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Acute Rejection Modulates Expression in the Collecting Duct

Bayram Edemir, Stefan Reuter, Reka Borgulya, Rita Schro¨ter, Ute Neugebauer, Gert Gabrie¨ls, and Eberhard Schlatter

Medizinische Klinik und Poliklinik D, Experimentelle Nephrologie, Universita¨tsklinikum Mu¨nster, Mu¨nster, Germany

ABSTRACT Kidney transplantation, especially when associated with acute rejection, leads to changes in the expres- sion of many , 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 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 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 down- regulation of aquaporin-2 and epithelial sodium channel, even though many other transporter genes remain downregulated.

J Am Soc Nephrol 19: 538–546, 2008. doi: 10.1681/ASN.2007040513

Renal transplantation (TX) in is often ac- and AQP2.7 The expression and activity of AQP2 companied by increased transport capacities of the are regulated by the antidiuretic hormone (vaso- graft and disturbances in salt and water homeosta- pressin, AVP) via the G-protein coupled AVP-2 re- sis.1–3 Half-life of grafts with normal function after ceptor (V2R).8 The binding of AVP to the V2R leads TX is 11.5 yr compared with 7.2 yr for those with to the activation of the G-protein Gs, followed by impaired renal function.4 A better understanding of activation of adenylate (AC), increased cy- the underlying cellular and molecular mechanisms clic adenosine monophosphate (cAMP) levels, acti- leading to such changes may help to increase long- vation of protein kinase A (PKA), and finally phos- term graft survival. phorylation of AQP29 and translocation to the Previously, using an allogeneic rat renal TX

(aTX) model, we have demonstrated acute changes Received April 28, 2007. Accepted October 24, 2007. in expression and function of several transporters 5,6 Published online ahead of print. Publication date available at and receptors after aTX. For example, the expres- www.jasn.org. sion of Naϩ/Hϩ-exchanger-3 (NHE3), aqua- ϩ Correspondence: Dr. Bayram Edemir, Medizinische Klinik und porin-2 (AQP2), and the epithelial Na channel Poliklinik D, Experimentelle Nephrologie, Domagkstra␤e 3a, (ENaC) were downregulated at the protein and 48149 Mu¨nster, Germany. Phone: 49-251-83-56912; Fax: ϩ49- mRNA level. Major transporters for water and Naϩ 251-83-56973; E-mail: [email protected] reabsorption in the collecting duct (CD) are ENaC Copyright © 2008 by the American Society of Nephrology

538 ISSN : 1046-6673/1903-538 J Am Soc Nephrol 19: 538–546, 2008 www.jasn.org BASIC RESEARCH

luminal membrane.10 AVP also induces mRNA and protein expression of AQP2.11 Several other factors also regulate ϩ AQP2.12 The activity of ENaC limits Na reabsorption in the ϩ CD.13 Aldosterone activates ENaC, decreasing urinary Na ex- ϩ ϩ cretion and increasing K and H excretion.14 AVP also in- duces 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 Naϩ/Hϩ exchanger regulatory factor (NHERF2), scaf- folding various proteins close to NHE3.18 NHERF2 is needed for the cGMP-mediated inhibition of NHE3 by the cGMP ki- Figure 1. Representative histologic lesions of control, aTX and nase II (Prkg2).19 In contrast to AQP2 and ENaC, PKA medi- aTX ϩ CsA kidneys. Hematoxylin and eosin staining of kidney ates an inhibition of NHE3.20 from control rats shows normal histologic morphology. The he- To investigate possible transplant-related changes in the ex- matoxylin and eosin-stained kidney after aTX showed an in- pression of these effectors and transporters important for renal creased number of infiltrating inflammatory cells indicating an function, we have performed gene expression analysis using activation of the immune system. The treatment of the aTX kidney microarrays. To separate possible effects mediated by the sur- with CsA (aTX ϩ CsA) blocked the infiltration of the graft signif- gery itself (e.g., ischemia/reperfusion or denervation) from the icantly. Original magnification ϫ400. mechanisms induced by the rejection process, we also per- formed syngeneic TX (sTX). Real-time polymerase chain reac- assessment of the renal function parameters is shown in Table tion (PCR) was used to validate the microarray results for se- 1. Urine volume and Naϩ and Kϩ excretion increased and lected genes, and the expression data were correlated with creatinine clearance decreased after aTX. After sTX, only frac- overall renal function. To understand the influence of immu- tional Naϩ excretion was reduced. CsA treatment after aTX nosuppression on aTX-dependent gene expression, selected resulted in normal volume and Kϩ excretion; however, the genes were also analyzed from animals that underwent aTX urinary concentrating ability was still impaired compared with and were treated with cyclosporine A (aTX ϩ CsA). control. Naϩ excretion was decreased, whereas protein excre- tion and serum Kϩ were increased. No significant changes in blood pressure were observed. RESULTS Graft recipients were bilaterally nephrectomized immedi- ately before TX. After sTX, the creatinine clearance was nor- Histologic and Functional Data mal, indicating that an adaptation and activation of compen- Changes in function and expression started on day 1 and were satory mechanisms such as increased GFR of the grafted kidney similar or even higher on day 4 after TX, after day 5 marked have taken place. The decreased creatinine clearances after aTX necrosis was observed.5,6 When compared with the control and aTX ϩ CsA indicate that such compensatory mechanisms group, the aTX model displayed signs of severe acute rejection were not present after aTX and aTX ϩ CsA. These observations characterized by massive leukocyte infiltration, as shown in suggest that the rejection processes and even the treatment of Figure 1 and previously reported.6 These changes were signif- rejection for 4 d with CsA inhibited the activation of compen- icantly reduced in kidneys of rats treated with CsA. After aTX, satory mechanisms. histologic signs of infiltration were already evident on day 2.6 To assess renal function, blood and urine samples were col- Differentially Expressed Genes lected before surgery and at the end of the experiment. The After aTX, 3871 probe sets were upregulated and 3483 of a

Table 1. Functional data Control aTX sTX aTX ؉ CsA Urine volume (ml/24 h) 14.5 Ϯ 0.8 (13) 36.8 Ϯ 4.8 (6)b 13.67 Ϯ 2.11 6 17.25 Ϯ 4.84 (4) Urine concentration (mOsmol/kg) 1860 Ϯ 154 (5) 348 Ϯ 27.8(4)b 1509 Ϯ 152 (4) 875 Ϯ 63.3(3)b ϩ FEa Na (%) 0.44 Ϯ 0.09 (10) 1.7 Ϯ 0.59(6)b 0.165 Ϯ 0.05(5)b 0.04 Ϯ 0.02(4)b ϩ FE K (%) 22.0 Ϯ 3.4 (10) 43.7 Ϯ 7.2 (6)b 16.18 Ϯ 2.16 (5) 18.39 Ϯ 1.9 (4) Aldosterone serum (pg/ml) 41.7 Ϯ 17.4 (9) 32.6 Ϯ 12 (5) 71.13 Ϯ 47.2 (3) 149.25 Ϯ 39.3(4)b Aldosterone urine (pg/ml) 7.5 Ϯ 0.6 (12) 27.9 Ϯ 5.8 (5)b 16.50 Ϯ 7.04 (5) 9.20 Ϯ 2.36 (4) Protein excretion (mg/24 h) 16.5 Ϯ 1.1 (13) 19.4 Ϯ 2.4 (6) 18.98 Ϯ 1.32 (6) 25.30 Ϯ 4.22(4)b Creatinine clearance (ml/min) 2.3 Ϯ 0.4 (12) 0.87 Ϯ 0.3 (6)b 2.54 Ϯ 0.51 (5) 0.87 Ϯ 0.06(6)b Data are mean Ϯ SEM. Blood and urine samples were collected for 24 h. One-way ANOVA was used to identify significant differences in aTX, sTX, and aTX ϩ CsA compared with control. aFE, fractional excretion. bSignificantly different values compared with control (P Ͻ 0.05).

J Am Soc Nephrol 19: 538–546, 2008 Gene Expression of Transporters 539 BASIC RESEARCH www.jasn.org

Table 2. terms over-represented after aTX GO Term Count P Carrier activity 127 6.38E-19 Sodium 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 Figure 2. Gene ontology terms that were over-represented after Sodium ion transport 26 0.0024 aTX. Selection of over-represented gene ontology terms with transporter activity 7 0.0032 importance for kidney function on level 3 related to “biologic Cellular physiological process 1179 0.0036 process,” “cellular component,” or “molecular function” in the Organic cation transporter activity 6 0.0111 Ͻ set of genes that were significantly downregulated after aTX (P Physiologic process 1288 0.0127 0.05, Fisher’s exact test). The numbers indicate the amount of Anion/cation symporter activity 10 0.0161 genes corresponding with the gene ontology terms. The P values Organic anion transport 7 0.0216 are presented in log scale. Anion transport 26 0.0225 Proton transport 17 0.0360 total of 31,000 were downregulated. After sTX, 564 probe Solute/cation symporter activity 15 0.0380 sets were downregulated and 1291 were upregulated. The Amino acid transporter activity 17 0.0438 complete output files are provided on our homepage Monovalent inorganic cation transport 51 0.0481 (http://medd.klinikum.uni-muenster.de/forschung/array- All terms are related to Љbiological process,ЉЉcellular component,Љ or tools_output.zip). After aTX 82 gene ontology (GO) terms Љmolecular functionЉ with kidney-related function in the set of genes that were significantly down-regulated after aTX (P Ͻ 0.05, Fisher’s exact test). were enriched in the upregulated and 55 in the downregu- The ЉCountЉ indicates the amount of genes corresponding with the GO lated group of genes. Enriched GO terms within the upregu- terms. lated group of genes indicate a massive activation of the immune response and infiltration of the graft by immuno- Slc13a2, SLC5a2, Slc34a2, or Slc34a3) was also decreased after logic active cells. Over-represented GO terms within the aTX (Figure 2). The majority of transporters showed lower genes downregulated after aTX indicate a depression of expression after sTX or aTX ϩ CsA, with AQP2 and ENaC metabolic and transport processes. Nine GO terms identi- being an exception. Their expression was normal after sTX. As fied genes with functions specifically related to kidney func- mentioned above, AQP2 and ENaC are the major transporters tion (Figure 2). A table with the list of genes classified in the for water and Naϩ reabsorption in the CD. The majority of the GO term transport is provided (http://medd.klinikum.uni- analyzed receptors were also decreased in expression after aTX muenster.de/forschung/go_transport.xls). Table 2 shows a compared with control (Figure 3). The V2R and mineralocor- selection of over-represented GO terms with significant im- ticoid receptor (MIR) are involved in the regulation of AQP2 portance for renal transport function. The complete lists and ENaC, respectively. In contrast, the expression of Adora2a, are provided (http://medd.klinikum.uni-muenster.de/for- Ptger2, and GCA was increased after aTX compared with con- schung/DAVID_chart.xls). Downregulation of such genes trol. These genes were not affected after sTX, indicating that leads to substantially decreased tubular function. the increased expression after aTX is related to the rejection process. Normally, activation of these receptors initiates signal Real-Time PCR transduction pathways inducing other factors such as kinases. For genes encoding for transporters, receptors, or signaling The expression of AC type 4 (AC4) was downregulated after factors with renal relevance, expression was validated by real- aTX compared with control. On the other hand, the expression time PCR. The majority of genes showed decreased expression of (PDE)21 was up-regulated. However, af- levels in all TX models compared with control and, thus, are ter sTX, the expression of these factors were downregulated independent of rejection. All analyzed aquaporins showed a with the exception of AC4, Prkcb1, and Cnp1. This indicates decreased expression after aTX (Figure 2). The expression of that the increased expression of these factors, after aTX, was transporters involved in Naϩ retention (i.e., NHE3, ENaC, induced because of acute rejection.

540 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 538–546, 2008 www.jasn.org BASIC RESEARCH

expression of V2R and MIR compared with controls (Figure 4). Following CsA treatment, the expression of AC4 was in- creased and that of PDEs were decreased (Figure 5). This indi- cates 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 Naϩ excretion observed in aTX ϩ CsA. Genes that showed increased expression after aTX, like GCA, Adora2a, or Ptger2 were decreased in expression after aTX ϩ 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) ϩ CsA group to identify direct effects of the immunosuppres- sant. Surprisingly, CsA led to decreased expression of ENaC, NHE3, GCA, and SGK1 (Figures 2 through 5).

Expression and Localization of AQP2 The expression and localization of AQP2 were analyzed in cryosections of control, sTX, aTX, and aTX ϩ 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 ϩ CsA when compared with control. The localization of AQP2 after aTX and aTX ϩ 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 compen- sate for the decreased expression of AQP1 and probably de-

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- Ct method. The changes in gene expression after aTX (light gray columns), sTX (white columns), aTX ϩ CsA (dark gray columns), and for selected genes after Ctr ϩ CsA (striped columns) are shown. Significantly different gene expressions are marked by an asterisk. Data are presented as mean Ϯ SEM values, and a P value Ͻ0.05 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 expres- sion of the majority of the transporters was still decreased, despite improved renal function in aTX ϩ CsA. For example, the expression of AQP1, AQP3, and AQP4 was still decreased, Figure 4. Changes in gene expression for selected receptors. whereas the expression of AQP2 was increased, compared with The expression for selected receptors was validated by real-time PCR using specific primer pairs or TaqMan gene expression as- aTX (Figure 2). However, the majority of the transporters in- -⌬⌬Ct ϩ says. Relative changes were evaluated using the 2 method. volved in Na retention still showed decreased expression. Changes in gene expression after aTX (light gray columns), sTX One exception was ENaC, which was upregulated by CsA com- (white columns), after aTX ϩ CsA (dark gray columns), and for pared with aTX. These results are comparable with the sTX selected genes after Ctr ϩ CsA (striped columns) are shown. data, where CD proteins AQP2 and ENaC showed normal ex- Significantly different expressed genes are marked by an asterisk pression. CsA treatment also led to normal or even increased (one-way ANOVA, P Ͻ 0.05).

J Am Soc Nephrol 19: 538–546, 2008 Gene Expression of Transporters 541 BASIC RESEARCH www.jasn.org

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 Naϩ excretion within the first 4 d after aTX and massively disturbed urinary concentrating capacity of the graft com- pared 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-repre- sented in the list of genes downregulated after aTX (Figure 2). Several transport systems are involved in water and Naϩ reten- tion, 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 Figure 5. Changes in gene expression for selected regulatory downregulation may contribute to a disturbed ATII signaling factors. The expression for selected regulatory factors was vali- and thereby a decreased NHE3 function. dated by real-time PCR using specific primer pairs or TaqMan Water transport in the kidney is facilitated by the aquapor- gene expression assays. Relative changes were evaluated using ins-1 to -4.23 We observed a downregulation of AQP1 to AQP4 ⌬⌬ the 2- Ct method. Changes in gene expression after aTX (light after aTX (Figure 2). This downregulation might contribute to gray columns), sTX (white columns), aTX ϩ CsA (dark gray col- the increased urinary volume observed after aTX resulting umns), and for selected genes after Ctr ϩ CsA (striped columns) from decreased reabsorption capacity. Expression of factors are shown. Significantly different gene expressions are marked by involved in activation or expression of AQP2, such as V2R, Ͻ an asterisk (one-way ANOVA, P 0.05). 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 dem- onstrated 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 downregu- lated after aTX, leading to an increased Naϩ excretion. A dis- turbed cAMP-signaling due to decreased AC4 and increased PDE expression may contribute to the disturbed ENaC func- tion. 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, Figure 6. Representative immunohistochemical staining of the expression of AQP2, ENaC, MIR, V2R, and AC4 was not AQP2. The expression of AQP2 was not altered after sTX, affected after sTX, indicating that downregulation of these whereas a reduction in expression was observed after aTX and genes after aTX was induced by mechanisms related to the aTX ϩ CsA compared with control. The localization was not altered after aTX and aTX ϩ CsA, whereas after sTX AQP2 seems rejection process. Furthermore, normal expression of AQP2, be localized predominantly on the luminal membrane. ENaC, and their corresponding receptors after sTX seemed to be sufficient for a normal overall kidney function despite the creased reabsorption in the proximal nephron resulting in nor- decrease of several other transporter genes. Renal function af- mal urine concentrating capacity. ter sTX, including creatinine clearance, was similar to that of the control group with both kidneys indicating a compensa- DISCUSSION tory mechanism of the grafted kidney. The CD seems to be most important for the observed tubu- In recent studies, we have demonstrated decreased function lar dysfunction after aTX. Further evidence for this hypothesis and/or expression of renal trasporters, such as NHE3, ENaC, is provided by the data obtained after aTX ϩ CsA, which had

542 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 538–546, 2008 www.jasn.org BASIC RESEARCH normal urinary volume excretion compared with aTX. The with positive effects on AQP2 or ENaC expression were down- expression of AQP1, AQP3, and AQP4, which are not under regulated after aTX followed by an increased Naϩ and water the control of AVP, remained decreased, whereas the expres- excretion. CsA treatment was followed by normal water excre- sion of V2R was normal and the expression of AQP2 and AC4 tion, increased Naϩ retention correlating with normal expres- was even higher than in controls. sion of AQP2, ENaC, and the majority of their regulatory fac- Treatment with CsA led to reduced expression of PDEs. tors. But the urinary concentrating ability was impaired. After This may increase cAMP levels, leading to a pronounced acti- sTX, the expression of AQP2 and ENaC was normal. vation of PKA and thereby of AQP2 and ENaC. CsA inhibits the calcium/calmodulin-dependent (PP2B).25 In- hibition of PP2B is followed by increased cAMP-mediated in- CONCISE METHODS sulin secretion in RINm5F cells.26 This could also apply for AQP2 and ENaC regulation by cAMP. Downregulation of Kidney Transplantation AVP signaling in the CD is likely to be responsible for the Experiments were approved by a governmental committee were per- observed increased volume excretion. Furthermore, aTX ϩ formed in accordance with national animal protection guidelines CsA led to normal AQP2 expression and possibly normal acti- (Westfa¨lische-Wilhems Universita¨t Mu¨nster, Mu¨nster, Germany). vation of AQP2. These results are in contrast to the observed Renal TXs were performed as described previously.5,6,35 For the long-term effects of CsA.27 This could be explained by differ- present study, all recipients were bilaterally nephrectomized immedi- ences in the used models, transplanted versus native kidney, ately before TX. For aTX and aTX ϩ CsA rats treated with CsA (5 and time points (i.e.,4dinthis study versus 4 wk in the previ- mg/kg per d), kidneys of LBN rats (n ϭ 5) were transplanted into LEW ously reported study27). The control group treated with CsA rats. The Ctr ϩ CsA animals were treated with CsA (5 mg/kg per d) showed a slight reduction of AQP2 and a significant reduction without TX (n ϭ 5). For sTX, kidneys from LBN-rats were trans- of ENaC expression. The creatinine clearance was similar to planted into LBN rats. The second kidney of the LBN donors (n ϭ 5) the aTX group. CsA treatment was not followed by a complete served as control. compensation of the grafted kidney as observed after sTX. CsA treatment after aTX was followed by increased expres- Histology and Immunofluorescence sion of ENaC and MIR and a massive decrease in fractional Histology and immunofluorescence were performed as described be- ϩ Na excretion. From the examined transporters involved in fore.6 AQP2 was detected using a specific antibody directed against ϩ Na retention, only ENaC showed an altered expression. The AQP2, kindly provided by Dr. Enno Klussmann.36 The bound pri- increased expression of AQP2 and ENaC after aTX ϩ CsA was mary antibody detected using a secondary goat antirabbit Alexa 488 not induced by a direct effect of CsA because CsA treatment labeled antibody (Invitrogen, Carlsbad, CA). alone did not induce such increases (Figure 2). An underlying ϩ mechanism for the increased ENaC expression and Na reten- General Functional Data tion could be an increased aldosterone level after aTX ϩ CsA. Overall functional data were obtained as described previously.5,6 Twenty- This would lead to an increased ENaC activity. Aldosterone four hours before TX surgery and at the endpoint, before kidney organ also induced a decreased apical and a pronounced basolateral harvest, animals were housed in metabolic cages. Urine and blood sam- sorting of AQP2 in the CD.28 The same study also reported a ples were analyzed for protein (Bradford Blue, Bio-Rad Laboratories, reduction in urine osmolality following an aldosterone treat- Mu¨nchen, Germany), creatinine (Enzym-Pap; Roche Diagnostics, ment that is comparable to our findings. Mannheim, Germany), and electrolytes by flame photometry (Instru- Renal TX is followed by hypertension in up to 80% of trans- mentation Laboratory 943, Kirchheim, Germany). Aldosterone was plant recipients with poor graft survival, reduced life expectancy, quantified by RIA (Aldosterone MAIA, Adaltis, Freiburg, Germany). and increased cardiovascular mortality.29–32 The pathogenesis of hypertension is complex and may reflect the influence of the pri- RNA Analysis mary renal disease, vascular injury, graft dysfunction, and the ef- After graft removal, the total RNA was isolated using an RNeasy-kit (Qia- fect of immunosuppressive therapy. AQP2 and ENaC can con- gen, Hilden, Germany) and used to prepare biotinylated target cDNA. tribute to the development of hypertension. For example, Target cDNAs were processed as per manufacturer’s instructions (http:// spontaneously hypertensive rats showed increased expression of www.affymetrix.com). Data were analyzed using Affymetrix GCOS array ϩ AQP2 and ENaC, thereby mediating increased water and Na analysis software. All data have been deposited in NCBIs Gene Expression retention.33 A prolonged change in expression beyond day 4 of Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through these transporters in aTX ϩCsA may contribute to a similar water GEO series accession number GSE6497. and Naϩ retention as described in these hypertensive rats and probably in patients treated with CsA.34 Identification of Differentially Expressed Genes In conclusion, our gene expression study showed that sev- Significant changes in the gene expression after aTX were identi- eral factors may contribute to changes in expression and activ- fied using class comparison with the BRB ArrayTools developed by ity of AQP2 and ENaC, the major transporters for water and R. Simon and A. Peng (http://linus.nci.nih.gov/BRB-ArrayTools. ϩ Na in the CD, after aTX and aTX ϩ CsA. Regulatory factors html). Nominal significance level of each univariate test was set to

J Am Soc Nephrol 19: 538–546, 2008 Gene Expression of Transporters 543 BASIC RESEARCH www.jasn.org

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 1 Cnp1 NM_022388.1 FXYD domain-containing ion transport regulator 4 Fxyd4 NM_012770.1 1, soluble, beta 2 Gucy1b2 NM_022284.1 Guanylate cyclase activator 2b Guca2b ϩ NM_176080.2 Na -dependent 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 A2, group IVA (cytosolic, calcium-dependent) Pla2g4a NM_017023.1 Potassium inwardly-rectifying channel, subfamily J, member 1 Kcnj1 NM_031088.1 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 13 (sodium-dependent dicarboxylate transporter), member 2 Slc13a2 NM_177962.2, Solute carrier family 14 (), member 2 Slc14a2 NM_031672.1 Solute carrier family 15 (Hϩ/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 ), 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 ), 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 ϩ 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

0.001. Confidence level of false discovery rate assessment used was ment analysis was performed for the lists of upregulated or downregu- 90%, and the maximum allowed numbers of false-positive genes lated after aTX to identify GO terms38 over-represented within a given were set to 10. candidate list (P Ͻ 0.05, Fishers exact test).

Functional Annotation Real-Time PCR The Database for Annotation, Visualization, and Integrated Discov- Real-time PCR was performed using the SYBR Green PCR Master ery (DAVID) was used for functional classification.37 Gene enrich- Mix or TaqMan Universal PCR Master Mix with the ABI PRISM 7700

544 Journal of the American Society of Nephrology J Am Soc Nephrol 19: 538–546, 2008 www.jasn.org BASIC RESEARCH

Sequence Detection System. Specific primer pairs or TaqMan Gene rat. Proc Natl Acad SciUSA91: 8984–8988, 1994 expression assays were used. All instruments and reagents were pur- 12. Noda Y, Sasaki S: Regulation of aquaporin-2 trafficking and its binding chased by Applied Biosystems (Darmstadt, Germany). Relative gene protein complex. Biochim Biophys Acta 1758: 1117–1125, 2006 -⌬⌬Ct 13. Garty H, Palmer LG: Epithelial sodium channels: function, structure, expression values were evaluated with the 2 method using and regulation. Physiol Rev 77: 359–396, 1997 GAPDH or 18s-RNA as housekeeping genes.39 A list with the gene 14. Pacha J, Frindt G, Antonian L, Silver RB, Palmer LG: Regulation of Na names, accession numbers, and gene symbols is provided in Table 3. channels of the rat cortical collecting tubule by aldosterone. J Gen Physiol 102: 25–42, 1993 15. Butterworth MB, Edinger RS, Johnson JP, Frizzell RA: Acute ENaC Statistics stimulation by cAMP in a kidney cell line is mediated by exocytic Data were tested with one-way analysis of variance using GraphPad- insertion from a recycling channel pool. J Gen Physiol 125: 81–101, Prism 4.0 (San Diego, CA). 2005 16. Pearce D: SGK1 regulation of epithelial sodium transport. Cell Physiol Biochem 13: 13–20, 2003 17. Snyder PM: Minireview: regulation of epithelial Naϩ channel traffick- ACKNOWLEDGMENTS ing. Endocrinology 146: 5079–5085, 2005 18. Donowitz M, Cha B, Zachos NC, Brett CL, Sharma A, Tse CM, Li X: The authors thank Zerina Lokmic for critically reading the manu- NHERF family and NHE3 regulation. J Physiol 567: 311, 2005 script. This work was supported by the fund “Innovative Medical 19. Cha B, Kim JH, Hut H, Hogema BM, Nadarja J, Zizak M, Cavet M, Lee-Kwon W, Lohmann SM, Smolenski A, Tse CM, Yun C, de Jonge Research” of the University of Mu¨nster Medical School (ED210404) HR, Donowitz M: cGMP inhibition of Naϩ/Hϩ antiporter 3 (B.E.) and Else-Kro¨ner-Fresenius-Foundation (P22/05//A43/05// (NHE3) requires PDZ domain adapter NHERF2, a broad specificity F00) (E.S.). protein kinase G-anchoring protein. J Biol Chem 280: 16642–16650, 2005 20. Weinman EJ, Minkoff C, Shenolikar S: Signal complex regulation of renal transport proteins: NHERF and regulation of NHE3 by PKA. Am J DISCLOSURES Physiol Renal Physiol 279: F393–F399, 2000 The authors have no financial conflict of interest. 21. Dousa TP: Cyclic-3Ј,5Ј-nucleotide phosphodiesterase isozymes in cell biology and pathophysiology of the kidney. Kidney Int 55: 29–62, 1999 22. Geibel J, Giebisch G, Boron WF: Angiotensin II stimulates both REFERENCES Naϩ-Hϩ exchange and Naϩ/HCO3- cotransport in the rabbit proximal tubule. 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