Endoplasmic Reticulum Calcium Homeostasis in Kidney Disease

Abstract

Calcium (Ca²⁺) homeostasis is a crucial determinant of cellular function and survival. Endoplasmic reticulum (ER) acts as the largest intracellular Ca²⁺ store that maintains Ca²⁺ homeostasis through the ER Ca²⁺ uptake pump, sarco/ER Ca²⁺ ATPase, ER Ca²⁺ release channels, inositol 1,4,5-trisphosphate receptor channel, ryanodine receptor, and Ca²⁺-binding proteins inside of the ER lumen. Alterations in ER homeostasis trigger ER Ca²⁺ depletion and ER stress, which have been associated with the development of a variety of diseases. In addition, recent studies have highlighted the role of ER Ca²⁺ imbalance caused by dysfunction of sarco/ER Ca²⁺ ATPase, ryanodine receptor, and inositol 1,4,5-trisphosphate receptor channel in various kidney diseases. Despite progress in the understanding of the importance of these ER Ca²⁺ channels, pumps, and binding proteins in the pathogenesis of kidney disease, treatment is still lacking. This mini-review is focused on: i) Ca²⁺ homeostasis in the ER, ii) ER Ca²⁺ dyshomeostasis and apoptosis, and iii) altered ER Ca²⁺ homeostasis in kidney disease, including podocytopathy, diabetic nephropathy, albuminuria, autosomal dominant polycystic kidney disease, and ischemia/reperfusion-induced acute kidney injury.

The endoplasmic reticulum (ER) is the main intracellular calcium (Ca²⁺) store and plays an essential role in intracellular Ca²⁺-mediated cell signaling.¹ Intracellular Ca²⁺ is a ubiquitous and versatile signaling molecule that controls many fundamental cellular processes, including proliferation, differentiation, secretion, metabolism, contraction, and cell death.^2,3 Thus, the maintenance of ER Ca²⁺ homeostasis is crucial for cell function and survival.

The ER maintains intracellular Ca²⁺ homeostasis through the integrated and coordinated processes of Ca²⁺ uptake, release, and binding, which are controlled by an ER-localized Ca²⁺ pump [sarco/ER Ca²⁺ ATPase (SERCA)], two Ca²⁺-release channels [inositol 1,4,5-trisphosphate receptor channel (IP₃R) and ryanodine receptor (RyR)],^4,5 and Ca²⁺-binding proteins, respectively. The lumen of the ER has a greater Ca²⁺ concentration than does the cytosol.⁶ The Ca²⁺ stored in the ER lumen is essential for the regulation of protein post-translational modification, folding, and transport. ER Ca²⁺ released to the cytosol provides sustained and precise Ca²⁺-mediated cellular responses,^3,4 the dysregulation of which may lead to cell death.

Alterations in ER Ca²⁺ homeostasis result in the accumulation of unfolded proteins, which in turn causes ER stress and activates the unfolded-protein response.⁴ Depending on the duration and severity of the stress, activation of the unfolded protein response can lead to either cell survival or cell death. The unfolded-protein response triggers an adaptive response, through the activation of activating transcription factor 6, protein kinase RNA–like ER kinase (PERK), or inositol-requiring enzyme (IRE)-1 signaling pathway, to relieve stress and restore ER function.⁷ However, if ER stress is too severe or prolonged, the unfolded-protein response switches to proapoptotic pathways through the activation of CCAAT/enhancer-binding protein homologous protein (CHOP), Jun N-terminal kinase (JNK), or caspase 12. In addition, ER Ca²⁺ depletion can induce a subsequent increase in cytosolic Ca²⁺, which in turn activates cytosolic Ca²⁺-dependent cysteine proteases (calpains), which cleave ER-resident procaspase 12, leading to the activation of caspase cascades and, ultimately, cellular dysfunction and cell death.⁸

Disturbances in ER Ca²⁺ homeostasis contribute to the pathophysiology of various diseases, including diabetes mellitus, neurologic disorders, cancer, and kidney disease.2, 3, 4^,6 The present review discusses the molecular mechanisms underlying Ca²⁺ dyshomeostasis–mediated kidney disease, with a focus on ER Ca²⁺ pumps, receptor channels, and Ca²⁺-binding proteins.

Ca²⁺ Homeostasis in the ER

Intracellular free Ca²⁺ concentration varies widely depending on its location. Cytosolic Ca²⁺ concentration is maintained at low levels (10 to 100 nmol/L) against a high (10,000-fold) Ca²⁺ concentration in the ER and in the extracellular milieu.^4,9 Inside of the cell, Ca²⁺ levels in the nuclear matrix and in the mitochondria matrix are similar to that in the cytosol.⁹ The largest intracellular Ca²⁺ store is the ER that can accumulate Ca²⁺ and maintain a high Ca²⁺ concentration (100 to 500 μmol/L). The Ca²⁺ gradient is conserved between different intracellular organelles and the cytosol, which facilitates a variety of Ca²⁺-mediated cell signaling.³ The tight regulation of ER and cytosolic Ca²⁺ levels is achieved by the orchestrated action of Ca²⁺ pumps, channels, and Ca²⁺-binding ER luminal proteins, which are located in the ER membrane, ER lumen, and ER–mitochondrial contact sites^3,4 (Figure 1).

Figure 1.

ER Ca²⁺ dynamics and homeostasis. ER Ca²⁺ homeostasis is controlled by the orchestrated action of SERCA, IP₃R, RyR, and Ca²⁺-regulating proteins. Ca²⁺ uptake into the ER from the cytosol is mainly driven by SERCA. Phosphorylation of phospholamban (PLN) activates SERCA through stimulation of the β-adrenergic receptor (AR)/protein kinase (PK)-A signaling pathway. When the Ca²⁺ stores in the ER are full, calnexin (CNX) and CRT reduce the activity of SERCA. Meanwhile, Ca²⁺ release from the lumen of the ER into the cytosol is mediated mainly by IP₃R and RyR. IP₃R is activated by binding of IP₃ that is produced through G protein-coupled receptor (GPCR)-mediated activation of PLC. IP₃R is inhibited by ER resident protein ERp44, depending on ER Ca²⁺ concentration. RyR activity is modulated by phosphorylation of serine and calstabin binding. PKA activation via β-AR stimulation directly phosphorylates RyR and increases its activity, inducing dissociation from calstabins. Once Ca²⁺ is depleted in the ER lumen, dissociation of Ca²⁺ from the STIM proteins leads to STIM oligomerization and interaction with the plasma membrane Ca²⁺ release–activated Ca²⁺ channel protein (Orai) to increase Ca²⁺ influx from the extracellular milieu. SERCA colocalizes with STIM–Orai complex and pumps Ca²⁺ directly from the cytosol to allow ER Ca²⁺ store refilling. ERp57 interacts with CNX, CRT, and STIM, regulating SERCA activity and SOCE. Furthermore, Ca²⁺ store in the ER is closely associated with ER protein homeostasis. Alteration in ER homeostasis activates unfolded protein response through three ER stress sensors [inositol-requiring enzyme (IRE)-1, protein kinase RNA–like ER kinase (PERK), and activating transcription factor (ATF)-6] that are inactive and bound to BiP in the absence of stress. AC, adenylyl cyclase; ATF, activating transcription factor; Gs, stimulatory G-protein; MCU, mitochondrial Ca²⁺ uniporter.

ER Calcium Uptake Pump SERCA

Ca²⁺ uptake into the ER from the cytosol is mainly driven by SERCA pumps. The SERCA pumps are integral transmembrane proteins of the ER that consume the energy of ATP to drive Ca²⁺ across the membrane against electrochemical gradient.⁵ The SERCA pumps are encoded by a family of three genes (SERCA1 to SERCA3) with different expression and Ca²⁺ affinities in a cell-specific manner.^2,3 SERCA1 is expressed in skeletal muscles, and SERCA2a is expressed in cardiac and skeletal muscles, while SERCA2b and SERCA3 are present in nonmuscle tissues.¹⁰ The function of the SERCA pump is modulated by membrane proteins phospholamban (PLN) and sarcolipin.^3,10 The phosphorylated form of PLN activates cardiac SERCA pump through a β-adrenergic response by either protein kinase (PK) A or calmodulin-dependent kinase (CaMK) II, whereas the dephosphorylated form of PLN inhibits SERCA activity.^5,10 A similar regulation of the SERCA pump in skeletal muscle is accomplished by sarcolipin.⁵ SERCA activity is regulated by certain ER luminal chaperones such as calnexin (CNX) and calreticulin (CRT).^3,11,12 When the Ca²⁺ store in the ER is full, CNX and CRT physically interact with SERCA2b and inhibit its activity, thereby controlling Ca²⁺ homeostasis.^11,12 It has been shown that overexpression of CRT promotes inactivation and degradation of SERCA2a in oxidative stress, leading to an alteration in Ca²⁺ homeostasis and enhanced susceptibility to apoptosis.¹²

ER Calcium-Release Channels IP₃R and RyR

Ca²⁺ release from the lumen of the ER into the cytosol is mediated mainly by IP₃R and RyR channels localized on the ER membrane.^3,4 Both channels exist in three isoforms (IP₃Rs 1 to 3 and RyRs 1 to 3), which are assembled to produce large hetero- and homotetrameric Ca²⁺-release channels. IP₃R and RyR channels have different Ca²⁺ affinities and are expressed in distinct tissues.^2,3 IP₃R channels are ubiquitously expressed in most cell types.¹³ IP₃R channels are activated by IP₃ that is produced when phospholipase (PL) C hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂).^13,14 Binding of a ligand or other agonists to a G-protein–coupled receptor results in G-protein–subunit activation, which in turn triggers PLC-mediated cleavage of PIP₂ to IP₃ and diacylglycerol.^4,14 The IP₃ acts as an intracellular messenger and binds to IP₃R, stimulating Ca²⁺ release from the ER to the cytosol.^2,4 Ca²⁺ release by IP₃R is inhibited by ER resident protein (ERp)-44 (depending on pH), ER Ca²⁺ concentration, and redox state. ERp44, a novel ER luminal oxidoreductase of the thioredoxin family, senses the environment in the ER lumen and inhibits IP₃R1 activity through direct interaction with the ER luminal domain of IP₃R1.¹⁵ Numerous modulatory proteins have been shown to bind to IP₃R and modulate its function, including IP₃R-binding protein released with IP₃, calmodulin, and caldendrin.^5,14

RyR is a member of the same gene family as IP₃R, and there is approximately 40% homology between RyR and IP₃R in their putative transmembrane regions.¹⁴ Although structurally related, RyRs and IP₃Rs have distinct physiologic profiles. The RyR family has three isoforms: i) RyR1 in skeletal muscle, ii) RyR2 in heart, and iii) RyR3 in brain. Recently, these isoforms have also been found in other tissues.^5,16 Although the expression level of RyR is much lower than that of IP₃R in most cell types, the role of RyR is important; the opening of RyR releases about 20-fold more Ca²⁺ ions than does that of IP₃R.¹³ In addition, RyR has high-affinity Ca²⁺-binding sites involved in triggering Ca²⁺-induced Ca²⁺ release to the cytosol.^17,18 The open probability of RyR depends on the cytosolic Ca²⁺ concentration; RyR is normally closed at low cytosolic Ca²⁺ levels (approximately 100 to 200 nmol/L), and opened at sub-μM cytosolic Ca²⁺ levels due to Ca²⁺ binding to high-affinity binding sites on RyR, which increases the open probability. The channel activity is maximal at a cytosolic Ca²⁺ concentration of approximately 10 μmol/L, while elevated levels beyond this point lead to a reduction in open probability.^14,18 Ca²⁺ influx across cell membrane via the voltage-gated L (long lasting)-type Ca²⁺ channel (CaV1.2) activates RyR2 and triggers Ca²⁺-induced Ca²⁺ release, leading to myocyte contraction.^16,18

RyR activity is modulated by phosphorylation and multiple binding proteins. β-Adrenoceptor stimulation leads to stimulatory G-protein–mediated activation of adenylyl cyclase (AC) and further cAMP-dependent activation of PKA. PKA can directly phosphorylate RyR1 at S2844 and RyR2 at S2808, causing reductions in the binding affinities to Ca²⁺ channel–stabilizing binding proteins (calstabins) 1 and 2, respectively.^19,20 Calstabins stabilize the closed state of RyR channels and prevent excessive ER Ca²⁺ leak.¹⁴ Hyperphosphorylation of RyR2 S2808 by PKA results in a diastolic sarcoplasmic reticulum Ca²⁺ leak in cardiomyocytes that contributes to chronically decreased sarcoplasmic reticulum Ca²⁺ contents and progressive cardiac dysfunction.²⁰ Recently, it was also found that ER-stressed podocytes undergo phosphorylation at S2808, causing leaky RyR2 and podocyte apoptosis.²¹ On the other hand, increased cytosolic Ca²⁺ levels activate CaMK II, which directly phosphorylates RyR2 at S2814 and increases open probability of RyR2, not via calstabin 2 dissociation.²² Other regulatory proteins, such as calmodulin, calsequestrin, triadin, and junctin, interact with RyR and regulate its function.^14,16

ER Calcium Refill

ER Ca²⁺ depletion promotes the entry of Ca²⁺ from the extracellular space into the cell to refill intracellular Ca²⁺ stores. This process is known as capacitive or store-operated Ca²⁺ entry (SOCE) and is activated by the ER sensor, stromal-interacting molecule (STIM).^2,3 Two homologous STIMs, 1 and 2, are ER-resident proteins containing a single transmembrane domain and two EF-hand domains for sensing ER Ca²⁺ depletion.⁵ After Ca²⁺ is dissociated from the EF-hand domains of the STIM proteins, STIM oligomerizes and interacts with the plasma membrane channel, Ca²⁺ release–activated Ca²⁺ channel protein (Orai), to open Ca²⁺ entry from the extracellular milieu to the cytosol.^2,4,5 In turn, SERCA co-localizes with the STIM–Orai complex, which allows Ca²⁺ to be pumped directly from the cytosol into the ER lumen to replenish the deficit.²³

ER Ca²⁺-Binding Proteins

In the ER, Ca²⁺ is bound to luminal proteins, the most abundant of which are CRT and calsequestrin. CRT predominates in nonmuscle cells, whereas calsequestrin is more restricted to the muscle cells. In addition to these ER Ca²⁺-storage proteins, there are a number of Ca²⁺-binding molecular chaperones. These include CNX, CRT, Ig-binding protein/78-kDa glucose–regulated protein (BiP/GRP78), and GRP94. A third class of Ca²⁺-binding proteins includes several oxidoreductases in the ER lumen, which comprise protein disulfide isomerases (PDIs), ERp44 and ERp57 (also known as PDI3), as well as ER oxidoreductin 1.^2,4,24 These molecular chaperones and folding enzymes can not only buffer ER free Ca²⁺ molecules, but also catalyze protein folding and processing.

Moreover, some of these proteins are involved in the regulation of the activities of Ca²⁺ pumps and channels, which contribute to the maintenance of a high free Ca²⁺ environment in the ER lumen.³ The ER luminal ERp57, a glycoprotein-specific thiol-disulfide oxidoreductase, can inhibit SOCE by interacting with the ER luminal domain of STIM1²⁵. ERp57 also interacts with CNX and CRT to modulate the activity of SERCA2b. Taken together, the integration of these Ca²⁺-handling proteins is essential for the control of steady-state ER luminal Ca²⁺ levels, which contribute to the maintenance of intracellular ER Ca²⁺ homeostasis.

ER Ca²⁺ Dyshomeostasis and Apoptosis

ER Ca²⁺ homeostasis critically controls cell survival and cell death.1, 2, 3, 4^,26,27 Physiologic Ca²⁺ release from the ER induces Ca²⁺ uptake by mitochondria, which in turn results in increased mitochondrial respiration and ATP production, enhancing mitochondrial bioenergetics and regulating cell survival.²⁶ The subsequent reduction in ER luminal Ca²⁺ is restored via SOCE and mediated by the STIM–Orai complex and the activation of SERCA.³ In contrast, pathologic conditions may cause an imbalance between ER Ca²⁺ release and uptake mechanisms (Figure 2). Impaired functioning of mitochondrial uptake, SOCE, or SERCA; increased activity of IP₃R or RyR; or altered modulation by Ca²⁺-regulating proteins may contribute to both decreased ER Ca²⁺ and increased mitochondrial and cytosolic Ca²⁺, thus failing to maintain normal ER luminal Ca²⁺ levels.2, 3, 4 The reduced ER luminal Ca²⁺ can immediately provoke ER stress and apoptosis by disrupted Ca²⁺-dependent chaperone function and subsequent accumulation of unfolded proteins in the ER.²⁸ The increased mitochondrial and cytosolic Ca²⁺ influx may augment apoptotic cell death through the activation of Ca²⁺-dependent mitochondrial permeability transition pore and enzymes, such as CaMK II and calpain.^1,4,26,27,29

Figure 2.

ER Ca²⁺ dysregulation and apoptosis. Enhanced chronic ER Ca²⁺ release elevates mitochondrial and cytosol Ca²⁺, which causes apoptosis through dysfunction of mitochondrial permeability transition pore (mPTP), CaMK II and calpain. Increased mitochondrial PTP opening results in mitochondrial cytochrome c release. CaMK II activation stimulates RyR phosphorylation and the Jun N-terminal kinase (JNK)-induced apoptotic pathway. Calpain activation cleaves truncated B-cell lymphoma 2 homology 3–interacting domain death agonist (tBid) and caspase 12, mediating apoptosis. Moreover, excessive ER Ca²⁺ depletion results in accumulation of misfolded proteins, which triggers ER stress-induced apoptosis through activation of CCAAT/enhancer-binding protein homologous protein (CHOP), JNK, and caspase12. ER stress and increased ER Ca²⁺ efflux can amplify each other. ATF, activating transcription factor; BAK, B-cell lymphoma 2 homologous antagonist/killer; CNX, calnexin; IRE, inositol-requiring enzyme; MCU, mitochondrial Ca²⁺ uniporter; PERK, protein kinase RNA–like ER kinase; PLN, phospholamban.

Calcium Dysregulation and ER–Mitochondria Crosstalk

Regulated transfer of Ca²⁺ from the ER to mitochondria occurs through the opening of the IP₃R on the ER membrane and the mitochondrial Ca²⁺ uniporter complex on the inner mitochondrial membrane. Excessive Ca²⁺ release from the ER can accumulate in the mitochondria.²⁷ Mitochondrial Ca²⁺ overload increases the production of reactive oxygen species and the opening of the mitochondrial permeability transition pore. The permeability transition pore opening causes mitochondrial swelling, collapse of mitochondrial membrane potential, rupture of the outer mitochondrial membrane, and subsequent release of proapoptotic factor cytochrome c.^1,4,26,27,29 Cytochrome c released into the cytosol binds to apoptosis-activating factor 1 to form the apoptosome complex, which can then recruit and activate the initiator caspase, caspase 9, and its downstream effector caspases, triggering apoptosis.^26,27,29 Furthermore, sarcoplasmic reticulum Ca²⁺ leak via RyR2 triggers mitochondrial Ca²⁺ overload and increases reactive oxygen species production, which in turn further oxidizes RyR2 and enhances sarcoplasmic reticulum Ca²⁺ efflux, thereby contributing to heart failure after myocardial infarction.³⁰

Cytosolic Ca²⁺ Signaling and CaMK II

Increased cytosolic Ca²⁺ activates CaMK II, which promotes inflammation and apoptosis.^4,31 In toxicant-treated bone-marrow B cells, CaMK II is activated by IP₃R-mediated Ca²⁺ release, leading to p38 and JNK phosphorylation^4,32 and the subsequent induction of intrinsic apoptosis, accompanied by a loss of mitochondrial membrane potential, the release of cytochrome c, and the activation of caspase 3.³³ In cholesterol-loaded macrophages, CaMK II, which is activated by accelerated ER to cytosol Ca²⁺ leakage, enables ER stress-induced apoptosis through the activation of the JNK-induced Fas apoptotic pathway and the promotion of mitochondrial Ca²⁺ uptake, followed by mitochondrial membrane permeabilization.³⁴ Moreover, activated CaMK II in cardiomyocytes after ischemia/reperfusion (I/R) injury results in NF-κB activation, elevated inflammatory response, and apoptosis.³¹ Sustained, excessive CaMK II activation is also implicated in obesity, advanced atherosclerotic lesions, and myocardial hypertrophy.^4,31, 32, 33, 34

Cytosolic Ca²⁺ Signaling and Calpain

Cytosolic Ca²⁺ elevation can also activate calpain by an autocatalytic cleavage, leading to the activation of key downstream apoptotic factors, such as caspase 12 and truncated BH3–interacting domain death agonist (tBid).^26,35 For instance, cadmium-mediated inhibition of SERCA disrupts ER Ca²⁺ homeostasis and causes apoptosis through the calpain–caspase 12 pathway.³⁶ In addition, calpain induces the cleavage of tBid, which promotes the oligomerization of BAX and/or BAK, and the release of cytochrome c,³⁵ as occurs in cisplatin-treated melanoma cells.³⁷

Altered ER Ca²⁺ Homeostasis in Kidney Disease

The functional units of the kidneys are the nephrons, which filter the blood, reabsorb water and solutes, and secrete wastes to the urine to maintain body homeostasis. Approximately 1 million nephrons are contained in the renal parenchyma, the functional portion of the kidney consisting of cortex and medulla. Each nephron is composed of a glomerulus and renal tubules, and the filtration barrier of the glomerulus is composed of glomerular endothelial cells, glomerular basement membrane, and podocytes. Each cell type contains a unique combination of Ca²⁺ pumps and channels to elicit the cell type–specific Ca²⁺ signaling required for specific functions.⁹ The role of ER Ca²⁺ dyshomeostasis in the pathogenesis of various kidney diseases is still not well understood. This review focuses on the role of dysfunctional ER Ca²⁺ channels, pumps, and Ca²⁺-handling proteins in the pathogenesis of a variety of kidney diseases, including podocytopathy, diabetic nephropathy, albuminuria, autosomal dominant polycystic kidney disease (ADPKD), and I/R-induced tubular injury. Also included are novel targeted therapies for the treatment of ER stress–induced kidney disease.

Monogenic Podocytopathy and RyR2

Familial focal segmental glomerulosclerosis and nephrotic syndrome are a primary podocytopathy caused by podocyte-specific mutations in genes including NPHS1, NPHS2, WT1, LAMB2, CD2AP, TRPC6, ACTN4, and INF2.³⁸ Hereditary familial focal segmental glomerulosclerosis has drawn considerable attention recently due to the molecular insights into the disease pathogenesis. Laminin (LAM)-β2, encoded by LAMB2, is synthesized and secreted from both podocytes and glomerular endothelial cells, and is a major component of the mature glomerular basement membrane. LAMB2 is also one of the most commonly mutated disease-causing genes, and C321R-LAMB2 missense mutation causes congenital nephrotic syndrome.³⁹ Our study has shown that podocyte ER stress induced by C321R-LAMB2 mutation causes ER Ca²⁺ leak and cytosolic Ca²⁺ elevation through phosphorylation of RyR2 at Ser2808.²¹ The cytosolic Ca²⁺ overload accelerates calpain 2 activation and cleavage of its downstream apoptotic molecule procaspase 12 and podocyte cytoskeletal protein talin 1, resulting in podocyte apoptosis and injury. Importantly, the ER Ca²⁺ stabilizer K201 can block RyR2 phosphorylation–mediated ER Ca²⁺ depletion and subsequent calpain 2–caspase 12 activation in the C321R-mutant podocytes, as well as mitigate albuminuria in the Lamb2^−/− mice expressing the mutant C321R-Lamb2 in podocytes. In addition, mesencephalic astrocyte–derived neurotrophic factor, as a biotherapeutic ER protein, can fix leaky RyR2 and inhibit ER stress–induced podocyte injury.²¹

Gene mutations in transient receptor potential channel subfamily C member 6 (TRPC6) gene, the main Ca²⁺-permeable ion channel in the plasma membrane of nonexcitable cells, including podocytes, also cause inherited familial focal segmental glomerulosclerosis. R895C and E897K TRPC6 mutations enhance channel activity and elevate cytosolic Ca²⁺ levels, which can activate cytosolic phosphatase calcineurin, resulting in dephosphorylation and translocation of nuclear factor of activated T cells to the nucleus to activate calcineurin-dependent transcription.⁴⁰ Podocyte nuclear factor of activated T-cell activation in mice reduces the expression of podocyte slit-diaphragm proteins, such as podocin, synaptopodin, and nephrin, and thus causes podocyte dysfunction and impairment of glomerular filtration barrier, eventually leading to progressive proteinuria and familial focal segmental glomerulosclerosis. Thus, Ca²⁺-regulated nuclear factor of activated T-cell signaling in podocytes may be a key intermediate factor in the pathogenesis of TRPC6 mutation–induced familial focal segmental glomerulosclerosis.⁴¹

Diabetic Nephropathy and SERCA

Diabetic nephropathy is the most common cause of end-stage renal disease worldwide. Convergent evidence reveals that SERCA2 activity and expression are diminished in islet, heart, and liver of animal models of diabetes, implying a potential pathologic role of SERCA2 dysfunction in the development of diabetic complications.42, 43, 44 In addition, significant reductions in SERCA2 activity and expression have been noted in the kidney cortex of db/db mice, a mouse model of type 2 diabetes.⁴⁵ The impaired activity and expression of SERCA2 caused ER Ca²⁺ depletion, which triggered ER stress and activation of ER stress– and mitochondria-mediated apoptotic pathways. Importantly, astragaloside (AS)-IV, a small molecular bioactive saponin isolated from Astragalus membranaceus, restored SERCA2 activity and expression, and suppressed aberrant ER Ca²⁺–induced apoptosis in db/db mouse kidneys. Moreover, AS-IV ameliorated albuminuria, glomerulosclerosis, and renal inflammation, as well as improved renal function through the up-regulation of SERCA2 function.⁴⁵

Albuminuria and ER Ca²⁺ Depletion

When the permeability of glomerular capillary wall increases, macromolecules such as albumin leak into urinary space, leading to proteinuria. Proteinuria is a clinical marker and a potent predictor of the progression of chronic kidney disease.⁴⁶ In addition, it is an active player in the development of chronic kidney disease.⁴⁷ In podocytes, albumin can stimulate Ca²⁺ release from the ER Ca²⁺ store and Ca²⁺ influx through TRPC6, the overexpression of which has been noted in human proteinuric kidney disease. TRPC6-mediated Ca²⁺ entry triggers ER stress–induced apoptosis and F-actin cytoskeleton disruption in podocytes.⁴⁶ In tubular cells, it has been shown that albumin rapidly elevated cytosolic Ca²⁺ by triggering Ca²⁺ efflux from the ER, which in turn activated SOCE to maintain the rise of cytosolic Ca²⁺.⁴⁷ The albumin-induced ER Ca²⁺ depletion up-regulated activating transcription factor 4–dependent lipocalin (LCN)-2 expression, which induced apoptosis by augmenting reactive oxygen species generation.⁴⁷ Consistently, Lcn2 has also been remarkably increased in the renal tubules of proteinuric chronic kidney disease patients and proteinuric WT1^+/mut mice. Treatment with a chemical chaperone, 4-phenylbutyric acid, or Lcn2 deficiency, has been reported to alleviate ER stress–induced apoptosis and tubulointerstitial damage in WT1^+/mut or acquired proteinuric nephropathy mice. Moreover, 4-phenylbutyric acid dramatically reduced urinary LCN2 excretion in proteinuric patients.⁴⁷

ADPKD and Disturbed Polycystin–Mediated Ca²⁺ Signaling

ADPKD is characterized by the formation and progressive enlargement of fluid-filled renal cysts that lead to end-stage renal disease in more than half of patients by the age of 60 years. Loss-of-function mutations in PKD1 or PKD2, which encode polycystins (PCs) 1 and 2, respectively, are the most common causes of ADPKD.^48,49 PCs 1 and 2 form a heterodimeric complex through their C-terminal regions in the plasma membrane, primary cilia, and ER membrane. PC1 is a large, 4302–amino acid transmembrane protein that serves as a sensor. PC2 is a 968–amino acid transmembrane protein primarily localized in the ER. Known as a member of the TRPC superfamily, PC2 is a nonselective cation channel.⁴⁸ The co-assembly of PC1/PC2 mediates Ca²⁺ influx at the plasma membrane,⁵⁰ and is involved in mechanosensation and flow-dependent Ca²⁺ signaling in the primary cilium.⁵¹ Furthermore, PC1 binds to IP₃R and STIM1 in the ER. PC2 interacts with TRPC subfamily C member 1 in the primary cilium and plasma membrane, TRPC subfamily V member 4 in the cilia, as well as IP₃R and RyR2 in the ER. Thus, PCs 1 and 2 also act as regulators of other Ca²⁺ channel proteins, ultimately modulating Ca²⁺ fluxes and ER Ca²⁺ stores.⁴⁹

Disrupted Ca²⁺ homeostasis through aberrant functioning of polycystins is linked to the development of ADPKD, in which cyst-lining epithelial cells show increased rates of both proliferation and apoptosis. Loss of PC1 and PC2 function results in a decrease in cytosolic Ca²⁺, and an increase in cAMP levels via the activation of Ca²⁺-dependent AC, which activates PKA and stimulates its downstream Src/Ras/B-Raf/MEK/ERK pathway to promote the growth and proliferation of cystic cells.^52,53 The compound deletion of both PC1 and AC6 in collecting duct attenuates PKD.⁵⁴ Moreover, Ca²⁺ restriction affects PI3K/AKT, Wnt, and mechanistic target of rapamycin signaling to augment cystic cell proliferation.

ER Ca²⁺ signaling regulated by polycystins plays a key role in the increased apoptosis in ADPKD. The disruption of PC1 function down-regulates the PI3K/AKT pathway. AKT can directly phosphorylate IP₃R1 at S2681, thereby inhibiting its activity.⁵⁵ Suppressed AKT activity in cystic cells relieves the inhibition of the IP₃R and contributes to the increase in IP₃-induced Ca²⁺ release.^48,49,55 On the other side, PC1 expression stimulates PI3K/AKT, which enhances the interaction between IP₃R and STIM1, thus inhibiting IP₃-induced Ca²⁺ release. PC1 can also bind to IP₃R, mitigating ER Ca²⁺ release, as well as bind to STIM1, sequestering it to the ER membrane. The dissociation of STIM1 from Orai in the plasma membrane inhibits SOCE. In contrast, the association of PC2 with IP₃R prolongs IP₃-induced Ca²⁺ release. Kidney epithelial cells overexpressing PC2 show markedly augmented ER Ca²⁺ release that is lost after C-terminal truncation or by the introduction of a disease-causing PKD2 missense mutation.^50,56 Thus, PC1 or STIM1 competes with PC2 for binding to IP₃R, which may be involved in controlling intracellular Ca²⁺ homeostasis.^49,57 Increased IP₃-induced Ca²⁺ release, particularly at the contact sites of the ER and mitochondria, mitochondria-associated membranes, constitutes a strong apoptotic signal. Recently, PC2 was shown to be enriched at mitochondria-associated membranes. In PC2-knockdown kidney epithelial cells, collecting duct–specific Pc2-knockdown mice, and cyst-lining epithelial cells from human ADPKD kidneys, the expression of the ER–mitochondrial tethering protein mitofusin 2, an outer mitochondrial membrane GTPase, is enhanced, and the ER to mitochondria Ca²⁺ transfer is increased.⁵⁸ Thus, PC2 also acts as a checkpoint for ER-mediated mitochondrial Ca²⁺ signaling, and loss of this regulation may contribute to ADPKD.

I/R-Induced Acute Kidney Injury and Intracellular Calcium Imbalance

Acute kidney injury is estimated to account for 2 million deaths worldwide each year and is a growing global health concern.⁵⁹ It has become clear that an episode of acute kidney injury is associated with an increased risk for chronic kidney disease, as well as for both short- and long-term mortality in children.⁶⁰ Renal I/R injury after hypotension due to various clinical problems is the leading cause of acute kidney injury in both native and transplanted kidneys.

Renal ischemia causes the accumulation of massive unfolded and misfolded proteins in the ER of renal tubular cells due to cellular ATP depletion and changes in Ca²⁺ homeostasis.^61,62 The high-energy demand of proximal tubular cells makes them especially vulnerable to ischemia. In a rat model of renal I/R injury, Szebenyi et al⁶³ demonstrated that renal ischemia caused a rapid and transient increase in cytoplasmic Ca²⁺ levels in proximal tubular cells, which returned to the basal level before the end of the ischemic period. Notably, reperfusion led to a secondary proximal tubular Ca²⁺ load, which was significantly decreased by a specific and potent blocker of Na–Ca exchanger (Ncx1), KB-R7943.

NCX1 is a plasma membrane anti-transporter that is also located in mitochondrial and ER membrane. In normal tubular cells, it uses ATP that is stored in the electrochemical gradient of Na⁺ by allowing Na⁺ to flow down its gradient across the plasma membrane in exchange for intracellular Ca²⁺ extrusion. In contrast, in ischemic tubular cells, the intracellular Na⁺ concentration rises from inhibition of the Na⁺/K⁺ ATPase activity due to decreased ATP production, and activation of the Na⁺/H⁺ exchanger due to intracellular acidosis. In turn, reperfusion may cause a large Ca²⁺ influx because of the reverse mode of the NCX1.⁶⁴ Yamashita et al⁶⁴ showed that NCX1^+/– mice were more resistant to I/R-caused kidney injury compared with wild-type mice. Meanwhile, Ca²⁺ deposition in necrotic tubular epithelium was less marked in heterozygous mice than in wild-type mice. In addition, both pre- and postischemic treatments with the NCX1 inhibitor KB-R7943 attenuated I/R-induced renal injury, suggesting that NCX1 is a potential therapeutic target in I/R injury.

Renal tubular Ca²⁺ homeostasis is also fine-tuned by ER Ca²⁺ channels, pumps, and Ca²⁺-binding proteins.^3,5 It has been reported that with hypoxia induced by 6 hours of exposure to 8% oxygen, mRNA levels of IP₃R1 and RyR2 in mouse kidneys were increased.⁶⁵ Furthermore, a study in a rat model reported that with pretreatment with an IP₃R blocker, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), but not with the RyR2 inhibitor dantrolene, 15 minutes before renal ischemia⁶⁶ kidney function and findings on histologic examination were improved. In vitro, with TMB-8, but not dantrolene, cytosolic Ca²⁺ elevation and tubular cell apoptosis were significantly suppressed during antimycin A–induced chemical anoxia.

Using biochemical assays, it has been shown that IP₃R1 is physically associated with the carboxy terminus of ER-localized PC2, and that IP₃R-mediated intracellular Ca²⁺ signaling is modulated by PC2.⁶⁷ Interestingly, it has been reported that Pc2^+/– mouse kidneys are more sensitive to I/R injury, followed by enhanced tubular and interstitial proliferation, tubular apoptosis, inflammation, as well as interstitial fibrosis after I/R surgery.⁶⁸ These effects might be attributable to misregulated intracellular Ca²⁺ signaling stemming from altered activity levels of PC2 and IP₃R1, which was not examined in the study.⁶⁸

BiP, the activity of which is induced in mouse kidney after bilateral I/R injury, is also an essential player in the modulation of ER Ca²⁺ homeostasis and Ca²⁺-related signal transduction.⁶⁹ BiP has been associated with the highly conserved ER chaperone protein sigma-1 receptor (Sig-1R) at the ER–mitochondrial contact sites, mitochondria-associated membranes. When excessive Ca²⁺ is released from the ER, Sig-1R is dissociated from BiP and binds to IP₃R3, which attenuates IP₃R3 degradation, stabilizes IP₃R3 at mitochondria-associated membranes, and prolongs Ca²⁺ signaling to the mitochondria.⁷⁰ It has been reported in a rat model that pretreatment with fluvoxamine, a Sig-1R agonist, improved survival and renal function, as well as ameliorated renal inflammatory response after renal I/R injury via Akt-mediated nitric oxide signaling and increased peritubular vasodilation and renal perfusion.⁷¹ However, the role of ER or mitochondrial Ca²⁺ homeostasis in this rat I/R injury model was not checked. Additionally, BiP regulates ER Ca²⁺ homeostasis by closing the Sec61 channel during protein translocation. The Sec61 translocon of the ER membrane forms an aqueous pore that mediates controlled ER Ca²⁺ efflux during protein translocation. Moreover, to seal the translocon, BiP must assume the ADP-bound conformation, and reopening of the pore requires an ATP binding–induced conformational change.⁷² Functions of these ER Ca²⁺-binding proteins further support the crucial role of Ca²⁺ in the pathogenesis of renal I/R injury.

Conclusion

Ca²⁺ fluctuation and homeostasis in the ER, the largest intracellular Ca²⁺ store, are implicated in a myriad of coordinated cellular processes, including protein folding and secretion, post-translational modification, and lipid and sterol biosynthesis. Modulation of ER Ca²⁺ pumps, channels, and Ca²⁺-binding proteins is tightly controlled at the molecular level to affect ER Ca²⁺ stores, signaling, and interorganelle communication. Disturbed ER Ca²⁺ balance affects cell function and survival in a variety of kidney diseases. Understanding the ER Ca²⁺–handling molecular mechanism and regulation, in combination with the precision intracellular delivery of ER modulators based on novel nanoplatforms, will change the therapeutic landscape for kidney disease.