Kidney transplantation, especially when associated with acuterejection, 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 increasedfractional excretion of sodium were observed. Gene array analysisrevealed that these findings were associated with significantdownregulation 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 ofrenal transport systems were observed. Syngeneic transplantationor treatment with cyclosporine A following allogeneic transplantationdid not impair graft function but did lead to the downregulationof aquaporin-1, -3, and -4 and several solute transporters.However, expression of aquaporin-2 and the epithelial sodiumchannel did not change, suggesting that the downregulation ofthese transporters following allogeneic transplantation is rejection-dependent.In conclusion, changes in gene expression may explain the impairedhandling of solute and water after allogeneic transplantation,especially during acute rejection. Treatment with cyclosporineA improves the regulation of solute and water by preventingthe downregulation of aquaporin-2 and epithelial sodium channel,even though many other transporter genes remain downregulated.
Renal transplantation (TX) in humans is often accompanied byincreased transport capacities of the graft and disturbancesin salt and water homeostasis.1–3 Half-life of graftswith normal function after TX is 11.5 yr compared with 7.2 yrfor those with impaired renal function.4 A better understandingof the underlying cellular and molecular mechanisms leadingto such changes may help to increase long-term graft survival.
Previously, using an allogeneic rat renal TX (aTX) model, wehave demonstrated acute changes in expression and function ofseveral 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 atthe protein and mRNA level. Major transporters for water andNa+ reabsorption in the collecting duct (CD) are ENaC and AQP2.7The expression and activity of AQP2 are regulated by the antidiuretichormone (vasopressin, AVP) via the G-protein coupled AVP-2 receptor(V2R).8 The binding of AVP to the V2R leads to the activationof 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 phosphorylationof AQP29 and translocation to the luminal membrane.10 AVP alsoinduces mRNA and protein expression of AQP2.11 Several otherfactors also regulate AQP2.12 The activity of ENaC limits Na+reabsorption in the CD.13 Aldosterone activates ENaC, decreasingurinary Na+ excretion and increasing K+ and H+ excretion.14AVP also induces cAMP-mediated translocation of ENaC to theluminal membrane.15 Furthermore, ENaC is regulated by Nedd4–2and SGK116 and several other factors.17 The NHE3 is regulatedby the Na+/H+ exchanger regulatory factor (NHERF2), scaffoldingvarious proteins close to NHE3.18 NHERF2 is needed for the cGMP-mediatedinhibition of NHE3 by the cGMP kinase II (Prkg2).19 In contrastto AQP2 and ENaC, PKA mediates an inhibition of NHE3.20
To investigate possible transplant-related changes in the expressionof 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 mechanismsinduced by the rejection process, we also performed syngeneicTX (sTX). Real-time polymerase chain reaction (PCR) was usedto validate the microarray results for selected genes, and theexpression data were correlated with overall renal function.To understand the influence of immunosuppression on aTX-dependentgene expression, selected genes were also analyzed from animalsthat underwent aTX and were treated with cyclosporine A (aTX+ CsA).
Histologic and Functional Data
Changes in function and expression started on day 1 and weresimilar or even higher on day 4 after TX, after day 5 markednecrosis was observed.5,6 When compared with the control group,the aTX model displayed signs of severe acute rejection characterizedby massive leukocyte infiltration, as shown in Figure 1 andpreviously reported.6 These changes were significantly reducedin kidneys of rats treated with CsA. After aTX, histologic signsof 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 x400.
To assess renal function, blood and urine samples were collectedbefore surgery and at the end of the experiment. The assessmentof the renal function parameters is shown in Table 1. Urinevolume and Na+ and K+ excretion increased and creatinine clearancedecreased after aTX. After sTX, only fractional Na+ excretionwas reduced. CsA treatment after aTX resulted in normal volumeand K+ excretion; however, the urinary concentrating abilitywas still impaired compared with control. Na+ excretion wasdecreased, whereas protein excretion and serum K+ were increased.No significant changes in blood pressure were observed.
Graft recipients were bilaterally nephrectomized immediatelybefore TX. After sTX, the creatinine clearance was normal, indicatingthat an adaptation and activation of compensatory mechanismssuch as increased GFR of the grafted kidney have taken place.The decreased creatinine clearances after aTX and aTX + CsAindicate that such compensatory mechanisms were not presentafter aTX and aTX + CsA. These observations suggest that therejection processes and even the treatment of rejection for4 d with CsA inhibited the activation of compensatory mechanisms.
Differentially Expressed Genes
After aTX, 3871 probe sets were upregulated and 3483 of a totalof 31,000 were downregulated. After sTX, 564 probe sets weredownregulated and 1291 were upregulated. The complete outputfiles 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 upregulatedand 55 in the downregulated group of genes. Enriched GO termswithin the upregulated group of genes indicate a massive activationof the immune response and infiltration of the graft by immunologicactive cells. Over-represented GO terms within the genes downregulatedafter aTX indicate a depression of metabolic and transport processes.Nine GO terms identified genes with functions specifically relatedto kidney function (Figure 2). A table with the list of genesclassified 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 withsignificant importance for renal transport function. The completelists are provided (http://medd.klinikum.uni-muenster.de/forschung/DAVID_chart.xls).Downregulation of such genes leads to substantially decreasedtubular 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
Real-Time PCR
For genes encoding for transporters, receptors, or signalingfactors with renal relevance, expression was validated by real-timePCR. The majority of genes showed decreased expression levelsin all TX models compared with control and, thus, are independentof rejection. All analyzed aquaporins showed a decreased expressionafter aTX (Figure 2). The expression of transporters involvedin Na+ retention (i.e., NHE3, ENaC, Slc13a2, SLC5a2, Slc34a2,or Slc34a3) was also decreased after aTX (Figure 2). The majorityof transporters showed lower expression after sTX or aTX + CsA,with AQP2 and ENaC being an exception. Their expression wasnormal after sTX. As mentioned above, AQP2 and ENaC are themajor transporters for water and Na+ reabsorption in the CD.The majority of the analyzed receptors were also decreased inexpression after aTX compared with control (Figure 3). The V2Rand mineralocorticoid receptor (MIR) are involved in the regulationof AQP2 and ENaC, respectively. In contrast, the expressionof Adora2a, Ptger2, and GCA was increased after aTX comparedwith control. These genes were not affected after sTX, indicatingthat the increased expression after aTX is related to the rejectionprocess. Normally, activation of these receptors initiates signaltransduction pathways inducing other factors such as kinases.The expression of AC type 4 (AC4) was downregulated after aTXcompared with control. On the other hand, the expression ofphosphodiesterases (PDE)21 was up-regulated. However, aftersTX, the expression of these factors were downregulated withthe exception of AC4, Prkcb1, and Cnp1. This indicates thatthe increased expression of these factors, after aTX, was inducedbecause 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-Ct 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 treated4 for d with CsA using real-time PCR. Interestingly, the expressionof the majority of the transporters was still decreased, despiteimproved renal function in aTX + CsA. For example, the expressionof AQP1, AQP3, and AQP4 was still decreased, whereas the expressionof AQP2 was increased, compared with aTX (Figure 2). However,the majority of the transporters involved in Na+ retention stillshowed decreased expression. One exception was ENaC, which wasupregulated by CsA compared with aTX. These results are comparablewith the sTX data, where CD proteins AQP2 and ENaC showed normalexpression. CsA treatment also led to normal or even increasedexpression of V2R and MIR compared with controls (Figure 4).Following CsA treatment, the expression of AC4 was increasedand that of PDEs were decreased (Figure 5). This indicates thata normal or increased expression of receptors and downstreamsignaling factors after CsA treatment is followed by a normalor increased expression or activity of AQP2 and ENaC in theCD. An increased ENaC activity may explain the reduced fractionalNa+ excretion observed in aTX + CsA. Genes that showed increasedexpression after aTX, like GCA, Adora2a, or Ptger2 were decreasedin 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 inthe control (Ctr) + CsA group to identify direct effects ofthe immunosuppressant. Surprisingly, CsA led to decreased expressionof 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-Ct 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-Ct 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 cryosectionsof control, sTX, aTX, and aTX + CsA kidneys by immunofluorescence(Figure 6). No change in total protein expression (as judgedby fluorescence intensity) was observed after sTX while a reducedexpression was evident after aTX and aTX + CsA when comparedwith control. The localization of AQP2 after aTX and aTX + CsAwas similar to control. After sTX, the AQP2 was mainly foundon the luminal membrane. This would suggest an increased AQP2translocation to the luminal membrane, which could be a mechanismto compensate for the decreased expression of AQP1 and probablydecreased reabsorption in the proximal nephron resulting innormal 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.
In recent studies, we have demonstrated decreased function and/orexpression of renal trasporters, such as NHE3, ENaC, and AQP2on day 4 after aTX.5,6 The changes in expression levels of AQP2,ENaC, and NHE3 mRNA reported in this study confirmed our previouslypublished observations.5,6
In this study, we have observed an increased urinary volumeand Na+ excretion within the first 4 d after aTX and massivelydisturbed urinary concentrating capacity of the graft comparedwith sTX, indicating that this functional deterioration is solelythe result of rejection. Microarray analysis showed that geneswith functions related to transport were over-represented inthe list of genes downregulated after aTX (Figure 2). Severaltransport systems are involved in water and Na+ retention, andvarious pathways regulate the expression and activity of thesetransporters.
Angiotensin-II (ATII) for example is an important activatorof NHE3 via the ATII-type-1-receptor (Agtr1a)22; thus, its downregulationmay contribute to a disturbed ATII signaling and thereby a decreasedNHE3 function.
Water transport in the kidney is facilitated by the aquaporins-1to -4.23 We observed a downregulation of AQP1 to AQP4 afteraTX (Figure 2). This downregulation might contribute to theincreased urinary volume observed after aTX resulting from decreasedreabsorption capacity. Expression of factors involved in activationor expression of AQP2, such as V2R, AC4, or PKA, as describedabove, was downregulated after aTX. These data indicate a grosslydisturbed AVP-mediated signal transduction in the CD after aTX.In addition to AQP2, AVP also regulates the expression of severalother genes, such as AQP3, AQP4, or casein kinase II.24 AVPtreatment was demonstrated to increase expression of 137 genes.Of these genes, more than 70% were downregulated in the presentstudy, after aTX again indicating a disturbed AVP signaling.
ENaC is one of the major transporters along the CD. Factorsthat have a positive influence on the activity or expressionof EnaC, such as MIR, SGK1, AC4, PKA, or V2R, were downregulatedafter aTX, leading to an increased Na+ excretion. A disturbedcAMP-signaling due to decreased AC4 and increased PDE expressionmay contribute to the disturbed ENaC function.
The majority of the analyzed genes were also downregulated aftersTX, which suggests an underlying mechanisms induced by ischemia/reperfusion,denervation or other factors related to the surgery and notdue to the acute rejection. Interestingly, the expression ofAQP2, ENaC, MIR, V2R, and AC4 was not affected after sTX, indicatingthat downregulation of these genes after aTX was induced bymechanisms related to the rejection process. Furthermore, normalexpression of AQP2, ENaC, and their corresponding receptorsafter sTX seemed to be sufficient for a normal overall kidneyfunction despite the decrease of several other transporter genes.Renal function after sTX, including creatinine clearance, wassimilar to that of the control group with both kidneys indicatinga compensatory mechanism of the grafted kidney.
The CD seems to be most important for the observed tubular dysfunctionafter aTX. Further evidence for this hypothesis is providedby the data obtained after aTX + CsA, which had normal urinaryvolume excretion compared with aTX. The expression of AQP1,AQP3, and AQP4, which are not under the control of AVP, remaineddecreased, whereas the expression of V2R was normal and theexpression of AQP2 and AC4 was even higher than in controls.
Treatment with CsA led to reduced expression of PDEs. This mayincrease cAMP levels, leading to a pronounced activation ofPKA and thereby of AQP2 and ENaC. CsA inhibits the calcium/calmodulin-dependentphosphatase (PP2B).25 Inhibition of PP2B is followed by increasedcAMP-mediated insulin secretion in RINm5F cells.26 This couldalso apply for AQP2 and ENaC regulation by cAMP. Downregulationof AVP signaling in the CD is likely to be responsible for theobserved increased volume excretion. Furthermore, aTX + CsAled to normal AQP2 expression and possibly normal activationof AQP2. These results are in contrast to the observed long-termeffects of CsA.27 This could be explained by differences inthe used models, transplanted versus native kidney, and timepoints (i.e., 4 d in this study versus 4 wk in the previouslyreported study27). The control group treated with CsA showeda slight reduction of AQP2 and a significant reduction of ENaCexpression. The creatinine clearance was similar to the aTXgroup. CsA treatment was not followed by a complete compensationof the grafted kidney as observed after sTX.
CsA treatment after aTX was followed by increased expressionof ENaC and MIR and a massive decrease in fractional Na+ excretion.From the examined transporters involved in Na+ retention, onlyENaC showed an altered expression. The increased expressionof AQP2 and ENaC after aTX + CsA was not induced by a directeffect of CsA because CsA treatment alone did not induce suchincreases (Figure 2). An underlying mechanism for the increasedENaC expression and Na+ retention could be an increased aldosteronelevel after aTX + CsA. This would lead to an increased ENaCactivity. Aldosterone also induced a decreased apical and apronounced basolateral sorting of AQP2 in the CD.28 The samestudy also reported a reduction in urine osmolality followingan aldosterone treatment that is comparable to our findings.
Renal TX is followed by hypertension in up to 80% of transplantrecipients with poor graft survival, reduced life expectancy,and increased cardiovascular mortality.29–32 The pathogenesisof hypertension is complex and may reflect the influence ofthe primary renal disease, vascular injury, graft dysfunction,and the effect of immunosuppressive therapy. AQP2 and ENaC cancontribute to the development of hypertension. For example,spontaneously hypertensive rats showed increased expressionof AQP2 and ENaC, thereby mediating increased water and Na+retention.33 A prolonged change in expression beyond day 4 ofthese transporters in aTX + CsA may contribute to a similarwater and Na+ retention as described in these hypertensive ratsand probably in patients treated with CsA.34
In conclusion, our gene expression study showed that severalfactors may contribute to changes in expression and activityof AQP2 and ENaC, the major transporters for water and Na+ inthe CD, after aTX and aTX + CsA. Regulatory factors with positiveeffects on AQP2 or ENaC expression were downregulated afteraTX followed by an increased Na+ and water excretion. CsA treatmentwas followed by normal water excretion, increased Na+ retentioncorrelating with normal expression of AQP2, ENaC, and the majorityof their regulatory factors. But the urinary concentrating abilitywas impaired. After sTX, the expression of AQP2 and ENaC wasnormal.
Kidney Transplantation
Experiments were approved by a governmental committee were performedin accordance with national animal protection guidelines (Westfälische-WilhemsUniversität Münster, Münster, Germany). RenalTXs were performed as described previously.5,6,35 For the presentstudy, all recipients were bilaterally nephrectomized immediatelybefore TX. For aTX and aTX + CsA rats treated with CsA (5 mg/kgper d), kidneys of LBN rats (n = 5) were transplanted into LEWrats. The Ctr + CsA animals were treated with CsA (5 mg/kg perd) without TX (n = 5). For sTX, kidneys from LBN-rats were transplantedinto LBN rats. The second kidney of the LBN donors (n = 5) servedas control.
Histology and Immunofluorescence
Histology and immunofluorescence were performed as describedbefore.6 AQP2 was detected using a specific antibody directedagainst AQP2, kindly provided by Dr. Enno Klussmann.36 The boundprimary antibody detected using a secondary goat antirabbitAlexa 488 labeled antibody (Invitrogen, Carlsbad, CA).
General Functional Data
Overall functional data were obtained as described previously.5,6Twenty-four hours before TX surgery and at the endpoint, beforekidney organ harvest, animals were housed in metabolic cages.Urine and blood samples were analyzed for protein (BradfordBlue, Bio-Rad Laboratories, München, Germany), creatinine(Enzym-Pap; Roche Diagnostics, Mannheim, Germany), and electrolytesby 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 targetcDNA. Target cDNAs were processed as per manufacturer's instructions(http://www.affymetrix.com). Data were analyzed using AffymetrixGCOS array analysis software. All data have been deposited inNCBIs 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 identifiedusing class comparison with the BRB ArrayTools developed byR. Simon and A. Peng (http://linus.nci.nih.gov/BRB-ArrayTools.html).Nominal significance level of each univariate test was set to0.001. Confidence level of false discovery rate assessment usedwas 90%, and the maximum allowed numbers of false-positive geneswere set to 10.
Functional Annotation
The Database for Annotation, Visualization, and Integrated Discovery(DAVID) was used for functional classification.37 Gene enrichmentanalysis was performed for the lists of upregulated or downregulatedafter aTX to identify GO terms38 over-represented within a givencandidate list (P < 0.05, Fishers exact test).
Real-Time PCR
Real-time PCR was performed using the SYBR Green PCR MasterMix or TaqMan Universal PCR Master Mix with the ABI PRISM 7700Sequence Detection System. Specific primer pairs or TaqMan Geneexpression assays were used. All instruments and reagents werepurchased by Applied Biosystems (Darmstadt, Germany). Relativegene expression values were evaluated with the 2-Ct method usingGAPDH or 18s-RNA as housekeeping genes.39 A list with the genenames, accession numbers, and gene symbols is provided in Table 3.
The authors have no financial conflict of interest.
Acknowledgments
The authors thank Zerina Lokmic for critically reading the manuscript.This work was supported by the fund "Innovative Medical Research"of the University of Münster Medical School (ED210404)(B.E.) and Else-Kröner-Fresenius-Foundation (P22/05//A43/05//F00)(E.S.).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
