당뇨병성 신장병증 모델 쥐에서 Granulocyte-Colony Stimulating Factor의 치료 효과

Abstract

1.20 ± 0.00 vs. 2.39 ± 0.00, P < 0.05; 0.42 ± 0.030 vs. 1.25 ± 0.07, P < 0.05). 고찰: 본 연구에서는 G-CSF가 당뇨병성 신장병증 초기 동물 모델에서 사구체 손상을 완화시킨다는 사실을 확인하였다. G-CSF는 골수 줄기세포의 가동화, 항염증 및 항섬유화 작용으로 당뇨병성 신장병증의 초기 사구체 손상을 완화시키고, 단백뇨 감소에 기여했을 것으로 추정된다. 현재 당뇨병성 신장병증에 관한 만족스러운 약제가 없는 실정에서 새로운 대안으로 제시 될 수 있을 것으로 예상된다.|Background: Diabetic nephropathy is a serious complication of diabetes and a major cause of end stage renal disease. However, no effective treatment for diabetic nephropathy has been developed. Recently, granulocyte-colony stimulating factor (G-CSF), a member of the hematopoietic growth factor family has been shown to exert beneficial effects on various tissues. However, the beneficial effect of G-CSF on diabetic nephropathy is still unknown. In this study, we investigated the effects of G-CSF on diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rat. Method: OLETF and Long-Evans Tokushima Otsuka (LETO) rats were used for the type 2 diabetic animals and non-diabetic normal controls, respectively. Diabetic nephropathy-induced OLETF rats were injected intraperitoneally with G-CSF (n=4) or saline (n=4) (200 ug/kg/day) for 5 days. The blood glucose level at 4-week intervals from 16 weeks, and the urine albumin/creatinine ratio (UACR) at 4-week intervals from 28 weeks in diabetes-induced OLETF rats were measured. At 43 weeks, the kidney/body weight ratio was measured. The areas of glomerular and glomerular basement membrane (GBM) thickness were evaluated using light and electron microscopy, respectively. Quantitative reverse transcription PCR (qRT-PCR) analyses were performed for transforming growth factor-β1 (TGF-β1), collagen IV and interleukin-1β (IL-1β). Result: The blood glucose in the G-CSF treated group was not significantly different from that in the saline-treated group (P > 0.05). The UACR in the G-CSF treated group was reduced by 41.9% compared with that in the saline-treated group (489.20 ± 176.45 mg/g vs. 841.38 ± 249.04 mg/g, P < 0.05). However, CrCl was not significantly different between the two groups (P > 0.05). The kidney/body weight ratio in the G-CSF treated group was reduced by 16.3% compared with that in the saline-treated group (0.36 ± 0.03% vs. 0.43 ± 0.04%, P < 0.05). In the G-CSF treated group, the mesangial matrix expansion was reduced by 52.6% compared with that in the saline-treated group (23.30 ± 7.17% vs. 49.17 ± 17.64%, P < 0.05). The GBM thickness in the G-CSF treated group was reduced by 24.8% compared with that in the saline-treated group (299.25 ± 43.32 nm vs. 397.81 ± 86.85 nm, P < 0.05). Besides TGF-β1, collagen IV and IL-1β mRNA expression in the G-CSF treated group were reduced by 44.5%, 49.8% and 67.1%, respectively, compared with that in the saline-treated group (3.50 ± 0.96 vs. 6.31 ± 1.58, P < 0.05; 0.42 ± 0.030 vs. 1.25 ± 0.07, P < 0.05). Conclusion: We showed that G-CSF could ameliorate early diabetic nephropathy in OLEFT rats. There results may be suggestive of the potential use of G-CSF as a novel therapeutic drug in diabetic nephropathy.; Background: Diabetic nephropathy is a serious complication of diabetes and a major cause of end stage renal disease. However, no effective treatment for diabetic nephropathy has been developed. Recently, granulocyte-colony stimulating factor (G-CSF), a member of the hematopoietic growth factor family has been shown to exert beneficial effects on various tissues. However, the beneficial effect of G-CSF on diabetic nephropathy is still unknown. In this study, we investigated the effects of G-CSF on diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rat. Method: OLETF and Long-Evans Tokushima Otsuka (LETO) rats were used for the type 2 diabetic animals and non-diabetic normal controls, respectively. Diabetic nephropathy-induced OLETF rats were injected intraperitoneally with G-CSF (n=4) or saline (n=4) (200 ug/kg/day) for 5 days. The blood glucose level at 4-week intervals from 16 weeks, and the urine albumin/creatinine ratio (UACR) at 4-week intervals from 28 weeks in diabetes-induced OLETF rats were measured. At 43 weeks, the kidney/body weight ratio was measured. The areas of glomerular and glomerular basement membrane (GBM) thickness were evaluated using light and electron microscopy, respectively. Quantitative reverse transcription PCR (qRT-PCR) analyses were performed for transforming growth factor-β1 (TGF-β1), collagen IV and interleukin-1β (IL-1β). Result: The blood glucose in the G-CSF treated group was not significantly different from that in the saline-treated group (P > 0.05). The UACR in the G-CSF treated group was reduced by 41.9% compared with that in the saline-treated group (489.20 ± 176.45 mg/g vs. 841.38 ± 249.04 mg/g, P < 0.05). However, CrCl was not significantly different between the two groups (P > 0.05). The kidney/body weight ratio in the G-CSF treated group was reduced by 16.3% compared with that in the saline-treated group (0.36 ± 0.03% vs. 0.43 ± 0.04%, P < 0.05). In the G-CSF treated group, the mesangial matrix expansion was reduced by 52.6% compared with that in the saline-treated group (23.30 ± 7.17% vs. 49.17 ± 17.64%, P < 0.05). The GBM thickness in the G-CSF treated group was reduced by 24.8% compared with that in the saline-treated group (299.25 ± 43.32 nm vs. 397.81 ± 86.85 nm, P < 0.05). Besides TGF-β1, collagen IV and IL-1β mRNA expression in the G-CSF treated group were reduced by 44.5%, 49.8% and 67.1%, respectively, compared with that in the saline-treated group (3.50 ± 0.96 vs. 6.31 ± 1.58, P < 0.05; 배경: 당뇨병성 신장병증(diabetic nephropathy, DN)은 망막병증(diabetic retinopathy), 신경병증(diabetic neuropathy)과 더불어 당뇨병의 3대 합병증 중 하나이며, 말기 신부전증의 가장 흔한 원인 질환이다. 당뇨병성 신장병증 유발 시 사구체 메산지움의 기질의 증가와 사구체여과율이 감소되며 단백뇨가 나타난다. 최근 본 연구실에서는 당뇨 합병증 동물모델에서 granulocyte-colony stimulating factor (G-CSF)의 효과를 연구하였다. 그 결과 G-CSF에 의해 간 조직의 손상완화 및 신경세포의 재생효과가 있는 것을 확인하였고, 신장병증에서 G-CSF 효과를 본 연구는 없어 이번 실험을 계획하였다. 본 연구의 목적은 G-CSF가 당뇨병성 신장병증 동물 모델에서 손상된 사구체 내 세포 손상을 완화시키는지 확인하는 것이다. 방법: 실험동물은 제 2형 당뇨병이 유발되는 otsuka long evans tokushima fatty (OLETF) 쥐와 정상 대조군인 long-evans tokushima otsuka (LETO) 쥐를 이용하였다. OLETF 쥐를 무작위로 두 군으로 나누어 G-CSF (n=4) 혹은 saline (n=4)을 200 ug/kg/day 용량으로 매일 5일간 실험동물의 복강 내에 투여하였다. 4주 후에 모든 동물을 희생시켜 G-CSF의 당뇨병성 신장병증 초기 동물 모델에서 손상된 사구체 치료 효과를 확인하였다. 당뇨병성 신장병증의 발생 및 호전 여부를 생리학적 검사 및 조직학적인 검사로 확인하였고, 소변검사를 통해 urine albumin to creatinine ratio (UACR)와 creatinine clearance (CrCl)를 확인하였다. 추가적으로 섬유화를 유발하는 cytokine인 transforming growth factor-β1 (TGF-β1)와 사구체의 메산지움, 족세포, 기저막을 구성하고 있는 collagen IV, 염증 반응을 일으키는 interleukin-1β (IL-1β)의 유전자 발현을 quantitative reverse transcription PCR (qRT-PCR) 검사로 확인하였다. 결과: 생리적 검사 결과, 혈당은 G-CSF 투여군과 saline 투여군 간 통계적으로 유의한 차이는 없었다 (P > 0.05). G-CSF 투여군의 UACR은 saline 투여군에 비해 41.9% 유의하게 감소하였고 (489.20 ± 176.45 mg/g vs. 841.38 ± 249.04 mg/g, P < 0.05), 사구체여과율을 측정하는 CrCl에서는 G-CSF 투여군과 saline 투여군 간 유의한 차이는 없었다 (P > 0.05). G-CSF 투여 군의 신장 무게(체중당 백분율)도 16.3% 유의하게 감소하였다 (0.36 ± 0.03% vs. 0.43 ± 0.04%, P < 0.05). 조직학적 검사 결과, glomerular area에서 G-CSF 투여 군의 mesangial matrix expansion은 saline 투여 군에 비해 52.6% 유의하게 감소 하였고 (23.30 ± 7.17% vs. 49.17 ± 17.64%, P < 0.05), G-CSF 투여군의 사구체 기저막 두께는 saline 투여군에 비해 24.8% 유의하게 감소하였다 (299.25 ± 43.32 nm vs. 397.81 ± 86.85 nm, P < 0.05). qRT-PCR 분석 결과에서도 G-CSF 투여군의 TGF-β1, collagen-IV 및 IL-1β 발현량은 saline 투여군에 비해 각각 44.5%, 49.8% 및 67.1%으로 유의하게 감소하였다 (3.50 ± 0.96 vs. 6.31 ± 1.58, P < 0.05