http://informahealthcare.com/ceh ISSN: 1064-1963 (print), 1525-6006 (electronic) Clin Exp Hypertens, Early Online: 1–11 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10641963.2014.954711

Differential transcriptional activity of kidney genes in hypertensive ISIAH and normotensive WAG rats Olga Evgenievna Redina, Svetlana Eduardovna Smolenskaya, Tatiana Olegovna Abramova, Liudmila Nikolaevna Ivanova, and Arcady L’vovich Markel

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Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russian Federation

Abstract

Keywords

Transcriptional activity of the kidney genes was compared in hypertensive ISIAH and normotensive WAG rats using the oligonucleotide microarray technique. Most of differentially expressed genes were downregulated in ISIAH kidney both in renal cortex and medulla. According to functional annotation the kidney function in ISIAH rats is based on altered expression of many genes working in stress-related mode. The alterations in gene expression are likely related to both pathophysiological and compensatory mechanisms. The further studies of genes differentially expressed in ISIAH and WAG kidney will help to reveal new hypertensive genes and mechanisms specific for stress-induced arterial hypertension.

ISIAH rats, kidney, microarray, stress-induced arterial hypertension, transcriptional activity of genes

Introduction Hypertension is the most common disease in industrialized societies. Several clinical and experimental studies indicate that essential hypertension is inherited as a multifactorial disease with a significant genetic and environmental component (1,2). Despite many decades of study, the ultimate cause of essential hypertension remains uncertain. A number of studies highlight the role of the kidney in regulation of blood pressure and the pathogenesis of hypertension (3,4). Renal function plays a major role in long-term control of arterial pressure and sodium balance. All forms of hypertension are thought to be a consequence of abnormal water–electrolyte balancing by kidney (1,5). Because of the multifactorial etiology of essential hypertension, the use of experimental animal models provides valuable information regarding many aspects of the disease, which include etiology, pathophysiology, and treatment (6). The Inherited Stress-Induced Arterial Hypertension (ISIAH) rat strain was developed to study the mechanisms of the stress-induced hypertension and its complications. The ISIAH rats were selected from an outbred Wistar population for a systolic arterial blood pressure (SABP) increase induced by 30 min restraint stress in a small cylindrical wire mesh cage. More than 30 generations of close inbreeding by brother–sister mating resulted in a high genetic homogeneity of the ISIAH strain (7). The ISIAH rats have elevated SABP at basal condition (175.0 ± 3.5 mmHg in males and Correspondence: Olga Evgenievna Redina, Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, 10 Lavrentieva Ave., Novosibirsk 630090, Russian Federation. E-mail: [email protected]

History Received 18 May 2014 Revised 4 July 2014 Accepted 22 July 2014 Published online 30 September 2014

165.0 ± 3.0 mmHg in females) and exhibit a dramatic increase in SABP when restrained (8). The ISIAH rats also show a number of other characteristic features of hypertensive state: hypertrophy of the left ventricle, increase in the wall thickness of the small arteries, and changes in the ECG pattern (9). ISIAH rats are characterized by increased kidney mass (10) and some alterations in kidney histology (11,12). These features, the genetically determined enhanced responsiveness to stressful stimulation, and the predominant involvement of the neuroendocrine hypothalamic–pituitary–adrenocortical and sympathetic adrenal medullary systems during the disease development let to consider the ISIAH rat strain as an advantageous model of the human hypertensive disease (13). The aim of the present work was the search for genetic determinants of the hypertensive state in ISIAH rats using kidney gene-expression studies. The oligonucleotide microarray technique was used to compare the transcriptional activity of genes in kidney of hypertensive ISIAH and normotensive WAG rats. The study has been concentrated on identifying differentially expressed genes between the strains and on revealing the molecular genetic mechanisms defining the functional differences between hypertensive and normotensive kidney.

Materials and methods Animals The work was carried out on hypertensive ISIAH and normotensive Wistar Albino Glaxo (WAG) rats bred in the Laboratory of Experimental Animals at the Institute of Cytology and Genetics (Novosibirsk, Russia). All rats were maintained in the standard conditions with free access to food

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and water. The SABP was measured indirectly by the tail-cuff method. The blood pressure level was determined under short-term ether anesthesia to exclude the effect of psychological stress induced by the measuring procedure. In microarray experiments, the 6-month-old ISIAH (n ¼ 3) and WAG (n ¼ 3) males were used. Their SABP was 173.67 ± 1.86 mmHg in ISIAH and 124.67 ± 2.67 mmHg in WAG males. Renal cortex and renal medulla were analyzed separately. All animal experiments were approved by the Institute’s Animal Care and Use Committee.

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Samples preparation The kidney of the decapitated rats was immediately removed and sectioned to get the test samples of renal cortex and medulla. Samples (50 mg) were homogenized in 1 ml of TRIzol (Invitrogen Life Technologies, Carlsbad, CA) in glass homogenizers, removed to 1.5-ml Eppendorf tubes and stored at 70  C until RNA isolation.

error model, which provides an expression difference score (Diff-Score) taking into account background noise and sample variability. Genes were considered significantly changed at a jDifferential Scorej of more than 20, which was equivalent to a p value of less than 0.01. Fold changes were calculated as ratio of gene expression value in ISIAH to gene expression value in WAG. The functional analysis of differentially expressed genes was performed using DAVID (The Database for Annotation, Visualization and Integrated Discovery) Bioinformatic Resources gene annotation tool (http://david.abcc.ncifcrf. gov/) (14,15). The Gene Ontology (GO) option was utilized to determine the most significant biological processes and molecular functions possibly related to blood pressure regulation. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database was used to identify pathways that were most significant to the data set. Real-time PCR

Microarray experiments The collected samples were sent to JSC Genoanalytika (Moscow, Russia), where total RNA was extracted and processed. Four-hundred nanograms of total RNA was used for complementary RNA in vitro transcription, followed by a T7 RNA polymerase-based linear amplification and labeling with the TotalPrep RNA Labeling Kit using Biotinylated-UTP (Ambion, Austin, TX). The signal was developed by staining with Cy3-streptavidin. The hybridization was performed on Illumina RatRef-12 Expression BeadChip microarray platform containing 22,524 probes for a total of 22,228 rat genes selected primarily from the National Center for Biotechnology Information RefSeq database (Release 16; Illumina, San Diego, CA). Hybridization, washing and staining were carried out according to the Illumina Gene Expression Direct Hybridization Manual. The BeadChip was scanned on a high-resolution Illumina BeadArray reader. Microarray data extraction, normalization, and analyses The primary statistical analysis of the hybridization results was performed by JSC Genoanalytika (Moscow, Russia). The Illumina GenomeStudio software was used to extract fluorescence intensities and normalize the expression data. Data acquisition and analysis were done using gene expression module and rank invariant normalization. After normalization, genes were filtered by their ‘‘detection’’ p value, which had to be less then 0.01 (significantly detected), in both samples. Subsequently, the differentially expressed genes were identified using the Illumina Custom

Two genes Ephx2 and Comt were selected for technical validation using real-time PCR (RT-PCR). Samples of renal cortex and renal medulla were analyzed in 6-month old ISIAH and WAG rats. Each group contained 5 rats. According to microarray data, the Ppia (peptidylprolyl isomerase A) had a nearly constant expression level in all samples and was used as endogenous control. The forward primers for target genes were designed to span exon–exon junctions to avoid amplification from contaminating genomic DNA. Primer’s sequences are given in Table 1. Total RNA extraction and DNAse treatment was performed using the SV total RNA isolation System (Promega, Madison, WI), according to the manufacturer’s instructions. cDNA was synthesized using 1 mg of total RNA, a mixture of reverse primers (75 ng each) and the M-MLV (Promega, Madison, WI, Cat.#M1705) according to the manufacturer’s guidelines. Real-time PCR was performed in a final volume of 25 ml. Reaction volume contained master mix with SYBR Green and an internal reference dye ROX (Cat.# R-414, Syntol, Moscow, Russia), forward primer (200 nM), reverse primer (200 nM), distilled water, and the cDNA template. Appropriate cDNA dilutions were empirically determined in preliminary experiments. All samples were analyzed in duplicates in 96-well plates (Cat.# PCR-96-AB-C, Axygen, Union City, CA). Real-time PCR was carried out using ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA). Cycling parameters were as follows: 95  C for 5 min, and 40 cycles of PCR reaction at 95  C for 15 s, and 62  C for 40 s, and a final dissociation step at 82  C for 40 s.

Table 1. Primers used in real-time PCR. Primers Gene Comt Ephx2 Ppia #

Forward 0

#

Reverse 0

5 -GACAA AGTCACCATCCTCAAT-3 50 -TATGTGACAGTGAAG#CCAGG-30 50 -ACCGTGTTCTTCGACATCAC-30

Position of exon–exon junction site in mRNA.

0

Length of PCR fragment (bp) 0

5 -GAAGTGTGTCTGGAAGGTAG-3 50 -GAAGATGAGTCTCCATAGCC-30 50 -GAACCCTTATAGCCAAATCCT-30

122 192 140

Differentially expressed genes in ISIAH and WAG kidney

DOI: 10.3109/10641963.2014.954711

Dissociation curves for each amplicon were analyzed to verify the specificity of each amplification reaction. To quantify gene expression differences in control and experimental samples, the comparative DDCt threshold change method (relative quantification) was used (16). Threshold cycle (Ct) and baseline were detected, and quantitative PCR fold differences were calculated by ABI 7000 SDS software. Calculations made on renal samples from normotensive WAG rats at rest were used as calibrator. Differences were tested with non-parametric Wilcoxon’s test for paired observations.

Results

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The interstrain gene expression differences Comparative analysis of kidney gene expression profiling in hypertensive ISIAH and normotensive WAG rats revealed 126 differentially expressed genes in renal cortex and 65 differentially expressed genes in renal medulla. The lists of genes differentially expressed in kidney of hypertensive ISIAH and normotensive WAG rats are available on the site of Institute of Cytology and Genetics SB RAS (http://icg.nsc.ru/ isiah/en/). The majority of differentially expressed genes were downregulated in ISIAH rats (61.1% in renal cortex and 78.5% in renal medulla) as compared to WAG (Table 2). Forty-one genes were differentially expressed in both renal tissues (Figure 1), the other, 85 genes in renal cortex and 24 genes in renal medulla, were tissue specific. The differentially expressed genes were annotated in two main categories (biological process and molecular function) of the GO classification. Table 3 contains most significant GO terms which may be related to hypertensive state of ISIAH rats. Renal cortex and renal medulla were characterized by several GO terms common to both renal tissues. These were ‘‘catalytic activity’’ and ‘‘ion binding’’ in molecular function category and ‘‘metabolic process’’, ‘‘response to stress’’, and ‘‘transport’’ in category for biological processes.

Table 2. The number of genes differentially expressed in ISIAH and WAG rat kidney. Number of genes Renal structure

ISIAH5WAG

ISIAH4WAG

Renal cortex Renal medulla

77 51

49 14

ISIAH5WAG – genes downregulated in ISIAH rats; ISIAH4WAG – genes upregulated in ISIAH rats.

3

In renal cortex most groups of genes in molecular function category were represented by approximately equal number of up- and downregulated genes, except the group ‘‘ion binding’’ where 20 out of 27 genes (74.1%) were downregulated in ISIAH rats. In biological process category mostly downregulated genes were associated with terms ‘‘transport’’, ‘‘regulation of apoptosis’’ and ‘‘regulation of cell proliferation’’. In renal medulla, only ‘‘lipid metabolic process’’ group contained a comparable number of up- and downregulated genes in ISIAH rats, in other groups genes were mostly downregulated in hypertensive kidney. Among the genes differentially transcribed in renal cortex of the ISIAH and WAG rats, 38 genes (30.2%) were associated with the GO term ‘‘response to stimulus’’. This group consisted of several subgroups describing specificity of stimulus: ‘‘response to stress’’, ‘‘response to external stimulus’’, ‘‘response to hormone (endogenous) stimulus’’, and some other. The genes related to response to stress were revealed both in renal cortex and renal medulla. In renal cortex the group ‘‘response to stress’’ contained genes for response to oxidative stress, hypoxia, inflammatory response and regulation of response to stress. The numerous genes in group ‘‘response to stress’’ were the same in two renal tissues (Table 4). The differentially expressed genes associated with GO term ‘‘transport’’ were also found in both renal tissues. The genes related to ion transport, lipid transport, and organic alcohol transport were revealed in this group (Table 5). KEGG analysis showed that the interstrain differences in gene expression both in renal cortex and renal medulla were related to cell adhesion molecules and tyrosine metabolic pathways (Table 6). Candidate genes for hypertension Among genes found to be differentially expressed in kidney of ISIAH and WAG rats, 12 genes were reported in Rat Genome Database (RGD) as genes related to hypertension (Table 7). Most of these genes (8 out of 11) were downregulated in hypertensive kidney. The genes Comt and Ephx2 had the most contrast ratios and were the only genes differentially expressed both in renal cortex and renal medulla. These genes were chosen for technical validation of the difference in their transcriptional activity in ISIAH and WAG kidney by real-time PCR (Figure 2). The other 9 genes showed the tissue-specific pattern of expression. Genes associated with renal diseases Among genes differentially expressed in kidney of ISIAH and WAG rats there were 14 genes referred in Rat Genome Database as associated with renal diseases (Table 8). Most of these genes (9 out of 14) were downregulated in hypertensive kidney. Nine genes were common to both hypertension and renal diseases.

Discussion Figure 1. Number of genes differentially expressed in kidney of hypertensive ISIAH and normotensive WAG rats.

A large-scale transcript profiling using RatRef-12 Expression BeadChip helped to detect genes with altered transcriptional activity in hypertensive kidney of ISIAH rats as compared to

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Table 3. Results of functional annotation (GO terms) clustering of genes differentially expressed in ISIAH and WAG rat kidney. Number of genes

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Gene Ontology terms

Number of genes

p Value

ISIAH5WAG

ISIAH4WAG

Renal cortex Molecular function Catalytic activity Hydrolase activity Oxidoreductase activity Ion binding Protein dimerization activity

42 19 12 27 9

5.5E3 5.6E2 4.9E3 4.0E2 1.8E2

25 9 7 20 5

17 10 5 7 4

Biological process Metabolic process Catabolic process Lipid metabolic process Alcohol metabolic process Oxidation reduction Steroid metabolic process Sulfur metabolic process Anti-apoptosis Triglyceride metabolic process Cholesterol metabolic process Response to stimulus Response to stress Response to external stimulus Response to hormone stimulus Response to oxidative stress Response to hypoxia Inflammatory response Regulation of response to stress Transport Ion transport Regulation of apoptosis Regulation of cell proliferation

55 13 12 9 11 5 4 4 3 3 38 22 15 8 6 6 5 5 19 8 10 9

3.2E6 6.0E3 2.0E3 2.6E3 3.5E3 1.8E2 2.8E2 5.6E2 3.6E2 6.9E2 6.7E4 4.1E5 3.0E4 3.3E2 4.8E3 5.8E3 4.1E2 6.3E2 7.6E2 9.4E2 2.0E2 4.5E2

32 5 4 3 6 1 4 3 1 0 23 16 8 4 3 5 4 2 14 7 8 7

23 8 8 6 5 4 0 1 2 3 15 6 7 4 3 1 1 3 5 1 2 2

Renal medulla Molecular function Catalytic activity Ion binding Lipid binding

20 16 6

9.3E2 3.0E2 5.4E3

15 14 5

5 2 1

Biological process Metabolic process Lipid metabolic process Cellular amino acid and derivative metabolic process Steroid metabolic process Response to stress Transport Lipid transport Organic alcohol transport

30 9 4 3 9 11 3 2

7.8E5 6.9E4 7.8E2 9.0E2 4.9E2 9.2E2 5.1E02 7.5E02

24 4 3 2 8 10 2 2

6 5 1 1 1 1 1 0

ISIAH5WAG – genes downregulated in ISIAH rats; ISIAH4WAG – genes upregulated in ISIAH rats. Generalized GO terms are given in bold.

normotensive WAG rat strain. Most of these genes were repressed in hypertensive kidney (Table 2). The down-regulation of many genes (67% of the 505 geneset) was found in old kidneys which, as compared to adult kidney, had more glomerulosclerosis, tubular atrophy, interstitial fibrosis, and fibrous intimal thickening in small arteries (17). Comparative electron microscopic study of glomerular apparatus in 6-month old ISIAH and Wistar rats showed hypertrophy of renal corpuscles in hypertensive kidney, accompanied by multiple structural changes. Complex of these changes was indicative of the increase in filtration barrier functional load, and of initial stages of glomerular (11) and renomedullary sclerosis (12). The downregulation in gene expression was also reported for induced models of hypertension where the

blood pressure elevated due to angiotensin II (AngII) infusion (18,19) or salt loading (20). The reduced expression in the salt-loaded rats was considered as potentially protective against hypertension (20). An increase in blood pressure and maintenance of hypertension caused by different influences (the Ang II increment or glutathione depletion) may occur through the stimulation of oxidative stress (21–24). The ISIAH rats were selected for significant elevation of arterial blood pressure in response to mild emotional stress. Success in selection led to the increased basal level of systolic blood pressure, too. A genetic drift during selection process might result in concentration of the specific alleles of many genes responsible for the stress-induced hypertension.

Differentially expressed genes in ISIAH and WAG kidney

DOI: 10.3109/10641963.2014.954711

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Table 4. Genes differentially expressed in ISIAH and WAG kidney and associated with GO term ‘‘response to stress’’. Ratio ISIAH/WAG, p50.01

Gene symbol

Acc.#

Gene name

Renal cortex Apoh*x $ Becn1xß$ Bnip3*Dß Cat #ß Cd24Dyxß $ Clcnka Clu# $ Comt*$ Cst3#Dß$ Ephx2*y Fads1ß Fn1y Gpx2#xß Ldha#Dß Nbnß$ Nupr1y $ P2rx4*ß Poll* Rfc4 ß Smad4*Dß$ Tf Dyß Txnrd2#

NM NM NM NM NM NM XM NM NM NM NM NM NM XM NM NM NM NM XM NM NM NM

001009626.1 053739.1 053420.2 012520.1 012752.2 053327.1 001053033.1 012531.1 012837.1 022936.1 053445.1 019143.1 183403.1 001080828.1 138873.1 053611.1 031594.1 001014168.1 001056509.1 019275.1 001013110.1 022584.1

Apolipoprotein H (beta-2-glycoprotein I) Beclin 1 BCL2/adenovirus E1B 19 kDa-interacting protein 3 Qatalase CD24 antigen Chloride channel Ka Clusterin Catechol-O-methyltransferase Cystatin C Epoxide hydrolase 2, cytoplasmic Fatty acid desaturase 1 Fibronectin 1 Glutathione peroxidase 2 Lactate dehydrogenase A Nibrin Nuclear protein 1 Purinergic receptor P2X, ligand-gated ion channel 4 (P2rx4), mRNA Polymerase (DNA directed), lambda Replication factor C (activator 1) 4 (predicted) (Rfc4_predicted), mRNA MAD homolog 4 (Drosophila) Transferrin Thioredoxin reductase

0.18 2.21 0.11 1.75 0.49 0.49 0.23 0.17 0.62 16.68 0.59 0.58 2.62 1.46 2.75 0.33 0.32 0.41 0.55 0.40 0.49 0.61

Renal medulla Apoh*x $ Bnip3*Dß Comt*$ Ephx2*y P2rx4*ß Poll* RT1 -Ba Slc14a2 Smad4*Dß$

NM NM NM NM NM NM NM NM NM

001009626.1 053420.2 012531.1 022936.1 031594.1 001014168 001008831.1 019347.1 019275.1

Apolipoprotein H (beta-2-glycoprotein I) BCL2/adenovirus E1B 19 kDa-interacting protein 3 Catechol-O-methyltransferase Epoxide hydrolase 2, cytoplasmic Purinergic receptor P2X, ligand-gated ion channel 4 (P2rx4), mRNA Polymerase (DNA directed), lambda RT1 class II, locus Ba Solute carrier family 14 (urea transporter), member 2 MAD homolog 4 (Drosophila)

0.43 0.03 0.22 20.63 0.29 0.27 0.27 0.36 0.15

*Genes are differentially expressed both in renal cortex and renal medulla of ISIAH and WAG rats. #Genes associated with GO term ‘‘response to oxidative stress’’. DGenes associated with GO term ‘‘response to hypoxia’’. yGenes associated with GO term ‘‘inflammatory response’’. xGenes associated with GO term ‘‘regulation of response to stress’’. ßGenes changing their expression in vascular smooth muscle cells exposed to fluid shear stress (30). Genes associated with GO term ‘‘regulation of apoptosis’’. $Genes associated with GO term ‘‘regulation of cell proliferation’’.

The results of functional annotation of differentially expressed genes in ISIAH and WAG kidney revealed the groups of genes associated with response to stress both in the renal cortex and renal medulla (Table 3). Genes from these groups were involved in response to oxidative stress, hypoxia and inflammation. The oxidative stress is considered to be the pathogenic outcome of oxidant overproduction, which occurs as a result of imbalance between prooxidants and antioxidants (25). The results of our work revealed that some genes with oxidoreductase activity had enhanced expression in hypertensive kidney (Cat, Gpx2, Ldha) and some were downregulated (Cst3, Fads1, Txnrd2) (Table 4). So, the interstrain differences in ISIAH and WAG renal function are probably the result of the imbalance in processes leading to the development of pathology from one side and the processes trying to restore the homeostasis from the other side. In the renal cortex, among the genes related to response to stimulus there were several involved in response to inflammation. The strong association between essential hypertension and inflammatory process is now well documented (26). The inflammation and oxidative stress in particular may

contribute to remodeling process and to hypertensionassociated vascular damage (25). The vascular wall remodeling in hypertension may also be related to growth and apoptosis (27). In our study several differentially expressed genes associated with the GO terms ‘‘regulation of apoptosis’’ and ‘‘regulation of cell proliferation’’ in renal cortex were also related to response to stimulus (Table 4). It is known that patients with essential hypertension are characterized by endothelial dysfunction (28). The expression of genes and proteins in vascular endothelial cells (29) and vascular smooth muscle cells (30) may be modulated by the shear stress. The exposure of vascular smooth muscle cells to shear stress significantly alters the expression of many genes associated with response to oxidative stress and response to hypoxia (30). In the current paper 13 genes out of 24 differentially expressed in ISIAH and WAG kidney and related to response to stress (Table 4) have been reported earlier as genes changing their expression in vascular smooth muscle cells exposed to shear stress (30). These genes may serve as candidates for further studies of kidney vascular smooth muscle cell function in hypertensive ISIAH rats.

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Table 5. Genes differentially expressed in ISIAH and WAG kidney and associated with GO term ‘‘transport’’.

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Gene symbol Renal cortex Adhfe1 Apoh# Bnip3 Cd24 Chchd4 Clcnka* Cldn16* Cubn Fads1 G6pc Gm2a# Myh14 P2rx4* Pctp# Slc17a3* Slc22a8* Slc5a3*D Tf* Tmprss8* Renal medulla Apoh# Bnip3 CadpsD Gc Gm2a# LOC361914* P2rx4* Pctp# Slc14a2* Slc5a3*D Tmprss8*

Ratio ISIAH/WAG, p50.01

Acc.#

Gene name

NM_001025423.1 NM_001009626.1 NM_053420.2 NM_012752.2 NM_001013431.1 NM_053327.1 NM_131905.1 NM_053332.2 NM_053445.1 NM_013098.1 NM_172335.2 XM_001080622.1 NM_031594.1 NM_017225.1 NM_153622.1 NM_031332.1 NM_053715.2 NM_001013110.1 NM_199371.1

Alcohol dehydrogenase, iron containing, 1 Apolipoprotein H (beta-2-glycoprotein I) BCL2/adenovirus E1B 19 kDa-interacting protein 3 CD24 molecule Coiled-coil-helix-coiled-coil-helix domain containing 4 Chloride channel Ka Claudin 16 Cubilin (intrinsic factor-cobalamin receptor) Fatty acid desaturase 1 Glucose-6-phosphatase, catalytic subunit GM2 ganglioside activator myosin, heavy chain 14 Purinergic receptor P2X, ligand-gated ion channel 4 Phosphatidylcholine transfer protein Solute carrier family 17 (sodium phosphate), member 3 Solute carrier family 22 (organic anion transporter 3), member 8 Solute carrier family 5 (inositol transporters), member 3 Transferrin Transmembrane protease, serine 8 (intestinal)

0.06 0.18 0.11 0.49 1.53 0.49 0.51 1.54 0.59 1.50 0.42 0.36 0.32 2.88 0.61 1.91 0.09 0.49 0.46

NM_001009626.1 NM_053420.2 NM_013219.1 NM_012564.1 NM_172335.2 NM_001017465.1

Apolipoprotein H (beta-2-glycoprotein I) BCL2/adenovirus E1B 19 kDa-interacting protein 3 Ca++-dependent secretion activator Group specific component GM2 ganglioside activator Similar to solute carrier family 7 (cationic amino acid transporter, y+ system), member 12 Purinergic receptor P2X, ligand-gated ion channel 4 Phosphatidylcholine transfer protein Solute carrier family 14 (urea transporter), member 2 Solute carrier family 5 (inositol transporters), member 3 Transmembrane protease, serine 8 (intestinal)

0.43 0.03 0.28 0.32 0.38 0.25

NM_031594.1 NM_017225.1 NM_019347.1 NM_053715.2 NM_199371.1

0.29 5.12 0.36 0.09 0.41

*Genes associated with GO term ‘‘ion transport’’. #Genes associated with GO term ‘‘lipid transport’’. D Genes associated with GO term ‘‘organic alcohol transport’’.

Table 6. Metabolic pathways with genes differentially expressed in ISIAH and WAG kidney. Gene symbol

Acc.#

Gene name

Ratio ISIAH/WAG, p50.01

Renal cortex: Cell adhesion molecules Cldn9 NM_001011889.1 Cldn16 NM_131905.1 RT1-A1 NM_001008827.1 RT1-CE15 NM_001008838.1

Claudin 9 Claudin 16 RT1 class Ia, locus A1 RT1 class I, CE15

0.39 0.51 6.09 8.43

Tyrosine metabolism Aox1 Comt

Aldehyde oxidase 1 Catechol-O-methyltransferase

0.29* 0.17

Renal medulla: Cell adhesion molecules Cldn9 NM_001011889.1 RT1-A1 NM_001008827.1 RT1-Ba NM_001008831.1

Claudin 9 RT1 class Ia, locus A1 RT1 class II, locus Ba

0.30 7.82 0.27

Tyrosine metabolism Aox1 Comt

Aldehyde oxidase 1 Catechol-O-methyltransferase

0.25 0.22

NM_019363.2 NM_012531.1

NM_019363.2 NM_012531.1

*p Value 50.05.

Sustained hypertension can occur if there is an abnormality of kidney function that shifts pressure natriuresis so that sodium balance is maintained at elevated blood pressure level (31). Tubular sodium reabsorption depends on the activity of

ion transport systems, which are modulated by neural, endocrine, paracrine, and physical factors (32). The alterations in ion transport are associated with cell membrane lipid disturbances (33) and may play an important role in the

Differentially expressed genes in ISIAH and WAG kidney

DOI: 10.3109/10641963.2014.954711

Table 7. Genes differentially expressed in ISIAH and WAG kidney and referred in Rat Genome Database as associated with hypertension.

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Gene symbol Renal cortex: Becn1 Cat Cldn16 Clu Comt Cst3 Ephx2 Fads1 Fn1 Sncg Tf Renal medulla: Comt Ephx2 RT1-Ba

Acc.#

Gene name

Ratio ISIAH/WAG, p50.01

NM_053739.1 NM_012520.1 NM_131905.1 XM_001053033.1 NM_012531.1 NM_012837.1 NM_022936.1 NM_053445.1 NM_019143.1 NM_031688.1 NM_001013110.1

Beclin 1 (coiled-coil, myosin-like BCL2-interacting protein) Catalase Claudin 16 Clusterin Catechol-O-methyltransferase Cystatin C Epoxide hydrolase 2, cytoplasmic Fatty acid desaturase 1 Fibronectin 1 Synuclein, gamma (breast cancer-specific protein 1) Transferrin

2.21 1.75 0.51 0.23 0.17 0.62 16.7 0.59 0.58 0.49 0.49

NM_012531.1 NM_022936.1 NM_001008831.1

Catechol-O-methyltransferase Epoxide hydrolase 2, cytoplasmic RT1 class II, locus Ba

0.22 20.6 0.27

Figure 2. Confirmation of microarray data by real-time PCR. The y axis represents the relative quantification.

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Table 8. Genes differentially expressed in ISIAH and WAG kidney and referred in Rat Genome Database as associated with renal diseases.

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Gene symbol Renal cortex: Apoh Becn1 Cat Clcnka Cldn16 Comt Cst3 Cubn Ephx2 Fn1 Jund Smad4 Tf Renal medulla: Apoh Comt Ephx2 RT1-Ba Smad4

Acc.#

Gene name

NM_001009626.1 NM_053739.1 NM_012520.1 NM_053327.1 NM_131905.1 NM_012531.1 NM_012837.1 NM_053332.2 NM_022936.1 NM_019143.1 XM_001070425.1 NM_019275.1 NM_001013110.1

Apolipoprotein H (beta-2-glycoprotein I) Beclin 1 (coiled-coil, myosin-like BCL2-interacting protein) Catalase Chloride channel Ka Claudin 16 Catechol-O-methyltransferase Cystatin C Cubilin (intrinsic factor-cobalamin receptor) Epoxide hydrolase 2, cytoplasmic Fibronectin 1 Jun D proto-oncogene MAD homolog 4 Transferrin

0.18 2.21 1.75 0.49 0.51 0.17 0.62 1.54 16.7 0.58 1.64 0.40 0.49

NM_001009626.1 NM_012531.1 NM_022936.1 NM_001008831.1 NM_019275.1

Apolipoprotein H (beta-2-glycoprotein I) Catechol-O-methyltransferase Epoxide hydrolase 2, cytoplasmic RT1 class II, locus Ba MAD homolog 4

0.43 0.22 20.6 0.27 0.15

etiology and/or pathophysiology of hypertension (34,35). The analysis of the genome sequence of spontaneously hypertensive rats (SHR) showed that the list of genes having alterations in their coding sequence was highly enriched by genes related to ion transport (36). Ion transport is one of the major processes that are vital to the functions of kidney and also to the function of the cells in nervous and muscular systems highly responsible for the blood pressure regulation (37). In our study, the differentially expressed genes were enriched for genes associated with the GO term ‘‘transport’’ both in renal cortex and renal medulla. These groups consisted of genes related to ion transport, lipid transport, and organic alcohol transport (Table 5). The alterations in ion transport in hypertensive kidney may be related to several genes contributing to different mechanisms of osmoregulation. Slc5a3 is sodium/myo-inositol co-transporter (38). P2rx4 is encoding the extracellular ATP-gated cation activity (39). Tmprss8 regulates sodium balance by activating the epithelial sodium channel (40). Slc17a3 is known as sodium/ phosphate co-transporter (41), and is considered as common renal secretory pathway for drugs and urate (42). Slc14a2 possesses urea transmembrane transporter activity (43) and may have an important function in urea reabsorption (44). Another gene, helping to create the osmolality gradient is Clcnka. It is involved in chloride transport and may be stimulated by dehydration (45). In Clcnk1/ mice both NaCl and urea accumulation are impaired (46). The downregulation of Clcnka in ISIAH kidney may play a mitigating role against hypertension. Nine genes out of 10 related to ion transport were repressed and only one gene (Slc22a8) was upregulated. Slc22a8 (organic anion transporter 3, Oat3) participates in the renal basolateral transport of glutathione (47). Earlier in study of essential hypertension the significant inverse correlation was obtained between blood pressure and antioxidants (glutathione and super oxide dismutase) level (48). It was

Ratio ISIAH/WAG, p50.01

also shown that induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats (24). OAT3 was found to be significantly elevated in the early stage of renal ischemia/reperfusion injury which may have substantial impact on renal excretion of some drugs and toxic compounds (49). Slc22a8 is localized on chromosome 1 (231.73 Mb) in the most significant QTL for BP (BP306) detected in analysis of ISIAH rats (10). Taking that into consideration, we may suggest that upregulation of Slc22a8 in ISIAH kidney may reflect the increased oxidative stress in ISIAH kidney which may contribute to stress-induced hypertension development. Two genes related to ion transport were annotated in RGD as hypertensive genes. These were transferrin (Tf) and claudin 16 (Cldn16). Transferrin (Tf) is the main iron-transporting protein in blood. Transferrin binds free iron and minimizes its ability to generate the reactive oxygen species considered as an important cause of renal injury (50). The claudins belong to the family of tight junction proteins that define the intercellular space between adjacent endo- and epithelial cells. Claudins are especially important for the regulation of paracellular ion permeability. Mutations in Cldn16 causes disturbances in magnesium and calcium homeostasis (51,52). Genes involved in tight cell junctions are associated with cell adhesion molecules pathway. This was one of the main metabolic pathways defined in analysis of genes differentially expressed in ISIAH and WAG kidney (Table 6). The second metabolic pathway associated with tyrosine metabolism involved two genes (Comt and Aox1) differentially expressed both in renal cortex and renal medulla. Tyrosine is the physiological precursor for catecholamine (dopamine, norepinephrine and epinephrine) synthesis. Renal dopamine acts as a paracrine substance in the control of sodium homeostasis and blood pressure (53). The enzyme catechol-O-methyltransferase (COMT) metabolizes catecholamines and plays an important role in determining the

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DOI: 10.3109/10641963.2014.954711

natriuretic effects of the renal dopamine system (54). In current study Comt was one of the 12 genes found to be differentially expressed in ISIAH and WAG kidney and reported in RGD as genes related to hypertension (Table 7). The downregulation of Comt in ISIAH kidney may lead to increased sympathetic nervous system activity and that is known to contribute to the renal malfunction and hypertension development (55,56). Aox1 (aldehyde oxidase 1) is considered to be a part of the tyrosine degradation pathway (pathway: ko00350) (57). AOX1 produces hydrogen peroxide and can catalyze the formation of superoxide. The decreased level of AOX1 was found earlier in old kidney and was considered as one of the genes which may contribute to decline in renal function (17). Another hypertensive gene – clusterin (Clu) is known to play a protective role, its upregulation attenuates renal fibrosis in obstructive nephropathy (58). Alternatively, the loss of clusterin expression worsens renal ischemia-reperfusion injury (59). The downregulation of Clu expression in ISIAH kidney is in good agreement with the presence of some kidney histological alterations described earlier in ISIAH rats (11,12). Several genes differentially expressed in kidney of the ISIAH and WAG rats were referred in Rat Genome Database as associated with both hypertension and renal diseases. In ISIAH kidney the most strongly enhanced expression was found for Ephx2 encoding the soluble epoxide hydrolase (sEH) which metabolizes the epoxyeicosatrienoic acids (EETs) having antihypertensive properties. EETs possess anti-inflammatory actions that could protect the kidney vasculature from injury during renal and cardiovascular diseases (60,61). The increased expression of Ephx2 may contribute to hypertension development in ISIAH rats through the elevated level of EETs catabolism. Beclin 1 (Becn1), a protein required for early autophagosome formation. Oxidative stress induces autophagy through multiple mechanisms. Autophagy constitutes a major protective mechanism that allows cells to survive in stressful conditions (62). Beclin 1 overexpression heightened autophagic activity and accentuated pathological heart remodeling induced by severe pressure stress (63). In mouse model associated with severe hepatic redox stress, the up-regulation of beclin 1 was protective against liver injury and excessive oxidative stress (64). Ischemia/reperfusion significantly increased renal beclin-1 (65). Based on that, the increased expression of Becn1 in ISIAH kidney may be considered as playing a protective role against the kidney injury. The enhanced expression of catalase (Cat) may play a protective role in ISIAH hypertensive kidney as it is known that catalase overexpression prevents hypertension and tubular apoptosis in angiotensinogen transgenic mice (66). Alternatively, the decreased level of expression of Cst3 and Fads1 genes with protective effects against various oxidative stresses that induce cell death (67,68) may contribute to the oxidant overproduction.

Conclusion The current study demonstrates that the development of essential hypertension as complex disease may be promoted

Differentially expressed genes in ISIAH and WAG kidney

9

through a wide range of mechanisms including neurohormonal changes, sympathetic activation, cardiovascular remodeling, and changes in kidney function and sodium balance. The alterations in gene expression levels are likely related to both pathophysiological and compensatory mechanisms. The further studies should be conducted to see if the altered expression of hypertension-relevant genes in the ISIAH kidney is based on functionally important mutations or other mechanisms with regulatory function. As soon as the kidney function in ISIAH rats is influenced by altered expression of many genes working in stress-related mode, we consider that further studies of genes differentially expressed in ISIAH and WAG kidneys will help to reveal new hypertensive genes and mechanisms specific for stressinduced arterial hypertension.

Acknowledgements The authors are grateful to JSC Genoanalytika (Moscow, Russia) for conducting the array hybridization experiment and the primary statistical analysis of the hybridization results.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work has been supported by the Russian Scientific Foundation, grant No. 14-15-00118.

References 1. Mullins LJ, Bailey MA, Mullins JJ. Hypertension, kidney, and transgenics: a fresh perspective. Physiol Rev 2006;86:709–46. 2. Hauck C, Frishman WH. Systemic hypertension: the roles of salt, vascular Na+/K+ ATPase and the endogenous glycosides, ouabain and marinobufagenin. Cardiol Rev 2012;20:130–8. 3. Guyton AC, Coleman TG, Cowley AW, et al. A systems analysis approach to understanding long-range arterial blood pressure control and hypertension. Circ Res 1974;35:159–76. 4. Herrera M, Coffman TM. The kidney and hypertension: novel insights from transgenic models. Curr Opin Nephrol Hypertens 2012;21:171–8. 5. Hall JE. The kidney, hypertension, and obesity. Hypertension 2003; 41:625–33. 6. Dornas WC, Silva ME. Animal models for the study of arterial hypertension. J Biosci 2011;36:731–7. 7. Adarichev VA, Korokhov NP, Ostapchuk IV, et al. Characterization of rat lines with normotensive and hypertensive status using genomic fingerprinting. Genetika 1996;32:1669–72. 8. Markel AL. Development of a new strain of rats with inherited stress-induced arterial hypertension. In: Sassard J, ed. Genetic hypertension. Paris: Colloque INSERM; 1992:405–7. 9. Markel AL, Maslova LN, Shishkina GT, et al. Developmental influences on blood pressure regulation in ISIAH rats. In: McCarty R, Blizard DA, Chevalier RL, eds. Development of the hypertensive phenotype: basic and clinical studies. Amsterdam: Elsevier; 1999:493–526. 10. Redina OE, Machanova NA, Efimov VM, Markel AL. Rats with inherited stress-induced arterial hypertension (ISIAH strain) display specific quantitative trait loci for blood pressure and for body and kidney weight on chromosome 1. Clin Exp Pharmacol Physiol 2006;33:456–64. 11. Shmerling MD, Filiushina EE, Lazarev VA, et al. Ultrastructural changes of kidney corpuscles in rats with hereditary stress-induced arterial hypertension [Article in Russian]. Morfologiia 2001;120: 70–4.

Clin Exp Hypertens Downloaded from informahealthcare.com by Laurentian University on 11/24/14 For personal use only.

10

O. E. Redina et al.

12. Filyushina EE, Shmerling MD, Buzueva II, et al. Structural characteristics of renomedullary interstitial cells of hypertensive ISIAH rats. Bull Exp Biol Med 2013;155:408–12. 13. Markel AL, Redina OE, Gilinsky MA, et al. Neuroendocrine profiling in inherited stress-induced arterial hypertension rat strain with stress-sensitive arterial hypertension. J Endocrinol 2007;195: 439–50. 14. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4:44–57. 15. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res 2009;37:1–13. 16. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006;1:1559–82. 17. Melk A, Mansfield ES, Hsieh SC, et al. Transcriptional analysis of the molecular basis of human kidney aging using cDNA microarray profiling. Kidney Int 2005;68:2667–79. 18. Makhanova NA, Crowley SD, Griffiths RC, Coffman TM. Gene expression profiles linked to AT1 angiotensin receptors in the kidney. Physiol Genom 2010;42A:211–18. 19. Yuan B, Liang M, Yang Z, et al. Gene expression reveals vulnerability to oxidative stress and interstitial fibrosis of renal outer medulla to nonhypertensive elevations of ANG II. Am J Physiol Regul Integr Comp Physiol 2003;284:R1219–30. 20. Hopcroft LE, McBride MW, Harris KJ, et al. Predictive responserelevant clustering of expression data provides insights into disease processes. Nucleic Acids Res 2010;38:6831–40. 21. Romero JC, Reckelhoff JF. Role of angiotensin and oxidative stress in essential hypertension. Hypertension 1999;34:943–9. 22. Reckelhoff JF, Romero JC. Role of oxidative stress in angiotensininduced hypertension. Am J Physiol Regul Integr Comp Physiol 2003;284:R893–912. 23. Kimura S, Zhang GX, Abe Y. Malfunction of vascular control in lifestyle-related diseases: oxidative stress of angiotensin II-induced hypertension: mitogen-activated protein kinases and blood pressure regulation. J Pharmacol Sci 2004;96:406–10. 24. Vaziri ND, Wang XQ, Oveisi F, Rad B. Induction of oxidative stress by glutathione depletion causes severe hypertension in normal rats. Hypertension 2000;36:142–6. 25. Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension: what is the clinical significance? Hypertension 2004;44:248–52. 26. Androulakis E, Tousoulis D, Papageorgiou N, et al. Inflammation in hypertension: current therapeutic approaches. Curr Pharm Des 2011;17:4121–31. 27. Intengan HD, Schiffrin EL. Vascular remodeling in hypertension: roles of apoptosis, inflammation, and fibrosis. Hypertension 2001; 38:581–7. 28. Ghiadoni L, Taddei S, Virdis A. Hypertension and endothelial dysfunction: therapeutic approach. Curr Vasc Pharmacol 2012;10: 42–60. 29. Chiu JJ, Usami S, Chien S. Vascular endothelial responses to altered shear stress: pathologic implications for atherosclerosis. Ann Med 2009;41:19–28. 30. Ekstrand J, Razuvaev A, Folkersen L, et al. Tissue factor pathway inhibitor-2 is induced by fluid shear stress in vascular smooth muscle cells and affects cell proliferation and survival. J Vasc Surg 2010;52:167–75. 31. Hall JE, Louis K. Dahl Memorial Lecture. Renal and cardiovascular mechanisms of hypertension in obesity. Hypertension 1994;23: 381–94. 32. Strazzullo P, Galletti F, Barba G. Altered renal handling of sodium in human hypertension: short review of the evidence. Hypertension 2003;41:1000–5. 33. Bing RF, Heagerty AM, Thurston H, Swales JD. Ion transport in hypertension: are changes in the cell membrane responsible? Clin Sci (Lond) 1986;71:225–30. 34. Trevisan M, Ostrow D, Cooper R, et al. Abnormal red blood cell ion transport and hypertension. The People’s Gas Company study. Hypertension 1983;5:363–7. 35. Haddy FJ. Abnormalities of membrane transport in hypertension. Hypertension 1983;5:V66–72.

Clin Exp Hypertens, Early Online: 1–11

36. Atanur SS, Birol I, Guryev V, et al. The genome sequence of the spontaneously hypertensive rat: analysis and functional significance. Genome Res 2010;20:791–803. 37. Ko JH, Ko EA, Gu W, et al. Expression profiling of ion channel genes predicts clinical outcome in breast cancer. Mol Cancer 2013; 12:106–22. 38. Chauvin TR, Griswold MD. Characterization of the expression and regulation of genes necessary for myo-inositol biosynthesis and transport in the seminiferous epithelium. Biol Reprod 2004;70: 744–51. 39. Luo J, Yin GF, Gu YZ, et al. Characterization of three types of ATP-activated current in relation to P2X subunits in rat trigeminal ganglion neurons. Brain Res 2006;1115:9–15. 40. Okumura Y, Nishikawa M, Cui P, et al. Cloning and characterization of a transmembrane-type serine protease from rat kidney, a new sodium channel activator. Biol Chem 2003; 384:1483–95. 41. Puschett JB, Whitbred J, Ianosi-Irimie M, et al. Molecular effects of volume expansion on the renal sodium phosphate cotransporter. Am J Med Sci 2003;326:1–8. 42. Jutabha P, Anzai N, Kitamura K, et al. Human sodium phosphate transporter 4 (hNPT4/SLC17A3) as a common renal secretory pathway for drugs and urate. J Biol Chem 2010; 285:35123–32. 43. Karakashian A, Timmer RT, Klein JD, et al. Cloning and characterization of two new isoforms of the rat kidney urea transporter: UT-A3 and UT-A4. J Am Soc Nephrol 1999;10: 230–7. 44. Su H, Carter CB, Frohlich O, et al. Glycoforms of UT-A3 urea transporter with poly-N-acetyllactosamine glycosylation have enhanced transport activity. Am J Physiol Renal Physiol 2012; 303:F201–8. 45. Uchida S, Sasaki S, Furukawa T, et al. Molecular cloning of a chloride channel that is regulated by dehydration and expressed predominantly in kidney medulla. J Biol Chem 1993; 268:3821–4. 46. Akizuki N, Uchida S, Sasaki S, Marumo F. Impaired solute accumulation in inner medulla of Clcnk1/ mice kidney. Am J Physiol Renal Physiol 2001;280:F79–87. 47. Lash LH, Putt DA, Xu F, Matherly LH. Role of rat organic anion transporter 3 (Oat3) in the renal basolateral transport of glutathione. Chem Biol Interact 2007;170:124–34. 48. Sagar S, Kallo IJ, Kaul N, et al. Oxygen free radicals in essential hypertension. Mol Cell Biochem 1992;111:103–8. 49. Zhang R, Yang X, Li J, et al. Upregulation of rat renal cortical organic anion transporter (OAT1 and OAT3) expression in response to ischemia/reperfusion injury. Am J Nephrol 2008;28: 772–83. 50. Haase M, Bellomo R, Haase-Fielitz A. Novel biomarkers, oxidative stress, and the role of labile iron toxicity in cardiopulmonary bypass-associated acute kidney injury. J Am Coll Cardiol 2010;55: 2024–33. 51. Hou J, Shan Q, Wang T, et al. Transgenic RNAi depletion of claudin-16 and the renal handling of magnesium. J Biol Chem 2007;282:17114–22. 52. Haisch L, Almeida JR, Abreu da Silva PR, et al. The role of tight junctions in paracellular ion transport in the renal tubule: lessons learned from a rare inherited tubular disorder. Am J Kidney Dis 2011;57:320–30. 53. Carey RM. Theodore Cooper Lecture: renal dopamine system: paracrine regulator of sodium homeostasis and blood pressure. Hypertension 2001;38:297–302. 54. Eklof AC, Holtback U, Sundelof M, et al. Inhibition of COMT induces dopamine-dependent natriuresis and inhibition of proximal tubular Na+,K+-ATPase. Kidney Int 1997;52:742–7. 55. DiBona GF. Sympathetic nervous system and the kidney in hypertension. Curr Opin Nephrol Hypertens 2002;11:197–200. 56. Grassi G, Bertoli S, Seravalle G. Sympathetic nervous system: role in hypertension and in chronic kidney disease. Curr Opin Nephrol Hypertens 2012;21:46–51. 57. Garattini E, Fratelli M, Terao M. Mammalian aldehyde oxidases: genetics, evolution and biochemistry. Cell Mol Life Sci 2008;65: 1019–48. 58. Jung GS, Kim MK, Jung YA, et al. Clusterin attenuates the development of renal fibrosis. J Am Soc Nephrol 2012;23:73–85.

DOI: 10.3109/10641963.2014.954711

Clin Exp Hypertens Downloaded from informahealthcare.com by Laurentian University on 11/24/14 For personal use only.

59. Zhou W, Guan Q, Kwan CC, et al. Loss of clusterin expression worsens renal ischemia-reperfusion injury. Am J Physiol Renal Physiol 2010;298:F568–78. 60. Imig JD. Epoxide hydrolase and epoxygenase metabolites as therapeutic targets for renal diseases. Am J Physiol Renal Physiol 2005;289:F496–503. 61. Jung O, Brandes RP, Kim IH, et al. Soluble epoxide hydrolase is a main effector of angiotensin II-induced hypertension. Hypertension 2005;45:759–65. 62. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Mol Cell 2010;40:280–93. 63. Zhu H, Tannous P, Johnstone JL, et al. Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest 2007; 117:1782–93. 64. Sun Q, Gao W, Loughran P, et al. Caspase 1 activation is protective against hepatocyte cell death by up-regulating

Differentially expressed genes in ISIAH and WAG kidney

65.

66. 67. 68.

11

beclin 1 protein and mitochondrial autophagy in the setting of redox stress. J Biol Chem 2013;288:15947–58. Yeh CH, Hsu SP, Yang CC, et al. Hypoxic preconditioning reinforces HIF-alpha-dependent HSP70 signaling to reduce ischemic renal failure-induced renal tubular apoptosis and autophagy. Life Sci 2010;86:115–23. Godin N, Liu F, Lau GJ, et al. Catalase overexpression prevents hypertension and tubular apoptosis in angiotensinogen transgenic mice. Kidney Int 2010;77:1086–97. Nishiyama K, Konishi A, Nishio C, et al. Expression of cystatin C prevents oxidative stress-induced death in PC12 cells. Brain Res Bull 2005;67:94–9. Zolfaghari R, Cifelli CJ, Banta MD, Ross AC. Fatty acid delta(5)desaturase mRNA is regulated by dietary vitamin A and exogenous retinoic acid in liver of adult rats. Arch Biochem Biophys 2001;391: 8–15.

Differential transcriptional activity of kidney genes in hypertensive ISIAH and normotensive WAG rats.

Transcriptional activity of the kidney genes was compared in hypertensive ISIAH and normotensive WAG rats using the oligonucleotide microarray techniq...
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