Macrophage Migration Inhibitory Factor Is a Possible Candidate for the Induction of Microalbuminuria in Diabetic db/db Mice

Abstract

Preventing the onset of microalbuminuria in diabetic nephropathy is a problem that needs urgent rectification. The use of a mouse model for diabetes is vital in this regard. For example, db/db mice exhibit defects in the leptin receptor Ob-Rb sub-type, while the ob/ob strain exhibits defects in the leptin ligand. These mouse strains demonstrate type 2 diabetes, either with or without microalbuminuria, respectively. The purpose of the present study was to use DNA microarray technology to screen for the gene responsible for the onset of diabetic microalbuminuria. Using Affymetrix Mouse Gene ST 1.0 arrays, microarray analysis was performed using total RNA from the kidneys of ob control, ob/ob, db/m, and db/db mice. Microarray and quantitative reverse transcription-polymerase chain reaction (RT-PCR) indicated that transcription of the macrophage migration inhibitory factor (MIF) gene was significantly enhanced in the kidneys of db/db mice. Western blotting showed that levels of MIF protein was enhanced in the kidneys of both diabetic db/db and ob/ob mice. On the other hand, elevation of urinary MIF excretion detected by enzyme-linked immunosorbent assay (ELISA) was only in db/db mice and preceded the onset of microalbuminuria. Immunofluorescence studies revealed that MIF was expressed in mouse kidney glomeruli. While MIF expression was enhanced in the diabetic kidneys of both mouse strains, the elevated secretion from db/db mouse kidneys may be responsible for initiating the onset of microalbuminuria in diabetic nephropathy.

Diabetic nephropathy is the primary causative disease leading to dialysis in Japan.¹⁾ The cumulative incidence of nephropathy after 30 years of post-pubertal diabetes was reported to be significantly higher in type 2 diabetic patients (44.4%) than in type 1 diabetic patients (20.2%).²⁾ However, diabetic nephropathy does not occur in all patients with type 2 diabetes mellitus, implying that there might be an unknown underlying mechanism to prevent the onset of diabetic nephropathy. Establishing the identity of such a mechanism is of vital importance.

It is thought that the onset of microalbuminuria, considered to be a marker of early diabetic nephropathy, must be suppressed in order to inhibit the onset and aggravation of nephropathy in type 2 diabetes. Two murine strains are available as an animal model for type 2 diabetes: db/db mice in which microalbuminuria develops at 8 weeks of age³⁾; and ob/ob mice that do not develop microalbuminuria at all. It is thought that a gene cluster associated with the onset of microalbuminuria could therefore be identified by comparing differences in gene expression between these two mouse strains, without considering a specific disease-developing mechanism underlying microalbuminuria.

The purpose of this study was therefore to identify a gene associated with the onset of microalbuminuria in the type 2 diabetic model mouse using a DNA microarray approach. Macrophage migration inhibitory factor (MIF) was identified as a gene for which expression changed in db/db mice, but not in ob/ob mice. Since earlier reports indicate that MIF is associated with glomerulonephritis and proteinuria,⁴⁾ the present study also aimed to analyze the expression of MIF in the kidneys of db/db and ob/ob mice.

Materials and Methods

Animals

Experiments involved male db/db and ob/ob mice and non-diabetic controls (db/m and ob control mice) that were 2 months of age. Urine was sampled using metabolic cages. Mice were anesthetized by intra-peritoneal injections of 50 mg/kg pentobarbital in order to obtain blood and kidney samples. Serum glucose was measured enzymatically using Glucose CII-test Wako (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Serum insulin and leptin was assayed using Mouse Insulin enzyme-linked immunosorbent assay (ELISA) kit (H-type) and Mouse Leptin ELISA kit, respectively (Shibayagi, Gunma, Japan). Urinary albumin was assayed using Albuwell M kit (Exocell Inc., Philadelphia, U.S.A.). Macrophage Migration Inhibitory Factor in plasma or urine was assayed by ELISA kit for mouse MIF (Uscn Life Science Inc., Houston, U.S.A.). Serum and urinary creatinine was assayed using HPLC as described previously.⁵⁾ All animal experiments were carried out in accordance with Teikyo University Guide for the Care and Use of Laboratory Animals.

Microarray Analysis

Total RNA was extracted from the kidneys of ob control, ob/ob, db/m, and db/db mice and purified as described previously.⁶⁾ Microarray analysis was performed using Affymetrix Mouse Gene ST 1.0 arrays and analyzed using Expression Console (PharmaFrontier Co., Ltd., Kyoto, Japan).

Real-Time Polymerase Chain Reaction (PCR)

cDNA was prepared from total RNA using a High-Capacity cDNA Reverse Transcription Kit. Real-time PCR analyses were subsequently conducted as described previously⁶⁾ using TaqMan probes corresponding to macrophage migration inhibitory factor (Mm01611157_gH), and 18s ribosomal RNA (rRNA) as an internal standard.

Western Blotting

Kidneys were homogenized in a specific solution (20 mm Tris, 3 m Urea, 0.5% CHAPS, 0.1 m NaCl, pH 7.4) containing protease inhibitors (Complete Mini, Roche Diagnostics Japan, Tokyo). Homogenates (5 µg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a Nitrocellulose membrane. After blocking, membranes were stained with anti-mouse MIF antibody (0.5 µg/mL; FL-115 (sc-20121); Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) and horseradish peroxidase (HRP) conjugated anti-rabbit immunoglobulin G (IgG) antibody (1 : 10000, ZYM 65–6120, Life Technologies Japan Ltd., Tokyo). An HRP-conjugated anti-β actin antibody (1 : 40000, β actin C4, sc-47778, Santa Cruz Biotechnology, Inc.) was used to calibrate sample loading. Products were detected with an ECL kit (GE Healthcare UK Ltd., Little Chalfont, U.K.).

Immunofluorescence

Paraffin sections were prepared from the kidneys of ob control, ob/ob, db/m, and db/db mice which had been perfused with 4% paraformaldehyde in 30 mm N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES) buffer. Deparaffinized sections were stained with anti-mouse MIF antibody (1 : 50, Santa Cruz Biotechnology, Inc.) and Cy5-conjugated anti-rabbit IgG (H+L) (1 : 100, A10523, Life Technologies Japan Ltd., Tokyo, Japan).⁷⁾

Statistical Analyses

All data, except that arising from DNA microarrays, are expressed as mean±standard error (S.E.M.). Differences between groups were analyzed statistically using the student’s t-test. p values <0.05 were considered statistically significant.

Results and Discussion

Serum glucose levels of db/db mice was significantly higher than those of db/m mice (p=0.017), while those of ob/ob mice tended to be higher than those of ob control mice (p=0.056). However, the urinary volume, serum insulin, and body weight of db/db and ob/ob mice were significantly higher than those of control mice. Therefore, type 2 diabetes-induced polyuria and obesity appears to have been demonstrated in both db/db and ob/ob mice. The obesity of ob/ob mice was caused by defective leptin, which normally suppresses over-eating by stimulating the satiety center in the hypothalamus. Obesity in db/db mice is known to be caused by defects in leptin signaling at the satiety center⁸⁾ (Table 1). Microalbuminuria were clearly observed in db/db mice (332.4±79.9 µg/d, p<0.01 vs. ob/ob mice), however, neither db/m mice (45.5±11.3 µg/d) nor ob/ob mice (31.5±9.5 µg/d) were free from microalbuminuria (Table 1). This concurs with an earlier report³⁾ which reported that all db/db mice develop microalbuminuria at 8 weeks of age, while ob/ob mice did not develop microalbuminuria at all. Therefore, these two strains of diabetic mice are appropriate models for DNA microarray analysis in an attempt to identify microalbuminuria-related genes.

Table 1. Comparison of Clinical State of Diabetes

	dm/m	db/db	ob control	ob/ob
Body weight (g)	24.2±0.5	41.1±0.9**	27.5±0.4	45.9±0.7**
Volume of urine (mL/d)	1.0±0.1	11.9±1.3**	1.3±0.2	6.5±0.8**
Serum glucose (mg/dL)	214±11	615±52**	308±21	366±28
Serum insulin (ng/mL)	2.3±0.6	11.5±3.3	0.9±0.3	30.6±6.3*
Creatinine clearance (mL/d/cm2)	0.23±0.04	0.53±0.05**	0.26±0.05	0.49±0.04**
Urinary albumin (µg/d)	45.5±11.3	332.4±79.9*,##		31.5±9.5
In-blood leptin (pg/mL)	49±21	7828±1232*,##		n.d.

* p<0.05, ** p<0.01 vs. control, ^## p<0.01 vs. ob/ob (n=4–10, mean±S.E.M.).

Creatinine clearance of db/db (0.53±0.05 mL/d/cm²) and ob/ob (0.49±0.04 mL/d/cm²) mice was significantly higher (p=0.002, 0.043) than those of db/m (0.23±0.04 mL/d/cm²) and ob control (0.26±0.05 mL/d/cm²) mice, respectively (Table 1). These results imply that microalbuminuria may not always be induced by glomerular hyper-filtration. Consequently, this indicates that there may be another candidate underlying the cause of microalbuminuria, other than glomerular hyper-filtration.

In order to screen candidates for microalbuminuria-related genes with db/db and ob/ob strains, we conducted three microarray data analysis steps (Fig. 1A). In STEP 1, up or down-regulated genes were determined as candidates for microalbuminuria-related genes. Genes for which the expression ratio of (db/db)/(db/m) was higher than (mean+2S.D.) or lower than (mean−2S.D.) were screened. In STEP 2, genes for which the expression ratio of (ob/ob)/(ob control) was higher than the mean+1S.D. or lower than the mean−1S.D. were excluded as genes for which expression changed not by microalbuminuria, but by diabetes. In STEP 3, genes for which the expression ratio of (ob control)/(db/m) was higher than the mean+1S.D. or lower than the mean−1S.D. were excluded as genes for which expression changed not by microalbuminuria, but by differences involving the genetic background of db/db and ob/ob strains. Of the 25527 genes expressed in the mouse kidney, the expression of 169 and 19 genes were enhanced or suppressed as candidates for microalbuminuria-related genes in db/db mouse kidney, respectively. Table 2 demonstrates the 169 up-regulated genes and the 19 down-regulated genes in the order of expression levels of db/db mouse kidney.

Fig. 1. Screening of Candidate Genes Underlying Microalbuminuria in Diabetic db/db Mice

(A) Microarray study and data analysis. STEP1: Genes for which the expression ratio of (db/db)/(db/m) was higher than the mean+2S.D. or lower than the mean−2S.D. were screened. Since the gene expression ratio of (db/db)/(db/m) was 1.020±0.193 (mean±S.D.) from a total of 25527 genes, 476 genes were induced by over 1.406-fold and 258 genes were suppressed below 0.634-fold. STEP 2: Genes for which the expression ratio of (ob/ob)/(ob control) was higher than the mean+1S.D. or lower than the mean−1S.D. were excluded as genes for which expression changed by diabetes. Since the expression ratio of (ob/ob)/(ob control) was 1.026±0.168 from 25527 genes, 280 genes in 476 genes, and 53 genes in the 258 genes for which the expression ratio of (ob/ob)/(ob control) was between 0.853- and 1.194-fold were screened. STEP 3: Genes for which the expression ratio of (ob control)/(db/m) was higher than the mean+1S.D. or lower than the mean−1S.D. were excluded as genes for which expression changed by strain differences. The expression ratio for (ob control)/(db/m) was 1.010±0.253 from 25527 genes. Consequently, 169 genes of the 280 genes, and 19 genes of the 53 genes, for which the expression ratio of (ob/ob)/(ob control) was between 0.757- and 1.263-fold were screened as candidates of microalbuminuria-related genes. (B) Messenger RNA levels of MIF determined by quantitative PCR. ob control vs. ob/ob and the db/m vs. db/db strains were compared. The amount of MIF transcription in the kidney was elevated in db/db mice (n=4–6, mean±S.E.M., * p<0.05).

Table 2. DNA Microarray Data

Gene	Full name	Ratio
Up-regulated genes
Gm16379	macrophage migration inhibitory factor-like	1.556
Mif	macrophage migration inhibitory factor	1.625
Mif	macrophage migration inhibitory factor	1.640
Nr1d1	nuclear receptor subfamily 1, gr. D, mbr. 1	1.558
Aqp2	aquaporin 2	1.626
Prodh	proline dehydrogenase	1.732
Efhd1	EF hand domain containing 1	1.628
Scnn1a	sodium channel, nonvoltage-gated 1 alpha	1.447
Vamp2	vesicle-associated membrane protein 2	1.651
Acot2	acyl-CoA thioesterase 2	1.527
Cyp2d9	cytochrome P450, fam. 2d, polypeptide 9	1.469
Hspa1a	heat shock protein 1A	1.520
Tspan7	tetraspanin 7	1.464
Ifi30	interferon gamma inducible protein 30	1.642
Pctk3	PCTAIRE-motif protein kinase 3	1.478
Cabc1	chaperone, ABC1 activity of bc1 complex like	1.545
Mfge8	milk fat globule-EGF factor 8 protein, tr-var. 1	1.416
Dmgdh	dimethylglycine dehydrogenase precursor	1.523
Cyp27a1	cytochrome P450, fam. 27a, polypeptide 1	1.492
Paqr5	progestin and adipoQ receptor family mbr. V	1.419
Aqp3	aquaporin 3	1.467
Pdxk	pyridoxal kinase	1.569
Dnajb4	DnaJ (Hsp40) homolog, subfam. B, mbr. 4	1.914
Scnn1g	sodium channel, nonvoltage-gated 1 gamma	1.421
Sec14l1	SEC14-like 1	1.517
Hspa1a	heat shock protein 1A	2.004
Scnn1b	sodium channel, nonvoltage-gated 1 beta	1.425
Brd2	bromodomain containing 2, var. 2	1.467
Nicn1	nicolin 1	1.408
Gstt3	glutathione S-transferase, theta 3	1.713
Gys1	glycogen synthase 1, muscle	1.578
Hk1	hexokinase 1, var. 2	1.517
Abhd6	abhydrolase domain containing 6	1.454
Gsta4	glutathione S-transferase, alpha 4	1.551
Rnf186	ring finger protein 186	1.835
Angptl3	angiopoietin-like 3	1.890
Myof	myoferlin	1.619
Foxi1	forkhead box I1	1.696
Fam38a	Fam38a protein gene	1.421
Fmo4	flavin containing monooxygenase 4	1.491
Sfxn2	sideroflexin 2	1.430
Bag3	BCL2-associated athanogene 3	1.490
Slc14a1	solute carrier fam. 14 (urea), mbr. 1	2.437
Fbxo44	F-box protein 44, tr-var. 1	1.442
Deaf1	deformed epidermal autoregulatory factor 1, tr-var. 2	1.426
4930572J05Rik	RIKEN cDNA	1.638
Abp1	amiloride binding protein 1, tr-var. 3	1.706
Wsb1	WD repeat & SOCS box-containing 1, tr-var. 1	1.928
Gyltl1b	glycosyltransferase-like 1B	1.572
Mpzl2	myelin protein zero-like 2	1.605
Sulf2	sulfatase 2 (Sulf2)	1.422
Dnajb1	DnaJ (Hsp40) homolog, subfam. B, mbr. 1	1.413
Fus	fusion, from t(12;16) malignant liposarcoma	1.426
Htra2	HtrA serine peptidase 2	1.425
Shmt1	serine hydroxymethyltransferase 1	1.474
Abtb2	ankyrin repeat and BTB domain containing 2	1.488
Sycn	syncollin	1.440
Slc45a3	solute carrier family 45, mbr. 3	1.487
2310081J21Rik	adult male tongue cDNA, RIKEN	1.447
2310081J21Rik	adult male tongue cDNA, RIKEN	1.447
2310081J21Rik	adult male tongue cDNA, RIKEN	1.447
Cyp2s1	cytochrome P450, fam. 2s, polypeptide 1	1.421
Aldoc	aldolase C, fructose-bisphosphate	1.543
Mgll	monoglyceride lipase	1.419
Unc45a	unc-45 homolog A	1.425
Pfkp	phosphofructokinase, platelet	1.503
Prkag3	AMP kinase, gamma 3, non-catatlytic subunit	1.790
Shpk	sedoheptulokinase	1.496
D430041B17Rik	RIKEN cDNA	1.425
Fstl3	follistatin-like 3	1.463
Tubb2a	tubulin, beta 2A	1.411
Bcl6	B-cell CLL/lymphoma 6	1.522
Upp2	uridine phosphorylase 2	1.452
Pappa	pregnancy-associated plasma protein A	1.532
Arrdc4	arrestin domain containing 4, tr-var. 1	1.503
Ier5l	immediate early response 5-like	1.452
Acot1	acyl-CoA thioesterase 1	1.606
Ppm1k	protein phosphatase 1K	1.452
2610301F02Rik	MCG142258, isoform CRA	1.485
AK220484	cDNA sequence	1.600
Atp2b4	ATPase, Ca2+ transporting, plasma mbr. 4	1.616
Snrpg	small nuclear ribonucleoprotein polypeptide G	1.450
Ddit4l	DNA-damage-inducible transcript 4-like	1.507
Snta1	syntrophin, acidic 1	1.511
Aff1	AF4/FMR2 fam., mbr. 1	1.407
Cuedc1	CUE domain containing 1	1.476
Rasl11b	RAS-like, fam. 11, mbr. B	1.487
Gprc5b	G protein-coupled receptor, fam. C, gr. 5, mbr. B	1.423
Brms1	breast cancer metastasis-suppressor 1	1.415
Lypd6	LY6/PLAUR domain containing 6	1.462
1810010H24Rik	RIKEN cDNA	1.550
Hsd17b14	hydroxysteroid (17-beta) dehydrogenase 14	1.432
Tspan1	tetraspanin 1	1.411
Fbp2	fructose bisphosphatase 2	1.488
Th1l	TH1-like homolog	1.465
Penk	preproenkephalin	1.638
Gpt	glutamic pyruvic transaminase, soluble	1.527
Freq	frequenin homolog	1.513
1110006O24Rik	RIKEN cDNA	1.576
Cck	cholecystokinin	1.407
Nes	nestin	1.440
Dph2	DPH2 homolog	1.419
Aen	apoptosis enhancing nuclease, tr-var. 1	1.557
Krt34	keratin 34	1.407
Fosb	FBJ osteosarcoma oncogene B	1.599
Cadps2	Ca2+-dep. activator protein for secretion 2	1.482
Tmem54	transmembrane protein 54	1.477
Nfil3	nuclear factor, interleukin 3, regulated	1.466
Spag5	sperm associated antigen 5	1.947
Gm9743	predicted gene 9743	1.430
Chkb	choline kinase beta	1.425
Grem2	gremlin 2 homolog, cysteine knot superfamily	1.663
Bhlhe41	basic helix–loop–helix family, mbr. e41	1.788
Zfp69	zinc finger protein 69	1.569
Olfr373	olfactory receptor 373	1.566
Zfp395	zinc finger protein 395	1.409
Apoc1	apolipoprotein C-I, tr-var. 1	1.424
Tspan2	tetraspanin 2	1.462
1700047G07Rik	putative uncharacterized protein	1.576
Chd7	chromodomain helicase DNA binding protein 7	1.548
Gabarapl2	GABA(A) receptor-associated protein like 2	1.593
Cyp17a1	cytochrome P450, fam. 17a, polypeptide 1	1.640
Tbx18	T-box18	1.610
Slc15a1	solute carrier fam. 15 (oligopeptide), mbr. 1	1.676
Bex4	brain expressed gene 4	1.427
Epha3	Eph receptor A3	1.442
Itgad	integrin, alpha D	1.411
Rgn	regucalcin	1.568
Olfr9	olfactory receptor 9	1.477
Trpc7	TRP cation channel, subfam.C, mbr. 7	1.490
Gm7039	predicted gene similar to calmodulin.	1.435
Bicc1	bicaudal C homolog 1	1.495
Gpr101	G protein-coupled receptor 101	1.450
Olfr312	olfactory receptor 312	1.558
Eda2r	ectodysplasin A2 isoform receptor, tr-var. 1	1.419
Spata18	spermatogenesis associated 18	1.472
Gabrb1	GABA-A receptor, subunit beta 1	1.643
Vip	vasoactive intestinal polypeptide	1.438
V1ra3	vomeronasal 1 receptor, A3	1.484
Olfr1164	olfactory receptor 1164	1.812
Rai2	retinoic acid induced 2, tr-var. 1	1.616
Lcn3	lipocalin 3	1.429
Olfr1459	olfactory receptor 1459	1.618
Spt1	salivary protein 1	1.407
Olfr611	olfactory receptor 611	1.588
A530021J07Rik	putative uncharacterized protein	1.429
Olfr1166	olfactory receptor 1166	1.482
Gm10667	putative uncharacterized protein	1.661
Hsd3b1	hydroxy-Δ-5-steroid dehydrogenase, 3β-steroid-Δ-isomerase 1	1.714
Gm5793	pseudogene	1.457
Olfr1255	olfactory receptor 1255	1.513
Mup20	predicted gene	1.764
Nlrp4c	NLR family, pyrin domain containing 4C	1.557
Svs3b	seminal vesicle secretory protein 3B	1.641
5530400C23Rik	putative uncharacterized protein	1.542
Olfr495	olfactory receptor 495	1.494
Cyp3a57	cytochrome P450, fam. 3a, polypeptide 57	1.490
Olfr1000	olfactory receptor 1000	1.686
Ssxb10	synovial sarcoma, X member B, breakpoint 10	1.545
Vmn2r78	vomeronasal 2, receptor 78	1.742
V1rc21	vomeronasal 1 receptor, C21	1.419
Olfr516	olfactory receptor 516	1.636
Zfp811	zinc finger protein 811	1.413
Cyp2c68	cytochrome P450, fam. 2c, polypeptide 68	1.549
V1rd4	vomeronasal 1 receptor, D4	1.517
LOC100045278	predicted similar to ribosomal protein S25	1.513
Wfdc6b	WAP four-disulfide core domain 6B	1.467
9830004L10Rik	adult male bone cDNA	1.698
Gm10848	putative uncharacterized protein	1.469
Down-regulated genes
Inmt	indolethylamine N-methyltransferase	0.607
Slco1a6	solute carrier organic anion transporter fam., mbr. 1a6	0.581
C730048C13Rik	RIKEN cDNA	0.306
Tmigd1	transmemrane & immunoglobulin domain containing 1	0.631
Etv1	ets variant gene 1	0.588
Stxbp5l	syntaxin binding protein 5-like, tr-var.xb	0.633
Aplnr	apelin receptor	0.632
Sp100	nuclear antigen Sp100	0.615
Asxl3	isoform 2 of putative polycomb gr. protein	0.553
Gbp6	guanylate binding protein 6	0.629
Gm4802	predicted gene	0.524
V1rc26	vomeronasal 1 receptor, C26	0.615
Gm11272	predicted gene	0.633
Prkar2b	cAMP dependent protein kinase, type II beta	0.600
Hmcn1	hemicentin 1	0.567
Lepr	leptin receptor, tr-var. 3	0.613
V1rc10	vomeronasal 1 receptor, C10	0.539
F730021E23Rik	B6-derived CD11 +ve dendritic cells cDNA	0.619
Gm9443	predicted: similar to LNR42	0.611

Abbreviations: gr, group; mbr, member; fam, family; tr-var., transcription variant; dep, dependent.

Microarray studies demonstrated that macrophage migration inhibitory factor gene (MIF) was the most expressed gene in the 169 up-regulated microalbuminuria-related genes. Using quantitative PCR, MIF mRNA levels in db/db mice were significantly higher than those in db/m mice (Fig. 1B). Moreover, the production of MIF is known to increase at the crescentic anti-glomerular basement membrane glomerulonephritis with nephrosis⁹⁾ while proteinuria is known to occur in glomerular epithelium-specific MIF transgenic mice.¹⁰⁾ Thus, MIF was screened as a possible candidate for the induction of microalbuminuria in diabetic db/db mice by up-regulated transcription in the kidneys and by its own proteinuric properties.

Among the other genes in Table 2, muscle glycogen synthase 1 gene (Gys1) was reported that its polymorphism is related to hypertension and microalbuminuria.¹¹⁾ Fructose-bisphosphate aldolase C gene (Aldoc) was reported that its expression was enhanced together with MIF gene in breast cancer.¹²⁾ Integrin alpha D (Itgad) was reported to be related to macrophage migration.¹³⁾ Although these genes may be revealed as further candidate genes for the induction of microalbuminuria, we decided to further investigate the protein profile of MIF in diabetic db/db and ob/ob mice.

Urinary excretion of MIF protein increased significantly only in db/db mice (Fig. 2A). Moreover, the increase of urinary secretion of MIF was observed significantly from 6-week-old (Fig. 2B). However, the urinary albumin was increased significantly from 8-week-old, as described in earlier report. Namely, the urinary secretion of MIF preceded microalbuminuria. Consequently, the urinary secretion of MIF is not induced by microalbuminuria, but a possible candidate for the inducer of microalbuminuria in diabetic db/db mice. Contrary to the MIF mRNA, MIF protein levels were significantly higher in the kidneys in both db/db and ob/ob mice (Fig. 2C). MIF exists in cells in preformed pools and secreted in response to a variety of stimuli including inflammation and hypoxia.¹⁴⁾ Consequently, MIF protein was secreted only from the kidneys of db/db mice, but was accumulated in the kidneys of both db/db and ob/ob mice. Another possible mechanism underlying the observed increase in urinary MIF protein is that MIF protein in the glomerular filtrate may not have been reabsorbed by the tubule and was thus excreted into the urine. However, MIF protein reabsorption failure at the tubule cannot explain the elevated levels of MIF mRNA in the kidneys of db/db mice. Consequently, it is possible that the observed increase in urinary MIF protein could be due to secretion of MIF protein from the kidneys of db/db mice.

Fig. 2. Expression Profile of MIF Protein

Ob control vs. ob/ob, and the db/m vs. db/db strains were compared. Urinary excretion of MIF protein (A) and MIF protein levels in the kidney (C) (n=4–6, mean±S.E.M., * p<0.05, ** p<0.01). The amount of MIF excretion in the urine was elevated in db/db mice. Weekly profile of urinary excretion of MIF (closed circle and continuous line) and albumin (open circle and dashed line) in db/db mice (B) (n=3, mean±S.E.M., * p<0.05, ** p<0.01). The urinary excretion of MIF preceded the onset of microalbuminuria.

The MIF receptor CD74 is increased in tubular cells and podocyte in human diabetic nephropathy. MIF engagement of CD74 activated mitogen activated protein (MAP) kinase and increased tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) expression in podocyte, which promote a modest increase in podocyte injury and cell death.¹⁵⁾ Podocyte overexpression of MIF caused podocyte injury and proteinuria in mice without macrophage migration.¹⁰⁾ Thus, we consider that the observed secretion of urinary MIF protein may be associated with the onset of microalbuminuria in diabetic db/db mice. Although it was suggested that in type 2 diabetes macroalbuminuria is the main predictor of mortality, independently of both eGFR and cardiovascular risk factors,¹⁶⁾ the spectrum of diabetic nephropathy has recently expanded, as lack of significant albuminuria is present in 30% of diabetics with kidney function impairment.¹⁷⁾ The MIF-induced TRAIL expression in tubular cell and podocyte also causes renal cell loss and impairment of kidney function. Thus, further study is necessary to clarify the mechanism of secretion of urinary MIF protein observed in diabetic db/db mice.

It has already been reported that MIF production in the kidneys of 8-month-old (32-week-old) db/db mice invaded by macrophages results in an increase in proinflammatory cytokines, MCP-1 and TNF-α.¹⁸⁾ In the same report, the invasion of MIF-producing macrophages and an increase in proinflammatory cytokines were not observed in the kidneys of 2-month-old db/db mice. Our current study also did not result in the invasion of MIF-producing macrophages in the kidneys of 2-month (8-week)-old db/db mice. Rather, MIF was localized to the Bowman’s capsule epithelium in the kidneys of both db/db and ob/ob mice (Fig. 3), in agreement with previous data concerning normal human kidneys.¹⁹⁾ Furthermore, chemokines, which are normally induced by macrophages such as MCP-1 and TNF-α, were not elevated in our microarrays. In other words, there was no evidence of proinflammatory cytokine involvement in relation to microalbuminuria in the kidney of early 8-weeks-old db/db mice.

Fig. 3. Localization of MIF Protein in the Kidney

Expression of MIF protein was demonstrated in the tubules or interstitium (A, D) of the kidneys from control mice. MIF appeared to be localized to the Bowman’s capsule in the kidneys of ob/ob mice (B, C) and db/db mice (E, F).

In conclusion, we suggest that the onset of microalbuminuria in early diabetic nephropathy may be associated with the urinary secretion of intra-renal MIF.

Acknowledgment

The authors thank Mai Suzuki and Nana Iwakawa for technical assistance. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant No. 23591340) and from the Gout Research Foundation.

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