Elevated ALOX12 in renal tissue predicts progression in diabetic kidney disease

Figures & data

Figure 1. Identification of DEGs in transcriptome sequencing data. (A) PCA of sequencing results. (B) Volcano plot of the DEGs. (C) Heat map of the DEGs.

Figure 2. Functional enrichment analysis and pathway of DEGs. (A) KEGG enrichment analysis. (B) KEGG enrichment analysis in the lipid metabolism pathway. (C) PPI network analysis in the AA metabolism pathway. (D) GO enrichment analysis. (E) Bubble plot. (F) RT-PCR detection of ALOX12 mRNA in db/m and db/db mouse models, **p < .01. (G, H) The diagnostic efficacy of ALOX12 for DKD was verified using ROC curves in dataset GSE1009 and dataset GSE7253.

Figure 3. Pathological staining and ALOX12 expression in mouse kidney tissues. (A) H&E staining in db/m and db/db kidney tissue. (B) PAS staining in db/m and db/db kidney tissue. (C) IHC detection of NGAL protein expression in db/m and db/db kidney tissue. (D) IHC detection of ALOX12 protein expression in db/m and db/db kidney tissues. Scale bar = 50 μm. *p < .05, **p < .01.

Figure 4. Expressions of ALOX12 in the body. (A) ALOX12 levels in serum. (B) ALOX12 levels in urine. *p < .05, **p < .01, and ***p < .001.

Table 1. Demographics of patients with hematuria.

Parameter	NC (n = 6)	MCD (n = 6)	DKD		p Value
			DKD1 (n = 6)	DKD2 (n = 6)
Age (years)	48.667 ± 12.307	59.000 ± 10.445	55.333 ± 12.176	54.833 ± 9.109	.4251
Sex (F/M)	2/4	2/4	2/4	1/5	.901
U-ACR (mg/g)	11.128 ± 9.740	779.170 ± 493.270*	124.110 ± 91.336	3615.100 ± 2278.700*^,&	<.01
Serum ALOX12 (ng/mL)	73.403 ± 8.113	72.877 ± 11.387	92.048 ± 4.799**^,#	111.620 ± 20.224*^,#	<.01
Urine ALOX12/urine creatinine (ng/mmol)	338.7 ± 81.44	260.2 ± 77.12	340.2 ± 132.3	604.5 ± 174.5**^,###,&&	<.001

NC group: the normal patients; MCD group: patients with microscopic renal disease patients; DKD1 group: the diabetic kidney disease patients with UACR <300 mg/g; DKD2 group: the diabetic kidney disease patients with UACR ≥300 mg/g; U-ACR: urine-albumin/urine creatinine ratio.

vs. NC *p < .05, **p < .01; vs. MCD ^#p < .01, ^###p < .001; vs. DKD1 ^&p < .05; vs. DKD1 ^&&p < .01.

Table 2. Baseline patient demographics and renal tissue ALOX12 protein expression.

Parameter	NC (n = 6)	MCD (n = 10)	DKD		p Value
			DKD1 (n = 17)	DKD2 (n = 25)
Age (years)	51.500 ± 11.540	47.900 ± 18.180	52.820 ± 7.748	52.600 ± 10.000	.932
Sex (F/M)	2/4	5/5	1/16	6/19	.097
ALOX12 (%)	8.436 ± 0.785	7.433 ± 1.970	13.130 ± 3.458***^,###	17.400 ± 4.908***^,###,&	<.001

vs. NC ***p < .001; vs. MCD ^###p < .001; vs. DKD1 ^&p < .05; the protein expression level of ALOX12 was expressed as AOD (%).

Figure 5. Expression of ALOX12 protein in human kidney tissue. (A) IHC detection of NGAL protein expression in human kidney tissue. (B) IHC detection of ALOX12 protein expression in human kidney tissue. (C) Expression results of ALOX12 levels and NGAL levels. (D) Correlation analysis of ALOX12 levels with NGAL levels. (E) IF double staining of ALOX12 and specific tubular markers in renal biopsy tissues of patients with DKD. Scale bar = 50 μm. *p < .05, **p < .01, and ***p < .001.

Table 3. Clinical data of patients with DKD.

Parameter	DKD		t(Z)	p Value
	DKD1 (n = 17)	DKD2 (n = 25)
DM duration (years)	8.441 ± 5.396	8.940 ± 6.604	−0.258	.798
BMI	26.500 ± 3.532	26.320 ± 3.712	0.151	.881
BUN (mmol/L)	8.420 (6.985, 9.395)	9.206 ± 3.354	(–.602)	.547
CRE (µmol/L)	119.200 ± 27.180	135, 500 (115.1, 191.9)	(–1.627)	.104
UA (µmol/L)	390.200 ± 114.40	365.800 ± 87.720	0.780	.440
eGFR, mL/(min*1.73 m^2)	60.000 (52.000, 115.000)	49.880 ± 25.620	(–2.576)	.010
HbA1c (%)	6.900 (6.450, 7.315)	7.200 (6.300, 8.400)	(–.744)	.457
ALB (g/L)	42.200 (39.350, 43.000)	34.46 ± 7.866	(–2.460)	.014
TC (mmol/L)	4.006 ± 1.121	5.465 ± 1.612	−3.232	.002
TG (mmol/L)	2.110 (1.215, 3.430)	1.590 (1.200, 2.300)	(–1.012)	.311
HDLC (mmol/L)	0.943 ± 0.258	1.174 ± 0.339	−2.373	.023
LHDL (mmol/L)	2.038 ± 0.785	3.312 ± 1.634	−3.370	.002
VLDL (mmol/L)	0.780 (0.400, 1.105)	0.710 (0.450, 1.370)	(–.372)	.710
Apo A (g/L)	1.115 ± 0.178	1.290 ± 0.290	−2.224	.032
Apo B (g/L)	0.794 ± 0.182	1.110 (0.925, 1.305)	(–3.872)	.000
U-RBP (mg/L)	0.460 (0.270, 1.200)	5.500 (2.360, 8.220)	(–3.524)	.001
U-CYC (mg/mL)	0.150 (0.009, 0.200)	0.670 (0.265, 2.040)	(–3.345)	.001
Ucr (mmol/L)	11.59 ± 5.901	7.028 ± 2.726	2.979	.007
U-ACR (mg/g)	73.200 (24.660, 120.800)	2762.000 ± 1955.000	(–5.445)	.000

BUN: blood urea nitrogen; CRE: serum creatinine; UA: uric acid; eGFR: estimated glomerular filtration rate; HbA1c: hemoglobin A1c; ALB: albumin; TC: total cholesterol; TG: triglyceride; HDLC: high-density lipoprotein cholesterol; LDLC: low-density lipoprotein cholesterol; VLDL: very low density lipoprotein; Apo A: apolipoprotein A; Apo B: apolipoprotein B; U-RBP: urinary retinol-binding protein; U-CYC: urinary cystatin c; U-Cr: urinary creatinine; U-ACR: urine-albumin/urine creatinine ratio.

Table 4. Pathological data of patients with DKD.

Parameter	DKD		Z	p Value
	DKD1 (n = 17)	DKD2 (n = 25)
Glomerular lesions, n (n, %)			−4.179	.000
I	0 (0.000)	0 (0.000)
II
IIa	13 (76.471)	4 (16.000)
IIb	3 (17.647)	4 (16.000)
III	1 (5.882)	13 (52.000)
IV	0 (0.000)	4 (16.000)
IFTA, n (n, %)			−3.156	.002
Score 0	1 (5.882)	0 (0.000)
Score 1	9 (52.941)	4 (16.000)
Score 2	7 (41.765)	16 (64.000)
Score 3	0 (0.000)	5 (20.000)
Interstitial inflammation, n (n, %)			−2.215	.027
Score 0	1 (5.882)	0 (0.000)
Score 1	16 (94.118)	20 (80.000)
Score 2	0 (0.000)	5 (20.000)
Arteriolar hyalinosis, n (n, %)
Score 0	1 (5.882)	0 (0.000)	−2.063	.039
Score 1	9 (52.941)	7 (28.000)
Score 2	7 (41.765)	18 (72.000)
Arteriosclerosis, n (n, %)
Score 0	0 (0.000)	0 (0.000)	−.695	.487
Score 1	12 (70.588)	15 (60.000)
Score 2	5 (29.412)	10 (40.000)

IFTA: interstitial fibrosis and tubular atrophy.

Table 5. Correlation analysis of renal tubular ALOX12 protein levels and clinical and pathological data.

Parameter	r	p Value
DM duration (years)	−.098	.538
BMI	−.006	.970
BUN (mmol/L)	−.169	.286
CRE (µmol/L)	−.037	.816
UA (µmol/L)	−.084	.595
eGFR, mL/(min*1.73 m^2)	.046	.774
HbA1c (%)	0.074	.639
ALB (g/L)	−.109	.491
TC (mmol/L)	0.438**	.004
TG (mmol/L)	0.186	.238
HDLC (mmol/L)	.327*	.034
LHDL (mmol/L)	.316*	.041
VLDL (mmol/L)	.263	.092
Apo A(g/L)	.177	.262
Apo B (g/L)	.405*	.008
U-RBP (mg/L)	.412**	.007
U-CYC (mg/mL)	.374*	.015
Ucr (mmol/L)	−.088	.577
UACR (mg/g)	.432**	.004
Glomerular lesions	.211	.181
IFTA	.347*	.024
Interstitial inflammation	.317*	.041
Arteriolar hyalinosis	.088	.578
Arteriosclerosis	−.039	.807

BUN: blood urea nitrogen; CRE: serum creatinine; UA: uric acid; eGFR: estimated glomerular filtration rate; HbA1c: hemoglobin A1c; ALB: albumin; TC: total cholesterol; TG: triglyceride; HDLC: high-density lipoprotein cholesterol; LDLC: low-density lipoprotein cholesterol; VLDL: very low density lipoprotein; Apo A: apolipoprotein A; Apo B: apolipoprotein B; U-RBP: urinary retinol-binding protein; U-CYC: urinary cystatin c; U-Cr: urinary creatinine; U-ACR: urine-albumin/urine creatinine ratio; IFTA: interstitial fibrosis and tubular atrophy.

* p < .05.

** p < .01.

Table 6. Binary logistic regression analysis.

Variables	Univariate analysis		Multivariate analysis
	p	OR (95% CI)	p	OR (95% CI)
ALOX12	.010	1.059–1.535	.046	1.007–2.186
eGFR	.040	0.941–0.999	–	–
ALB	.024	0.799–0.984	–	–
TC	.008	1.244–4.208	–	–
HDLC	.037	1.193–268.120	–	–
LDLC	.012	1.187–3.891	–	–
Apo A	.048	1.028–947.893	–	–
Apo B	.002	10.822–35518.698	–	–
U-RBP	.009	1.093–1.869	–	–
U-CYC	.033	1.210–93.704	–	–
Cr	.009	0.628–0.937	.052	0.564–1.002
Glomerular lesions	.000	2.257–17.542	.005	1.751–23.723
Arteriolar hyalinosis	.038	1.076–12.789	–	–
IFTA	.004	1.831–24.649	–	–

eGFR: estimated glomerular filtration rate; ALB: albumin; TC: total cholesterol; TG: triglyceride; HDLC: high-density lipoprotein cholesterol; LDLC: low-density lipoprotein cholesterol; Apo A: apolipoprotein A; Apo B: apolipoprotein B; U-RBP: urinary retinol-binding protein; U-CYC: urinary cystatin c; U-Cr: urinary creatinine; IFTA: interstitial fibrosis and tubular atrophy.

Figure 6. ROC curve analysis to determining diagnostic value. Area under the ROC curve (95% confidence interval (CI)) for the presence of the AUC for macroalbuminuria.

Table 7. ROC curve analysis.

	Cut off	AUG (95% CI)	p Value	Sensitivity	Specificity	Youden Index
ALOX12	12.342	0.736 (0.583–0.890)	.010	0.880	0.529	0.409
Model	0.613	0.953 (0.896–1.000)	.000	0.880	0.941	0.821

Model: In(p/1 – p) = –8.157 + 1.930 × glomerular lesions + 0.318 × ALOX12.

Data availability statement

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.