Gene expression profiling in the remnant kidney model of wild type and kinin B1 and B2 receptor knockout mice
2007; Elsevier BV; Volume: 72; Issue: 4 Linguagem: Inglês
10.1038/sj.ki.5002172
ISSN1523-1755
AutoresJoost P. Schanstra, Magdalena Bachvarova, Eric Neau, Jean‐Loup Bascands, Dimcho Bachvarov,
Tópico(s)Kruppel-like factors research
ResumoAngiotensin-converting enzyme inhibitors are the most efficient pharmacologic agents to delay the development of end-stage renal disease (ESRD). This is a multipharmacologic approach that inhibits angiotensin II formation while increasing kinin concentrations. Considerable attention has been focused on the role of decreased angiotensin II levels; however, the role of increased kinin levels is gaining in interest. Kinins affect cellular physiology by interacting with one of two receptors being the more inducible B1 and the more constitutive B2 receptors. This study utilizes the mouse remnant kidney of 20 weeks duration as a model of ESRD. Whole mouse genome microarrays were used to evaluate gene expression in the remnant kidneys of wild type, B1 and B2 receptor knockout animals. The microarray data indicate that gene families involved in vascular damage, inflammation, fibrosis, and proteinuria were upregulated, whereas gene families involved in cell growth, metabolism, lipid, and protein biosynthesis were downregulated in the remnant kidneys. Interestingly, the microarray analyses coupled to histological evaluations are suggestive of a possible protective role of kinins operating through the B2 receptor subtype in this model of renal disease. The results highlight the potential of microarray technology for unraveling complex mechanisms contributing to chronic renal failure. Angiotensin-converting enzyme inhibitors are the most efficient pharmacologic agents to delay the development of end-stage renal disease (ESRD). This is a multipharmacologic approach that inhibits angiotensin II formation while increasing kinin concentrations. Considerable attention has been focused on the role of decreased angiotensin II levels; however, the role of increased kinin levels is gaining in interest. Kinins affect cellular physiology by interacting with one of two receptors being the more inducible B1 and the more constitutive B2 receptors. This study utilizes the mouse remnant kidney of 20 weeks duration as a model of ESRD. Whole mouse genome microarrays were used to evaluate gene expression in the remnant kidneys of wild type, B1 and B2 receptor knockout animals. The microarray data indicate that gene families involved in vascular damage, inflammation, fibrosis, and proteinuria were upregulated, whereas gene families involved in cell growth, metabolism, lipid, and protein biosynthesis were downregulated in the remnant kidneys. Interestingly, the microarray analyses coupled to histological evaluations are suggestive of a possible protective role of kinins operating through the B2 receptor subtype in this model of renal disease. The results highlight the potential of microarray technology for unraveling complex mechanisms contributing to chronic renal failure. The prevalence and incidence of end-stage renal disease (ESRD) has dramatically increased over the last decade.1.U.S. Renal Data System USRDS 2005 Annual Data Report: Atlas of End-Stage Renal Disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD2004Google Scholar Regardless of the underlying etiology, chronic renal insufficiency is accompanied by a permanent loss of functional nephrons leading to a progressive loss of renal function.1.U.S. Renal Data System USRDS 2005 Annual Data Report: Atlas of End-Stage Renal Disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD2004Google Scholar The remnant kidney model (subtotal or 5/6 nephrectomy (SNX)) is an experimental animal model of chronic renal disease progression in which the development of segmental glomerulosclerosis and tubulointerstitial fibrosis are both clearly established after 20 weeks following SNX.2.Kren S. Hostetter T.H. The course of the remnant kidney model in mice.Kidney Int. 1999; 56: 333-337Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar It is now well admitted that angiotensin-converting enzyme inhibitors (ACEi) are the most efficient drugs to delay ESRD development.3.Heart Outcome Prevention Evaluation (HOPE) investigators Effects of ramipril on cardiovascular and microvsacular outcomes in people with diabetes mellitus: results of the HOPE study and MICRO-HOPE substudy.Lancet. 2000; 355: 253-259Abstract Full Text Full Text PDF PubMed Scopus (3093) Google Scholar ACE inhibition decreases angiotensin II (vasoconstrictor peptide) and increases kinin (vasodilator peptides) concentrations.4.Chambell D.J. Kladis A. Duncan A.M. Effects of converting enzyme inhibitors on angiotensin and bradykinin peptides.Hypertension. 1994; 23: 439-449Crossref PubMed Scopus (300) Google Scholar The beneficial effects of ACE inhibitors have been well determined; kinin implications are less clear, although in several studies a role for kinins was proposed.5.Uehara Y. Hirawa N. Numabe A. et al.Long-term infusion of kallikrein attenuates renal injury in Dahl salt-sensitive rats.Am J Hypertens. 1997; 10: 83S-88SCrossref PubMed Google Scholar,6.Wang D.Z. Chao L. Chao J. Hypotension in transgenic mice overexpressing human bradykinin B2 receptor.Hypertension. 1997; 29: 488-493Crossref PubMed Google Scholar Kinins execute their physiological function through the activation of two types of kinin receptors, the B1 (B1R) which is overexpressed in pathological states and the B2 (B2R) which is constitutively expressed.7.Marceau F. Hess J. Bachvarov D. The B1 receptors for kinins.Pharmacol Rev. 1998; 50: 357-386PubMed Google Scholar A number of reports have shown involvement of kinin receptors, as components of the renal kallikrein–kinin system, in disease states that lead to renal failure (reviewed in Leeb-Lundberg et al.8.Leeb-Lundberg L.M. Marceau F. Muller-Esterl W. et al.International union of pharmacology. XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences.Pharmacol. Rev. 2005; 57: 27-77Crossref PubMed Scopus (759) Google Scholar). We have recently demonstrated that a number of genes relevant to renal physiology/pathology were differentially expressed in B1R-knockout (B1R-KO) and B2R-knockout (B2R-KO) mice, which is indicative for the important role of both kinin receptors in renal function.9.Bachvarov D. Bachvarova M. Koumangaye R. et al.Renal gene expression profiling using kinin B1 and B2 receptor knockout mice reveals comparable modulation of functionally related genes.Biol Chem. 2006; 387: 15-22Crossref PubMed Scopus (11) Google Scholar To date, kidney disease-oriented research has focused on mechanisms responsible for ESRD initiation and progression. However, because ESRD is a complex disease, interruption of a single pathway is unlikely to result in significant therapeutic benefit. Further understanding of the pathogenesis of renal failure and the development of new therapies will thus require global expression analysis of disease states using genomics and/or proteomics tools. In this study, we examined changes of gene expression in 20-week remnant kidneys of SNX wild-type (SNX-WT) mice, SNX-B1R-KO mice, and SNX-B2R-KO mice to comprehend molecular mechanisms of advanced renal disease better and possible implications of kinin receptors in renal failure. Numerous genes that encode proteins previously implicated in ESRD displayed drastic altered expression in 20-week remnant WT kidneys. However, and importantly, we observed strong variations in a number of genes and gene families (cytochrome P450 (CYPs), major urinary proteins (MUPs), serpins, glutathione S-transferase (GSTs), and enzymes of the urea cycle) and a novel pathway (linked with hepatocyte nuclear factor 4-alpha (Hnf4a) signaling) not previously linked with renal disease progression. The altered expression of some of these genes is also associated with possible protective and compensatory mechanisms. Additionally, our results are indicative for implications of kinin receptors, and especially the kinin B2R subtype, in chronic renal disease. As expected with mice on a C57BL6 genetic background, we found that our WT- and KO-SNX mice are rather resistant to glomerulosclerosis, proteinuria, and hypertension.2.Kren S. Hostetter T.H. The course of the remnant kidney model in mice.Kidney Int. 1999; 56: 333-337Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,10.Ma L.J. Fogo A.B. Model of robust induction of glomerulosclerosis in mice: importance of genetic background.Kidney Int. 2003; 64: 350-355Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar Indeed all animals survived 20 weeks of SNX. Consistent with previously described data,11.Milia A.F. Gross V. Plehm R. et al.Normal blood pressure and renal function in mice lacking the bradykinin B(2) receptor.Hypertension. 2001; 37: 1473-1479Crossref PubMed Scopus (54) Google Scholar,12.Xu J. Carretero O.A. Sun Y. et al.Role of the B1 kinin receptor in the regulation of cardiac function and remodeling after myocardial infarction.Hypertension. 2005; 45: 747-753Crossref PubMed Scopus (54) Google Scholar no significant change in blood pressure was observed along the study period in the SNX-WT and SNX-KO animals (Figure 1a). However, the biochemical analyses performed on urine and blood samples collected before killing of all SNX mice were confirmative for ESRD progression (Figure 1b). Serum creatinine was significantly increased and urinary creatinine was significantly decreased in SNX animals compared with control animals. Proteinuria was not significantly different between WT and SNX animals, but a tendency was observed. No significant difference was observed in the biochemical parameters between SNX-WT and SNX-KO animals (Figure 1b). Initially, we identified global changes of renal gene expression in WT remnant kidneys (20-week post-SNX). The microarray experiments were performed in triplicates, as the renal gene expression was compared at random between three separate pairs of SNX-WT versus WT-sham-operated (WT-C) animals (SNX-WT/WT-C). A subset of 1131 differentially expressed genes was selected from all triplicate microarray data by filtering on expression level (≥2-fold). Using these selection criteria, we found 543 genes to be upregulated and 588 genes to be downregulated in the 20-week remnant kidneys, compared to that of the control kidneys. Table 1A shows list of selected functional groups of genes that were upregulated (≥2-fold) in the 20-week remnant kidneys. As seen from Table 1A, there is substantial number of upregulated genes with previously shown implication in mechanisms of renal pathology, including altered lipid metabolism, inflammation, fibrosis, vascular homeostasis, and/or reactive oxygen species formation. Some of them displayed very strong upregulation (>5-fold) in remnant kidneys (indicated in bold). These included albumin (Alb), fatty acid-binding protein 1 (Fabp1), retinol-binding protein 4 (Rbp4), carbamoylphosphate synthetase 1 (Cps1), apolipoproteins Apoa1, Apoa2, Apoh, and Apoc4, kininogen (Kng1), vitronectin (Vtn), fetuin-A (Ahsg), esterase 1 (Es1), paraoxonase 1 (Pon1), fibrinogen (Fg), orosomucoid 1 (Orm1), hemopexin (Hpxn), and different members of the cytochrome P450 gene family (Cyp2c39, Cyp2c40, Cyp3a44, Cyp2a12, Cyp2c29, Cyp3a11, and Cyp2c40). Interestingly, four members of the major urinary protein family (Mup1, Mup3, Mup4, and Mup5) displayed similar high range (>5-fold) of gene expression. Table S1 shows the complete list of upregulated genes (≥2-fold) in the 20-week remnant WT kidneys.Table 1Selected functionally related groups of differentially expressed genes (≥2-fold)aGenes in bold are differentially expressed at least ≥5-fold in all microarray experiments. in 20-week remnant WT kidneys(A) Upregulated genes Cytochrome P450 family (vascular homeostasis and/or ROS formation)Cyp1a2,Cyp2a12, Cyp2b9, Cyp2b10,Cyp2b13, Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c54, Cyp2c70, Cyp2f2,Cyp3a11, Cyp3a16, Cyp3a25, Cyp3a44, Cyp4a14 Lipid metabolism and transportApoa1, Apoa2, Apoa5, Apoc1, Apoc3, Apoc4, Apoe,Apof, Apoh, Fabp1, Sult2a2, Sth2a1, Slc27a5, Pon1, Sth2, Acdc Serpins; proteolysis and peptidolysisSerpina1b, Serpina1d, Serpina1e, Serpina3c, Serpina3k, Serpina3n, Serpina6, Serpina10, Serpinc1, Serping1,F2, F10 Solute carriers and other transportersSlc7a12, Slc10a1, Slc38a4, Slco1b2, Car3, Slc16a4, Slc26a8, Slc39a4, Ambp Inflammation; complement activationEs1, C4bp, Orm1, Hpxn, Es31, C3, Hamp, Orm2, Hp, Cfh, Saa4, Cpb2, Cfi, Cfhl1, Adn, Mbl2, Retn, Plg, Hpgd, C1r, C9, C1s, Masp, Crp Immune responseH2-Bl, Ig-γ1, Was, Igh-VJ558, Igl-6G14-F6, H2-Q10, Cd1d1, Igj, Igl-11, C6-E3, Ig-κV-V, Wasf3, Igg1, Igh-L2pecB, Mug2 FibrosisFgg, Fgb, Fga, Fgl1, Ppp1r9a, Fgfr1, Fn1 ApoptosisCard14, Csnk2a1, Il21r, Clu ECM proteinsCsf3r, Vtn, Lum, Ceacam2 Cell growth and proliferationIgf2, Rgn, Igf1b, Igf1, Hel308 Oxidative stress (pro- and antioxidants)Maob, Uox, Hao3, Cp, Hao1,Trf, Trfr2,Ucp1, Gsta3, Gsta2, Gsta1,Akr1c20, Akr1c6, Akr1c12, Cbr1 Enzymes of the urea cycleCps1, Arg1, Arg2, Otc Major urinary proteinsMup1, Mup3, Mup4, Mup5 Other genes relevant to renal pathologyAlb1, Ahsg, Rbp4, Kng1, Agxt(B) Downregulated genes Cell growth and metabolismFgf5, Fgf2,Odc, Odc1,Tph1, Dhrs8, Dhrs1,Ldhd, Ldh1,Atp11a, Mod1, B4galt5, Psap,Sah, Slc27a2, Ugt8, Pcyt1a, Crot, Cyb5, Tgfbr2,Acox1, Acox3,Mapk6 TransportCacna2d2, Uty, Pitpnm1, Kcnk5, Slco1a1, Slco1a5, Slc7a13, Slco1a1, Tnpo3 Protein biosynthesisEif2s3y, Eif5a,Polr3e, Gfm, AK018591, Mrpl12 Cell adhesionPcdhb9, Pcdh8, Itga2b, Lama1, Dscaml1,Gjc1, Mmp5 Cytochrome P450 familyCyp2j13, Cyp4b1, Cyp7b1 ApoptosisBcl2I1, Cideb, Dnase1, Pdcd8 Genes relevant to renal pathologyHbα-a, Hb-β, Hb-β-b1, Pkd2, Nppb, AceIECM, extracellular matrix; ROS, reactive oxygen species.a Genes in bold are differentially expressed at least ≥5-fold in all microarray experiments. Open table in a new tab Download .xls (.1 MB) Help with xls files Table S1 ECM, extracellular matrix; ROS, reactive oxygen species. Five hundred and eighty-eight genes were subject to at least twofold downregulation in the remnant WT kidneys (a list of selected downregulated genes is presented in Table 1B). Among the genes with known functions, major classifications comprised metabolism (35%), transport (17%), cell growth and maintenance (11%), transcription (11%), signal transduction (10%), protein turnover (8%), immune response (4%), apoptosis (2%), proteolysis and peptidolysis (2%), and more than half (323 genes) had unknown function. Table S2 displays the complete list of the 588 downregulated genes (≤2-fold) in the 20-week remnant WT kidneys. Download .xls (.1 MB) Help with xls files Table S2 A network analysis based on the 1131 gene list with twofold altered expression identified 26 highly significant networks with score ≥11 (see Table S3). The four top-scoring networks are shown in Table 2 and were associated with the functions of cell-to-cell signaling and interaction, cellular growth and proliferation, lipid metabolism, molecular transport, and immune response. A common network obtained upon merging the four top-scoring networks (Figure 2) recognized several important nodes linked with numerous interaction partners, including the v-fos FBJ murine osteosarcoma viral oncogene homolog (Fos), thrombin (Thr), fibronectin 1 (Fn1), C-reactive protein (Crp), plasminogen (Plg), insulin-like growth factor 1 (Igf1), and Hnf4a. As the majority of these genes and related pathways were previously associated with renal pathology, the Hnf4a signaling represents a novel pathway (see also Figure 3), possibly linked with the mechanisms of advanced renal disease. Download .xls (.02 MB) Help with xls files Table S3Table 2The four top-scoring genetic networks in the 20-week remnant WT mouse kidneysNetworkGenes in ingenuity networksaUpregulated (↑) and downregulated (↓) genes are indicated with arrows.ScorebA score >3 was considered significant.Functions1↑ANG, ↑APOH, CDCP1, ↑CEACAM1, ↑CISH, ↑CP, ↑CPB2, ↓CSDA, ↑CSF3R, ↑DCN, ↑DF, ↑F10, ↑FABP4, ↓FOXE1, ↑IGF1, ↑IGF2, ↓IGFBP3, ↓INS, ↑LYZ, ↓MMP2, ↑PLG, ↑PRLR, ↓PTHR1, ↑RARRES2, ↑SERPINA3, ↑SERPINA10, ↑SERPINC1, ↓SERPINF2, ↓SLC20A1, ↑SOCS2, ↑TF, ↑TFPI, ↑TFR2, ↑VTN, ↓ZFP3546Cell-to-cell signaling and interaction, cellular growth, and proliferation2↑APOA1, ↑APOA2, ↑APOC1, ↑APOC3, ↑APOE, ↓BLMH, ↑C9, ↑C4A, ↑C8G, ↑CLU, ↑CUGBP2, ↓ENO1, ↓FARS2, ↑FGA, ↑FGB, ↑FOS, ↑GCNT1, ↓HNF4A, ↑HPD, ↑LCAT, ↓LDHA, ↓NP, ↓OAZ1, ↓OCM, ↓ODC1, ↑PON1, ↑PRKAR2B, ↑PYGL, ↓RNH, ↓RPS6KA3, ↓SLC27A2, ↓SMARCD1, ↓SPN, ↑TDO2, ↓TEGT46Lipid metabolism, molecular transport, small molecule biochemistry3↑ABCC3, ↓ACE, ↑ALB, ↑C3, ↑C1R, ↓CD9, ↓CD63, ↑CFH, ↓CYBA, ↑CYP2B2, ↑DGKQ, ↑F2, ↓FGF2, ↑GLUL, ↑GPNMB, ↑IF, ↑ITGA6, ↓ITGA2B, ↑KNG1, ↑MAFB, ↑MASP1, ↑MASP2, ↑MBL2, ↓MEP1B, ↓NAGK, ↓NOX3, ↓PBP, ↓PIK4CA, ↓PITPNM1, ↑PRKCB1, ↑SCD, ↓SERPINA5, ↑SERPING1, ↓SMPD2, ↓TNS46Cardiovascular disease, tissue morphology, lipid metabolism4↓ACOX1, ↑ADIPOQ, ↑APOA5, ↓ATP6V0C, ↑B2M, ↑CANX, ↑CD1D, ↓CPT1A, ↑CRP, ↑CSNK2A1, ↑CXCL9, ↑DCT, ↑FASN, ↑FCER1G, ↑FN1, ↑HLA-A, ↑HLA-B, ↑HLA-E, ↑IGHG1, ↑IGHM, ↑IL2, ↓IL2RA, ↑KLRC2, ↑LOC56628, ↓LPL, ↑MUG1, ↓MYH1, ↓NPPB, ↓PDCD8, ↑PTPN6, ↑SERPINA1, ↓SNRPN, ↓U2AF2, ↑UCP1, ↓ZBTB746Cell-to-cell signaling and interaction, immune responsea Upregulated (↑) and downregulated (↓) genes are indicated with arrows.b A score >3 was considered significant. Open table in a new tab Figure 3The Hnf4a signaling pathway. Figure legends are as described in Figure 2. Edge labels: A, activation; B, binding; E, expression; PP, protein–protein binding; RB, regulation of binding; LO, localization.View Large Image Figure ViewerDownload (PPT) In parallel, we studied global gene expression changes in 20-week remnant kidneys of SNX-B1R-KO and SNX-B2R-KO mice. Gene expression analyses were performed at random in five separate pairs (biological replicates) of SNX-B1R-KO mice versus SNX-WT mice (SNX-B1R-KO/SNX-WT) and six biological replicates of SNX-B2R-KO versus SNX-WT animals (SNX-B2R-KO/SNX-WT). A subset of differentially expressed genes was selected displaying at least twofold difference in four of the five B1R-KO microarray experiments and in four of the six B2R-KO microarray experiments. Using these selection criteria, we found 73 genes to be upregulated and 118 genes to be downregulated in B1R-KO remnant kidneys, compared with that of WT remnant kidneys, whereas 71 genes were upregulated and 52 genes were downregulated in B2R-KO remnant kidneys, when compared with WT remnant kidneys (Table S4 and Table S5). Download .xls (.04 MB) Help with xls files Table S4 Download .xls (.04 MB) Help with xls files Table S5 Interestingly, a substantial number of genes (43 upregulated and 30 downregulated) were commonly differentially expressed in the SNX-B1R-KO/SNX-WT experiments and in the SNX-WT/WT-C experiments (Figure 4a). In contrast, the number of the differentially expressed genes that are common in the SNX-B2R-KO/SNX-WT experiments and in the SNX-WT/WT-C experiments was considerably lower (18 upregulated and 0 downregulated genes – see Figure 4b). The number of overlapping genes in both experimental conditions including kinin receptors KO mice was also negligible (Figure 4c). However, some functionally important renal pathology genes, including genes implicated in renal fibrosis (Fga, Fgg, Saa2, Edn1, and Ren1) were uniquely overexpressed only in the B2R-KO remnant kidneys. This also holds for a subset of genes, functionally implicated in control of cell proliferation (Nrg3, Sycp3, Plec1, Ptn, Kif11, and Kif13a), found to be exclusively downregulated in remnant kidneys of the B2R-KO mice (Table S5). Table 3 displays the classifications by function of the differentially expressed genes in both experimental conditions involving kinin receptors-KO mice (SNX-B1R-KO/SNX-WT and SNX-B2R-KO/SNX-WT). These data are indicative for some similarities, as well for some differences in the functional classes that are specific for each mouse KO strain. Thus among the upregulated genes, major classifications in the B1R-KO remnant kidneys include transport, metabolism (especially lipid transport and metabolism), proteolysis and peptidolysis, whereas in B2R-KO remnant kidneys, the top functional classes are associated with metabolism, signal transduction, transport, and acute-phase response (Table 3A). As for the downregulated genes, functional groups in the B1R-KO remnant kidneys mostly comprise metabolism, transcription, and signal transduction, whereas in the B2R-KO remnant kidneys these mainly include signal transduction, transcription, cell proliferation, and transport (Table 3B). The differences between the two SNX-KO strains were further confirmed by clustering and pathway analysis. Supervised clustering based on a selected list of 65 genes revealed formation of two major cluster groups that perfectly distinguish between SNX-B1R-KO and SNX-B2R-KO kidney samples (Figure 5). The 65 gene list is presented in Table S6. Twenty genes from the 65 genes list were upregulated in the B2R-KO remnant kidneys. Major classifications of these genes include metabolism and regulation of transcription. Genes, upregulated in B1R-KO remnant kidneys (45 genes) are mainly involved in signal transduction, metabolism, transport, and apoptosis.Table 3Functional distribution of the differentially expressed genes (twofold) in remnant kidneys of SNX kinin receptors KO mice compared with SNX-WT miceSNX-B1R-KO: functional groups%SNX-B2R-KO: functional groups%(A) Upregulated genes (≥2-fold) Transport21.9Metabolism15.3 Metabolism17.2Signal transduction12.5 Proteolysis and peptidolysis14.1Transport12.5 Lipid metabolism and transport10.9Acute phase response9.7 Immune response9.4Transcription8.3 Inflammation7.8Inflammation5.6 Other6.2Immune response5.5 Unknown function12.5Vasoconstriction2.8Unknown function27.8(B) Downregulated genes (≤2-fold) Metabolism12.4Signal transduction17.3 Transcription8.3Transcription13.5 Signal transduction6.6Cell proliferation11.5 Cell growth and proliferation5.8Transport11.5 Transport5.8Metabolism7.7 Immune response4.9Immune response5.8 Apoptosis3.3Unknown function32.7 Unknown function52.9KO, knockout; SNX, subtotal or 5/6 nephrectomy; WT, wild type. Open table in a new tab Download .xls (.04 MB) Help with xls files Table S6 KO, knockout; SNX, subtotal or 5/6 nephrectomy; WT, wild type. Network analysis identified three significant gene networks in both up and down-differentially expressed genes in the B1R-KO remnant kidneys, whereas in the B2R-KO remnant kidneys three networks for the upregulated genes and two networks for the downregulated genes displayed a significant score (Table 4). The discrepancies were mostly evident in the networks obtained with downregulated genes, as the SNX-B1R-KO networks were associated with gene expression and cellular growth and proliferation (Table 4B), whereas the SNX-B2R-KO networks were mainly linked to immune response, lipid metabolism, and transport (Table 4D).Table 4Genetic networks in remnant kidneys of the kinin receptors-KO miceNetworkGenes in ingenuity networksaGenes marked with asterisk are present in more than one network. Bold genes are present in networks identified by both experimental conditions (including B1R-KO and B2R-KO mice).ScorebThe table displays only networks with significant scores (score >3 is considered as significant).Focus genescOnly genes identified by microarray analysis (focus genes) that are differentially expressed (≥2-fold) are listed.Functions(A) Networks in remnant kidneys of SNX-B1R-KO mice (upregulated genes) 1AGXT, APOA1, APOC1, APOC3, COX8B, CYP3A2, F2, F10*,FGB, ITGA6, MUP4, PLG, PON1, SERPINC1, SERPIND1, TFR22916Lipid metabolism, molecular transport 2AHSG,ARG1*, C5, CIDEA, DF*, ELOVL6, HP,HPX, MBL2, MYO5A,ORM1, SAA4, SERPINAC3 (Kalbp)2213Development, lipid metabolism 3AMBP,ARG1*, BAAT,CYP3A7, DF*, F10*, HDC, HLA-B, IGJ, MGC27165,ORM2, SERPINA3K, UCP12213Cellular growth and proliferation(B) Networks in remnant kidneys of SNX-B1R-KO mice (downregulated genes) 1BCL2L2, BMF, BRCA2, CD36*, HDAC2, ITGB1, MEOX2, NFKBIZ, PLF2, PTPN4, SDHA, SH3GLB1, TM4SF8, TSC22D12414Gene expression, cellular movement 2ABHD5, CDH17, CYP4B1, G0S2, MPRA, POLR3E, RAP1B, SFRS1, SLAMF6, SLC20A1, SLC30A1, SLCO1A1, SNX6, UGT2B72414Cellular growth and proliferation 3AKR1C2, BRF1, CD36*, CENPB, CES1, DARS, EIF5A, ENPEP, HSPA8, LACTB, NBR1, TP53BP2, TSC22D32113Gene expression(C) Networks in remnant kidneys of SNX-B2R-KO mice (upregulated genes) 1ANTXR1, APCS,ARG1*, CD163, CPB2, DHCR7, FGG*, HP, ITIH3, LCAT, LRG1, MAT1A,ORM1*, SAA2, SERPINA32715Immune response, cellular growth and proliferation 2ALAD, CD3D, CPS1, CYP2F1, EDN1, FASN, GSTM2, HAND1, HAO1, HCK,HPX, ORM1, SOX182213Lipid metabolism, molecular transport 3ARG1*, CDGAP,CYP3A7, FGA,FGB, FGG*, FKBP5, GSTM1,ORM2, PC4, PSMD11, RPS6KA12012Cell signaling(D) Networks in remnant kidneys of SNX-B2R-KO mice (downregulated genes) 1ABCD3, CAMP, CCL27, CTSC, ERCC2, GHSR, IGH1A, PHIP, PLEC1, PRKACA, PRKCI, SP1002312Immune response, cellular growth and proliferation 2CAST (ERC2), KIF11, NRG3, NT5E, PTN, RASSF4, SYCP3, TFRC, ZBTB33169Lipid metabolism, molecular transportB1R, kinin receptor, which is overexpressed in pathological states; B2R, kinin receptor, which is constitutively expressed; KO, knockout; SNX, subtotal or 5/6 nephrectomy.a Genes marked with asterisk are present in more than one network. Bold genes are present in networks identified by both experimental conditions (including B1R-KO and B2R-KO mice).b The table displays only networks with significant scores (score >3 is considered as significant).c Only genes identified by microarray analysis (focus genes) that are differentially expressed (≥2-fold) are listed. Open table in a new tab B1R, kinin receptor, which is overexpressed in pathological states; B2R, kinin receptor, which is constitutively expressed; KO, knockout; SNX, subtotal or 5/6 nephrectomy. To validate microarray results, we arbitrarily selected 15 differentially expressed genes in 20-weeks remnant WT kidneys and quantified their expression by semi-quantitative real time-polymerase chain reaction (sqRT-PCR) in the available WT remnant and WT control kidney samples. Additionally, we validated a number of genes by RT-PCR, found to be differentially expressed in 20-week remnant kidneys of SNX-B1R-KO or SNX-B2R-KO mice compared with SNX-WT control animals. All sqRT-PCR analyses were performed in samples previously used for the microarray experiments. Table 5 summarizes the gene expression measurements of all validated genes. We found that both methods (microarray analysis and sqRT-PCR) detected similar patterns for the upregulated and downregulated genes selected for validation.Table 5sqRT-PCR validation of microarray dataGenBank No.Common nameFold expression (SNX-WT/WT-C)MicroarraysqRT-PCRaThe sqRT-PCR data represent medium values of triplicate RT-PCR experiments, as the specific gene expression was compared at random between three separate pairs of experimental animals.(A) Comparison of the qualitative results obtained by microarray analysis and conventional method (sqRT-PCR) in kidney tissues from SNX-WT mice versus WT control mice NM_009654Albumin 1 (Alb1)+267.50+55.29 NM_031188Major urinary protein 1 (Mup1)+94.96+10.30 NM_011548Serpin clade A, 3K (Serpina3k)+94.27+43.60 NM_017399Fatty acid-binding protein 1 (Fabp1)+64.35+21.46 AK008765Retinol-binding protein 4 (Rbp4)+63.18+9.73 NM_009692Apolipoprotein A-1 (Apoa1)+59.52+23.12 NM_080852Solute carrier family 7, member 12 (Slc7a12)+52.90+10.15 NM_023125Kininogen 1 (Kng1)+52.84+4.87 NM_177380Cytochrome P450 3a44 (Cyp3a44)+34.86+37.59 NM_011134Paraoxonase 1 (Pon1)+36.51+7.83 NM_133862Fibrinogen, gamma (Fgg)+24.68+5.75 NM_007576Complement 4 binding protein (C4bp)+22.67+17.89 NM_023114Apolipoprotein C-III (Apoc3)+19.53+8.04 NM_019946Microsomal glutathione S-transferase 1 (Mgst1)+8.94+3.07 NM_013797Solute carrier, member 1a1 (Slco1a1)-55.25-26.01Fold expression (SNX-B1R-KO/SNX-WT)GenBank No.Common nameMicroarraysqRT-PCRaThe sqRT-PCR data represent medium values of triplicate RT-PCR experiments, as the specific gene expression was compared at random between three separate pairs of experim
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