ABC Transporter Expression Profiling After Ischemic Reperfusion Injury in Mouse Kidney M Huls1, JJMW Van Den Heuvel1, HBPM Dijkman2, FGM Russel1 and R Masereeuw1

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provided by Elsevier - Publisher Connector original article http://www.kidney-international.org & 2006 International Society of Nephrology

ABC transporter expression profiling after ischemic reperfusion injury in mouse kidney M Huls1, JJMW van den Heuvel1, HBPM Dijkman2, FGM Russel1 and R Masereeuw1

1Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands and 2Department of Pathology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Renal ATP binding cassette (ABC) transporters have an Members of the ATP binding cassette (ABC) superfamily of important role in the elimination of metabolic waste transporters are expressed in almost all tissues and cells where products and compounds foreign to the body. The kidney many of them are involved in the cellular defense against has the ability to tightly control the expression of these efflux endogenous and exogenous xenobiotics, and their meta- transporters to maintain homeostasis, and as a major bolites.1,2 Their overexpression in tumor cells is associated mechanism of adaptation to environmental stress. In the with multidrug resistance, which is a major complication for present study, we investigated the expression of 45 ABC successful cancer chemotherapy. The transport proteins transporter genes in the mouse kidney under basal couple ATP hydrolysis to the transport of specific substrates. conditions, after induction of ischemia and after In excretory organs, like the kidney, ABC transporters have regeneration. Two days after clamping, mice showed a 76% an important role in the elimination of metabolic waste decrease in renal creatinine clearance, which improved products and compounds foreign to the body. The kidney has clearly within 7 days. This was confirmed by histological the ability to optimize the effectiveness of the excretory examinations. Seven days after ischemia, real-time systems through a dynamic functional regulation at trans- quantitative Polymerase chain reaction data showed that criptional and post-transcriptional levels in order to maintain transcript abundance of abcb1, abcb11, and abcc4 was homeostasis.3 Under pathological conditions, waste products increased, and that of abca3, abcc2, and abcg2 decreased. and xenobiotics may accumulate in the kidney owing to Expression of all transporters returned to baseline after 14 tubular dysfunction,4 and there is increasing evidence from days, except for abcb11, which was reduced. Abcb11 is the animal models that the expression level of efflux transporters major liver canalicular bile salt export pump. Here we show is tightly controlled as a major mechanism of adaptation to for the first time expression in the kidney and localization of environmental stress.5 the transporter to the apical membrane of proximal tubules. In addition to their efflux function, ABC transporters have The presence of another novel renal transporter, abca3, was been implicated in cellular protection against apoptosis and confirmed by Western blotting. Immunohistochemistry hypoxic damage.6,7 Furthermore, they have been associated showed that abca3 is localized to the peritubular capillaries with cell proliferation and differentiation in regeneration of, andapicalmembraneofproximaltubules.Inconclusion, among others, liver, heart, and skeletal muscle9–11 but it is after inducing ischemic reperfusion injury in the kidney, ABC unknown whether they play a role in kidney repair after renal transporters appear to be differentially regulated, which injury. For a few ABC transport proteins, differential might be associated with the renal regeneration process. expression was found following renal injury. After induction Furthermore, we showed for the first time expression and of acute renal failure in rats, a decrease in Abcb1 protein subcellular localization of abcb11 and abca3 in mouse levels was observed in the kidney, whereas the expression in kidney. liver and brain was unchanged.12 In contrast, using Kidney International (2006) 69, 2186–2193. doi:10.1038/sj.ki.5000407; nephrectomy as a model of chronic renal failure in rats published online 12 April 2006 in vivo, Laouari et al.13 showed that renal Abcb1 mRNA KEYWORDS: acute renal failure; renal protection; ABC transporter; expression expression levels were unchanged 3 weeks and significantly increased 6 weeks after nephrectomy. However, protein levels remained unchanged during both periods. On the other hand, Abcc2 protein and mRNA levels were clearly elevated Correspondence: R Masereeuw, Department of Pharmacology and in kidney and liver, possibly as an adaptive response to Toxicology, 149, Nijmegen Centre for Molecular Life Sciences, Radboud 13 University Nijmegen Medical Centre, PO Box 9101, Nijmegen 6500 HB, elevated levels of circulating toxins. The Netherlands. E-mail: [email protected] The present study was designed to evaluate the renal Received 13 January 2006; revised 20 February 2006; accepted 21 expression of 45 different ABC transporters after damage of February 2006; published online 12 April 2006 the mouse kidney. One of the most frequently occurring

2186 Kidney International (2006) 69, 2186–2193 M Huls et al.: Differential expression of ABC transporters original article

clinical problems concerning the kidney is acute kidney ab injury, which is associated with a high morbidity and P mortality. Because of the necrosis of renal tubules observed, P P the clinical phenomenon is termed acute tubular necrosis. Renal tubular cells are very sensitive to oxygen deprivation, G therefore, ischemic insults, especially in combination with potentially toxic drugs, are the most important causes of acute tubular necrosis. Ischemia may be a result of operative crossclamping of the aorta and/or renal arteries leading to cd necrosis in certain segments of the proximal tubule and to a lesser extent the thick ascending limb.4 Despite the high vulnerability of the kidney to ischemia and toxicity, the organ has a natural ability to recover and regenerate after acute damage. Dedifferentiation of glomerular and tubular cells to a more embryonic/mesenchymal phenotype represents a key process for recovery in response to damage.14 Whether ABC transporters play a role in the renal recovery process is yet Figure 1 | Histopathology of the mouse kidney. Characteristic undeﬁned. After applying ischemia–reperfusion injury to histologic signs of renal injury and regeneration after an ischemic insult. (a) Control kidney slice showing a glomerulus (G) surrounded mouse kidneys, we found that a number of ABC transporters by proximal tubules (P). (b) Two days after bilateral clamping of the are differentially regulated, which might be associated with renal artery and vein for 30 min, serious damage to the proximal renal regeneration. Furthermore, we show for the ﬁrst time tubules (P) is observed with debris in tubular lumen (indicated by arrows). (c) Seven days after the insult, the tubules are partially expression and localization of abcb11 and abca3 in mouse recovered as can be concluded from repaired brush border kidney. membranes (indicated by arrows). (d) Almost completely restored tubular segments (indicated by arrows) were found after 14 days. RESULTS Original magnification, (all the figures) Â 400 . Ischemic–reperfusion injury in the kidney

Histological examination 2 days after inducing renal ischemia FVB revealed serious damage to the proximal tubules, compared 0.075 Sham-operated mice to the control (Figure 1a). Loss of proximal tubule cells and loss of brush border membranes caused tubular obstruction 0.050 (arrows in Figure 1b). After 7 days, recovery of the proximal tubules was observed. New brush border membranes were 0.025 formed but also cast formation was seen (arrows in Figure *** 1c). Finally, after 14 days, the proximal tubules were almost Clearance (ml/min) fully recovered (arrows in Figure 1d). 0.000 Blood and urine samples were collected 24 h before and 2 0.0 2.5 5.0 7.5 and 7 days after surgery, and creatinine concentrations were Time (days) determined. After 2 days, creatinine clearance decreased Figure 2 | Effect of kidney ischemia–reperfusion on renal signiﬁcantly by 75% (one-way analysis of variance), and creatinine handling. Creatinine clearances from sham-operated mice and mice that underwent bilateral ischemia for 30 min followed recovered partially to a level of 60% 7 days after ischemia as by 7 days of recovery. Data are presented as mean7s.e.m. of eight compared to the normal baseline creatinine clearance in independent experiments. *Significantly different from basal value sham-operated mice (Figure 2). (Po0.001).

Gene expression in the mouse kidney before and after expression remained stable after inducing ischemia ischemia (CT ¼ 19.870.32, n ¼ 16) and during the recovery period In mice, 51 members of the abc family have been identiﬁed, (CT ¼ 19.170.19, n ¼ 16). Other housekeeping genes, which show high concordance with their corresponding glucuronidase and hypoxanthine guanine phosphoribosyl human gene orthologs. The ABC family is divided into transferase 1, gave comparable values to glyceraldehyde-3- seven classes (A–G) of which the A and C classes contain the phosphate dehydrogenase (data not shown). Table 1 presents largest number of members (12 each). The expression the expression values of the transporter genes before patterns of 48 different transporter genes were assessed (control) and 7 and 14 days after the induction of ischemia. by real-time quantitative PCR (RQ-PCR). Their expression Measuring the expression values 2 days after ischemia was not was normalized for the average cycle threshold (CT) value possible because of the poor quality of the mRNA. for the housekeeping gene, glyceraldehyde-3-phosphate Transporters that showed a high gene expression in controls dehydrogenase (CT ¼ 19.270.48, n ¼ 16), and expressed as are abca3, abcc2, abcd3, abcd4, and abcg2 with DCT values delta CT values. Glyceraldehyde-3-phosphate dehydrogenase varying between 2.1 and 4.1. After ischemic injury, several

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Table 1 | ABC transporter gene expression along the kidney after inducing ischemia

a b ABC transporter Delta CT values

Gene symbol Assay Ids FVB controls FVB 7 days after ischemia FVB 14 days after ischemia Abca1 Mm00442646_m1 6.470.11 4.170.42 6.970.10 Abca2 Mm00431553_m1 6.270.02 7.470.95 7.070.66 Abca3 Mm00550501_m1 2.170.56 3.970.82 2.270.25 Abca4 Mm00492004_m1 9.370.12 12.570.38 8.870.46 Abca5 Mm00461656_m1 6.970.18 7.570.34 7.570.44 Abca6 Mm00461636_m1 11.070.07 8.770.54 11.670.11 Abca7 Mm00497010_m1 5.670.06 5.470.94 6.670.77 Abca8a Mm00462440_m1 9.570.26 8.270.32 7.970.13 Abca8b Mm00457361_m1 10.170.04 9.470.68 10.970.06 Abca9 Mm00461704_m1 8.070.22 7.270.31 8.270.65 Abcb1a Mm00440761_m1 9.270.03 9.470.07 9.470.25 Abcb1b Mm00440736_m1 11.070.35 8.770.13 8.770.41 Abcb4 Mm00435630_m1 14.370.02 9.070.03 12.670.62 Abcb6 Mm00470049_m1 6.170.28 7.370.19 6.970.97 Abcb9 Mm00498197_m1 8.170.44 9.570.15 8.570.58 Abcb10 Mm00497927_m1 7.570.26 8.470.23 8.470.60 Abcb11 Mm00445168_m1 11.470.23 5.470.56 21.270.09 Tap1 Mm00443188_m1 6.870.03 4.370.06 8.070.17 Tap2 Mm00441668_m1 6.570.26 5.770.35 7.070.58 Abcc1 Mm00456156_m1 6.770.29 5.670.28 7.470.12 Abcc2 Mm00496899_m1 4.170.08 6.170.98 4.570.37 Abcc3 Mm00551550_m1 10.170.18 7.170.11 11.170.07 Abcc4 c 7.170.25 6.070.21 6.870.67 Abcc5 Mm00443360_m1 7.370.23 7.470.04 8.370.09 Abcc8 Mm00803450_m1 11.070.19 12.370.39 14.570.83 Abcc9 Mm00441638_m1 6.770.21 6.770.09 7.070.10 Abcc10 Mm00467403_m1 6.570.32 7.570.08 8.070.28 Abcd1 Mm00431749_m1 6.370.51 6.370.05 7.570.24 Abcd2 Mm00496455_m1 8.570.21 8.670.21 10.170.18 Abcd3 Mm00436150_m1 3.570.31 4.870.18 3.270.21 Abcd4 Mm00436180_m1 4.170.47 5.770.32 4.470.19 Abce1 Mm00649858_m1 3.570.05 5.570.59 6.770.14 Abcf2 Mm00457400_g1 6.370.21 6.470.36 6.170.13 Abcf3 Mm00658695_m1 6.370.17 7.070.21 7.970.44 Abcg1 Mm00437390_m1 6.770.28 5.370.09 7.470.27 Abcg2 Mm00496364_m1 2.270.18 4.870.31 3.370.28 Abcg3 Mm00446072_m1 12.970.63 9.970.57 13.170.13 Abcg4 Mm00507250_m1 14.570.18 12.370.27 15.570.33 Abcg5 Mm00446249_m1 15.270.09 11.770.48 15.070.31 dAbcg8 Mm00445970_m1 20.670.41 15.970.26 21.270.78 ABC, ATP binding cassette; FVB, friend leukaemia virus B strain. aGene expression values of different ABC transporters (http://nutrigene.4t.com/humanabc.htm) in the mouse kidney before inducing ischemia and 7 and 14 days after inducing ischemia. bData of 16 pooled samples, measured in duplicate, are given. cPrimers and probe designed by PrimerExpress Software, version 1.5 (Applied Biosystems; see Materials and Methods section). d Low-expressed transport protein: CT value is not considered as valid.

transporter genes were upregulated (i.e. at least a two times increased expression of NF-kB was observed (DCT: higher expression) or downregulated (at least a two times 1.6670.36). lower expression) compared to control values from sham- operated animals.15 Fourteen days after ischemia, the Protein expression expression levels of all genes returned to baseline except for Seven days after inducing ischemia, we performed Western abcb11, which was downregulated. Based on their variable blotting to determine the protein expression of abca3, expression pattern or remarkably high expression in the abcb1a/b, abcb11, abcc2, abcc4, and abcg2. The presence kidney, the transporter gene products of abca3, abcb1a/b, and localization of abca3 (150 kDa) in the kidney has, as yet, abcc2, abcc4, and abcb11 were investigated further. not been described. Figure 3a shows that after inducing Furthermore, the expression of the transcription factor, ischemia, abca3 was downregulated. On the other hand, nuclear factor-kB (NF-kB), was investigated. Gene expression abcb1, with a size of approximately 140 kDa, was clearly was induced 7 days after ischemia (DCT: 1.9670.52 vs upregulated (Figure 3b). Abcb11 is the major liver canalicular 4.2670.98). Fourteen days after the ischemic insult, still an bile salt export pump; however, its expression in the kidney

2188 Kidney International (2006) 69, 2186–2193 M Huls et al.: Differential expression of ABC transporters original article

ab ab

200 40 *** *** 30 * * 20 100 B

10 Mean pixel intensity Mean pixel intensity 0 0 FVB FVB ischemia FVB FVB ischemia cd c d 250 250 *** *** 200 200 150 150 100 100 50 50

Mean pixel intensity 0 Mean pixel intensity 0 FVB FVB ischemia FVB FVB ischemia ef e f

250 300 *** * 200 200 150 G 100 100 P 50 Mean pixel intensity Mean pixel intensity 0 0 FVB FVB ischemia FVB FVB ischemia Figure 4 | Renal distribution of mouse abca3, abcb11, and abcg2. Figure 3 | Immunoblot analyses of ABC proteins in FVB mice (a) Representative immunohistochemical images show that abca3 is before and 7 days after inducing ischemia. Total membrane expressed in the peritubulary capillaries (indicated by arrows) and the fractions of mice kidneys were isolated and analyzed for expression of apical membranes of the proximal tubules (*). (b) In addition, abca3 is (a) abca3, (b) abcb1, (c) abcb11, (d) abcc2, (e) abcc4, and (f) abcg2. expressed in Bowman’s capsule (B). (c and d) Abcb11 is expressed in Values are means7s.e., n ¼ 4, where pixel intensity (arbitrary units) the apical membrane of the proximal tubules, as observed using two was set at 1.0 in the control. Significantly different from control different antibodies (c: antibody from Kamiya Biomedical Company, (*P 0.05, **P 0.01, ***P 0.001). o o o Seattle, WA, USA; (d) antibody from professor Dr B Stieger, University Hospital Zu¨rich, Switzerland). (e) Abcc4 expression seen in the apical membrane of the proximal tubules. (f) After double staining with was not detected until now. Our Western blot results Na þ ,Kþ -ATPase, which is localized to the basolateral membrane, it is confirmed the RQ-PCR data and showed that the transport clear that abcg2 is expressed in the apical membrane of the proximal tubules. G, glomerulus; B, Bowman’s capsule, P, proximal tubule, D, protein is present in the mouse kidney, but, in contrast with distal tubule (original magnification, (a and b) Â 500; (c) Â 200; (d–f) its gene expression, the protein disappeared within 7 days Â 400). after renal injury (Figure 3c). Both abcc2 and abcc4 showed a slight downregulation (Figure 3d and e), whereas the expression of abcg2 was upregulated after inducing renal membranes of the proximal tubules (Figure 4c and d), injury (Figure 3f). which indicates that abcb11 may function as a urinary efflux pump. Immunohistochemistry The localization of Abcc4/abcc4 to the apical membranes Because RQ-PCR data and Western blotting revealed two of renal proximal tubules has been described previously;16,17 ABC transporters that have not been described before in the however, its segmental distribution is unknown. A represen- kidney, namely abca3 and abcb11, we investigated their renal tative immunohistochemical image (Figure 4e) indicates distribution in more detail. Immunohistochemical analysis that the protein is expressed in all the segments of the indicated that abca3 is localized in the peritubular capillaries proximal tubule. Finally, the expression of abcg2 was of the kidney (Figure 4a and b). After counterstaining against investigated. After counterstaining with Na þ ,K þ -ATPase,18 heparan sulfate (Figure 4a and b), a specific staining of the a clear apical staining of abcg2 in the proximal tubules is abca3 transporter was observed in the apical membrane of observed (Figure 4f). several segments of the proximal tubule. Furthermore, Bowman’s capsule appeared to express abca3. DISCUSSION Figure 4c and d shows expression of abcb11 in the kidney. In the present study, we investigated the gene expression The transporter was found to be localized to the apical profile of ABC transporters after ischemia–reperfusion injury

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in the mouse kidney. Histological and functional data in the present study may be an adaptive response to increased indicate that the postischemic kidney has the ability to urinary substrates in renal failure.17 restore its damaged nephron segmental structure and its A downregulation after ischemia at the mRNA level was function, which is in good agreement with previously established for abca3, abcc2, and abcg2. Abca3 and abcc2 published studies.19,20 Two days after ischemia, brush border protein levels were also decreased, in contrast to abcg2, which membranes are lost and cell debris is deposited in the lumen was upregulated. Abca3 is expressed in the lung in the of the proximal tubules. Seven days after ischemia, cast alveolar type II cells, where it is supposed to play an formation is seen but brush border membranes are nearly important role in the formation of pulmonary surfactant.38 intact, and after 14 days the kidney is almost completely Mutations in the human ABCA3 transporter have been recovered from injury. identified as a cause of acute respiratory distress syndrome Our RQ-PCR experiments demonstrated increased mRNA and can cause fatal surfactant deficiency in newborns.39 Our levels of the transporter genes abcb1, abcb11, and abcc4, 7 data demonstrate that abca3 is highly expressed in the kidney days after inducing ischemia. The upregulation of abcb1 is and that it is localized in the peritubular membranes, the likely to be an adaptive response in the regeneration process apical membranes of the proximal tubule, and in Bowman’s of the kidney after injury, and facilitates in this way the efflux capsule of the kidney. We can only speculate about the of waste products. Reoxygenation of the kidney following function of the transporter in the kidney. ABCA3 likely plays ischemia is accompanied by the production of free radicals, a role in lipid homeostasis, as it closely resembles other potentially leading to oxidative damage. In the brain, Abcb1 ABCA family members that are known to transport expression was also found to be upregulated after oxidative phospholipids. Reorganization of membrane lipids may be stress.21 Abcb1 is upregulated in other stress situations as an important function in the regeneration process of well, like cadmium toxicity and after insulin stimulation;22,23 damaged tissue.40 however, a downregulation was observed after chronic renal Abcc2 was downregulated after ischemia at both the failure.13 Under these conditions, the induction of the mRNA level and protein levels. This is different from the abcb1a/1b transporter was suggested to be mediated through study by Laouari et al.,13 who found an increase in Abcc2 signaling of the NF-kB pathway. It is known that reactive after chronic renal failure; however, a downregulation is in oxygen species are formed in proximal tubules after agreement with our findings in killifish renal tubules ischemia,24,25 which activate NF-kB.23,26 NF-kB gene expres- after short-term exposure to nephrotoxic compounds.41,42 sion was increased fourfold, 7 and 14 days after ischemia, Remarkably, in liver failure, the transport protein is down- which suggests an activation of this pathway. This issue will regulated in the hepatocyte but upregulated in the kidney, be addressed further in the near future. probably to facilitate the renal excretion of accumulating bile In contrast to their mRNA levels, Western blot analysis, salts.43,44 The expression of Abcc2 in the liver is under the however, showed that protein levels of abcb11 and abcc4 were control of nuclear receptor activation, which is impaired decreased after the ischemic insult, which indicated that these during cholestasis,29,37 but post-transcriptional mechanisms transporters are regulated by a post-transcriptional mecha- of downregulation have been reported as well.31 Similar to nism. This is in good agreement with previous observations abcc4, the expression of abcc2 in liver opposes from those in for abcb11 in the liver and abcc4 in the kidney.17,27,28 ABC the kidney in liver failure, indicating that regulation of transporters expression is clearly regulated at the transcrip- transport proteins in the kidney is clearly distinct from the tional level,29 but several mechanisms of post-transcriptional mechanisms observed in the liver. regulation have been described as well, including phosphory- Abcg2 or Breast Cancer Resistance Protein 1 is highly lation and protein routing.30,31 Using two different anti- expressed in the mouse kidney and is known to transport bodies, we showed by immunohistochemistry that abcb11 several anticancer drugs, like methotrexate, topotecan, and localizes to the apical membrane of the proximal tubular cells doxorubicin.45 After inducing an ischemic insult, mRNA of the kidney. Abcb11 has been studied intensively in the liver levels were downregulated in contrast to the protein levels where it is localized to the canalicular microvilli and to that were upregulated. Its localization in the apical mem- subcanalicular vesicles of the hepatocyte.32 The efflux pump brane of renal proximal tubules and substrate specificity mediates the ATP-dependent transport of conjugated mono- suggest that upregulation of abcg2 after injury is an adaptive valent bile salts, like glycocholate, taurochenodeoxycholate, response to accumulating harmful compounds that have to and tauroursodeoxycholate.33 In addition to these bile salts, be excreted into urine. In addition to its role in detoxification compounds like vinblastine, pravastatine, taxol, and calcein through efflux, ABCG2 has been shown to be a determinant have been reported to be Abcb11 substrates.34–36 Expression in the side population phenotype of progenitor cells,46,47 has in human and rat liver is variable under cholestatic been associated with regeneration and repair of skeletal conditions and in advanced cirrhosis, in which the protein muscle and heart tissue, and it was suggested that the protein and the gene were upregulated.37 In addition, Abcc4 was protects cells from hypoxic damage by controlling porphyrins upregulated in rat liver in obstructive cholestasis, whereas the and/or heme levels.6 Because of its high expression in the protein was downregulated in the kidney.17 As suggested for mouse kidney and upregulation after ischemic–reperfusion Abcc4, the downregulation of abcb11 and abcc4 in the kidney damage, we speculate that abcg2 may have a role in tissue

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defense and regeneration by cell differentiation in the kidney performed in a total mixture of 20 ml containing 50 mM Tris-HCl as well. (pH 8.3), 75 mM KCl, 3 mM MgCl2,10mM dithiothreitol, 12.5 mM In conclusion, our results show for the first time dNTP mix, 200 U Mo-MLV reverse transcriptase (Invitrogen, Breda, expression and subcellular localization of abcb11 and abca3 The Netherlands), 20 U RNAsin (Promega, Madison, WI, USA), and m in the kidney. Several transporter genes and proteins appear 5 g of random hexamers (Pharmacia, Uppsala, Sweden). This reaction mixture was incubated at 201C for 10 min, at 421C for to be differentially regulated after renal injury, which might 45 min, and at 951C for 10 min. be associated with the renal regeneration process. Further research is needed to elucidate the functional role of the Quantitative PCR transporters in renal tissue repair and regeneration. For RQ-PCR, two pooled samples obtained from 16 different mice were used. RQ-PCR was performed in duplo using the ABI/PRISM MATERIALS AND METHODS 7900HT Gene Expression Micro Fluidic Card (Applied Biosystems, Materials Foster City, CA, USA) according to the manufacturer’s instructions. Primer and probe sets for RQ-PCR were purchased from Applied Primer and probe set ID numbers are given in Table 1. Abcc4 was Biosystems (Zwijndrecht, The Netherlands). Primers for PCR performed separately because it was not available at the Micro experiment were obtained from Biolegio (Malden, The Nether- Fluidic Card, and the utilized primers and probe were designed lands). Paraplast was obtained from Amsterstad (Amsterdam, The by PrimerExpress Software (version 1.5, Applied Biosystems, Foster Netherlands). Nitrocellulose membranes were acquired from City, CA, USA). The primers used were as follows: mMRP4_RT_Fw, Amersham (Buckinghamshire, UK). Vectashield, mounting med- 50-CTGCAGGACGCCAACCTCTG-30; mMRP4_RT_Rev, 50-CTT ium, normal goat serum, avidin and biotin, and the vectastain ABC CTTCCAGTCTCCGCTTATGA-30. Probe sequence was TET-50- kit were obtained from Vector Laboratories Inc. (Burlingame, CA, CGCGCGTGTTCTTCTGGTGGC-30-TAMRA (Applied Biosystems, USA). Antibody directed against ABCB1 (C219), and peroxidase- Zwijndrecht, The Netherlands). cDNA amplification was performed labeled and biotinylated secondary antibodies were purchased from with TaqMans Universal PCR Master Mix (Applied Biosystems, Dako Cytomation (Heverlee, Belgium). Secondary fluorescent Zwijndrecht, The Netherlands), 5 pmol of TET-labeled probe, and antibodies were obtained from Molecular Probes Diaminobenzidine 15 pmol of forward and reverse primer in a total reaction volume of chromogene was acquired from Sigma-Aldrich (Zwijndrecht, 25 ml. The thermal cycling conditions were 2 min at 501C and 10 min Leiden, The Netherlands). The antibody directed against abcg2 at 951C, followed by 40 cycles of 9 s at 951C and 1 min at 601C. PCR was obtained from Kamiya Biomedical Company (Seattle, WA, reactions were analyzed using either ABI PRISM 7900HT Sequence USA). The abca3 antibody was a kind gift from Dr Nobohiro Ban Detection System or the 7700 Sequence Detection System (Applied (Akita, University School of Medicin, Akita, Japan). Antibodies Biosystems, Foster City, CA, USA). For normalization, the house- directed against abcb11 were obtained from Kamiya Biomedical keeping genes glyceraldehyde-3-phosphate dehydrogenase, glucur- Company (Seattle, WA, USA) and from Professor Dr Bruno Stieger. onidase, and hypoxanthine guanine phosphoribosyl transferase 1 The antibody used for heparan sulfate staining was obtained from were used. Dr TH van Kuppevelt48 and the antibody against Na-K-ATPase was from Dr JB Koenderink.18 All other chemicals used were of the Membrane preparation and Western blotting highest purity available and were obtained from Sigma-Aldrich Total membrane fractions were obtained using the micro-dismem- (St Louis, MO, USA). brator (Sartorius BBI Systems GmbH, Melsungen, Germany). To this end, frozen kidneys were pulverized (2000 r.p.m., 30 s) and Animals and ischemia induction transferred to ice-cold TS buffer (10 mM Tris-HCl, 250 mM sucrose) All procedures involving animals were approved by the Animal including protease inhibitors (1 mM phenylmethylsulfonyl fluoride, Experimental Committee of the Radboud University Nijmegen. 1 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin, and 10 mM Male FVB (Friend leukaemia virus B strain) mice (6–8 weeks) were E64). From these suspensions, a supernatant was prepared (12 000 g, obtained from Charles River (Germany), and were housed under 30 min, 41C), which was centrifuged for 75 min at 105 000 g at 41C. routine laboratory conditions at the Central Animal Facility The resulting pellet containing crude membrane fractions (30 mg) Nijmegen. Mice were anesthesized using isoflurane and ischemia was solubilized in Laemmli sample buffer, heated at 651C for 10 min, was induced by bilateral clamping of the renal artery and vein for separated on a 6 or 10% polyacrylamide gel, and transferred to 30 min; control mice were sham operated. Mice were placed in Hybond-C pure nitrocellulose membrane. Transfer of proteins was metabolic cages 2, 7, and 14 days after inducing ischemia to collect confirmed by the reversible staining of the membrane with Ponceau urine and blood. Mice were killed by exsanguination. Kidneys were Red. Subsequently, the blot was blocked for 60 min with 5% nonfat removed and divided in two parts: one part was used for histological dry milk powder in Tris-buffered saline supplemented with 0.1% examination and was fixed in Bouin’s solution. The other part was Tween 20 (TBS-T), after which the blot was washed twice in TBS-T. used for immunohistochemistry, RNA, and membrane isolation and The membrane was incubated at 41C overnight with the primary was stored at À801C until further use. Serum creatinine levels were antibody: Abca3 diluted 1:1000,50 Abcb1a/b diluted 1:200, Abcb11 determined using the Jaffe´ method.49 diluted 1:500, Abcc2 diluted 1:1000,51 Abcc4 diluted 1:1000,16 and Abcg2 diluted 1:50. After two washes for 5 min with TBS-T, the blot RNA isolation and cDNA reaction was blocked for 30 min as described above. The blot was then RNA isolation was performed using the micro-dismembrator washed twice with TBS-T and incubated at room temperature for (Sartorius BBI Systems GmbH, Melsungen, Germany). Total RNA 60 min with the secondary antibody goat anti-mouse, goat anti- was isolated with Trizol reagent (Invitrogen, Breda, The Nether- rabbit, and goat anti-rat diluted 1:5000. Finally, the blot was washed lands). RNA material from 16 mice was pooled in two groups and twice for 5 min with TBS-T and TBS, respectively. Proteins were used for reverse transcription in duplicate. Reverse transcription was visualized using enhanced chemiluminescence (Pierce, Rockford, IL,

Kidney International (2006) 69, 2186–2193 2191 original article M Huls et al.: Differential expression of ABC transporters

USA). For semiquantification, we measured the pixel intensity of the 7. Leslie EM, Deeley RG, Cole SP. Multidrug resistance proteins: role of P-glycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense. Toxicol bands using Scion image version beta 4.02 for Windows (Scion Appl Pharmacol 2005; 204: 216–237. Corp., Frederick, MD, USA). 8. Assmann KJ, van Son JP, Dijkman HB et al. Antibody-induced albuminuria and accelerated focal glomerulosclerosis in the Thy-1.1 transgenic mouse. Histological examination Kidney Int 2002; 62: 116–126. 9. Martin CM, Meeson AP, Robertson SM et al. Persistent expression of the For light microscopy, kidney fragments taken 2, 7, and 14 days after ATP-binding cassette transporter, Abcg2, identifies cardiac SP cells in the inducing ischemia were fixed in Bouin’s solution, dehydrated, and developing and adult heart. Dev Biol 2004; 265: 262–275. embedded in paraplast. Sections of 3 mm were stained with periodic 10. Meeson AP, Hawke TJ, Graham S et al. Cellular and molecular regulation acid-Schiff.8 of skeletal muscle side population cells. Stem Cells 2004; 22: 1305–1320. 11. Israeli D, Ziaei S, Gonin P et al. A proposal for the physiological significance of mdr1 and Bcrp1/Abcg2 gene expression in normal tissue Immunohistochemistry regeneration and after cancer therapy. J Theor Biol 2005; 232: 41–45. Serial tissue sections of 1.5 mm were obtained using a cryostat, air 12. Huang ZH, Murakami T, Okochi A et al. Expression and function of P-glycoprotein in rats with glycerol-induced acute renal failure. Eur J dried, and fixed in acetone for 10 min. Again, slices were air dried Pharmacol 2000; 406: 453–460. and incubated for 60 min with the first antibody in phosphate- 13. Laouari D, Yang R, Veau C et al. Two apical multidrug transporters, P-gp buffered saline supplemented with 1% bovine serum albumin. Slices and MRP2, are differently altered in chronic renal failure. Am J Physiol were rinsed with phosphate-buffered saline for 10 min and Renal Physiol 2001; 280: F636–F645. 14. Ricardo SD, Deane JA. Adult stem cells in renal injury and repair. incubated with the secondary antibody Alexa Fluor 488 or Alexa Nephrology (Carlton) 2005; 10: 276–282. Fluor 594 for 45 min, diluted 1:200. After rinsing, slices were 15. Winer J, Jung CK, Shackel I et al. Development and validation of real-time mounted in Vectashield mounting medium. For the antibodies quantitative reverse transcriptase-polymerase chain reaction for mon- abcb11 and abcc4,16 slices of 4 mm were obtained using a cryostat itoring gene expression in cardiac myocytes in vitro. Anal Biochem 1999; 270: 41–49. and fixed in acetone for 5 min. Endogenous peroxidase was 16. van Aubel RA, Smeets PH, Peters JG et al. The MRP4/ABCC4 gene encodes quenched by incubation with 5% H2O2 for 5 min. Sections were a novel apical organic anion transporter in human kidney proximal air dried, incubated with normal goat serum for 10 min, and tubules: putative efflux pump for urinary cAMP and cGMP. JAmSoc incubated with the primary antibody in phosphate-buffered saline Nephrol 2002; 13: 595–603. 17. Denk GU, Soroka CJ, Takeyama Y et al. Multidrug resistance-associated and 1% bovine serum albumin overnight. Slices were rinsed with protein 4 is up-regulated in liver but down-regulated in kidney in phosphate-buffered saline and incubated with avidin for 10 min and obstructive cholestasis in the rat. J Hepatol 2004; 40: 585–591. biotin for 10 min. Slices were rinsed and incubated with the 18. Koenderink JB, Geibel S, Grabsch E et al. Electrophysiological analysis of biotinylated goat anti-rabbit secondary antibody (dilution 1:200) for the mutated Na,K-ATPase cation binding pocket. J Biol Chem 2003; 278: 51213–51222. 30 min followed by incubation with the vectastain ABC kit for 19. Witzgall R, Brown D, Schwarz C et al. Localization of proliferating cell 45 min. Subsequently, color reaction was developed using diamino- nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. benzidine chromogene. Slices were counterstained with hematoxy- Evidence for a heterogenous genetic response among nephron lin, dehydrated, and mounted in mounting medium. Sections were segments, and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 1994; 93: 2175–2188. examined with a confocal laser-scanning microscope (CLSM; Leica 20. Thadhani R, Pascual M, Bonventre JV. Acute renal failure. N Engl J Med lasertechnik GmbH, Heidelberg, Germany). 1996; 334: 1448–1460. 21. Felix RA, Barrand MA. P-glycoprotein expression in rat brain endothelial cells: evidence for regulation by transient oxidative stress. J Neurochem Data analysis 2002; 80: 64–72. Data are given as means7s.e. Mean values were considered to be 22. Thevenod F, Friedmann JM, Katsen AD et al. Up-regulation of multidrug significantly different when Po0.05 by use of a Student’s t-test. resistance P-glycoprotein via nuclear factor-kappaB activation protects Software used for statistical analysis was GraphPad Prisms (version kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis. J Biol Chem 2000; 275: 1887–1896. 4.00 for Windows; Graph Pad Software, San Diego, CA, USA). 23. Zhou G, Kuo MT. NF-kappaB-mediated induction of mdr1b expression by insulin in rat hepatoma cells. J Biol Chem 1997; 272: 15174–15183. ACKNOWLEDGMENTS 24. Chien CT, Lee PH, Chen CF et al. De novo demonstration and co- We gratefully acknowledge Dr Bruno Stieger (University Hospital localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion. J Am Soc Nephrol 2001; 12: Zu¨rich, Switzerland) for providing us with the Abcb11 antibody, and 973–982. 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