The Potential Protective Effect and Underlying Mechanisms of Physiological Unconjugated Hyperbilirubinemia Mediated by UGT1A1 Antisense Oligonucleotide Therapy in a Mouse Model of Cyclosporine A-Induced Chronic Kidney Disease

Abstract

Cyclosporine A (CSA) is an immunosuppressive drug that has improved transplant survival rates. However, its use is often limited because it is thought to be linked to the development of chronic kidney disease after kidney transplants. This study aimed to investigate the protective effects and underlying mechanisms of physiological unconjugated (UC) hyperbilirubinemia mediated by UGT1A1 antisense oligonucleotide in a mouse model of CsA-induced chronic kidney disease, and match these with that of chitosan (CH) as a natural chelator against kidney injury. In the current study, CsA-treated mice were given an intravenous injection of UGT1A1 antisense morpholino oligonucleotide (16 µg/kg) every third day for 14 days. In serum samples, bilirubin, creatinine, and urea were determined. Markers of oxidative stress, antioxidant activities, and mRNA expression of target genes PPAR-α, cFn, eNOS, NF-B, AT1-R, ETA-R, Kim-1, and NGAL were measured in the kidney tissues. Moreover, histopathological examinations were carried out on the kidney tissue. Physiological UC hyperbilirubinemia could be a promising protective strategy against CsA-induced kidney disease in transplant recipients. UGT1A1 antisense oligonucleotide-induced physiological UC hyperbilirubinemia serum significantly protected against CsA-induced kidney dysfunction. UCB acts as a signaling molecule that protects against kidney disease through different mechanisms, including antioxidant, anti-inflammatory, and hormonal action, by activating nuclear hormone receptors (PPAR-α). Moreover, it significantly downregulated mRNA expression of NF-kB, ETA-R, iNOS, AT1-R, cFn, Kim-1, and NGAL in the kidney tissue and alleviated CsA-induced kidney histological changes in CsA-treated mice.

Keywords: UGT1A1 antisense; chronic kidney disease; cyclosporine A; mild unconjugated hyperbilirubinemia; oxidative stress.

Figure 1

UGT1A1 antisense oligonucleotide mediates physiological unconjugated hyperbilirubinemia in CsA-treated mice: (A) total bilirubin (TB); (B) direct bilirubin (conjugated bilirubin; CB); and (C) indirect bilirubin (unconjugated bilirubin; UCB). Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05) (ANOVA; Duncan’s post hoc analysis).

Figure 2

Physiological unconjugated hyperbilirubinemia protects against CsA-induced kidney dysfunction: (A) creatinine and (B) urea (U). Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05) (ANOVA; Duncan’s post hoc analysis).

Figure 3

Physiological unconjugated hyperbilirubinemia inhibits CsA-induced kidney oxidative stress: (A) malondialdehyde (MDA); (B) nitric oxide (NO); (C) reduced glutathione (GSH); (D) glutathione S-transferase (GST); (E) catalase (CAT); (F) glutathione peroxidase (GPx), and (G) superoxide dismutase (SOD). Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05) (ANOVA; Duncan’s post hoc analysis).

Figure 4

Unconjugated bilirubin, a signaling molecule, activates PPAR-α and regulates NF-kB, ETA-R, iNOS, AT1-R, cFn, Kim-1, and NGAL gene expression in CsA-treated mice: (A) peroxisome proliferator-activated receptor alpha (PPAR-α); (B) nuclear factor kappa B (NF-kB); (C) endothelin type A-receptor (ETA-R); (D) inducible nitric oxide synthase (iNOS); (E) angiotensin type1-receptor (AT1-R); (F) cellular fibronectin (cFN); (G) kidney injury molecule-1 (Kim-1); and (H) neutrophil gelatinase-associated lipocalin (NGAL). Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05). (ANOVA; Duncan’s post hoc analysis).

Figure 5

(A) Photomicrograph of kidney sections in different experimental groups (H&E; X: 400). Normal histological structure of cortical glomeruli (G) and tubules (T) beside normal medullary tubules (MT) in kidney sections of the control group (A1,A2), CH-treated group (B1,B2), and UGT1A1 antisense oligonucleotide-treated group (C1,C2). Glomerular hypertrophy dilated Bowman’s space (curved arrow) with mesangial proliferation (dashed arrows), tubular degeneration in the cortex (arrowheads), tubular degeneration (arrowheads), and necrosis (black arrows) in the medulla in kidney sections of the CsA-treated group (D1,D2). Narrowed Bowman’s space (curved arrow), mild to moderate tubular degeneration in the cortex (arrowheads), and tubular degeneration in the medulla (arrowheads) in kidney sections of the CsA + CH treated group (E1,E2). Very mild tubular dilation (black arrows) and degeneration (arrowheads) in cortex and medulla in kidney sections of the CsA + UGT1A1 antisense oligonucleotide-treated group (F1,F2). (B) Semiquantitative analysis of renal tissue lesions based on tubular damage score in different experimental groups. Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05). (ANOVA; Duncan’s post hoc analysis).

Figure 5

(A) Photomicrograph of kidney sections in different experimental groups (H&E; X: 400). Normal histological structure of cortical glomeruli (G) and tubules (T) beside normal medullary tubules (MT) in kidney sections of the control group (A1,A2), CH-treated group (B1,B2), and UGT1A1 antisense oligonucleotide-treated group (C1,C2). Glomerular hypertrophy dilated Bowman’s space (curved arrow) with mesangial proliferation (dashed arrows), tubular degeneration in the cortex (arrowheads), tubular degeneration (arrowheads), and necrosis (black arrows) in the medulla in kidney sections of the CsA-treated group (D1,D2). Narrowed Bowman’s space (curved arrow), mild to moderate tubular degeneration in the cortex (arrowheads), and tubular degeneration in the medulla (arrowheads) in kidney sections of the CsA + CH treated group (E1,E2). Very mild tubular dilation (black arrows) and degeneration (arrowheads) in cortex and medulla in kidney sections of the CsA + UGT1A1 antisense oligonucleotide-treated group (F1,F2). (B) Semiquantitative analysis of renal tissue lesions based on tubular damage score in different experimental groups. Values are expressed as mean ± SEM (n = 8). Means of different letters are statistically different (p < 0.05). (ANOVA; Duncan’s post hoc analysis).