Epigenetic memory of oxidative stress: does nephrilin exert its protective effects via Rac1?

Figures & data

Figure 1 Fibroblasts were grown and challenged with either UV-A (_uv subscript) or H₂O₂ (_ox subscript) and cell viability and collagen I synthesis measured as described in Methods. Values are averages of triplicate assays and are expressed relative to control (=100). C=control untreated cells; N=20 ug/ml nephrilin peptide. *, **: p<0.05 and p<0.01 respectively, N versus C pairing; #, ##: p<0.05 and p<0.01 respectively, C_uv (or C_ox) versus C pairing.

Figure 2 Renal PTECs were grown and challenged with H₂O₂ (_ox subscript) ± nicotine (Nic) and ROS generation measured as described in Methods. Values are averages of triplicate assays. C=control untreated cells; N=10 ug/ml nephrilin peptide; S=10 ug/ml scrambled peptide. *, **: p<0.05 and p<0.01 respectively, N (or S) versus C pairing; # = p<0.05 C_ox versus C.

Figure 3 Pathway analysis using Ingenuity Systems software. Major network (score=32) is shown. Dataset was 3057 genes exhibiting differential expression in the Agilent RNA arrays (see text).

Table 1 RNA arrays. RNAs were extracted from cells and analyzed on Agilent Rat Gene Expression Microarrays 4×44K (AMDID 014879) as described in Methods. Of 41,105 genes interrogated 49 upregulated (>3-fold threshhold) genes of known function for which treatment with H₂O₂ alone was >30% over control and treatment with the Nic+ H₂O₂ combination elevated expression an additional >30% were identified.

ProbeName	GeneSymbol	GenbankAccession	GeneName	FOLD UPREG
A_44_P812665	Mypn	NM_001107628	myopalladin	9.3
A_42_P812805	Smoc2	NM_001106215	SPARC related modular calcium binding 2	8.4
A_44_P704258	En2	NM_001109214	engrailed homeobox 2	8.2
A_43_P12905	Bik	NM_053704	BCL2-interacting killer (apoptosis-inducing)	8.2
A_44_P288548	Prex1	NM_001135718	phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1	8.0
A_44_P285988	Chrdl2	NM_001107537	chordin-like 2	7.6
A_44_P361951	Adssl1	XM_001072867	adenylosuccinate synthase like 1	7.2
A_44_P356161	Ppy	NM_012626	Pancreatic Polypeptide	7.1
A_43_P15474	Spef2	NM_022620	sperm flagellar 2	6.9
A_44_P572533	Cabyr	NM_001143893	calcium binding tyrosine-(Y)-phosphorylation regulated	5.8
A_44_P323349	Gria4	NM_017263	glutamate receptor, ionotropic, AMPA 4	5.7
A_42_P832832	Ckm	NM_012530	creatine kinase, muscle	5.6
A_44_P250467	Cd22	NM_001107503	CD22 molecule	5.2
A_42_P536266	Dnm3	XM_006250137	dynamin 3	5.2
A_44_P258225	Ly6h	NM_001134839	lymphocyte antigen 6 complex, locus H	5.2
A_44_P414340	Amelx	NM_019154	amelogenin, X-linked	5.0
A_44_P208532	ldo2	XM_006222273	indoleamine 2,3-dioxygenase 2	4.9
A_44_P145562	Dock2	XM_001068649	dedicator of cytokinesis 2	4.7
A_43_P13056	IllOra	NM_057193	interleukin 10 receptor, alpha	4.7
A_44_P665425	Rnfl65	NM_001164505	ring finger protein 165	4.5
A_44_P762053	Palld	XM_006253064	palladin, cytoskeletal associated Protein	4.4
A_44_P104630	Lat2	NM_173840	linker for activation of T cells family, member2	4.4
A_44_P236523	Hoxdl0	NM_001107094	homeo box D10	4.4
A_44_P212521	Gzmf	NM_153466	granzyme F	4.3
A_44_P497282	Rab11fip4	NM_001107023	RAB11 family interacting protein 4 (class II)	4.3
A_44_P416899	Npy4r	NM_031581	neuropeptide V receptor V4	4.3
A_44_P1043302	Crb3	NM_001025661	crumbs homolog 3 (Drosophila)	4.3
A_44_P1036769	Matn1	NM_001006979	matrilin 1, cartilage matrix Protein	4.2
A_42_P841193	Ddit4l	NM_080399	DNA-damage-inducible transcript 4-like	4.2
A_44_P291071	Rora	NM_001106834	RAR-related orphan receptor A	4.2
A_43_P18101	Eif3	NM_001024768	E74-like factor 3	4.1
A_42_P525876	Erg	NM_133397	v-ets avian erythroblastosis virus E26 oncogene	4.0
A_44_P372305	Syn3	NM_017109	synapsin 111	3.9
A_43_P19535	Mrgprb4	NM_001002287	MAS-related GPR, member B4	3.9
A_44_P520521	Slcl7a2	NM_001107353	solute carrier family 17, member 2	3.8
A_44_P1001317	Nabp1	NM_001014216	nucleic acid binding protein 1	3.8
A_44_P555417	Hoga1	NM_001106355	4-hydroxy-2-oxoglutarate aldolase 1	3.7
A_44_P147156	Rbm47	NM_001005882	RNA binding motif protein 47	3.7
A_44_P199028	Dkk1	NM_001106350	dickkopf WNT signaling Pathway inhibitor 1	3.5
A_44_P144788	Mtmr12	XM_006232046	myotubularin related protein 12	3.5
A_43_P18793	Gfpt2	NM_001002819	glutamine-fructose-6-phosphate transaminase 2	3.4
A_44_P485948	Cux2	NM_001271380	cut-like homeobox 2	3.3
A_44_P491864	Fgfl4	XM_006252526	fibroblast growth factor 14	3.1
A_44_P1020482	Nalcn	NM_153630	sodium leak channel, non-selective	3.0
A_42_P601961	Susd3	NM_001107341	sushi domain containing 3	3.0
A_44_P318628	Chrnb2	NM_019297	cholinergic receptor, nicotinic, beta 2 (neuronal)	3.0
A_44_P1035007	Chrne	NM_017194	cholinergic receptor, nicotinic, epsilon (muscle)	3.0
A_44_P178498	B3galt5	NM_001105887	UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, Polypeptide 5	3.0
A_44_P1048543	Ghrh	NM_031577	growth hormone releasing hormone	3.0

Figure 4 Kidney tissue extracts were prepared and analyzed by ELISA as described in Methods. S=sham group; B=burn+saline; N1=burn+4mg/kg/day SQ nephrilin for 1 week post-burn. **p<0.01, N1 versus B; #, ##: p<0.05 and p<0.01, B versus S.

Abbreviation: AU, arbitrary units.

Figure 5 All extracts were prepared and assays performed as described in Methods. (A) Urine samples were assayed by ELISA and standardized against Cystatin C. (B) Gene transcripts were standardized versus GAPDH as standard. (C) Histone-3 phosphorylation and acetylation, as well as total histone-3, were determined by ELISA. Kidney tissues from 6 (B6=B group 6 hr) and 24 hours (S24, B24, N24) post-burn were assayed for phosphorylation, as early event. (D) CpG methylation is expressed in arbitrary units; each bar is an average of 4 animals. S=sham group; B=burn+saline; N1=burn+4mg/kg/day SQ nephrilin for 1 week post-burn. *p<0.05, N1 versus B; # p<0.05, B versus S.