Sodium Depletion Enhances Renal Expression of (Pro)Renin Receptor via Cyclic GMP-Protein Kinase G Signaling Pathway
(Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal–regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.
Prorenin receptor (PRR) is one of the newly discovered components of the renin-angiotensin-aldosterone system1,2 and is expressed in renal vasculature, glomeruli, and tubules.1–4 PRR contributes to the conversion of angiotensinogen to angiotensin I.1 Recent studies also demonstrated involvement of PRR in the development of kidney diseases and inflammation.4–7 Overexpression of human PRR in transgenic rats resulted in an increase of aldosterone production and elevation of blood pressure.8 At the present time, the physiological regulation of PRR expression is unknown. The relationship between low sodium intake and increased activity of the renin-angiotensin-aldosterone system is well established. Low sodium intake is associated with increased production of renin9,10 and angiotensin II10–12 and enhanced expression of the angiotensin receptor type 113 and type 2.14 Similarly, a low-sodium diet (LS) enhances renal production of cGMP.12,15–18 Recent studies demonstrated upregulation of PRR in diabetic animals3 and renal cells exposed to high glucose medium.4,5 However, it is unknown whether PRR expression is regulated by sodium or cGMP. Defining the relationship between sodium and PRR could be the first step to elucidate the physiological role of PRR in the kidney. This study was conducted to evaluate whether LS, cGMP, or its messenger protein kinase G (PKG) influences PRR expression. We hypothesized that, in the kidney, sodium depletion enhances PRR expression via cGMP-PKG–mediated intracellular signaling pathway. We also identified the cellular signals, transcription factors, and their functional binding sites in the promoter region of PRR that may influence PRR expression in response to sodium depletion.
Materials and Methods
Animal Preparation, Salt Intake, and Renal Expression of PRR
Study protocols were approved by the University of Virginia Animal Care and Use Committee. Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 245 to 255 g were used in this study. The effects of low-sodium intake on the renal PRR expression were studied by placing the animals for 1 week on normal sodium diet (NS; 0.3% NaCl, Harlan Teklad, Madison, WI) or LS (0.05% NaCl, Harlan Teklad; n=8 for each group). At the end of this period, animals were euthanized, the kidneys were harvested for protein and total RNA extraction, and parts of kidney were also fixed with Bounin fixative. Renal PRR expression was evaluated with quantitative real-time PCR, Western blotting, and immunohistochemical staining.
Cell Culture, Sodium Depletion, and Inhibition of cGMP-PKG Signaling Cascade
Mouse renal inner medullary collecting duct epithelial cells (IMCDs) were obtained from the American Type Culture Collection (Manassas, VA) and cultured according to American Type Culture Collection recommended protocols. Proximal tubular epithelial cells from Wistar Kyoto rats (proximal tubular cell [PTCs]) were kindly provided by Dr John J. Gildea at the University of Virginia. Cells were grown to confluence in DMEM/Nutrient Mixture F12 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS and antibiotics. Serum starvation was conducted with Opti-MEM I (Invitrogen) for 12 hours.
NS and LS media were prepared according to the methods of Yang et al.19 LS medium was prepared by Opti-MEM I in a 1:1 mixture with 300 mmol/L of d-mannitol (to reduce Na+ concentration to 57.03±0.37 mmol/L). In control groups, cells were exposed to Opti-MEM I in a 1:1 mixture with isotonic saline (final Na+ concentration was ≈132.00±0.32 mmol/L). In time-response course studies, cells were serum-starved for 12 hours previously and then exposed to NS or LS medium for 1, 3, 6, 12, and 24 hours, respectively. At the end of experiments, cells were harvested for total RNA and protein extraction. For different treatments experiments, each drug was added to serum-free medium 30 minutes before the end of serum starvation. After 30 minutes of pretreatment, cells were refreshed with NS or LS medium with or without treatment, which included 1 of the following: cGMP analog (8-bromo-GMP; Calbiochem, La Jolla, CA), 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ; Sigma-Aldrich, St Louis, MO), or PKG inhibitor (Calbiochem).
Assessing Gene Expression and Protein Phosphorylation
Determination of gene expression and protein phosphorylation was conducted with real-time PCR and Western blotting assay. The details are as described in the online Data Supplement (please see http://hyper.ahajournals.org).
Measurement of Cell Viability, cGMP Production, PKG Activity, and Phosphatase Protein Phosphatase 2A Activity
The measurement of cell viability, cGMP production, PKG, and phosphatase protein phosphatase 2A (PP2A) activities was carried out as described in the online Data Supplement.
Real-Time Mapping and In Vitro Binding Activities of cAMP Response Element, Nuclear Factor-κB, and Activator Protein 1 Regulatory Elements in the PRR Promotor
The protocol for real-time mapping of transcription factors cAMP response element binding protein 1 (CREB-1), nuclear factor-κB (NF-κB) p65, and c-Jun to cAMP response element (CRE), NF-κB, and activator protein 1 (AP-1) elements and their in vitro binding activity is detailed in the online Data Supplement.
The data analysis was carried out using STATISTICA version 5.0 (StatSoft, Tulsa OK). Results are expressed as mean±SE. Comparisons among different treatment groups were evaluated by ANOVA with repeated measures and the Bonferroni correction method as a post hoc test. A P value of <0.05 was defined as statistically significant.
Sodium Depletion Increased PRR Expression in the Kidneys of Rats
Compared with NS-treated rats, PRR mRNA and protein expression were upregulated in the kidney of LS-treated rats (Figure 1A and 1B). Similarly, immunostaining of PRR was increased in the glomeruli, proximal and distal tubules, and collecting ducts in the LS group (Figure 1C).
Sodium Depletion Upregulated PRR Expression in a Time-Dependent Manner in PTCs and IMCDs
Compared with NS, PRR mRNA and protein were upregulated after 6 hours of LS exposure and reached the peak after 12 hours in PTCs and IMCDs, as demonstrated in Figure S1 (available in the online Data Supplement, at http://hyper.ahajournals.org).
Sodium Depletion Increased cGMP Production and Relative PKG Activity in PTCs and IMCDs
cGMP production was significantly increased in culture supernatants and cells of both PTCs and IMCDs after 12 hours of exposure to LS (Figure S2). cGMP analog, 8-bromo-cGMP, significantly enhanced relative PKG activity in PTCs and IMCDs exposed to NS (Figure S2). LS significantly increased relative PKG activity. Blockade of soluble guanylyl cyclase with ODQ inhibited an LS-induced increase of relative PKG activities in both PTCs and IMCDs (Figure S2).
Effect of cGMP Stimulation and Soluble Guanylyl Cyclase Blockade on PRR Expression in PTCs
8-Bromo-cGMP treatment upregulated PRR mRNA and protein expression in PTCs cells exposed to NS (Figure 2A and 2B). There was no dose-dependent effect of 8-bromo-cGMP on PRR mRNA and protein expression at the used doses.
Soluble guanylyl cyclase blockade with ODQ did not influence PRR expression in cells exposed to NS. In contrast, ODQ at the concentration of 100 nmol/L significantly attenuated PRR upregulation in PTCs exposed to LS (Figure 2C and 2D).
Effect of PKG Inhibition on PRR Expression, PP2A Activities, and Protein Phosphorylation of Signaling Molecules in PTCs
PKG inhibition did not influence PRR expression in PTCs exposed to NS but attenuated PRR expression in a dose-dependent manner in LS-treated PTCs cells (Figure 3A and 3B). Relative phosphatase PP2A activities were decreased in LS, and this process was reversed by PKG inhibition (Figure 3C).
LS significantly increased the phosphorylation of extracellular signal–regulated kinase (ERK; T185Y187), c-Jun N-terminal kinase 1/2 (T183Y185), c-Jun (S63), CREB-1 (S133), and NF-κB p65 (S276). These protein phosphorylations were attenuated by PKG inhibition (Figure 3C).
Sodium Depletion Increased CREB-1 Binding to CRE Elements in the PRR Promoter
Three CREs were predicted in the rat PRR promoter (Figure 4A). Chromatin immunoprecipitation (ChIP) results (Figure 4B through 4D) demonstrated that LS significantly increased CREB-1 binding to all 3 of the CRE elements. Compared with NS, electrophoretic mobility-shift assay results showed increased CREB-1/CRE complex formation and band shift in LS. CREB-1 antibody completely inhibited the formation of CREB-1/CRE complexes (Figure 4E through 4G).
Sodium Depletion Increased NF-κB p65 Binding to NF-κB Elements in the PRR Promoter
Two NF-κB binding elements were predicted in the rat PRR promoter (Figure 5A). Results from ChIP and electrophoretic mobility-shift assay demonstrated that LS significantly increased NF-κB p65 binding to the distal NF-κB element (Figure 5B), as well as NF-κB p65/NF-κB complex formation and band shift (Figure 5C). NF-κB p65 antibody completely inhibited the formation of NF-κB p65/NF-κB element complexes (Figure 5C).
Sodium Depletion Increases c-Jun Binding to AP-1 Elements in the PRR Promoter
Four AP-1 binding elements were predicted in the rat PRR promoter (Figure 6A). ChIP results demonstrated that LS significantly increased c-Jun binding to AP-1 elements (Figure 6B through 6E). In contrast to NS, electrophoretic mobility-shift assay results showed increased c-Jun/AP-1 complex formation with LS (Figure 6F through 6I). The c-Jun antibody completely inhibited the formation of c-Jun/AP-1 complexes (Figure 6F through 6I).
In the present study, our first finding demonstrated that LS intake significantly upregulated PRR mRNA and protein expression in the rat kidney. Although upregulation of PRR was observed in the whole kidney, it was more pronounced in proximal tubules and collecting ducts. Similarly, in cultured proximal tubular and inner medullary collecting duct epithelial cells, LS exposure also upregulated PRR expression. These results implied that PRR might play a physiological role in proximal tubules and collecting ducts during sodium depletion.
Our in vivo studies were designed to evaluate renal expression of PRR after 1 week of LS diet. However, PRR expression in cultured renal cells was significantly upregulated after 6 hours of LS exposure and reached a peak after 12 hours. These results suggested a rapid change in renal PRR expression in response to changes in sodium depletion. Because of a lack of availability of renal-specific PRR knockout animal model or specific antagonists, we were not able to determine the exact role of this receptor in the regulation of renal functions. Our previous studies demonstrated that PRR is functional because it enhances the intracellular signaling protein phosphorylation4,5 and contributes to renal production of inflammatory factors.4–7 A possible function of PRR, although not evaluated in the present study, is the regulation of renal sodium handling. A candidate for the link between PRR and sodium reabsorption is ERK. Previous studies demonstrated enhanced ERK phosphorylation by PRR.4,5 ERK was shown to regulate the expression/activity of Na+,K+-ATPase,20 Na+/H+ exchanger,21 and Na+ channel.22 Future studies should evaluate the link between PRR and sodium reabsorption.
Despite being exposed to different intratubular sodium concentrations, PRR expression in proximal tubules and inner-medullary collecting ducts is similar in response to low-sodium intake. It is likely that the sensing mechanism of sodium level that influences renal PRR expression may not be directly related to intratubular fluid sodium concentration. Both PTCs and IMCDs increased cGMP production and PKG activity in response to LS exposure. These results suggest that these cells mount similar PRR signaling pathways in response to sodium depletion.
Our second finding was that the increment of cGMP production by sodium depletion contributes to renal PRR expression. Our previous studies showed that LS intake increased cGMP production in the kidneys.12,15–18 In this study, we also demonstrated that LS increased cGMP production in PTCs and IMCDs. Previous studies12,15–18 demonstrated that cGMP is a primary messenger to activate downstream target molecules to initiate secondary response. In this study, PTCs treated with 8-bromo-cGMP in the presence of NS upregulated PRR mRNA and protein expression. In contrast, inhibition of soluble guanylyl cyclase attenuated PRR expression in cells exposed to LS. These results confirm that PRR is regulated by cGMP.
Similarly, we found that PKG activity was increased in cells exposed to LS and to 8-bromo-cGMP during NS exposure. PKG activity was attenuated by GC inhibition. These results confirm the existence of the LS-cGMP-PKG pathway. The involvement of PKG in the regulation of PRR expression was confirmed by PKG inhibition. PKG inhibition, in a dose-dependent manner, attenuated the observed increase in PRR mRNA and protein expression in cells exposed to LS. These results demonstrated that activated PKG participates in PRR transcriptional regulation in the kidney exposed to LS via the cGMP-PKG signaling pathway.
Multiple CRE, NF-κB, and AP-1 binding elements were found in the PRR promoter. In the present study, we confirmed the involvement of transcription factors CREB-1, NF-κB p65, and c-Jun in LS-stimulated PRR expression. We demonstrated that these regulatory elements were actively functional in PRR promoter in renal cells exposed to LS to enhance this receptor expression. Both ChIP and electrophoretic mobility-shift assay results showed that LS comprehensively enhanced dynamic binding of transcriptional factors CREB-1, NF-κB p65, and c-Jun to CRE, NF-κB, and AP-1 elements in vivo and in vitro, respectively. Previously we reported that NF-κB p65 and c-Jun positively enhance transcriptional regulation of PRR expression in renal mesangial cells exposed to high glucose concentration.5 In this study we further confirmed that CREB-1, NF-κB p65, and c-Jun enhance transcriptional regulation of PRR, and their binding to the PRR promoter was significantly amplified in renal PTCs exposed to LS.
Previous studies demonstrated the involvement of the cGMP-PKG signaling pathway in enhancing the effect of CRE,23,24 NF-κB,25 and AP-126,27 in different tissues or cells. In the present study, kinase phosphorylation of ERK1/2, c-Jun N-terminal kinase, CREB-1, p65, and c-Jun was observed in renal cells exposed to LS. The phosphorylation of these kinases was attenuated by PKG inhibition. These results suggested that PKG mediated the activation of CREB-1, NF-κB p65, and c-Jun and further influenced their binding to regulatory elements of the PRR promoter in renal cells.
Several studies demonstrated that phosphatase PP2A negatively regulates PKC activity in many experimental models.28,29 In this study, LS suppressed phosphatase PP2A activity. This effect was reversed by PKG. Thus, LS seems to decrease phosphatase PP2A and to increase PKC activities. These findings are in agreement with our previous study demonstrating the involvement of PKCs as regulators of mitogen-activated protein kinases and PRR expression in renal mesangial cells.5 Taken together, PKG regulates PRR expression by activating CREB-1, p65, and c-Jun via PKG-PP2A-PKC signaling pathways.
Our previous studies demonstrated that sodium depletion increases renal cGMP concentration by enhancing NO production by angiotensin type II and bradykinin B2 receptors.15,16 Combined with the data in this study, we conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway and enhancing the activities of transcription factors CREB-1, NF-κB p65, and c-Jun.
The present study confirms that sodium depletion significantly upregulates renal expression of the PRR via cGMP-PKG signaling pathways. These findings may help in identifying new mechanisms related to the regulation of this receptor expression under physiological conditions. In addition, this study suggests the possibility of involvement of PRR in the regulation of renal function. Elucidation of this effect could lead to better understanding of the importance of PRR in health and disease.
Sources of Funding
This study was supported by grants DK-078757 and HL091535 from the National Institutes of Health (to H.M.S.).
We thank Dr John J. Gildea for providing proximal tubular epithelial cell line and William Pitkin for technical assistance.
- Received October 12, 2011.
- Revision received November 6, 2011.
- Accepted November 30, 2011.
- © 2011 American Heart Association, Inc.
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