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BMC Nephrol. 2013;14:105.

Bilirubin attenuates the renal tubular injury by inhibition of oxidative stress and apoptosis.

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제1저자 오세원 · 교신저자 진호준

PMID 23683031 PMC3681641 DOI 10.1186/1471-2369-14-105 IF 2.9 신장내과
소속 (1)
  1. Department of Internal Medicine, Eulji General Hospital, Eulji University College of Medicine, Seoul, Korea.

Abstract

BACKGROUND Bilirubin (BIL) has been recognized as an endogenous antioxidant that shows a protective effect for cardiorenal diseases. We investigated whether administration of BIL had a protective effect on cyclosporine (CsA)-induced nephropathy (CIN), and examined the effects of BIL on the oxidative stress and apoptosis.

METHODS BIL was pretreated intraperitoneally three times for a week (60 mg/kg), and CsA was injected for 4 weeks (15 mg/kg/day, subcutaneous). Proximal tubular epithelial (HK2) cells were pretreated with 0.1mg/ml of BIL for 24 hours, and then treated with 20 μM of CsA for another 24 hours.

RESULTS CsA induced marked increases in urine kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL) concentrations (P < 0.05). BIL reduced urine Kim-1 in CIN (P < 0.05), while urine NGAL exhibited a decreasing tendency. In CsA-treated rat kidneys, the protein expression of NOX4 and p22phox was reduced by BIL (P < 0.05). BIL ameliorated CsA-induced arteriolopathy, tubulointerstitial fibrosis, tubular injury, and the apoptosis examined by TUNEL assay (P < 0.01). In HK2 cells, BIL reduced intracellular reactive oxygen species in CsA-treated cells. CsA increased the protein expression of bax, cleaved caspase-9, caspase-3 and the activity of caspase-3; however, the anti-apoptotic bcl-2 protein was reduced. These changes were recovered by BIL (P < 0.05).

CONCLUSIONS The direct administration of BIL protected against CsA-induced tubular injury via inhibition of oxidative stress and apoptosis.

Figures

Figure 1
Figure 1 Intraperitoneal injection of bilirubin (BIL) significantly increase the plasma BIL. *P < 0.05 vs. 0 hours.
Figure 2
Figure 2 Urine concentrations of kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (NGAL).A: The urine concentration of Kim-1 is markedly increased in CsA-only treated rats, and is significantly reduced by BIL administration. B: The urine concentration of NGAL is markedly increased in CsA-only treated rats, and urine NGAL shows a decreasing tendency in BIL-treated rats. The data are the means ± SD. *P < 0.05 vs. VH, †P < 0.05 vs. BIL, and ‡P < 0.05 vs. CsA.
Figure 3
Figure 3 Afferent arteriolopathy in CsA-treated rat kidneys.A: Vehicle (VH), B: Bilirubin (BIL) C: CsA, and D: BIL + CsA groups E: Semiquantitative scoring of arteriolopathy according to mean percentage of afferent arterioles injured, which was estimated in at least 100 preglomerular afferent arterioles from the A, B, C and D groups (PAS stain, ×200). Compared with CsA group, BIL + CsA group displayed afferent arteriolopathy. The data are the means ± SD. *P < 0.05 vs. VH, †P < 0.05 vs. BIL, and ‡P < 0.05 vs. CsA.
Figure 4
Figure 4 Tubulointerstitial fibrosis in CsA-treated rat kidneys.A: Vehicle (VH), B: Bilirubin (BIL) C: CsA, and D: BIL + CsA groups (MT staining, magnification × 200). Semiquantitative scoring of E: tubulointerstitial fibrosis and F: tubular injury according to mean percentage of injured area obtained in at least 20 fields from the A, B, C and D groups. Compared with CsA group, BIL + CsA group showed improved tubulointerstitial fibrosis. The data are the means ± SD. *P < 0.05 vs. VH, †P < 0.05 vs. BIL, and ‡P < 0.05 vs. CsA.
Figure 5
Figure 5 Bilirubin (BIL) suppresses CsA-induced increased expression of Nox4 and p22phoxin rat kidneys.A: Western blot analysis of Nox4 and p22phox molecules from VH, BIL, CsA, and BIL + CsA groups. B: The average densitometric ratio was calculated relative to β-actin for C: The average densitometric ratio was calculated relative to β-actin for p22phox. The data are the means ± SD. *P < 0.05 vs. VH, †P < 0.05 vs. BIL, and ‡P < 0.05 vs. CsA.
Figure 6
Figure 6 Bilirubin (BIL) ameliorates intracellular reactive oxygen species (ROS) in CsA-induced nephropathy (CIN). 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) was used to determine the intracellular production of ROS. A: Vehicle (VH), B: CsA, and C: BIL + CsA groups (magnification × 400). D: The number of cells with intracellular ROS obtained in at least 10 fields from the A, B and C groups. The data are the means ± SD. *P < 0.05 vs. control, †P < 0.05 vs. CsA.
Figure 7
Figure 7 Bilirubin (BIL) ameliorates apoptosis in CsA-induced nephropathy (CIN). Representative TUNEL staining of the renal cortex in CIN. A: Vehicle (VH), B: Bilirubin (BIL) C: CsA, and D: BIL + CsA groups (magnification × 400). E: The percentage of apoptotic cells (TUNEL-positive cells) was obtained in at least 10 fields from the A,B and C groups. The data are the means ± SD. *P < 0.05 vs. VH, †P < 0.05 vs. BIL, and ‡P < 0.05 vs. CsA.
Figure 8
Figure 8 Bilirubin (BIL) ameliorates apoptosis in CsA-treated HK-2 cells.A: Western blot analysis of bcl-2 and bax molecules from VH, CsA, and BIL + CsA groups in HK2 cells. B: The average densitometric ratio was calculated relative to β-actin for bcl-2. C: The average densitometric ratio was calculated relative to β-actin for bax. The data are the means ± SD. *P < 0.05 vs. control, †P < 0.05 vs. CsA.
Figure 9
Figure 9 Bilirubin (BIL) suppresses CsA-induced apoptosis in HK-2 cells.A: The protein expression of cleaved caspase-9 and cleaved caspse-3 from VH, CsA, BIL, and BIL+CsA groups in HK2 cells. B: The average densitometric ratio was calculated relative to β-actin for cleaved caspase-9. C: The protein expression of cleaved caspse-3 from VH, CsA, BIL, and BIL+CsA groups in HK2 cells. D: The average densitometric ratio was calculated relative to β-actin for cleaved caspase-3. E: Caspase-3 activity was determined by caspase-3 fluorometric assay in HK2 cells. The data are the means ± SD. *P<0.05 vs. control, †P<0.05 vs. CsA.

그림 출처: PubMed Central 공개 논문 (PMC3681641)

그 밖의 정보
출판 형태
Journal Article, Research Support, Non-U.S. Gov't
저널
BMC nephrology · eISSN 1471-2369
전자 출판
2013-05-17
MeSH
Acute Kidney Injury / metabolism / pathology / prevention & control · Animals · Apoptosis / drug effects / physiology · Bilirubin / administration & dosage · Cell Line · Humans · Injections, Intraperitoneal · Kidney Tubules / drug effects / metabolism / pathology · Male · Oxidative Stress / drug effects / physiology · Rats · Rats, Sprague-Dawley
Substances
Bilirubin
참고문헌
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