Summary
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The molecular pathways linking polycystins to cyst development in ADPKD are still unclear. Intracystic fluid secretion via ion transporters and channels plays a crucial role in cyst expansion in ADPKD. Unexpectedly, we observed significant and selective up-regulation of NHA2, a member of the SLC9B family of Na+/H+ exchangers that correlated with cyst size and disease severity in ADPKD patients. Using three-dimensional cultures of MDCK cells to model cystogenesis in vitro, we show that ectopic expression of NHA2 is causal to increased cyst size. Induction of PC1 in MDCK cells inhibited NHA2 expression with concordant inhibition of Ca2+ influx through store-dependent and independent pathways, whereas reciprocal activation of Ca2+ influx by a dominant negative, membrane-anchored C-terminal tail fragment of PC1 elevated NHA2. We show that NHA2 is a target of Ca2+/NFAT signaling and is transcriptionally induced by methylxanthine drugs such as caffeine and theophylline, which are contraindicated in ADPKD patients. Finally, we observe robust induction of NHA2 by vasopressin, which is physiologically consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in ADPKD. Our findings have mechanistic implications on the emerging use of vasopressin V2 receptor antagonists such as tolvaptan as safe and effective therapy for PKD and reveal a potential new regulator of transepithelial salt and water transport in the kidney.
Introduction
Autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent hereditary disease, affecting one in 400–1000 humans (1,2). ADPKD accounts for up to 10% of end-stage renal disease cases, making it one of the leading causes of kidney failure (1). A better understanding of the underlying pathophysiology is key to management of ADPKD and related disorders. ADPKD is caused by mutations in PKD1 and PKD2 encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 is a transmembrane protein with a large extra-cytoplasmic N-terminal domain that is thought to function as a receptor, and a C-terminal cytoplasmic tail (3,4). PC2 is a transmembrane protein that functions as a non-selective Ca2+ channel. PC1 interacts with PC2 via C-terminal coil-coiled domains that are required for proper function and trafficking (5,6). The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation leading to activation of STAT signaling (7). In renal tubular cells, the polycystins localize to the primary cilium where they regulate intracellular Ca2+ and cAMP levels in response to mechano-stimulating urinary flow. A disruption of PC1-PC2 interaction is thought to lead to cyst formation due to abnormal cellular proliferation and fluid secretion, although the specific molecular pathways that connect the underlying genetic defects to disease pathogenesis and cyst development are poorly understood (1,2).
In this context, increased plasma membrane Na+/H+ exchange (NHE) activity has been reported in cilium-deficient collecting duct cells from the Oak Ridge mouse model of polycystic kidney disease (8,9). Apical mislocalization of NHE1 in these cells leads to hyperabsorption of Na+ and epidermal growth factor-induced cell proliferation (8,10). The potential role of other Na+/H+ exchanger isoforms in PKD pathophysiology has not been explored. Na+/H+ exchangers belong to the superfamily of monovalent cation/proton antiporters (CPA) that share a common transmembrane organization of 12-14 hydropathic helices with detectable sequence similarity. The CPA1 subgroup includes the electroneutral NHE family of Na+(K+)/H+ exchangers represented by the well-characterized plasma membrane transporter NHE1 (11-13). More recently, a new clade of CPA2 genes was discovered in animals, sharing ancestry with electrogenic bacterial NapA and NhaA antiporters (14,15). Of the two human CPA2 genes, NHA2 has ubiquitous tissue distribution, whereas the closely related NHA1 isoform is restricted to testis (14-16). Despite the multiplicity of genes encoding Na+/H+ exchangers in mammals, emerging evidence indicates that CPA1 and CPA2 subtypes have distinct, non-redundant transport roles based on differences in chemiosmotic coupling, inhibitor sensitivity and pH activation (17). At the plasma membrane, NHE1 couples proton extrusion to the inwardly directed Na+ electrochemical gradient, established by the Na/K-ATPase, to mediate salt reabsorption in epithelia and recovery from acid load. Consistent with these functions, NHE isoforms are activated by low cytoplasmic pH. In contrast, NHA2 has an alkaline pH optimum and appears to couple inward transport of protons to mediate sodium (or lithium) efflux at the cell membrane, recapitulating the function of the bacterial CPA2 ortholog NhaA (12,15,17,18).
Human NHA2 has been implicated as a marker of essential hypertension, with potential roles in kidney diseases relating to salt and water balance (14,18,19). NHA2 expression in kidney is confined to the distal nephron and renal collecting duct, regions that play critical roles in salt, pH and volume homeostasis (15). NHA2 is localized to both principal cells and intercalated cells of the distal tubule in vivo. Principal cells maintain sodium and water balance and the intercalated cells control acid-base homeostasis (19). Although the physiological function of NHA2 is largely obscure, defining the pathways regulating NHA2 expression is an important first step towards deciphering its potential role in hypertension and kidney disease.
A key aspect of precision medicine is to harness patient databases to uncover novel risk factors and potential therapeutic targets. This approach led to our unexpected discovery of selective and significant up-regulation of NHA2 expression in polycystic kidney disease. Using an established MDCK model of in vitro cystogenesis we show that NHA2 expression correlates with cyst size, replicating patient data. PC1-mediated Ca2+ transients and NFAT signaling regulate NHA2 expression, providing a mechanistic link to PKD pathogenesis. To extend physiological relevance, we demonstrate robust vasopressin-mediated NHA2 expression, which is consistent with increased levels of circulating vasopressin and up-regulation of vasopressin V2 receptors in PKD. Importantly, pharmacological inhibition of the NHA2 transporter drastically attenuated cyst size. Taken together, our findings reveal a hitherto under-recognized role for Na+/H+ exchange activity in PKD progression and allow us to propose NHA2 as a potential chemotherapeutic target.
Results
NHA2 is up-regulated in polycystic kidney disease and promotes cyst development in vitro
We sought to investigate differences in transcript levels of plasma membrane isoforms of the SLC9 superfamily of Na+/H+ exchangers in cysts obtained from patients with autosomal dominant polycystic kidney disease (20). Transcripts of well-studied SLC9A (NHE) subfamily genes remained unchanged (NHE1 and NHE3) or showed modest repression (NHE2) (Fig. S1A-C). Unexpectedly, we observed significant up-regulation of a member of the SLC9B subfamily, NHA2, relative to normal kidney tissue that correlated with cyst size and disease severity (Fig. 1A): 4.6-fold in large cysts (p=0.002), 3.4-fold in medium cysts (p=0.001), 3.1-fold in small cysts (p=0.0004), and 1.2-fold in minimally cystic tissue (not significant, p=0.49). Based on this observation, we hypothesized that increased expression of NHA2 could contribute to pathophysiology of PKD.
To test this hypothesis, we used an established in vitro MDCK cell model of cystogenesis (21,22). Three-dimensional culturing of MDCK cells in Matrigel produces cysts with polarized, single-layer, thinned epithelium surrounding a fluid-filled lumen (Fig. 1B). Similar to PKD kidneys, MDCK cells in cysts undergo proliferation, fluid transport, and matrix remodeling. To model NHA2 up-regulation, we used MDCK cells stably expressing GFP-tagged NHA2 which colocalizes with the basolateral membrane marker E-cadherin(17) (Pearson’s correlation=0.66±0.11; Manders’ coefficient=0.64±0.17; n=25) (Fig. S1D-E). Three-dimensional culturing of these cells in Matrigel resulted in cyst formation with surface expression of NHA2-GFP (Fig. 1C), similar to monolayer cultures. Consistent with our hypothesis linking NHA2 up-regulation to cyst development, we observed significant expansion in cyst size with NHA2 expression relative to non-transfected controls (Fig. 1D-E). Notably, we documented a dramatic difference in the relative cyst diameter on day 10 of culture (Control: 100±66.4; n=363, NHA2 up-regulation: 232.3±213.7; n=367, p=4.2X10−22). Intriguingly, we also observed several mutilayered and multiloculated cysts in NHA2+ MDCK cells that were not seen in cysts derived from control MDCK cells (Fig. S1F-G). We classified cysts derived from control cells based on their relative diameter: small (<50 percentile), medium (50-90 percentile), and large (>90 percentile). We observed a significantly higher percentage of larger cysts in NHA2+ cells (control: 10% vs. NHA2 up-regulation: 44%; p<0.0001; Chi-square test; Fig. 1F). Taken together, these findings suggest that NHA2 up-regulation in polycystic kidney disease may promote cyst development.
Polycystin 1 downregulates Ca2+ influx and NHA2 expression
Previously, using a well-characterized MDCK cell model of inducible PC1 expression, we showed that PC1 negatively regulates PC2 levels post-translationally, by enhancing its degradation via the aggresome-autophagosome pathway (23). Using this model, we sought to determine if PC1 induction also alters expression of NHA2 (Fig. 2A). Tetracycline-inducible PC1 expression resulted in significant down-regulation of NHA2 protein levels by ~2.9-fold, relative to non-induced control (p=0.0353; Fig. 2B-C), similar to our previous findings with PC2 (23). Whereas transcript levels of PC2 remained unchanged upon PC1 induction (p=0.663; Fig. 2D), we observed significantly lower NHA2 mRNA levels, consistent with PC1-mediated transcriptional down-regulation of NHA2 (p=0.0029; Fig. 2D). Given that PC2 is a known calcium channel, we next determined if calcium influx is altered by PC1 induction in MDCK cells. We tested two modes of Ca2+ entry: store independent Ca2+ entry (SICE) and store operated Ca2+ entry (SOCE), both mechanisms that are implicated in normal physiology and pathological conditions (24,25). In the absence of PC1 induction, MDCK cells showed robust SICE that was significantly and proportionately attenuated upon induction of PC1 for 24 and 48 hours (Fig. 2E-F). Similarly, PC1 induction for 48 hours also reduced SOCE following thapsigargin-mediated release of store Ca2+ (Fig. 2G), consistent with previous reports of PC1 mediated inhibition of STIM1 translocation during store depletion (26). Given these concordant observations on NHA2 expression and Ca2+ influx, we investigated whether reduced Ca2+ influx in PC1 induced cells is causal to transcriptional down-regulation of NHA2. We first analyzed a publicly available microarray dataset from Ca2+-induced activation of primary T lymphoblasts following treatment with Ca2+ ionophore ionomycin and phorbol myristate (27). Intriguingly, we observed SLC9 gene expression changes similar to ADPKD cysts, including ~2.4-fold increase in NHA2 levels, no change in NHE1, and lower, but non-significant levels of NHE2 in response to Ca2+ influx in these cells (Fig. S2A-C). Ionomycin treatment increased cytoplasmic Ca2+ in PC1 induced MDCK cells (Fig. 2H), resulting in significant and dose dependent increase in NHA2 expression to levels similar to MDCK cells without PC1 induction (Fig. 2I). Taken together these findings are consistent with Ca2+-mediated regulation of NHA2 expression by PC1.
NFAT mediates Ca2+-dependent NHA2 expression
The NFAT family of transcription factors has been implicated in the regulation of genes that control numerous aspects of normal physiology including cell cycle progression and differentiation, and in pathological conditions such as inflammation and tumorigenesis (28). The TNF family member Receptor Activator of NF-kB Ligand (RANKL) elicits Ca2+ oscillations in differentiating osteoclasts that converge on the calcineurin/NFAT pathway(29). During RANKL-induced osteoclast differentiation, NHA2 ranked among top five NFATc1-dependent transcripts, although the physiological role of this up-regulation is yet to be determined (30). To confirm and extend these findings, we analyzed the microarray data from this study (30) and another study reporting NFAT-mediated osteoclast differentiation in vitro (31) to show profound, >95% down-regulation of NHA2 levels upon NFAT deletion (Fig. 3A) and in contrast, time-dependent up-regulation of NHA2 levels (~50-fold on day 2-3) upon NFAT activation (Fig. S3A). There was no change in expression of the NHE1 isoform (Fig. 3A). Expression patterns of well-known NFAT target genes, CLCN7 and MMP9 (32), are shown for comparison (Fig. 3A and S3B-C). Consistent with these results, an independent study revealed strong down-regulation of NFATc1 expression and consequently, >30-fold lower NHA2 levels in macrophage colony stimulation factor (M-CSF) and RANKL primed osteoclast precursor myeloid blasts seeded on plastic, relative to cells seeded on bone (33). Similarly, independent studies have reported NFAT-mediated regulation of NHA2 expression in T cells (34,35). Furthermore, NFAT activation might also underlie recent reports of TNF-alpha induced up-regulation of NHA2 expression in human endothelial cells (36).
Studies have established the importance of NFAT signaling in normal kidney development and in pathological conditions including polycystic kidney disease(37-39). Based on findings from the literature and our data so far, we propose a model that PC1 mediated modulation of Ca2+ homeostasis regulates NHA2 expression through NFAT signaling (Fig. 3B). To test this hypothesis, we used a membrane anchored C-terminal tail fragment of PC1 (PC1-MAT) that was previously shown to function as a dominant negative effector and mimic cellular pathologies associated with patient mutations in PC1, including activation of downstream AP-1, WNT, STAT3 and NFAT signaling (7,39-41) (Fig. 3C). We therefore asked if exogenous expression of PC1-MAT fragment could induce NHA2 expression in HEK293 cells. Using the firefly and Renilla luciferase reporter gene system, we first confirmed that ectopic expression of PC1-MAT resulted in significant, ~5-fold increase in NFAT reporter activity, relative to empty vector transfection control (p<0.0001; Fig. 3D). These results were independently validated by visualizing localization of GFP-tagged NFAT: in vector transfected cells, NFAT-GFP is predominantly localized in the cytoplasm, whereas increased nuclear translocation of NFAT-GFP was documented in cells expressing PC1-MAT, similar to cells expressing constitutively active NFAT-GFP (CA-NFAT) (Fig. 3E-F). In striking contrast, expression of full-length PC1 showed significant, ~4-fold down-regulation of NFAT reporter activity, relative to empty vector transfection control (p<0.0001; Fig. 3D). These results are consistent with down-regulation of Ca2+ influx and NHA2 expression by induction of full length PC1 in MDCK cells (Fig. 2B-C and 2E-F). We have previously shown that store independent Ca2+ entry (SICE) regulates NFAT nuclear translocation and promotes cell proliferation (25). We therefore analyzed SICE in cells transfected with PC1-MAT. In contrast to our SICE data in MDCK cells expressing full-length PC1 (Fig. 2E-F), we observed robust ~2.4-fold increase in Ca2+ entry, compared to empty vector control (p=0.02; Fig. 3G-H). We also observed a higher baseline with PC1-MAT, relative to the empty vector control, suggesting higher basal Ca2+ levels with PC1-MAT expression (Fig. 3G). Taken together, these data provide strong evidence for reciprocal down- and up-regulation of SICE/NFAT signaling by full length PC1 and PC1-MAT, respectively. Importantly, consistent with our proposed model, we observed ~2.6-fold higher NHA2 expression in cells expressing PC1-MAT (p=0.0002; Fig. 3I), and on the other hand, ~25%-lower NHA2 levels in cells expressing full-length PC1 (p=0.0016; Fig. 3I), relative to empty vector transfection. These findings are also consistent with our data from MDCK cells expressing full-length PC1 (Fig. 2B-D), and evidence from the literature showing significant up-regulation of NHA2 upon ectopic expression of engineered constitutively active NFAT1 in T cells (34). Finally, analysis of NFATc1 expression in ADPKD patient-derived cysts revealed significant up-regulation relative to normal kidney tissue that correlated with cyst size and disease severity (Fig. 3J): large cysts (3.5-fold, p=0.005), medium cysts (3.5-fold, p=0.235), small cysts (2.2-fold, p=0.003), and minimally cystic tissue (1.5-fold, p=0.003). These findings, taken together, suggest that polycystin 1 mediated modulation of Ca2+ influx regulates NHA2 expression through the calcineurin/NFAT pathway.
Potential modulation of kidney function by drug and hormonal regulation of NHA2
Public datasets can be leveraged to identify novel mechanisms of gene regulation, as described earlier (42). To gain new insights into the regulation of NHA2 expression and to predict a functional role of NHA2 in PKD, we performed an unbiased bioinformatics approach to analyze drug-induced gene expression signatures from 1078 microarray studies in human cells. We identified experimental conditions eliciting a minimum of ±2-fold change in NHA2 gene expression (Fig. 4A). Notably, the highest up-regulation of NHA2 (≥3-fold) was observed in response to methyl xanthine drugs: caffeine (7.5mM; 24h) and theophylline (10mM; 24h). Further analysis of these individual microarray studies showed that the ability of these drugs to induce NHA2 expression is dependent on dosage and duration of treatment (Fig. 4B-C). Interestingly, these methyl xanthine drugs are suggested to promote cyst development in PKD patients(43). Furthermore, both caffeine and theophylline are known to activate ryanodine sensitive receptors (RyR) and release Ca2+ stores in response to an initial Ca2+ entry through PC2 and other plasma membrane Ca2+ channels, thereby effectively amplifying cytoplasmic Ca2+ (44).
On the other hand, at least two drugs that resulted in maximal down-regulation of NHA2, namely valproic acid and dexamethasone, are known to reduce cytoplasmic Ca2+ concentration (45,46). Interestingly, the therapeutic potential of valproic acid has been shown in zebra fish and mouse models of PKD (47). Taken together, top hits in NHA2 gene expression (both up- and down-regulation) funneled into pathways that modulate cytoplasmic Ca2+, further bolstering the credibility of our model of Ca2+ mediated regulation of NHA2 expression. Our previous studies show that NHA2 is an important cellular lithium efflux transporter that might be involved in Li+ clearance in kidney (17). In this context it is important to note that methylxanthine drugs caffeine and theophylline that increase NHA2 levels are well known to promote Li+ clearance via kidney. These drugs enhance renal salt and water transport and are contraindicated for Li+ therapy (48,49). Using ratiometric Fura 2 Ca2+ imaging, we first confirmed significant increase in cytoplasmic Ca2+ with caffeine and theophylline treatment in MDCK cells (Fig. 4D). Furthermore, caffeine and theophylline treatment elicit significant, dose-dependent increase in NHA2 expression (Fig. 4E-F), relative to vehicle controls, validating the findings from bioinformatic studies (Fig. 4A-C).
Given that physiological concentrations of vasopressin, a hormone strongly implicated in PKD pathogenesis and a promising drug target, also functions via activation of RyR (50), we considered whether vasopressin regulates NHA2 expression (Fig. 4G). The MDCK cell line is a well-established model to study transport function changes in the distal renal tubule in response to hormones, including vasopressin and aldosterone (51,52). To test our hypothesis, we used the MDCK cell model and first documented robust increase in cytoplasmic Ca2+ following vasopressin treatment, relative to vehicle control (Fig. 4H). Compared with the quick (~50-100s) initial slope of the Ca2+ rise in caffeine and theophylline treatment, the slope of the Ca2+ rise was more gradual (~500-1000s) consistent with indirect activation of downstream signaling cascades by vasopressin (Fig. 4G). Of note, the peak heights of cytosolic [Ca2+] were comparable between caffeine, theophylline and vasopressin. It is worth noting that both caffeine and vasopressin have been shown to activate NFAT nuclear translocation and signaling (53,54). Therefore, to determine if rise in cytosolic Ca2+ with vasopressin is translated to increase in NHA2 expression, we treated MDCK cells with vasopressin for different intervals, ranging from 2-24h. We observed significant and phasic increase in NHA2 expression with vasopressin treatment that peaked (~6-fold) at 4-8h and reached baseline by 24h (Fig. 4I). Using Western blotting we confirmed robust elevation of NHA2 protein with vasopressin treatment for 6h (Fig. 4J).
These findings are consistent with our recent in vivo studies showing that high salt diet in mice significantly increased transcript and protein expression of NHA2 in renal tubules (19). High salt diet significantly raises circulating levels of vasopressin to retain water and help sustain normal plasma osmolarity (55). Likewise, increased vasopressin concentrations have been associated with PKD severity and disease progression and in experimental studies vasopressin has been shown to directly modulate transepithelial fluid transport and regulate cyst growth (50). Intriguingly, studies have documented that the NHA2 inhibitor phloretin attenuates vasopressin-stimulated solute movement (56). Thus, NHA2 might regulate salt and water transport across the epithelium and help contribute to the functions of vasopressin in PKD and high salt diet, both are associated with hypertension.
For comparison, we tested the effect of another hormone, aldosterone, on NHA2 expression in MDCK cells. Basic and clinical research have shown increased activation of the renin-angiotensin-aldosterone-system in ADPKD (57,58). Intriguingly, like vasopressin, aldosterone also increased NHA2 mRNA (Fig. S4A) and protein levels (Fig. 4J). Similar induction with both vasopressin and aldosterone expression has been reported in the literature for aquaporin-2 and gamma subunit of the epithelial sodium channel (ENaC)(59,60). Moreover, a synergistic effect of vasopressin and aldosterone has been described for Na+ channel and Na+/K+-ATPase activities (61,62). It is worth noting that aldosterone has been shown to enhance global Ca2+ transients, through activation of protein kinase A (PKA)(63). To evaluate functional consequences of the hormonal induction of NHA2, we monitored growth-sensitivity to lithium in MDCK cells. Previously, we showed that NHA2 is functionally coupled to plasma membrane V-ATPase in MDCK cells to mediate robust H+-driven efflux of Li+, resulting in selective survival and growth advantage in media with high concentration of lithium (17). Consistent with increased NHA2 mRNA and protein expression, treatment with either vasopressin or aldosterone for 8 h resulted in increased cell survival in media supplemented with 90 mM LiCl, relative to untreated control (Fig. S4B).
Vasopressin and aldosterone play key roles in the fine adjustment of transport of salt and water in the nephron (60-62). In order to test the potential role of NHA2 in regulating transepithelial fluid transport, we generated fluid-filled hemi-cysts or domes, a simple, yet powerful assay widely used over the last 40 years to determine vectorial salt and water transport in vitro (64,65). MDCK cells grown on impermeable substratum, such as plastic, spontaneously form hemicysts due to transepithelial transport and accumulation of fluid in focal regions, beneath the cell layer (Fig. 5A-B). Studies have established that cyclic AMP levels, Na+/H+ exchange activity and PC1 function regulate hemicyst formation (65-67). Importantly, vasopressin and aldosterone treatment promote transepithelial transport and robustly stimulate hemicyst formation in MDCK cells(68,69). Consistent with these findings, we show that ectopic expression of NHA2 in MDCK cells resulted in larger, ~2.8-fold increase in area of hemicyst (Fig. 5C-D), suggesting an increase in vectorial transport of salt and water with NHA2 expression that might, in part, contribute to the downstream effects of vasopressin and aldosterone in PKD.
Knockdown or inhibition of NHA2 attenuates cyst development in vitro
Given that fluid secretion and cyst expansion are major factors for the progressive and irreversible decline and renal failure in PKD, therapies targeting salt and water transport are of major interest as an alternative, or to complement anti-proliferative therapies in PKD (70). To evaluate the therapeutic potential of targeting NHA2 in PKD we used two complementary approaches: shRNA-mediated knockdown of NHA2 and pharmacological inhibition of NHA2 using phloretin. MDCK cells have low endogenous NHA2 expression (17), therefore we knocked down NHA2 in NHA2+ MDCK cells (Fig. S5A). We have previously shown that overexpression of NHA2 does not alter growth in MDCK cells (17). Consistent with these findings, we now show that lentivirally mediated knockdown of NHA2 also did not alter growth of MDCK cells (Fig. 6A). Next, we tested the effect of NHA2 knock down on cyst formation by MDCK cells (Fig. 6B-C). Specific knockdown of NHA2, using two different lentiviral shRNA constructs, resulted in formation of significantly smaller (~70-80% lower diameter) cysts (Fig. 6B-C).
NHA2 is insensitive to classical NHE inhibitors such as amiloride and its derivatives but is sensitive to phloretin (17). In contrast to the effect of pharmacological inhibition of NHE1, inhibition of NHA2 with phloretin did not significantly alter cytoplasmic pH (Fig. S5B-C). Therefore, to functionally validate the effect of phloretin, we turned to lithium sensitivity as a defining phenotype of NHA2 transport function. Treatment of NHA2+ MDCK cells with phloretin conferred growth sensitivity to lithium (Fig. 6D). Notably, no difference in lithium sensitivity was observed between phloretin treatment with or without NHA2 knockdown indicating the specificity of lithium sensitivity assay as a marker of NHA2 activity (Fig. 6D). Next, we analyzed the effect of phloretin (20-150μM) on MDCK cyst formation in Matrigel. Consistent with lower cyst size resulting from NHA2 knockdown (Fig. 6B-C), we observed dose-dependent attenuation of cyst size with NHA2 inhibition with phloretin (Fig. 6E-F). Taken together, these findings suggest that NHA2 may be a potential drug target for attenuating cyst development in PKD.
Discussion
The three key components of cystogenesis in PKD are abnormalities in tubular cell proliferation, matrix remodeling and fluid secretion (70). Cysts arise from the renal tubule as saccular expansions, and eventually become pinched off and anatomically separated from the tubule. Thus, the intracystic fluid is derived from transepithelial fluid secretion due to abnormal reversal of normally absorptive epithelium to a secretory one. This leads to dramatic expansion of cystic volume, which is the single most important predictor of progressive deterioration and renal failure in PKD (70,71). Therefore, a better understanding of pathways driving salt and water transport in PKD models could lead to novel strategies to halt disease progression. Although Na+/H+ exchangers are known to be of vital physiological importance for fluid and salt homeostasis in the kidney (11,12), their contribution to the pathology of PKD has yet to be fully explored or recognized. Using in vitro models of cystogenesis in conjunction with gene expression data from PKD patients, we now show that NHA2 expression is regulated by PC1 via Ca2+ and NFAT signaling to promote cyst development. A better understanding of a kidney-specific role for NHA2 in PKD awaits a mechanistic study of transport. It is as yet unclear whether the normal physiological role of NHA2 in renal tubular cell is in salt and water reabsorption or secretion or both depending on the coupling ion (sodium or protons) and localization (apical or basolateral)(19).
Although far from conclusive, there are tantalizing hints pointing to a role for NHA2 in renal function and hypertension that may be relevant to pathophysiology of PKD. First, SLC9B2, the gene encoding NHA2, lies within a limited (~300 genes) region of human chromosome 4q24 genetically linked to phloretin-sensitive and amiloride-insensitive sodium-lithium countertransport (SLC), an activity linked to essential hypertension (14,18). Indeed, NHA2 mediates phloretin-sensitive and amiloride-insensitive SLC activity in stably transfected MDCK cells (17). SLC activity has been associated with PKD and there is an intriguing, yet unexplained link between lithium treatment and formation of renal cysts in human and rodents (72-74). Second, a recent genome-wide association study (GWAS) of renal function identified NHA2 as a genetic risk locus for estimated glomerular filtration rate by serum creatinine. GFR, which shows heritability in the range of 36-75%, is commonly used to monitor kidney function decline in PKD (75). Third, NHA2 expression is predominant in the distal nephron where sodium reabsorption is under hormonal and dietary control and, consistent with the induction of renal NHA2 expression by a high salt diet in mouse (15,19). In addition to these findings, we now show that NHA2 is highly elevated in PKD, a disease, with predisposition to hypertension (57).
The dysregulation of two second-messengers, Ca2+ and cAMP, is central to the disease mechanism in PKD and there is evidence connecting NHA2 to both. Here we show that methylxanthine drugs (caffeine and theophylline) that activate Ca2+ release via the ryanodine receptor channel (RyR), increase NHA2 expression which is associated with cyst growth (Fig. 7). Furthermore, we show that NHA2 is up-regulated by vasopressin, a hormone that in physiological concentrations functions through activation of RyR and increases cytoplasmic Ca2+ levels. Thus, NHA2 is a novel vasopressin effector in renal epithelial cells with potential implications for PKD.
The precise NHA2-mediated regulation of transepithelial fluid transport and cystogenesis remains to be determined. Vasopressin is secreted in response to increased plasma osmolality resulting from high salt intake, consistent with our previous observation that high sodium diet in mice significantly increases expression of NHA2 in renal tubules (19,55). Previous studies have shown that membrane anchored C-terminal tail fragment of polycystin 1 (PC1-MAT) enhances caffeine-induced intracellular Ca2+ levels (39). Similarly, caffeine has been reported to synergize the effects of desmopressin, a synthetic analogue of vasopressin (43). Caffeine is also thought to promote cyst development in PKD patients (43). Furthermore, caffeine and theophylline are well known to promote clearance of lithium through kidney (48,49). Given our previous findings that NHA2 is an important cellular lithium efflux transporter (17), we suggest that induction of NHA2 expression might, in part, contribute to the downstream effects of caffeine in PKD and lithium clearance.
NHA2 may mediate cross-talk between cAMP and Ca2+ signaling. In spermatozoa, loss of NHA2, or the closely related NHA1 isoform, led to decreased soluble adenylyl cyclase and cAMP levels, and conversely, high levels of NHA antiporters stabilized soluble adenylyl cyclase expression (16). Given the central role of cAMP in cyst formation and growth, elevated NHA2 could exacerbate cAMP signaling in ADPKD patients, which might in turn modulate the activity of other transport proteins. PKD patients have high circulating levels of vasopressin. Treatment with vasopressin V2-receptor antagonist tolvaptan has been shown to inhibit cyst progression in preclinical studies and large clinical trials (50). The potential synergistic or additive effects of NHA2 inhibition with tolvaptan merits further study. In summary, our findings warrant a thorough in vivo investigation of NHA2 as a potential new player in cellular salt and pH regulation in the kidney and in the pathogenesis of PKD and hypertension.
Materials and Methods
Cell culture
Stable MDCK cell line with inducible PC1 expression was generated using Flp-In System (Invitrogen) by transfecting pcDNA5/FRT/TO-based wild-type mPkd1 expression plasmid according to the manufacturer’s protocol (23). These cells were cultured in DMEM supplemented with 10% fetal bovine serum, 150 μg/ml hygromycin, and 5 μg/ml blasticidin, with PC1 expression induced with tetracycline (2 μg/ml). Stable MDCK cell line with NHA2 expression was generated by transfecting pEGFPC2 vector carrying human NHA2 tagged to GFP at the N terminus and selecting for G418 antibiotic resistant clones (17). MDCK cells (control and NHA2+) were cultured in MEM supplemented with 10% fetal bovine serum. HEK293 cells were cultured in DMEM supplemented with 10% fetal bovine serum. Culture conditions were in a 5% CO2 incubator at 37°C. 3-dimensional cysts were cultured in 10% Matrigel with a 100% Matrigel basement membrane and grown in full media, which was changed every 2 days (21,22). MDCK hemicysts in monolayer cultures were generated as previously described (65,66). Hormonal treatment protocols (vasopressin-1μM or aldosterone-100nM) were previously reported to regulate transporter expression and function in MDCK cells (51,52). Lithium sensitivity was determined by measuring cell growth in the presence of media containing 90-100 mM LiCl. Cell growth was quantified using MTT assay (ThermoFisher Scientific), following the manufacturer’s instructions (17).
Plasmids and transfection
PC1 full-length plasmid was a gift from Dr Gregory Germino (Addgene plasmid #21369). Membrane anchored C-terminal tail fragment of PC1 (PC1-MAT) plasmid was a gift from Dr Thomas Weimbs (Addgene plasmid #41567). GFP-tagged NFAT1 (1–460) plasmid was described previously(25). Constitutively active NFAT (CA-NFAT) was a gift from Dr Anjana Rao (Addgene plasmid #11102). pGL3-NFAT-luc was a gift from Dr Jerry Crabtree (Addgene plasmid #17870). pSV40-RL was a gift from Dr Jennifer L. Pluznick (Johns Hopkins University). HsNHA2 short hairpin RNA (shRNA) sequence (5’-CACTGTAGGCCTTTGTGTTGTTCAAGAGACAACACAAAGGCCTACAGTTTTTTCAAT T-3’) was designed to knockdown NHA2 (ShRNA#1). An additional knockdown construct (ShRNA#2) targeting NHA2 with sequence (5’-CCGGGCATTGCAGTATTGATACGAACTCGAGTTCGTATCAATACTGCAATGCTTTTT TG-3’) was purchased from Sigma (#TRCN0000130075). MDCK cells were transfected using lentiviral packaging and expression. HEK293 cells were transfected using Lipofectamine 2000 reagent, as per the manufacturer’s instructions.
Antibodies and reagents
Specific rabbit polyclonal NHA2 antibody was raised against a 15-aa peptide of NHA2 (14,17). Mouse monoclonal antibodies used were Anti-PC1 (7E12) (#sc-130554, Santa Cruz Biotechnology), Anti-α-Tubulin (#T9026, Sigma), Anti-β-Actin (#A5441, Sigma), and Anti-e-cadherin (#610181, BD Transduction Laboratories). Aldosterone (A9477), Blasticidin (#15205), Caffeine (#C0750), Ionomycin (#I3909), Lithium chloride (#L9650), Phloretin (#P7912), Thapsigargin (#T9033), Theophylline (#T1633), and Vasopressin (#V9879) were obtained from Sigma. Tetracycline Hydrochloride (A1004-5) and Hygromycin B (#10843555001) were purchased from Zymo Research and Roche, respectively.
Bioinformatics
Public datasets were mined to uncover novel mechanisms of gene regulation, as described earlier (42). We analyzed drug-induced gene expression signatures from 1078 microarray studies in human cells to identify drugs that significantly altered NHA2 expression. Experimental conditions eliciting a minimum of ±2-fold change in gene expression were selected for further pathway analysis. Normalized gene expression data was obtained from Genevestigator (Nebion AG). Other mammalian gene expression datasets included in the study are GSE7869, GSE37219, GSE50971 and GSE57468.
Cytoplasmic pH measurement
Cytoplasmic pH was measured using pHrodo Red AM Intracellular pH Indicator (#P35372, Thermo Fisher Scientific), following manufacturer’s instructions. Briefly, cells were rinsed with serum-free medium and then incubated with 1 μL/ml of pHrodo Red AM at 37°C for 30 minutes. Cells were washed, trypsinized and pH was determined by flow cytometry analysis of −10,000 cells in biological triplicates using the FACSCalibur instrument (BD Biosciences). Cells were gated on a forward scatter and side scatter to obtain live single cells. A four-point calibration curve with different pH values (4.5, 5.5, 6.5 and 7.5) was generated using Intracellular pH Calibration Buffer Kit (#P35379, Thermo Fisher Scientific) in the presence of 10μM K+/H+ ionophore nigericin and 10μM K+ ionophore valinomycin.
Quantitative Real-time PCR (qPCR)
mRNA was isolated using RNeasy Mini kit (#74104, Qiagen) with DNase1 (#10104159001, Roche) treatment and complementary DNA was synthesized using the high-Capacity RNA-to-cDNA Kit (#4387406, Applied Biosystems), following the manufacturer’s instructions. Gene expression was assessed by quantitative real-time PCR using following Taqman gene expression assays: Dog: NHA2, Cf02729492_m1; PC2, Cf02690612_m1; GAPDH, Cf04419463_gH; Human: NHA2, Hs01104990_m1; GAPDH, Hs02786624_g1.
Western blot
Cells were harvested and lysed with 1% Nonidet P-40 (Sigma) supplemented with protease inhibitor cocktail (Roche). Following sonication, cell lysates were centrifuged for 15 min at 14,000 rpm at 4°C. The protein concentrations were measured using the BCA assay. Equal amounts of total protein were electrophoretically separated by polyacrylamide gel (NuPAGE) before transferring onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with 5% milk, followed by overnight incubation with primary antibodies and 1h incubation with HRP-conjugated secondary antibodies (GE Healthcare). Amersham 600 chemiluminescence imager system was used to capture images.
Confocal microscopy
To determine colocalization of NHA2-GFP with E-cadherin, cultured MDCK NHA2+ cells on polyornithine coverslips were pre-extracted with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.8) containing 0.025% saponin for 2 min, then washed twice for 2 min with PHEM buffer containing 0.025% saponin and 8% sucrose. Following fixation (4% paraformaldehyde and 8% sucrose in PBS) for 30min, cells were blocked with 1% BSA and 0.025% saponin in PBS for 1 hr. Cells were stained with primary Anti-e-cadherin antibody (overnight) and Alexa Fluor 568 conjugated secondary antibody (1 hr). On the other hand, to monitor nuclear translocation of NFAT, cultured HEK293 cells with NFAT-GFP transfection were fixed with a solution of 4% paraformaldehyde in PBS. Following DAPI staining, coverslips from both experiments were mounted onto slides using Dako fluorescent mounting medium and were imaged using LSM 700 Confocal microscope (Zeiss). Confocal imaging was performed with a ×63 oil immersion objective and the fractional colocalization was determined using the JACoP ImageJ plugin.
Calcium Imaging
Calcium imaging was performed as we previously described (25). Briefly, cells were cultured on 25 mm circular coverslips. Cells were briefly washed with PBS and loaded with Fura-2 AM at 1 mg/ml in calcium imaging buffer (126 mM NaCl, 4.5 mM KCl, 2 mM MgCl2, 10 mM Glucose, 20 mM HEPES, pH 7.4) containing 2 mM CaCl2. After incubation at room temperature for 30 minutes, cells were washed briefly in imaging buffer without Fura-2 AM. After baseline recordings were established, cells were excited at 340 nm and 380 nm, and Fura-2 AM emission at 505 nm was monitored.
Luciferase assay
Luciferase assay was performed as we previously described (42). Briefly, pGL3-NFAT-luc (encoding a 3x NFAT binding sequence and firefly luciferase) and pSV40-RL (encoding for a constitutively active SV40 promoter and Renilla luciferase) plasmids were transiently transfected into HEK293 cells using lipofectamine 2000 reagent, as per manufacturer’s instructions. The ratio of firefly luciferase and Renilla luciferase, a measure of NFAT activity, was determined using the Dual Luciferase Assay System (#E1910, Promega). Data were collected using a FLUOstar Omega automated plate reader (BMG LabTech).
Acknowledgements
We gratefully acknowledge the Baltimore Polycystic Kidney Disease (PKD) Research and Clinical Core Center for stable MDCK cells with inducible PC1 expression. This work was supported by grants from the National Institutes of Health to R.R. (DK108304) and to V.C. (DK103078). H.P. was a Fulbright Fellow supported by the International Fulbright Science and Technology Award. K.C.K. was a postdoctoral fellow of the American Heart Association.