Identification of common and specific fibrosis-related genes in three common chronic kidney diseases

Figures & data

Figure 1. Results of the pathological and NanoString analyses. (a) Representative images of Masson’s trichrome stained kidneys. Scale bar, 50 μm. (b) The volcano plots of the DEGs in DN. (c) The volcano plots of the DEGs in IgAN. (d) The volcano plots of the DEGs in MN. (e) The venn plot of the DEGs.

Table 1. qPCR Primer sequences.

Gene	Forward	Reverse
PTPRC	5′-ATGAAAGAGTGAGAGTGGACGAT-3′	5′-ATTCTGGTGTCTGCCTGCTTC-3′
ICAM1	5′-GGTAGCAGCCGCAGTCATAA-3′	5′-GATAGGTTCAGGGAGGCGTG-3′
VCAM1	5′-AAAGTTCTTGTTTGCCGAGC-3′	5′-CTGGAACACATCGCACAAGT-3′
IL10RA	5′-CAGACGCTCATGGGACAGAG-3′	5′-GGTGTCCAGTGGAGGATGTG-3′
CCR2	5′-AGTCAACTGGACCAAGCCAC-3′	5′-TGAAAAAGGCTTCTGAACTTCTCC-3′
PPARA	5′-TCCTCTCAGGAAAGGCCAGT-3′	5′-CTGGCAGCAGTGAAAGATGC-3′
LOX	5′-TTCACATTGCTTTGCAGTCC-3′	5′-CTGTATGCTGTACCTTCGCC-3′
SPP1	5′-AGCTTTACAACAAATACCCAGATGC-3′	5′-GGACTTACTTGGAAGGGTCTGTG-3′

Table 2. Demographic, clinical and laboratory features of study subjects*.

Variable	DN group	IgAN group	MN group	Control group	P Value
	(N = 3)	(N = 3)	(N = 3)	(N = 3)
Age (years)	62.67 ± 7.76	42.67 ± 14.66	50.33 ± 7.76	58.67 ± 10.34	0.370
BMI (kg/m^2)	26.67 ± 1.76	29.37 ± 2.04	24.90 ± 1.34	27.07 ± 2.44	0.238
SBP (mmHg)	172.33 ± 14.06	151.00 ± 26.09	130.33 ± 7.72	115.33 ± 7.04	0.004^a
DBP (mmHg)	82.33 ± 5.44	89.00 ± 8.52	91.00 ± 7.79	72.67 ± 8.22	0.160
Scr (µmol/L)	372.40 ± 87.42	140.93 ± 25.28	132.47 ± 28.50	68.73 ± 14.10	0.004^a
BUN (mmol/L)	14.02 ± 0.92	8.29 ± 0.96	8.49 ± 1.09	5.45 ± 1.04	0.004^a
24hUTP (g/24 h)	2.44 ± 0.80	2.47 ± 1.18	8.47 ± 4.15	0.05 ± 0.03	0.004^b
eGFR (mL/min/1.73 m^2)^c	11.93 ± 3.15	43.27 ± 16.04	37.08 ± 14.37	102.81 ± 8.84	0.004^a

* Kruskal–Wallis test was used for comparison among groups.

^a Adjusted p-value. There is a significant difference between DN and control group.

^b Adjusted p-value. There is a significant difference between MN and control group.

^c eGFR was estimated using the CKD-EPI formula.

Abbreviations: 24hUTP, 24-h urinary total protein; BMI, body mass index; BUN, blood urea nitrogen; CKD, chronic kidney disease; DBP, diastolic blood pressure; DN, diabetic nephropathy; eGFR, estimated glomerular filtration rate; IgAN, IgA nephropathy; MN, membranous nephropathy; SBP, systolic blood pressure; Scr, serum creatinine.

Figure 2. GO and KEGG pathway enrichment analysis of the DEGs. (a) GO result of the 64 common DEGs. (b) GO result of the 29 DN-specific DEGs. (c) GO result of the 29 IgAN-specific DEGs. (d) GO result of the 26 MN-specific DEGs. (e) KEGG result of the 64 common DEGs. (f) KEGG result of the 29 DN-specific DEGs. (g) GO result of the 29 IgAN-specific DEGs. (H) KEGG result of the 26 MN-specific DEGs.

Figure 3. PPI network analysis of the DEGs. (a) PPI network of the 64 common DEGs. (b) PPI network of the 29 DN-specific DEGs. (c) PPI network of the 29 IgAN-specific DEGs. (d) PPI network of the 26 MN-specific DEGs.

Figure 4. Validation of the key DEGs in the Nephroseq database. (a) Expression of PTPRC in CKD tissues and the normal controls. (b) Expression of ICAM1 in CKD tissues and the normal controls. (c) Expression of VCAM1 in CKD tissues and the normal controls. (d) Expression of IL10RA in CKD tissues and the normal controls. (e) Expression of CCR2 in CKD tissues and the normal controls. (f) Expression of PPARA in DN tissues and the normal controls. (g) Expression of SPP1 in MN tissues and the normal controls. (h) Expression of LOX in IgAN tissues and the normal controls. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Correlation between the expression of hub genes and GFR in different types of CKD patients. (a) Correlation between PTPRC and GFR in DN patients. (b) Correlation between PTPRC and GFR in IgAN patients. (c) Correlation between PTPRC and GFR in MN patients. (d) Correlation between ICAM1 and GFR in DN patients. (e) Correlation between ICAM1 and GFR in IgAN patients. (f) Correlation between VCAM1 and GFR in CKD patients. (g) Correlation between IL10RA and GFR in DN patients. (h) Correlation between IL10RA and GFR in IgAN patients. (i) Correlation between PPARA and GFR in DN patients. (j) Correlation between SPP1 and GFR in MN patients. X axis: Log2 GFR (MDRD) (ml/min/1.73m²); Y axis: Median-centered Log2 expression of the genes.

Figure 6. qRT-PCR analysis of the mRNA levels of the identified key genes in fibrotic CKD and normal tissues. (a) Relative expression of PTPRC, ICAM1, VCAM1, IL10RA and CCRA in DN, IgAN, MN and control groups. (b) Relative expression of PPARA in DN, IgAN, MN and control groups. (c) Relative expression of LOX in DN, IgAN, MN and control groups. (d) Relative expression of SPP1 in DN, IgAN, MN and control groups. n = 3. *p < 0.05, **p < 0.01. Student’s t-test was used for comparison between the groups.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.