely accepted that NADPH oxidases are the major source of ROS in the kidney (58, 64–68, 177). Among seven isoforms of NADPH oxidases, Nox4 and Nox2 have been found in podocytes and both of them regulated TRPC6 channel function through ROS (45, 106, 107, 212). In the study described previously, TRPC6 protein expression was upregulated in PAN-treated podocytes. This response was significantly attenuated by knockdown of Nox4 (212). Similarly, knockdown of Nox4 significantly reduced insulin-stimulated cell surface expression of TRPC6 in podocytes (106). However, Nox4 might not be the only NADPH oxidase to regulate TRPC6 in podocytes. In a recent study, Nox2 was found to physically interact with TRPC6 channels in podocytes, and the complexes contributed to TRPC6 activation by diacylglycerol, a product generated in G protein-coupled and receptor tyrosine kinase receptor signaling cascades (107).

Regulation of TRPC6 by ROS in tubular epithelial cells

As described previously, TRPC6 is highly expressed in principal cells of the collecting duct, located at both the apical (colocalized with aquaporin 2) and basolateral (colocalized with Na⁺-K⁺-ATPase) membranes (61). This existence suggests that TRPC6 may play a role in fine-tuning water and Na⁺ reabsorption in the collecting duct.

Using an ischemia–reperfusion rat model of acute kidney injury, Shen et al. found that the impaired water and Na⁺ excretion was associated with a reduced expression of TRPC6 protein in the collecting ducts (181). Pretreatment of the ischemia–reperfusion rats with recombinant human erythropoietin restored TRPC6 protein expression in the collecting ducts, which was accompanied with attenuation of acute renal tubular injury (181). Because ischemia–reperfusion injury is directly related to the formation of ROS (4, 53, 224), the decreased protein abundance of TRPC6 might be due to oxidative stress generated during the procedure. It has been well known that antioxidative stress is one of the major mechanisms for the cytoprotective effects of erythropoietin (36, 118, 229). An earlier study from our laboratory also showed that ROS reduced TRPC6 protein expression in MCs (72).

TRPC6 is also expressed in MDCK cells, a cell line of the intercalated cells of the renal cortical collecting duct (12). Treatment of MDCK cells with thymol, a natural essential oil present in many plants, elevated the intracellular ROS level and stimulated Ca²⁺ entry (31). The thymol-evoked Ca²⁺ influx was significantly inhibited by SK&F96365. Although the authors argued that the Ca²⁺ entry was mediated by store-operated Ca²⁺ channels, the compound of SK&F96365 in fact is a nonselective inhibitor of TRPC channels. Therefore, it is most likely that the thymol-induced Ca²⁺ entry was through one or more isoforms of TRPC channels. MDCK cells express redox-sensitive TRPC6 channels (12). Therefore, it is possible that TRPC6 channels were involved in the thymol-stimulated Ca²⁺ response.

It should be noted that MDCK cells also express TRPC1 and TRPC3 (12) and both may also be redox sensitive (56, 156, 223). Thus, it is uncertain whether TRPC6 was the only channel contributing to the thymol-induced increase in intracellular Ca²⁺.

In summary, ROS, primarily derived from NADPH oxidases, regulate TRPC6 signaling through multiple mechanisms in kidney cells. These mechanisms include alteration of TRPC6 protein abundance, stimulation of cell surface trafficking of cytosolic TRPC6 channel protein, and direct activation of the channel. The influence of ROS on TRPC6 channel function and the underlying mechanisms may be cell context dependent (Fig. 5).

FIG. 5.

Regulation of TRPC6 protein expression or channel activity by ROS in kidney cells.

Concluding Remarks

It is evident that the TRPC6-mediated signaling pathway in kidney cells is under control of ROS under both physiological and pathological conditions. However, the mechanism by which ROS regulate TRPC6 Ca²⁺ signals is poorly understood. ROS may directly or indirectly affect TRPC6 at any site of transcription, post-transcription, translation, and post-translation. If an indirect mechanism is involved, further study is needed to identify the intermediators, which may help search for a more specific target for treating ROS- and/or TRPC6-associated kidney diseases.

In terms of redox modulation of TRP channels, studies have focused on oxidative modifications of the redox-sensitive cysteine residues in proteins (125, 227). However, it is important to note that the oxidation of polyunsaturated fatty acids may also be a mechanism. This is particularly important for TRPC6 because some lipid messengers such as phosphoinositides and dicylglycerol are important regulators of TRPC6 channels (6, 7, 113, 116).

It should be noted that ROS have distinct effects on TRPC6 protein expression in different types of kidney cells. For instance, ROS decreased abundance of TRPC6 protein in MCs (72, 122), but increased TRPC6 protein expression in podocytes (212, 201, 106). Although the mechanism for the cell-type-specific effects of ROS is not known, one possibility is that ROS regulation of TRPC6 is cell context dependent. In MCs, the ROS/PKC or Akt/NF-κB pathway, which downregulates TRPC6 protein expression (211, 193), is predominant. However, this inhibitory pathway may not be very active in podocytes, instead the Ang II signaling pathway, which upregulates TRPC6 expression (9, 169, 231, 142), may play a major role.

In addition to TRPC6, several other isoforms of TRPC channels are also redox sensitive. These include TRPC1, TRPC3, TRPC4, and TRPC5 (56, 156, 223, 227). Although all those studies were carried out in nonrenal cells or the HEK293 cell line, the fact that those TRPC channels are also expressed in kidney cells and are involved in physiological processes in the kidney (12, 43, 59–62, 117, 141, 187, 202, 209, 210, 230) provides a possibility of those TRPC isoforms contributing to ROS-induced changes in renal structure and function.

Recent studies showed that reactive nitrogen species also regulated the function of TRPC channels (111, 203, 227). Like ROS, reactive nitrogen species such as nitric oxide may regulate TRPC signaling through site-specific modifications of amino acid residues such as the nitrosation or nitration of cysteine and tyrosine residues. Whether TRPC6 channel in kidney cells can be modulated by this type of redox is not known and needs to be further studied.

In this review, we focused on studies in which TRPC6 protein was a downstream target of ROS. However, emerging evidence suggests that there is a cross-talk between intracellular Ca²⁺ signals and ROS (21, 90, 203). Particularly, Duox 1 and 2 contain a Ca²⁺ binding domain (EF-hand) (8, 73, 170). It would be interesting to determine if this Ca²⁺-ROS interplay also exists between TRPC6 channel and ROS in kidney cells and, importantly, how it affects renal function.

Considering the ubiquitous nature of TRPC6 channels and ROS production in the kidney, it is conceivable that TRPC6 channel redox sensitivity may participate in multiple physiological and pathological processes. Hence, understanding the molecular mechanisms by which ROS alter TRPC6 channel activity and expression level will help develop specific drugs to treat TRPC6- and/or ROS-associated kidney diseases.

Abbreviations Used

Ang II

angiotensin II

Ank

ankyrin repeats

CBD

caveolin-1 binding domain

CC

coiled-coiled domain

CD

collecting duct

CIRB

calmodulin/IP₃R binding domain

ER

endoplasmic reticulum

ERK

extracellular signal-regulated kinase

FSGS

focal and segmental glomerulosclerosis

H₂O₂

hydrogen peroxide

IC

intercalated cell

IP₃

inositol 1,4,5-trisphosphate

IP₃R

inositol 1,4,5-trisphosphate receptor

JG

juxtaglomerular

MC

mesangial cell

MDCK

Madin–Darby canine kidney

NCX

Na⁺-Ca²⁺ exchanger

NFAT

nuclear factor of activated T cells

PAN

puromycin aminonucleoside

PC

principal cell

PKC

protein kinase C

PMCA

plasma membrane Ca²⁺ ATPase

ROS

reactive oxygen species

RyR

ryanodine receptor

SERCA

sarcoplasmic and endoplasmic reticulum Ca²⁺ ATPase

SR

sarcoplasmic reticulum

STZ

streptozotocin

TRP

transient receptor potential

TRPC

canonical transient receptor potential