鋅原卟啉在慢性腎臟病進程中之角色： 對鐵離子代謝及細胞死亡影響及機制之探討

Abstract

全球有超過十分之一的人口罹患慢性腎臟病，慢性腎臟病不僅是心血管疾病死亡的重要原因，也是導致末期腎衰竭的關鍵因素。儘管其盛行率持續上升，目前仍缺乏有效的治療策略以阻止疾病持續惡化。尿毒素的累積為慢性腎臟病的核心病理特徵，然而其對腎臟鐵代謝及細胞死亡機制的影響，至今仍未被充分闡明。本研究利用腎小管上皮細胞株NRK-52E 與 HK-2及腺嘌呤誘發之慢性腎臟病小鼠模型並配合人類基因資料庫分析及腎臟檢體染色，系統性的探討尿毒素如何破壞腎臟鐵離子平衡並促進腎臟損傷。在體外實驗中，硫酸吲哚酚誘發典型的鐵死亡特徵，包括鐵離子累積、活性氧大量生成及脂質過氧化反應，同時鐵離子的累積會伴隨著內質網壓力、細胞老化及促纖維化以共同加劇慢性腎臟病的腎損傷。值得注意的是，這些不利影響可分別透過在體外實驗使用鐵螯合劑 deferoxamine，或於體內以 AST-120 吸附尿毒素前驅物以降低腎臟病小鼠中尿毒素的含量而顯著改善，證實鐵離子是慢性腎臟病致病原因中的關鍵。我們接著進行進一步的機制探討，尿毒素所造成的鐵離子代謝干擾及由於抑制鐵自噬作用所導致的鐵利用障礙，會破壞慢性腎臟病腎臟中的血紅素合成，導致血紅素含量下降，並促使鋅原卟啉異常的累積在腎臟組織中。此外，本研究證實在腎小管上皮細胞中鋅原卟啉的累積會活化第一型血紅素加氧酶、擾亂細胞內鐵恆定，最終造成游離鐵大量沉積。與此結果一致，慢性腎臟病腎臟亦呈現血紅素加氧酶表現上升、鐵死亡活化，以及鐵代謝失衡。而上述病理變化可藉由 TPEN 進行鋅螯合而有效逆轉，不僅抑制鋅原卟啉形成、恢復鐵代謝平衡，亦可降低鐵死亡反應，並減輕腎臟纖維化與腎毒性。此外，我們的研究還發現過量的游離鐵除了會透過誘發內質網壓力、細胞老化及纖維化來加劇腎臟損傷，它甚至還會透過泛細胞凋亡，一種新被發現的整合型程序發炎性細胞死亡途徑，包含焦亡、凋亡與壞死性凋亡，共同促進腎臟損傷。綜合上述，本研究發現一條先前未被釐清的慢性腎臟病致病機制，顯示尿毒素可藉由造成鐵離子代謝及利用失調而干擾腎臟中血紅素的合成，導致鋅原卟啉累積、血紅素加氧酶活化，誘發鐵死亡與發炎性細胞死亡，最終加速慢性腎臟病的進展，並且在人類基因資料庫分析以及腎臟組織染色結果也顯示出鐵死亡與泛細胞凋亡為不同因素的慢性腎臟病患者的重要臨床特徵。 本研究結果指出，腎臟鐵恆定為慢性腎臟病治療中一個關鍵且具潛力的策略。包括鐵螯合、AST-120 尿毒素吸附劑，以及以清除鋅原卟啉為目標或鋅螯合之治療策略，皆有潛力作為未來醫學上延緩慢性腎臟病惡化與疾病進程的重要治療方向。

Over 10% of the global population is affected by chronic kidney disease, a major contributor to cardiovascular mortality and end-stage renal failure. Despite its growing burden, effective therapeutic strategies to halt chronic kidney disease progression remain limited. Accumulation of uremic toxins is a central pathological feature of chronic kidney disease; however, their impact on renal iron metabolism and regulated cell death has not been fully elucidated. In this study, we employed renal tubular epithelial cells (NRK-52E and HK-2) and an adenine-induced chronic kidney disease mouse model, combined with human genome database analysis and kidney specimen staining, to investigate how uremic toxins disrupt renal iron homeostasis and promote kidney injury. In vitro, indoxyl sulfate triggered canonical ferroptotic features, including increased iron influx, excessive reactive oxygen species generation, and lipid peroxidation. These changes were accompanied by endoplasmic reticulum stress, cellular senescence, and profibrotic phenotypic transitions. Importantly, these deleterious effects were markedly alleviated by iron chelation with deferoxamine in vitro and by AST-120-mediated adsorption of indoxyl sulfate precursors in vivo. Further mechanistic analyses revealed that uremic toxin-induced disturbances in iron utilization progressively impair renal heme biosynthesis and lead to both reduced heme production and the accumulation of zinc protoporphyrin in chronic kidney disease kidneys. Furthermore, we demonstrate that abnormal zinc protoporphyrin accumulation in tubular epithelial cells triggers heme oxygenase-1, which upsets intracellular iron homeostasis and ultimately leads to excessive labile iron deposition. Consistent elevation of HO-1 expression, strong ferroptotic activation, and extensive iron dysregulation are features of chronic kidney disease that are consistent with our mechanistic discoveries. These abnormalities were effectively reversed by zinc chelation using TPEN, which limited zinc protoporphyrin formation, normalized iron handling, suppressed ferroptosis, and attenuated renal fibrosis and nephrotoxicity. Our research also found that in addition to exacerbating kidney damage by inducing endoplasmic reticulum stress, cellular senescence, and fibrosis, excessive labile iron can even promote kidney damage through PANoptosis, a newly discovered integrated programmed inflammatory cell death pathway that includes pyroptosis, apoptosis, and necroptosis. In conclusion, this study uncovers a previously unrecognized pathogenic mechanism in chronic kidney disease, demonstrating that uremic toxins disrupt renal heme synthesis by dysregulating iron metabolism and utilization. This interference results in zinc protoporphyrin accumulation, heme oxygenase-1 hyperactivation, induction of ferroptosis and inflammatory cell death, and consequent acceleration of disease progression. Furthermore, analyses of human genomic databases combined with immunohistochemical staining of kidney tissues revealed that ferroptosis and PANoptosis represent critical clinical features in chronic kidney disease patients, highlighting their unique contributions to renal injury. These results identify renal iron homeostasis as a critical therapeutic target in chronic kidney disease. Interventions such as iron chelation, uremic toxin adsorption with AST-120, and zinc protoporphyrin -targeted or zinc-chelating strategies may represent promising approaches to mitigate chronic kidney disease progression and delay clinical disease advancement.