The regulatory role of miR-144-3p/Nrf2 axis in renal fibrosis

다국어 초록 (Multilingual Abstract)

Chronic kidney disease (CKD) is characterized by progressive fibrosis leading to irreversible loss of renal function. Among various regulatory mechanisms, microRNAs (miRNAs) have emerged as key post-transcriptional modulators of renal pathophysiology. This dissertation investigates the role of microRNA-144- 3p (miR-144-3p) as a negative regulator of the nuclear factor erythroid 2–related factor 2 (NRF2) antioxidant pathway in two representative models of kidney injury: the diabetic-hypertensive model and the unilateral ureteral obstruction (UUO) model. In the metabolic disease–associated renal injury model, miR-144-3p was markedly elevated in both kidney tissue and urine and was accompanied by repression of NRF2 and its antioxidant target genes, including heme oxygenase- 1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1), and glutamate–cysteine ligase catalytic subunit (GCLC). Mechanistically, TGF-β stimulation increased miR-144-3p expression via a SMAD-dependent pathway in tubular epithelial cells, and miR-144-3p directly bound the 3′-untranslated region of NRF2 mRNA to suppress NRF2 translation. Functional studies in NRK-52E cells demonstrated that miR-144-3p–mediated NRF2 inhibition increased intracellular reactive oxygen species (ROS) accumulation and promoted extracellular matrix (ECM) production. Pharmacological activation of NRF2 using cilostazol restored antioxidant responses and attenuated profibrotic gene expression in vitro and in vivo, supporting the pathological relevance of the TGF-β/miR-144-3p/NRF2 axis under metabolic stress. In the UUO model, miR-144-3p expression progressively increased during obstructive injury, whereas NRF2 signaling and its downstream antioxidant defenses were significantly suppressed. NRF2 activation with bardoxolone methyl ameliorated UUO-induced renal injury, improving biochemical indices and reducing tubular atrophy and interstitial collagen deposition, accompanied by restoration of NRF2 target genes and reduction of ECM markers. In contrast, renal lentiviral overexpression of miR-144-3p exacerbated tubular injury and fibrotic remodeling, establishing a causal role for miR-144-3p in the progression of obstruction-induced fibrosis. Moreover, exosomes released from TGF-β1–treated tubular epithelial cells were enriched in miR-144-3p. In complementary in vitro experiments, overexpression of miR-144-3p in renal fibroblasts was associated with increased fibrotic gene expression and suppression of NRF2 signaling, suggesting that miR-144-3p may influence fibroblast behavior under fibrotic conditions. Collectively, these findings identify miR-144-3p as a central molecular mediator that links TGF-β/SMAD signaling to impaired NRF2-dependent antioxidant defenses, thereby amplifying oxidative stress and fibrogenesis in both metabolic and obstructive nephropathy. The results further suggest that therapeutic restoration of NRF2 activity and/or inhibition of miR-144-3p may represent a rational strategy to attenuate renal fibrosis, while also highlighting a previously underappreciated role of exosome-mediated miR-144-3p transfer in intercellular—and potentially inter-organ—communication during chronic kidney injury. Keywords : Chronic kidney disease, Renal fibrosis, microRNA-144-3p, NRF2 antioxidant pathway, TGF-β signaling, Exosomes

국문 초록 (Abstract)

만성 신장질환(chronic kidney disease, CKD)은 진행성 신장 섬유화를 특징으로 하며, 궁극적으로 비가역적인 신기능 소실로 이어진다. 다양한 조절 기전 가운데, 마이크로RNA(microRNA, miRNA)는 신장 병태생리에서 중요한 전사 후 조절 인자로 주목받고 있다. 본 학위논문은 당뇨·고혈압성 대사성 신장질환 모델과 일측성 요관결찰(unilateral ureteral obstruction, UUO) 모델을 이용하여, microRNA-144-3p(miR-144-3p)가 항산화 전사인자인 nuclear factor erythroid 2–related factor 2(NRF2) 신호계를 억제하는 핵심 조절 인자로 작용하는지를 규명하고자 하였다.
대사성 신장 손상 모델에서 miR-144-3p는 신장 조직과 소변에서 유의하게 증가하였으며, 이는 NRF2 및 그 하위 항산화 표적 유전자들인 heme oxygenase-1(HO-1), NAD(P)H quinone dehydrogenase 1(NQO1), glutamate–cysteine ligase catalytic subunit(GCLC)의 발현 감소와 동반되었다. 기전적으로, TGF-β 자극은 세뇨관 상피세포에서 SMAD 의존적 경로를 통해 miR-144-3p 발현을 증가시켰으며, miR-144-3p는 NRF2 mRNA의 3′-비번역 부위에 직접 결합하여 NRF2의 번역을 억제하였다. NRK-52E 세포를 이용한 기능적 실험에서, miR-144-3p에 의한 NRF2 억제는 세포 내 활성산소종(reactive oxygen species, ROS) 축적을 증가시키고 세포외기질(extracellular matrix, ECM) 생성을 촉진하였다. 또한 NRF2 활성제인 cilostazol 처리는 시험관 내 및 생체 내에서 항산화 반응을 회복시키고 섬유화 관련 유전자 발현을 감소시켜, 대사성 스트레스 환경에서 TGF-β/miR-144-3p/NRF2 축의 병리학적 중요성을 입증하였다.
UUO 모델에서도 miR-144-3p 발현은 폐쇄성 손상 진행에 따라 점진적으로 증가한 반면, NRF2 신호 및 하위 항산화 방어 기전은 현저히 억제되었다. NRF2 활성제인 bardoxolone methyl 투여는 UUO로 유도된 신장 손상을 완화하여 생화학적 지표를 개선하고 세뇨관 위축 및 간질 콜라겐 침착을 감소시켰으며, NRF2 표적 유전자 발현 회복과 ECM 표지자 감소를 동반하였다. 반대로, 신장 내 lentiviral miR-144-3p 과발현은 세뇨관 손상과 섬유화 재형성을 더욱 악화시켜, 폐쇄성 신장 섬유화 진행에서 miR-144-3p의 인과적 역할을 확립하였다. 주목할 점으로, miR-144-3p 과발현은 순환 엑소좀 내 miR-144-3p 증가 및 간 조직 내 miR-144-3p 발현 증가와 연관되었으며, 이는 간에서의 NRF2 신호 억제와 섬유화 표지자 증가를 동반하여, 전신적 확산 및 잠재적인 신장 외 장기 영향 가능성을 시사하였다. 더 나아가, TGF-β1 처리된 세뇨관 상피세포에서 분비된 엑소좀은 miR-144-3p가 풍부하게 함유되어 있었으며, 이러한 엑소좀은 섬유아세포 활성화를 촉진하였다. 이에 부합하게, 섬유아세포에서 miR-144-3p를 직접 과발현시켰을 때 NRF2 억제와 함께 섬유화 표현형이 유도되어, 상피세포–섬유아세포 간의 측분비(paracrine) 기전을 통한 섬유화 전파 가능성을 지지하였다.
종합하면, 본 연구는 miR-144-3p가 TGF-β/SMAD 신호와 NRF2 의존적 항산화 방어 기전 억제를 연결하는 핵심 분자 매개체로 작용함을 규명하였으며, 이를 통해 산화 스트레스와 섬유화 반응이 증폭됨을 제시하였다. 또한 NRF2 활성 회복 또는 miR-144-3p 억제가 신장 섬유화를 완화하기 위한 합리적인 치료 전략이 될 수 있음을 시사함과 동시에, 엑소좀 매개 miR-144-3p 전달이 만성 신장 손상 과정에서 세포 간 나아가 장기 간 신호 전달에 관여할 수 있는 새로운 기전임을 제시한다.

목차 (Table of Contents)

• LIST OF FIGURES ⅳ
• LIST OF TABLES ⅴ
• LIST OF ABBREVIATIONS ⅵ
• ABSTRACT ⅷ
• Ⅰ INTRODUCTION 1
• 1 Kidney disease 1
• 1.1 chronic kidney disease (CKD) 1
• 1.2 Acute kidney injury (AKI) 2
• 1.3 transition from AKI to CKD 3
• 2 Renal fibrosis 4
• 3 The NRF2 Signaling Pathway 5
• 3.1 Overview of NRF2-Keap1 System 5
• 3.2 NRF2 in the Regulation of Oxidative Stress and Fibrotic Signaling 6
• 4 Nrf2 and kidney disease 7
• 5 miRNA 9
• 5.1 Overview of miRNA biogenesis and function 9
• 5.2 miRNAs in kidney disease and Fibrosis 10
• 5.3 Clinical applications of miRNAs 11
• 6 miRNA-mediated intercellular communication in renal fibrosis 13
• 7 Purpose 14
• Ⅱ MATERIALS AND METHODS 16
• 1 Materials 16
• 2 Establishment of Animal experiments 16
• 3 Measurement of Fasting blood glucose levels 20
• 4 Measurement of blood pressure 20
• 5 Urinary cystatin C levels 20
• 6 Histopathological analysis 20
• 7 Cell culture 21
• 8 Western blotting 22
• 9 RNA isolation and real-time reverse transcription polymerase chain reaction (RT-PCR) 23
• 10 Transfection with the miRNA mimic, miRNA inhibitor, and small interfering RNA (siRNA) 24
• 11 Luciferase reporter assay for the nrf2 3′UTR, SMAD response element (SRE), and ARE 25
• 12 ROS levels 25
• 13 Lentiviral vector construction and production 26
• 14 Exosome isolation from cell culture supernatants 26
• 15 Exosome size measurement by dynamic light scattering 27
• 16 Statistical analysis 28
• Ⅲ RESULTS 29
• 1. Renal microRNA-144-3p is associated with transforming growth factor-β1-induced oxidative stress and fibrosis by suppressing the NRF2 pathway in hypertensive diabetic kidney disease 29
• Summary 30
• 1.1 MiR-144-3p levels increase in urine and kidney from hypertensive diabetic rats 31
• 1.2 MiR-144-3p represses the NRF2 pathway in renal tubular epithelial cells 35
• 1.3 MiR-144-3p directly binds to the 3’-UTR of NRF2 gene 39
• 1.4 TGF-β1-induced NRF2 suppression is mediated through miR-144-3p 41
• 1.5 TGF-β1-induced fibrotic change associates with miR-144-3p/NRF2 47
• 1.6 NRF2 activating cilostazol ameliorates kidney damage in diabetic hypertensive rats 52
• 2. The Role of miR-144-3p in Obstructive Kidney Injury 59
• Summary 60
• 2.1 Upregulation of miR-144-3p and Suppression of NRF2 Signaling in UUO Rats 62
• 2.2 Bardoxolone Activates NRF2 Signaling and Attenuates Renal Fibrosis in UUO Rats 66
• 2.3 Lentiviral Overexpression of miR-144-3p Exacerbates Renal Fibrosis in the UUO Model 71
• 2.4 Systemic Dissemination of miR-144-3p and Hepatic Fibrotic Effects 75
• 2.5 TGF-β1–Treated Epithelial Cells Release miR-144-3p-Enriched Exosomes 78
• 2.6 Overexpression of miR-144-3p in Fibroblasts Induces a Fibrotic Phenotype 81
• Ⅳ DISCUSSION 83
• Ⅴ REFERENCES 90
• Appendix 96
• 국문초록 101