Zinc oxide nanoparticles and spironolactone-enhanced Nrf2/HO-1 pathway and inhibited Wnt/β-catenin pathway in adenine-induced nephrotoxicity in rats

Figures & data

Table 1. PCR primers.

Common name	Sequence (5′−3′)	Accession no.
TGF-β1	F: 5′-CACTCCCGTGGCTTCTAGTG-3′ R: 5′-GGACTGGCGAGCCTTAGTTT-3′	NM_021578.2
Wnt7a	F: 5′-GCCCACCTTTCTGAAGATCAAG-3′ R: 5′-TGGGTCCTCTTCACAGTAATTGG-3′	NM_001100473.1
β-catenin	F: 5′-ACAGCACCTTCAGCACTCT-3′ R: 5′-AAGTTCTTGGCTATTACGACA-3'	NM_053357.2
Fibronectin	F: 5′-GTGGCTGCCTTCAACTTCTC-3′ R: 5′-GTGGGTTGCAAACCTTCAAT-3′	NM_019143.2
Collagen IV	F: 5′-ATAGAGAGAAGCGAGATGTTCAAGA-3′ R: 5′-GGATATAATTCTAGGGTTCGTTGCT-3′	NM_001135009.1
α-SMA	F: 5′-TTCGTGACTACTGCTGAGCG-3′ R: 5′-AAGCGTTCATTCCCGATGGT-3′	NM_031004.2
TNF-α	F: 5′-TTCGGAACTCACTGGATCCC-3′ R: 5′-GGAACAGTCTGGGAAGCTCT-3′	NM_012675.3
IL-6	F: 5′-GCCCTTCAGGAACAGCTATGA-3′ R: 5′-TGTCAACAACATCAGTCCCAAGA-3′	NM_012589.2
Nrf2	F: 5′-ATTGCTGTCCATCTCTGTCAG-3′ R: 5′-GCTATTTTCCATTCCCGAGTTAC-3′	NM_001399173.1
HO-1	F: 5′-TGCTTGTTTCGCTCTATCTCC-3′ R: 5′-CTTTCAGAAGGGTCAGGTGTC-3′	NM_012580.2
GAPDH	F: 5′-TATCGGACGCCTGGTTAC-3′ R: 5′-CTGTGCCGTTGAACTTGC-3′	NM_017008.4

Table 2. Treatment of CKD rat model with ZnO-NP and SPL results in improvement of renal function.

Variables (mean ± SD)	Control	CKD	SPL	ZnO-NPs	SPL + ZnO-NPs
Serum creatinine (mg/dL)	0.68 ± 0.19	4.09 ± 0.9^a	2.74 ± 0.29^ab	1.53 ± 0.36^abc	0.8 ± 0.16^bc
BUN (mg/dL)	18.99 ± 1.95	54.36 ± 6.99^a	41.98 ± 6.73^ab	32 ± 7.05^abc	21.4 ± 2.33^abcd
Crcl (mL/min)	1.44 ± 0.16	0.54 ± 0.17^a	0.83 ± 0.09^ab	0.93 ± 0.11^ab	1.16 ± 0.12^abcd
Microalbuminuria (mg/24 h)	20.69 ± 1.6	96.8 ± 13.22^a	68.9 ± 10.2^ab	52.55 ± 7.74^abc	35.54 ± 5.45^abcd

Notes: Where control group: normal group, CKD group: adenine sulfate (positive control group), SPL group: SPL + adenine sulfate, ZnO-NPs group: ZnO-NPs + adenine sulfate, SPL + ZnO-NPs group: SPL + ZnO-NPs + adenine sulfate.

Significant difference compared to corresponding ^acontrol group, ^bCKD group,^cSPL group, ^dZnO-NPs group at p < 0.05 by ANOVA and Tukey test.

Table 3. Treatment of CKD rat model with ZnO-NP and SPL results in down-regulation of oxidative stress and enhanced antioxidants.

Groups	Control	CKD	SPL	ZnO-NPs	SPL + ZnO-NPs
GSH (mmol/g)	9.4 ± 2.5	2.5 ± 0.73^a	4.44 ± 1.41^a	5.78 ± 1.14^ab	8.39 ± 1.72^bc
SOD (U/g)	324.7 ± 41.21	121.6 ± 37.2^a	217.03 ± 34.7^ab	260.14 ± 33.8^ab	297.6 ± 21.27^bc
CAT (U/g)	8 ± 3.32	1.68 ± 0.51^a	5.25 ± 1.32^ab	6.03 ± 1.21^ab	7.36 ± 2.11^b
MDA (nmol/g)	11.58 ± 3.33	49.77 ± 11.05^a	32.37 ± 5.96^ab	24 ± 5.65^ab	15.9 ± 4.12^bc

Notes: Significant difference compared to corresponding ^acontrol group, ^bCKD group, ^cSPL group, ^dZnO-NPs group at p < 0.05 by ANOVA and Tukey test.

Figure 1. Treatment of CKD rat model with ZnO-NP and SPL results in down-regulation of fibrotic and inflammatory gene expression and enhanced antioxidant gene expression. Significant difference compared to corresponding ^acontrol group, ^bCKD group, ^cSPL group, ^dZnO-NPs group at p < 0.05 by ANOVA and Tukey test. Regarding levels of relative quantification of TGF-β1 mRNA, Wnt7a mRNA, and β-catenin mRNA (A), relative quantification of FN1 mRNA, Col-IV mRNA, and α-SMA mRNA (B), relative quantification of TNF-α mRNA and IL-6 mRNA (C), and relative quantification of Nrf2 mRNA and HO-1 mRNA (D).

Figure 2. Treatment of CKD rat model with ZnO-NP and SPL results in down-regulation of β-cateninin cytoplasm and up-regulation of Nrf2 in western blot analysis. Significant difference compared to *control group at p < 0.0001 where C: normal control group, D: CKD group, T1: SPL group, T2: ZnO-NPs group, T3: SPL + ZnO-NPs group.

Figure 3. Histopathological examination of the kidney tissues with H&E. (A) The score of tubulointerstitial damage. Microscopic pictures of kidney specimens showing: (B) normal glomerular and tubular kidney structure (normal group, 100×), (C) protein leakage in the tubular lumen (CKD group, 100×), (D) mild interstitial collagen proliferation (bold black arrow) and prominent nuclei (white arrow) (SPL group, 400×), (E) mild congestion (star), mitotic figure (arrowhead) and prominent nuclei (white arrow) (ZnO-NPs group, 400×) and (F) prominent nuclei (white arrow) and mitotic figure (arrowhead) (SPL + ZnO-NPs group, 400×). a: significant difference versus control, b: significant versus CKD, c: significant versus SPL, d: significant versus ZnO-NPs at p < 0.05.

Figure 4. Immunohistopathological examination for β-catenin expression in kidney tissues. (A) The score of β-catenin in all studied groups, (B) negative expression for beta catenin (control group, 400×), (C) marked cytoplasmic expression for beta catenin in both renal tubules and glomeruli (CKD group, 200×), (D) moderate expression of β-catenin in SPL group (200×), (E) mild expression of β-catenin in ZnO-NPs group (200×), and (F) mild expression in SPL + ZnO-NPs group (200×). a: significant difference versus control, b: significant versus CKD, c: significant versus SPL, d: significant versus ZnO-NPs at p < 0.05.

Figure 5. Immunohistopathological examination for TGF-β1 expression in kidney tissues. (A) The score of TGF-β1 in all studied groups, (B) negative expression for TGF-β1 (control group, 400×), (C) marked cytoplasmic expression for TGF-β1 in both renal tubules and glomeruli (CKD group, 200×), (D) moderate expression of TGF-β1 in SPL group (200×), (E) mild expression of TGF-β1 in ZnO-NPs group (200×), and (F) mild expression in SPL + ZnO-NPs group (200×). a: significant difference versus control, b: significant versus CKD, c: significant versus SPL, d: significant versus ZnO-NPs at p < 0.05.

Figure 6. Immunohistopathological examination for α-SMA expression in kidney tissues. (A) The score of α-SMA in all studied groups, (B) negative expression for α-SMA (control group, 400×), (C) marked cytoplasmic expression for α-SMA in both renal tubules and glomeruli (CKD group, 200×), (D) moderate expression of α-SMA in SPL group (200×), (E) mild expression of α-SMA in ZnO-NPs group (200×), and (F) mild expression in SPL + ZnO-NPs group (200×). a: significant difference versus control, b: significant versus CKD, c: significant versus SPL, d: significant versus ZnO-NPs at p < 0.05.

Figure 7. Immunofluorescence investigation of collagen III in kidney tissues showing: (A) mild expression in control group (200×), (B) marked expression in CKD group (200×), (C) moderate expression in SPL group (200×), (D) mild expression in ZnO-NPs group (200×), and (E) mild expression in SPL + ZnO-NPs group (200×).

Figure 8. Immunofluorescence investigation of collagen IV in kidney tissues showing: (A) mild expression in control group (200×), (B) marked expression in CKD group (200×), (C) moderate expression in SPL group (200×), (D) mild expression in ZnO-NPs group (200×), and (E) mild expression in SPL + ZnO-NPs group (200×).

Data availability statement

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