Acute Rejection Modulates Gene Expression in the Collecting Duct

Abstract

Kidney transplantation, especially when associated with acute rejection, leads to changes in the expression of many genes, including those encoding solute transporters and water channels. In a rat model of acute rejection after allogeneic renal transplantation, impaired renal function, increased urine volume, and increased fractional excretion of sodium were observed. Gene array analysis revealed that these findings were associated with significant downregulation of water channels (aquaporin-1, -2, -3, and -4) and transporters of sodium, glucose, urea, and other solutes. In addition, changes in expression of various receptors, kinases, and phosphatases that modulate the expression or activity of renal transport systems were observed. Syngeneic transplantation or treatment with cyclosporine A following allogeneic transplantation did not impair graft function but did lead to the downregulation of aquaporin-1, -3, and -4 and several solute transporters. However, expression of aquaporin-2 and the epithelial sodium channel did not change, suggesting that the downregulation of these transporters following allogeneic transplantation is rejection-dependent. In conclusion, changes in gene expression may explain the impaired handling of solute and water after allogeneic transplantation, especially during acute rejection. Treatment with cyclosporine A improves the regulation of solute and water by preventing the downregulation of aquaporin-2 and epithelial sodium channel, even though many other transporter genes remain downregulated.

Renal transplantation (TX) in humans is often accompanied by increased transport capacities of the graft and disturbances in salt and water homeostasis.^1–3 Half-life of grafts with normal function after TX is 11.5 yr compared with 7.2 yr for those with impaired renal function.⁴ A better understanding of the underlying cellular and molecular mechanisms leading to such changes may help to increase long-term graft survival.

Previously, using an allogeneic rat renal TX (aTX) model, we have demonstrated acute changes in expression and function of several transporters and receptors after aTX.^5,6 For example, the expression of Na⁺/H⁺-exchanger-3 (NHE3), aquaporin-2 (AQP2), and the epithelial Na⁺ channel (ENaC) were downregulated at the protein and mRNA level. Major transporters for water and Na⁺ reabsorption in the collecting duct (CD) are ENaC and AQP2.⁷ The expression and activity of AQP2 are regulated by the antidiuretic hormone (vasopressin, AVP) via the G-protein coupled AVP-2 receptor (V2R).⁸ The binding of AVP to the V2R leads to the activation of the G-protein Gs, followed by activation of adenylate cyclase (AC), increased cyclic adenosine monophosphate (cAMP) levels, activation of protein kinase A (PKA), and finally phosphorylation of AQP2⁹ and translocation to the luminal membrane.¹⁰ AVP also induces mRNA and protein expression of AQP2.¹¹ Several other factors also regulate AQP2.¹² The activity of ENaC limits Na⁺ reabsorption in the CD.¹³ Aldosterone activates ENaC, decreasing urinary Na⁺ excretion and increasing K⁺ and H⁺ excretion.¹⁴ AVP also induces cAMP-mediated translocation of ENaC to the luminal membrane.¹⁵ Furthermore, ENaC is regulated by Nedd4–2 and SGK1¹⁶ and several other factors.¹⁷ The NHE3 is regulated by the Na⁺/H⁺ exchanger regulatory factor (NHERF2), scaffolding various proteins close to NHE3.¹⁸ NHERF2 is needed for the cGMP-mediated inhibition of NHE3 by the cGMP kinase II (Prkg2).¹⁹ In contrast to AQP2 and ENaC, PKA mediates an inhibition of NHE3.²⁰

To investigate possible transplant-related changes in the expression of these effectors and transporters important for renal function, we have performed gene expression analysis using microarrays. To separate possible effects mediated by the surgery itself (e.g., ischemia/reperfusion or denervation) from the mechanisms induced by the rejection process, we also performed syngeneic TX (sTX). Real-time polymerase chain reaction (PCR) was used to validate the microarray results for selected genes, and the expression data were correlated with overall renal function. To understand the influence of immunosuppression on aTX-dependent gene expression, selected genes were also analyzed from animals that underwent aTX and were treated with cyclosporine A (aTX + CsA).

RESULTS

Histologic and Functional Data

Changes in function and expression started on day 1 and were similar or even higher on day 4 after TX, after day 5 marked necrosis was observed.^5,6 When compared with the control group, the aTX model displayed signs of severe acute rejection characterized by massive leukocyte infiltration, as shown in Figure 1 and previously reported.⁶ These changes were significantly reduced in kidneys of rats treated with CsA. After aTX, histologic signs of infiltration were already evident on day 2.⁶

Figure 1.

Representative histologic lesions of control, aTX and aTX + CsA kidneys. Hematoxylin and eosin staining of kidney from control rats shows normal histologic morphology. The hematoxylin and eosin-stained kidney after aTX showed an increased number of infiltrating inflammatory cells indicating an activation of the immune system. The treatment of the aTX kidney with CsA (aTX + CsA) blocked the infiltration of the graft significantly. Original magnification ×400.

To assess renal function, blood and urine samples were collected before surgery and at the end of the experiment. The assessment of the renal function parameters is shown in Table 1. Urine volume and Na⁺ and K⁺ excretion increased and creatinine clearance decreased after aTX. After sTX, only fractional Na⁺ excretion was reduced. CsA treatment after aTX resulted in normal volume and K⁺ excretion; however, the urinary concentrating ability was still impaired compared with control. Na⁺ excretion was decreased, whereas protein excretion and serum K⁺ were increased. No significant changes in blood pressure were observed.

Table 1.

Functional data

	Control	aTX	sTX	aTX + CsA
Urine volume (ml/24 h)	14.5 ± 0.8 (13)	36.8 ± 4.8 (6)b	13.67 ± 2.11 6	17.25 ± 4.84 (4)
Urine concentration (mOsmol/kg)	1860 ± 154 (5)	348 ± 27.8 (4)b	1509 ± 152 (4)	875 ± 63.3 (3)b
FEa Na+ (%)	0.44 ± 0.09 (10)	1.7 ± 0.59 (6)b	0.165 ± 0.05 (5)b	0.04 ± 0.02 (4)b
FE K+ (%)	22.0 ± 3.4 (10)	43.7 ± 7.2 (6)b	16.18 ± 2.16 (5)	18.39 ± 1.9 (4)
Aldosterone serum (pg/ml)	41.7 ± 17.4 (9)	32.6 ± 12 (5)	71.13 ± 47.2 (3)	149.25 ± 39.3 (4)b
Aldosterone urine (pg/ml)	7.5 ± 0.6 (12)	27.9 ± 5.8 (5)b	16.50 ± 7.04 (5)	9.20 ± 2.36 (4)
Protein excretion (mg/24 h)	16.5 ± 1.1 (13)	19.4 ± 2.4 (6)	18.98 ± 1.32 (6)	25.30 ± 4.22 (4)b
Creatinine clearance (ml/min)	2.3 ± 0.4 (12)	0.87 ± 0.3 (6)b	2.54 ± 0.51 (5)	0.87 ± 0.06 (6)b

Data are mean ± SEM. Blood and urine samples were collected for 24 h. One-way ANOVA was used to identify significant differences in aTX, sTX, and aTX + CsA compared with control.

^aFE, fractional excretion.

^bSignificantly different values compared with control (P < 0.05).

Graft recipients were bilaterally nephrectomized immediately before TX. After sTX, the creatinine clearance was normal, indicating that an adaptation and activation of compensatory mechanisms such as increased GFR of the grafted kidney have taken place. The decreased creatinine clearances after aTX and aTX + CsA indicate that such compensatory mechanisms were not present after aTX and aTX + CsA. These observations suggest that the rejection processes and even the treatment of rejection for 4 d with CsA inhibited the activation of compensatory mechanisms.

Differentially Expressed Genes

After aTX, 3871 probe sets were upregulated and 3483 of a total of 31,000 were downregulated. After sTX, 564 probe sets were downregulated and 1291 were upregulated. The complete output files are provided on our homepage (http://medd.klinikum.uni-muenster.de/forschung/arraytools_output.zip). After aTX 82 gene ontology (GO) terms were enriched in the upregulated and 55 in the downregulated group of genes. Enriched GO terms within the upregulated group of genes indicate a massive activation of the immune response and infiltration of the graft by immunologic active cells. Over-represented GO terms within the genes downregulated after aTX indicate a depression of metabolic and transport processes. Nine GO terms identified genes with functions specifically related to kidney function (Figure 2). A table with the list of genes classified in the GO term transport is provided (http://medd.klinikum.uni-muenster.de/forschung/go_transport.xls). Table 2 shows a selection of over-represented GO terms with significant importance for renal transport function. The complete lists are provided (http://medd.klinikum.uni-muenster.de/forschung/DAVID_chart.xls). Downregulation of such genes leads to substantially decreased tubular function.

Figure 2.

Gene ontology terms that were over-represented after aTX. Selection of over-represented gene ontology terms with importance for kidney function on level 3 related to “biologic process,” “cellular component,” or “molecular function” in the set of genes that were significantly downregulated after aTX (P < 0.05, Fisher's exact test). The numbers indicate the amount of genes corresponding with the gene ontology terms. The P values are presented in log scale.

Table 2.

Gene ontology terms over-represented after aTX

GO Term	Count	P
Carrier activity	127	6.38E-19
Sodium ion transporter activity	26	1.09E-12
Hydrogen ion transporter activity	49	3.02E-12
Transporter activity	280	1.77E-07
Porter activity	48	1.40E-04
Symporter activity	31	1.63E-04
Ion transporter activity	132	3.93E-04
Cation transporter activity	110	4.23E-04
Iron ion binding	43	7.94E-04
Vitamin transport	7	0.0013
Carboxylic acid transport	25	0.0014
Organic acid transport	25	0.0014
Water-soluble vitamin metabolism	16	0.0017
Sodium ion binding	24	0.0017
Carboxylic acid transporter activity	27	0.0022
Organic acid transporter activity	27	0.0022
Sodium ion transport	26	0.0024
Cofactor transporter activity	7	0.0032
Cellular physiological process	1179	0.0036
Organic cation transporter activity	6	0.0111
Physiologic process	1288	0.0127
Anion/cation symporter activity	10	0.0161
Organic anion transport	7	0.0216
Anion transport	26	0.0225
Proton transport	17	0.0360
Solute/cation symporter activity	15	0.0380
Amino acid transporter activity	17	0.0438
Monovalent inorganic cation transport	51	0.0481

All terms are related to “biological process,” “cellular component,” or “molecular function” with kidney-related function in the set of genes that were significantly down-regulated after aTX (P < 0.05, Fisher's exact test). The “Count” indicates the amount of genes corresponding with the GO terms.

Real-Time PCR

For genes encoding for transporters, receptors, or signaling factors with renal relevance, expression was validated by real-time PCR. The majority of genes showed decreased expression levels in all TX models compared with control and, thus, are independent of rejection. All analyzed aquaporins showed a decreased expression after aTX (Figure 2). The expression of transporters involved in Na⁺ retention (i.e., NHE3, ENaC, Slc13a2, SLC5a2, Slc34a2, or Slc34a3) was also decreased after aTX (Figure 2). The majority of transporters showed lower expression after sTX or aTX + CsA, with AQP2 and ENaC being an exception. Their expression was normal after sTX. As mentioned above, AQP2 and ENaC are the major transporters for water and Na⁺ reabsorption in the CD. The majority of the analyzed receptors were also decreased in expression after aTX compared with control (Figure 3). The V2R and mineralocorticoid receptor (MIR) are involved in the regulation of AQP2 and ENaC, respectively. In contrast, the expression of Adora2a, Ptger2, and GCA was increased after aTX compared with control. These genes were not affected after sTX, indicating that the increased expression after aTX is related to the rejection process. Normally, activation of these receptors initiates signal transduction pathways inducing other factors such as kinases. The expression of AC type 4 (AC4) was downregulated after aTX compared with control. On the other hand, the expression of phosphodiesterases (PDE)²¹ was up-regulated. However, after sTX, the expression of these factors were downregulated with the exception of AC4, Prkcb1, and Cnp1. This indicates that the increased expression of these factors, after aTX, was induced because of acute rejection.

Figure 3.

Changes in gene expression for selected transporters. The expression of selected transporters was validated by real-time PCR using specific primer pairs or TaqMan gene expression assays. Relative changes were evaluated using the 2^-ΔΔCt method. The changes in gene expression after aTX (light gray columns), sTX (white columns), aTX + CsA (dark gray columns), and for selected genes after Ctr + CsA (striped columns) are shown. Significantly different gene expressions are marked by an asterisk. Data are presented as mean ± SEM values, and a P value <0.05 was considered statistically significant.

Effects of CsA

We analyzed the gene expression in grafts from rats treated 4 for d with CsA using real-time PCR. Interestingly, the expression of the majority of the transporters was still decreased, despite improved renal function in aTX + CsA. For example, the expression of AQP1, AQP3, and AQP4 was still decreased, whereas the expression of AQP2 was increased, compared with aTX (Figure 2). However, the majority of the transporters involved in Na⁺ retention still showed decreased expression. One exception was ENaC, which was upregulated by CsA compared with aTX. These results are comparable with the sTX data, where CD proteins AQP2 and ENaC showed normal expression. CsA treatment also led to normal or even increased expression of V2R and MIR compared with controls (Figure 4). Following CsA treatment, the expression of AC4 was increased and that of PDEs were decreased (Figure 5). This indicates that a normal or increased expression of receptors and downstream signaling factors after CsA treatment is followed by a normal or increased expression or activity of AQP2 and ENaC in the CD. An increased ENaC activity may explain the reduced fractional Na⁺ excretion observed in aTX + CsA. Genes that showed increased expression after aTX, like GCA, Adora2a, or Ptger2 were decreased in expression after aTX + CsA compared with controls. Interestingly, CsA treatment was followed by a massive downregulation of SGK1 (Figure 4). For selected genes, we analyzed the expression in the control (Ctr) + CsA group to identify direct effects of the immunosuppressant. Surprisingly, CsA led to decreased expression of ENaC, NHE3, GCA, and SGK1 (Figures 2 through 5).

Figure 4.

Changes in gene expression for selected receptors. The expression for selected receptors was validated by real-time PCR using specific primer pairs or TaqMan gene expression assays. Relative changes were evaluated using the 2^-ΔΔCt method. Changes in gene expression after aTX (light gray columns), sTX (white columns), after aTX + CsA (dark gray columns), and for selected genes after Ctr + CsA (striped columns) are shown. Significantly different expressed genes are marked by an asterisk (one-way ANOVA, P < 0.05).

Figure 5.

Changes in gene expression for selected regulatory factors. The expression for selected regulatory factors was validated by real-time PCR using specific primer pairs or TaqMan gene expression assays. Relative changes were evaluated using the 2^-ΔΔCt method. Changes in gene expression after aTX (light gray columns), sTX (white columns), aTX + CsA (dark gray columns), and for selected genes after Ctr + CsA (striped columns) are shown. Significantly different gene expressions are marked by an asterisk (one-way ANOVA, P < 0.05).

Expression and Localization of AQP2

The expression and localization of AQP2 were analyzed in cryosections of control, sTX, aTX, and aTX + CsA kidneys by immunofluorescence (Figure 6). No change in total protein expression (as judged by fluorescence intensity) was observed after sTX while a reduced expression was evident after aTX and aTX + CsA when compared with control. The localization of AQP2 after aTX and aTX + CsA was similar to control. After sTX, the AQP2 was mainly found on the luminal membrane. This would suggest an increased AQP2 translocation to the luminal membrane, which could be a mechanism to compensate for the decreased expression of AQP1 and probably decreased reabsorption in the proximal nephron resulting in normal urine concentrating capacity.

Figure 6.

Representative immunohistochemical staining of AQP2. The expression of AQP2 was not altered after sTX, whereas a reduction in expression was observed after aTX and aTX + CsA compared with control. The localization was not altered after aTX and aTX + CsA, whereas after sTX AQP2 seems be localized predominantly on the luminal membrane.

DISCUSSION

In recent studies, we have demonstrated decreased function and/or expression of renal trasporters, such as NHE3, ENaC, and AQP2 on day 4 after aTX.^5,6 The changes in expression levels of AQP2, ENaC, and NHE3 mRNA reported in this study confirmed our previously published observations.^5,6

In this study, we have observed an increased urinary volume and Na⁺ excretion within the first 4 d after aTX and massively disturbed urinary concentrating capacity of the graft compared with sTX, indicating that this functional deterioration is solely the result of rejection. Microarray analysis showed that genes with functions related to transport were over-represented in the list of genes downregulated after aTX (Figure 2). Several transport systems are involved in water and Na⁺ retention, and various pathways regulate the expression and activity of these transporters.

Angiotensin-II (ATII) for example is an important activator of NHE3 via the ATII-type-1-receptor (Agtr1a)²²; thus, its downregulation may contribute to a disturbed ATII signaling and thereby a decreased NHE3 function.

Water transport in the kidney is facilitated by the aquaporins-1 to -4.²³ We observed a downregulation of AQP1 to AQP4 after aTX (Figure 2). This downregulation might contribute to the increased urinary volume observed after aTX resulting from decreased reabsorption capacity. Expression of factors involved in activation or expression of AQP2, such as V2R, AC4, or PKA, as described above, was downregulated after aTX. These data indicate a grossly disturbed AVP-mediated signal transduction in the CD after aTX. In addition to AQP2, AVP also regulates the expression of several other genes, such as AQP3, AQP4, or casein kinase II.²⁴ AVP treatment was demonstrated to increase expression of 137 genes. Of these genes, more than 70% were downregulated in the present study, after aTX again indicating a disturbed AVP signaling.

ENaC is one of the major transporters along the CD. Factors that have a positive influence on the activity or expression of EnaC, such as MIR, SGK1, AC4, PKA, or V2R, were downregulated after aTX, leading to an increased Na⁺ excretion. A disturbed cAMP-signaling due to decreased AC4 and increased PDE expression may contribute to the disturbed ENaC function.

The majority of the analyzed genes were also downregulated after sTX, which suggests an underlying mechanisms induced by ischemia/reperfusion, denervation or other factors related to the surgery and not due to the acute rejection. Interestingly, the expression of AQP2, ENaC, MIR, V2R, and AC4 was not affected after sTX, indicating that downregulation of these genes after aTX was induced by mechanisms related to the rejection process. Furthermore, normal expression of AQP2, ENaC, and their corresponding receptors after sTX seemed to be sufficient for a normal overall kidney function despite the decrease of several other transporter genes. Renal function after sTX, including creatinine clearance, was similar to that of the control group with both kidneys indicating a compensatory mechanism of the grafted kidney.

The CD seems to be most important for the observed tubular dysfunction after aTX. Further evidence for this hypothesis is provided by the data obtained after aTX + CsA, which had normal urinary volume excretion compared with aTX. The expression of AQP1, AQP3, and AQP4, which are not under the control of AVP, remained decreased, whereas the expression of V2R was normal and the expression of AQP2 and AC4 was even higher than in controls.

Treatment with CsA led to reduced expression of PDEs. This may increase cAMP levels, leading to a pronounced activation of PKA and thereby of AQP2 and ENaC. CsA inhibits the calcium/calmodulin-dependent phosphatase (PP2B).²⁵ Inhibition of PP2B is followed by increased cAMP-mediated insulin secretion in RINm5F cells.²⁶ This could also apply for AQP2 and ENaC regulation by cAMP. Downregulation of AVP signaling in the CD is likely to be responsible for the observed increased volume excretion. Furthermore, aTX + CsA led to normal AQP2 expression and possibly normal activation of AQP2. These results are in contrast to the observed long-term effects of CsA.²⁷ This could be explained by differences in the used models, transplanted versus native kidney, and time points (i.e., 4 d in this study versus 4 wk in the previously reported study²⁷). The control group treated with CsA showed a slight reduction of AQP2 and a significant reduction of ENaC expression. The creatinine clearance was similar to the aTX group. CsA treatment was not followed by a complete compensation of the grafted kidney as observed after sTX.

CsA treatment after aTX was followed by increased expression of ENaC and MIR and a massive decrease in fractional Na⁺ excretion. From the examined transporters involved in Na⁺ retention, only ENaC showed an altered expression. The increased expression of AQP2 and ENaC after aTX + CsA was not induced by a direct effect of CsA because CsA treatment alone did not induce such increases (Figure 2). An underlying mechanism for the increased ENaC expression and Na⁺ retention could be an increased aldosterone level after aTX + CsA. This would lead to an increased ENaC activity. Aldosterone also induced a decreased apical and a pronounced basolateral sorting of AQP2 in the CD.²⁸ The same study also reported a reduction in urine osmolality following an aldosterone treatment that is comparable to our findings.

Renal TX is followed by hypertension in up to 80% of transplant recipients with poor graft survival, reduced life expectancy, and increased cardiovascular mortality.^29–32 The pathogenesis of hypertension is complex and may reflect the influence of the primary renal disease, vascular injury, graft dysfunction, and the effect of immunosuppressive therapy. AQP2 and ENaC can contribute to the development of hypertension. For example, spontaneously hypertensive rats showed increased expression of AQP2 and ENaC, thereby mediating increased water and Na⁺ retention.³³ A prolonged change in expression beyond day 4 of these transporters in aTX + CsA may contribute to a similar water and Na⁺ retention as described in these hypertensive rats and probably in patients treated with CsA.³⁴

In conclusion, our gene expression study showed that several factors may contribute to changes in expression and activity of AQP2 and ENaC, the major transporters for water and Na⁺ in the CD, after aTX and aTX + CsA. Regulatory factors with positive effects on AQP2 or ENaC expression were downregulated after aTX followed by an increased Na⁺ and water excretion. CsA treatment was followed by normal water excretion, increased Na⁺ retention correlating with normal expression of AQP2, ENaC, and the majority of their regulatory factors. But the urinary concentrating ability was impaired. After sTX, the expression of AQP2 and ENaC was normal.

CONCISE METHODS

Kidney Transplantation

Experiments were approved by a governmental committee were performed in accordance with national animal protection guidelines (Westfälische-Wilhems Universität Münster, Münster, Germany). Renal TXs were performed as described previously.^5,6,35 For the present study, all recipients were bilaterally nephrectomized immediately before TX. For aTX and aTX + CsA rats treated with CsA (5 mg/kg per d), kidneys of LBN rats (n = 5) were transplanted into LEW rats. The Ctr + CsA animals were treated with CsA (5 mg/kg per d) without TX (n = 5). For sTX, kidneys from LBN-rats were transplanted into LBN rats. The second kidney of the LBN donors (n = 5) served as control.

Histology and Immunofluorescence

Histology and immunofluorescence were performed as described before.⁶ AQP2 was detected using a specific antibody directed against AQP2, kindly provided by Dr. Enno Klussmann.³⁶ The bound primary antibody detected using a secondary goat antirabbit Alexa 488 labeled antibody (Invitrogen, Carlsbad, CA).

General Functional Data

Overall functional data were obtained as described previously.^5,6 Twenty-four hours before TX surgery and at the endpoint, before kidney organ harvest, animals were housed in metabolic cages. Urine and blood samples were analyzed for protein (Bradford Blue, Bio-Rad Laboratories, München, Germany), creatinine (Enzym-Pap; Roche Diagnostics, Mannheim, Germany), and electrolytes by flame photometry (Instrumentation Laboratory 943, Kirchheim, Germany). Aldosterone was quantified by RIA (Aldosterone MAIA, Adaltis, Freiburg, Germany).

RNA Analysis

After graft removal, the total RNA was isolated using an RNeasy-kit (Qiagen, Hilden, Germany) and used to prepare biotinylated target cDNA. Target cDNAs were processed as per manufacturer's instructions (http://www.affymetrix.com). Data were analyzed using Affymetrix GCOS array analysis software. All data have been deposited in NCBIs Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO series accession number GSE6497.

Identification of Differentially Expressed Genes

Significant changes in the gene expression after aTX were identified using class comparison with the BRB ArrayTools developed by R. Simon and A. Peng (http://linus.nci.nih.gov/BRB-ArrayTools.html). Nominal significance level of each univariate test was set to 0.001. Confidence level of false discovery rate assessment used was 90%, and the maximum allowed numbers of false-positive genes were set to 10.

Functional Annotation

The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for functional classification.³⁷ Gene enrichment analysis was performed for the lists of upregulated or downregulated after aTX to identify GO terms³⁸ over-represented within a given candidate list (P < 0.05, Fishers exact test).

Real-Time PCR

Real-time PCR was performed using the SYBR Green PCR Master Mix or TaqMan Universal PCR Master Mix with the ABI PRISM 7700 Sequence Detection System. Specific primer pairs or TaqMan Gene expression assays were used. All instruments and reagents were purchased by Applied Biosystems (Darmstadt, Germany). Relative gene expression values were evaluated with the 2^-ΔΔCt method using GAPDH or 18s-RNA as housekeeping genes.³⁹ A list with the gene names, accession numbers, and gene symbols is provided in Table 3.

Table 3.

Accession number, gene names, and gene symbols of genes used for the validation by the real-time PCR

RefSeq	Gene Name	Gene Symbol
NM_053294.3	Adenosine A2a receptor	Adora2a
NM_019285	Adenylate cyclase 4	Adcy4
XM_001061777	Angiotensin/vasopressin receptor	Nalp6
NM_030985	Angiotensin II receptor, type 1	Agtr1a
NM_012778.1	Aquaporin 1	Aqp1
NM_012909.2	Aquaporin 2	Aqp2
NM_031703.1	Aquaporin 3	Aqp3
NM_012825.1	Aquaporin 4	Aqp4
NM_019136.1	Arginine vasopressin receptor 2	Avpr2
NM_012809.1	Cyclic nucleotide phosphodiesterase 1	Cnp1
NM_022388.1	FXYD domain-containing ion transport regulator 4	Fxyd4
NM_012770.1	Guanylate cyclase 1, soluble, beta 2	Gucy1b2
NM_022284.1	Guanylate cyclase activator 2b	Guca2b
NM_176080.2	Na+-dependent glucose transporter 1	Naglt1
NM_012613.1	Natriuretic peptide receptor 1	Npr1
NM_133583.1	N-myc downstream regulated gene 2	Ndrg2
NM_013131	Nuclear receptor subfamily 3, group C, member 2 (MIR)	Nr3c2
NM_020073.1	Parathyroid hormone receptor 1	Pthr1
NM_031079.1	Phosphodiesterase 2A, cGMP-stimulated	Pde2a
NM_017031.2	Phosphodiesterase 4B	Pde4b
NM_133551.1	Phospholipase A2, group IVA (cytosolic, calcium-dependent)	Pla2g4a
NM_017023.1	Potassium inwardly-rectifying channel, subfamily J, member 1	Kcnj1
NM_031088.1	Prostaglandin E receptor 2, subtype EP2	Ptger2
NM_012713.2	Protein kinase C, beta 1	Prkcb1
NM_022507.1	Protein kinase C, zeta	Prkcz
NM_019142.1	Protein kinase, AMP-activated, alpha 1 catalytic subunit	Prkaa1
NM_013012.1	Protein kinase, cGMP-dependent, type II	Prkg2
NM_031075.1	Purinergic receptor P2X, ligand-gated ion channel, 3	P2rx3
NM_017255.1	Purinergic receptor P2Y, G-protein coupled 2	P2ry2
NM_019232.1	Serum/glucocorticoid regulated kinase	Sgk
NM_031548.2	Sodium channel, nonvoltage-gated 1 alpha	Scnn1a
NM_175758.3	Sodium-dependent neutral amino acid transporter ASCT2	Slc1a5
NM_031746.1	Solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 2	Slc13a2
NM_177962.2,	Solute carrier family 14 (urea transporter), member 2	Slc14a2
NM_031672.1	Solute carrier family 15 (H+/peptide transporter), member 2	Slc15a2
NM_017102.1	Solute carrier family 2 (facilitated glucose transporter), member 3	Slc2a3
NM_031741.1	Solute carrier family 2, member 5	Slc2a5
NM_017224.1	Solute carrier family 22 (organic anion transporter), member 6	Slc22a6
NM_031332.1	Solute carrier family 22 (organic anion transporter), member 8	Slc22a8
NM_031664.1	Solute carrier family 28 (sodium-coupled nucleoside transporter), member 2	Slc28a2
NM_053380.1	Solute carrier family 34 (sodium phosphate), member 2	Slc34a2
NM_139338.1	Solute carrier family 34 (sodium phosphate), member 3	Slc34a3
NM_199111.1	Solute carrier family 35, member B2	Slc35b2
NM_053715.1	Solute carrier family 5 (inositol transporters), member 3	Slc5a3
NM_022590.1	Solute carrier family 5 (sodium/glucose cotransporter), member 2	Slc5a2
NM_053818.1	Solute carrier family 6 (neurotransmitter transporter, glycine), member 9	Slc6a9
NM_013111.1	Solute carrier family 7 (cationic amino acid transporter, y + system), member 1	Slc7a1
NM_053811.2	Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 regulator 2	Slc9a3r2
NM_012654.1	Solute carrier family 9 (sodium/hydrogen exchanger), member 3	Slc9a3

Statistics

Data were tested with one-way analysis of variance using GraphPad-Prism 4.0 (San Diego, CA).

DISCLOSURES

The authors have no financial conflict of interest.