Clinical Significance of Glomerular Autophagy in Evaluation of Diabetic Kidney Disease Progression

Figures & data

Figure 1 Identification and GO analysis of DEGs in glomeruli. (A) Sample normalization; (B) PCA of glomerular genes; (C) UMAP of glomerular genes; (D) volcanic map of DEGs in glomeruli; (E) GO analysis of DEGs in glomeruli (top 26).

Abbreviations: DKD group, group 1; NC group, group 2; DEGs, differential expression genes; DKD, diabetic kidney disease.

Figure 2 Identification and GO analysis of DEGs in renal tubular interstitium. (A) Sample normalization; (B) PCA of genes in renal tubulointerstitium; (C) UMAP of genes in renal tubulointerstitium; (D) volcanic map of DEGs in renal tubular interstitium; (E) GO analysis of DEGs in renal tubulointerstitium (top 26).

Abbreviations: DKD group, group 1; NC group, group 2; DEGs, differential expression genes; DKD, diabetic kidney disease.

Table 1 Baseline Demographic Data of Patients and p62 Protein Expression of Renal Tissue

Parameter	NC (n=6)	MCD (n=11)	DKD(n=43)		P
			DKD1(n=13)	DKD2(n=30)
Age (year)	50.83±2.27	48.36±12.83	45.00±10.46	48.00±11.92	0.214
M/F	4/2	6/5	8/5	24/6	0.376
ACR (mg/g)	–	1349.00(1102.00,2921.00) ***	89.24(52.00, 216.15)	1363.75(750.98,3560.50) ***
P62	0.33(0.31,0.47)	1.48(1.95,1.77)	1.87(1.75,2.83)	3.87(3.29,8.70) **^###$$$

Notes: 1: MCD group: minimal change disease group without diabetic kidney disease; DKD: diabetic kidney disease group, divided into DKD1 and DKD2; NC: non-diabetic renal cancer group. 2: The overall analysis of age was one-way ANOVA. Pairwise comparisons between groups were performed using the LSD-t test. Other indicators were calculated by Mann-Whitney U-test vs DKD1 **p < 0.01, ***p < 0.001; vs NC ^###p < 0.001; vs MCD ^$$$p < 0.001.

Figure 3 The expressions of the P62 protein in human renal tissues were significantly increased in the DKD group patients with macroalbuminuria, mainly in the podocytes. (A) IHC staining of P62 protein in human renal tissues; (B) double immunofluorescence staining of P62 and WT-1 in human renal tissue of DKD2 group. (C) Double immunofluorescence staining of P62 and CD31 in human renal tissue of DKD2 group. (D) Double immunofluorescence staining of P62 and PDGFR-β in human renal tissue of DKD2 group. Data are expressed as mean ± standard deviation (SEM). Scale bar = 50 μm; *p < 0.05, ***p < 0.001.

Table 2 Clinical Data of Patients with DKD

Parameter	DKD1(n=13)	DKD2(n=30)	t (Z) [χ2]	P value
DM duration (m)	48.00(1.00, 96.00)	78.00(36.00, 168.00)	(2.053)	0.041
Smoking [n (%)]	53.85	50.00	[0.023]	0.879
Weight	66.69±11.97	71.62±14.99	1.347	0.301
SBP (mmHg)	129.67±28.09	148.40±20.00	2.436	0.019
DBP (mmHg)	82.83±11.15	89.43±13.39	1.508	0.139
Scr (μmol/L)	85.55(55.25, 136.88)	111.40(82.98, 146.30)	(1.072)	0.284
BUN (mmol/L)	7.23(6.46, 9.75)	6.86(5.91, 10.78)	(0.014)	0.989
UA (μmol/L)	314.00(274.00, 373.00)	358.00(308.50, 427.50)	(1.629)	0.103
eGFR [mL/min·1.73 m^2]	86.42±38.33	73.70±31.22	1.117	0.271
FBG (mmol/L)	5.90(4.51, 6.50)	7.24(6.001, 8.26)	(2.395)	0.017
HbAc1(%)	7.15(6.73, 7.70)	7.50(6.70, 8.20)	(0.809)	0.419
CHO (mmol/L)	3.95±0.89	5.24±1.37	3.003	0.005
TG (mmol/L)	1.95(1.55, 2.34)	1.51(1.03, 2.32)	(0.849)	0.396
LDL (mmol/L)	2.21±0.99	3.23±1.21	2.560	0.014
VLDL (mmol/L)	0.72(0.58, 0.84)	0.56(0.38, 0.85)	(0.794)	0.427
HDL (mmol/L)	0.93±0.21	1.22±0.35	2.648	0.012
ALB (g/L)	42.89±3.57	36.28±7.52	3.852	0.000
Hb (g/L)	125.42±19.56	124.47±23.43	0.124	0.902
ApoA1(mmol/L)	1.03(0.98, 1.18)	1.23(1.04, 1.40)	(6.048)	0.014
ApoB (mmol/L)	0.80±0.17	1.04±0.28	2.687	0.011
ApoB/ApoA1	0.79±0.23	0.84±0.26	0.624	0.537
Lipoprotein α(mmol/L)	112.00(65.50, 244.50)	229.00(130.00, 487.00)	(1.727)	0.084
CRP (g/L)	0.89(0.60, 1.34)	1.44(0.91, 2.00)	(1.499)	0.134
U-TRF (mg/L)	6.43(5.07, 17.15)	59.90(31.60, 134.43)	(4.211)	0.000
U-RBP (mg/L)	0.54(0.17, 1.21)	1.60(0.55, 3.89)	(1.727)	0.084
U-NAG (U/L)	8.40(7.50, 9.75)	13.84(8.86, 18.70)	(1.985)	0.047
U-IgG (mg/L)	9.10(4.85, 18.03)	100.94(38.12, 228.57)	(4.242)	0.000
U-FDP (ug/mL)	0.15(0.10, 0.38)	0.19(0.09, 0.51)	(0.349)	0.727
U-cyc (mg/L)	0.15(0.11, 0.21)	0.26(0.15, 0.40)	(1.697)	0.090
U-Cr (mmol/L)	9.94(7.41, 13.30)	6.95(5.11, 9.44)	(2.060)	0.039
U-β2-MG (mg/L)	0.31(0.16, 1.09)	0.98(0.40, 2.15)	(1.727)	0.084
U-α1-MG (mg/L)	12.02(6.31, 18.15)	21.34(13.15, 30.61)	(1.848)	0.065
U-ALB/Cr	80.24(44.25, 192.91)	1324.85(503.39, 3202.47)	(4.696)	0.000
U-TP/Cr	358.41(142.98, 504.35)	1899.81(891.33, 3900.79)	(4.575)	0.000
U-ALB/β2-MG	428.05(45.53, 1048.60)	760.78(433.37, 2201.54)	(1.878)	0.06
24-h UTP (g)	80.24(44.25, 192.91)	1324.85(503.39, 3202.47)	(4.749)	0.000
24-h UTP/Cr (g/g)	0.39(0.34, 0.53)	2.77(1.54, 6.09)	(4.637)	0.000
U-ALB (g)	0.11(0.05, 0.20)	0.92(0.46, 2.27)	(4.720)	0.000
24-h UAER (g)	0.18(0.08, 0.27)	1.60(0.97, 4.09)	(4.657)	0.000

Notes: Data presented as means ± SD, medians (IQR), or n (%); t-tests for continuous data, Mann–Whitney U-tests for abnormally distributed variables, and χ2 tests for categorical data.

Abbreviations: DM, diabetic mellitus; SBP, systolic blood pressure; DBP, diastolic blood pressure; BUN, blood urea nitrogen; UA, uric acid; eGFR, estimated glomerular filtration rate; FBG, fasting blood glucose; CHO, cholesterol; LDL, low-density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein; ALB, albumin; ApoB, apolipoprotein B; ApoA1, apolipoprotein A1; CRP, c-reactive protein; U-TRF, urinary transferrin; U-NAG, N-acetyl β-glucosaminidase; U-IgG, urinary IgG; U-FDP, urinary fibrinogen; U-cyc, urinary cystatin C; U-β2-MG, urinary β2 microglobulin; U-α1-MG, urinary α1-microglobulin; U-Cr, urinary creatinine; 24-hUTP, 24-h urine protein quantification; U-ALB, urinary albumin; 24-hUAER, 24-h urinary albumin excretion.

Table 3 Comparison of Pathological Data of DKD Patients

Parameter	DKD1	DKD2	Z	P
Glomerular lesions [n (%)]			2.854	0.004
I	0 (0)	0 (0)
II
IIa	10 (76.92)	9 (30.00)
IIb	2 (15.38)	8 (26.67)
III	1 (7.69)	13 (43.33)
IV	0 (0)	0 (0)
IFTA (0/1/2/3)	3/7/3/0	1/10/17/2	2.395	0.028
Interstitial inflammation (0/1/2)	3/10/0	0/21/9	3.032	0.002
Arteriolar hyalinosis (0/1/2)	1/10/2	1/17/12	1.607	0.108
Arteriosclerosis (0/1/2)	2/10/1	2/18/10	1.965	0.049

Abbreviations: IFTA, interstitial fibrosis and tubular atrophy; DKD, diabetic kidney disease.

Table 4 Correlation Analysis of P62 Protein Level on Glomerulus and Clinicopathological Data of DKD Group

Parameter	Correlation Coefficient (r)	P
DM duration	0.198	0.202
SBP	0.199	0.207
CHO	0.545	0.000
HDL	0.335	0.030
LDL	0.547	0.000
FBG	0.123	0.437
ApoB	0.460	0.003
ApoA1	0.272	0.089
ALB	−0.590	0.000
U-TRF	0.713	0.000
U-IgG	0.772	0.000
U-NAG	0.371	0.018
U-Cr	−0.345	0.029
U-TP/Cr	0.755	0.000
U-ALB/Cr	0.761	0.000
U-ALB	0.784	0.000
24-h UAER	0.799	0.000
24-h UTP/Cr	0.700	0.000
24-h UTP	0.752	0.000
Glomerular lesions	0.544	0.000
Interstitial inflammation	0.310	0.043
IFTA	0.253	0.101
Arteriosclerosis	0.279	0.070

Note: Spearman’s test.

Abbreviations: DM, diabetic mellitus; SBP, systolic blood pressure; CHO, cholesterol; LDL, low-density lipoprotein; HDL, high density lipoprotein; FBG, fasting blood glucose; ApoB, apolipoprotein B; ApoA1, apolipoprotein A1; ALB, albumin; U-TRF, urinary transferrin; U-IgG, urinary IgG; U-NAG, N-acetyl β-glucosaminidase; U-Cr, urinary creatinine; U-ALB, urinary albumin; 24-hUAER, 24-h urinary albumin excretion; 24-hUTP, 24-h urine protein quantification; IFTA, interstitial fibrosis and tubular atrophy.

Table 5 Stepwise Regression Analysis

Parameter	Regression Coefficients	95% CI	VIF
Constant	−0.269 (t = −0.968)	−0.813 ~ 0.276	–
P62	0.077** (t = 3.911)	0.038 ~ 0.116	1.020
ApoA1	0.520* (t = 2.329)	0.082 ~ 0.957	1.020
Sample size	39
R ^2	0.397
Adjust R ^2	0.364
F value	F (2, 36) =11.859, p=0.000

Notes: D-W value: 0.476; *p < 0.05, **p < 0.01.

Abbreviation: ApoA1, apolipoprotein A1.

Table 6 Binary Logistic Regression Analysis

	β	Se	Wald χ2	P	OR	95% CI
ApoA1 P62 Constant	0.209	1.518	0.019	0.891	1.232	0.063–24.156 1.152–10.962
	1.268	0.575	4.871	0.027	3.554
	−3.519	2.134	2.719	0.099	0.030

Abbreviation: ApoA1, apolipoprotein A1.

Table 7 ROC Curve Analysis of P62 Protein on Glomerulus of DKD Patients

	Cut-off	AUC (95% CI)	P	Sensitivity (n/N)	Specificity (n/N)	Youden Index
P62	2.857	0.905 (0.800–1.000)	0.000	0.967 (29/30)	0.769 (10/13)	0.736

Figure 4 ROC curve analysis of glomerular P62 protein in DKD patients.