應用基因體學於慢性腎臟病貓篩選次世代益生菌及其腎臟保護功效之探討

Abstract

慢性腎臟病 (Chronic Kidney Disease, CKD) 是人類和寵物中最常見的腎臟疾病之一。CKD在年齡較大的貓隻中更為普遍，15歲以上的老年貓隻盛行率可達80%，並且是高齡貓隻的主要死因之一。目前尚無治療可以完全恢復腎臟功能，而CKD的管理重點在於延緩病程發展與改善病患的生活品質。因此，發展新型輔助療法仍是不可或缺的。近年研究顯示CKD與腸道菌相失衡有關，使得腸-腎軸的研究成為新型疾病預防或治療策略的重要方向。透過益生菌和益生質等非藥物輔助療法調節腸道菌相，有望成為CKD治療的新途徑。隨著基因體定序技術的發展，我們可以更全面的了解疾病與腸道菌相之間的關係，精準找到對於疾病進展的關鍵物種，進而改善疾病的發展。因此，本研究目的為建立次世代益生菌的篩選方法，透過第三代定序技術從健康與慢性腎臟病貓隻中篩選出候選菌株，並藉由絕對定量技術即時聚合酶鏈鎖反應進行驗證，進一步在慢性腎臟病動物模型中評估潛力益生菌改善慢性腎臟病的能力，以及其潛在作用機制。 菌相分析的結果顯示，慢性腎臟病貓隻的菌相組成嚴重失衡，不論是豐富度、均勻度和總物種數均顯著低於健康貓隻。此外，慢性腎臟病貓隻菌相中發現多種丁酸生成菌的相對豐度顯著低於健康貓隻，而丁酸的生成對於動物的腸道完整性與腸道環境穩定具有重要的調節作用，推測丁酸生成菌的減少對於疾病的發展具有重要意義。因此挑選其中Faecalibacterium prausnitzii，又稱為普拉梭菌作為候選菌株並透過即時定量聚合酶鏈鎖反應證實其含量在健康貓隻顯著高於慢性腎臟病貓隻，並以腺嘌呤誘導小鼠慢性腎臟病動物模型，探討其作為次世代益生菌的潛力。研究結果顯示，F. prausnitzii可恢復慢性腎臟病引起的菌相失調，提升丁酸生成菌的含量，增加腸道中丁酸的含量，緩解慢性腎臟病引起的腸滲漏，進而改善小鼠的飲食與體重變化。此外，F. prausnitzii雖然無法有效改善腺嘌呤誘導的慢性腎臟病損傷，但是可以降低腎損傷指標的濃度、纖維化前驅因子、發炎相關因子和氧化壓力指標的表現，顯示其具有緩解疾病的能力。同樣重要的是，給予小鼠F. prausnitzii不會對其健康造成任何負面影響，更突顯其作為益生菌的潛力。總結來說，透過基因體分析技術篩選出的F. prausnitzii可以改善腸道菌相失衡進而改善慢性腎臟病，提供以調節腸道菌相為基礎的治療策略一個新的研究方向，並促進慢性腎臟病新型輔助療法的發展。

Chronic Kidney Disease (CKD) is one of the most prevalent kidney diseases in human and pets. CKD is particularly common in older feline, with a prevalence rate reaching up to 80% in feline over 15 years old, making it one of the leading causes of death in aging feline. Currently, no treatment can fully restore kidney function, so CKD management focuses on slowing disease progression and improving the quality of life for patients. Therefore, developing novel adjuvant therapies is crucial. Recent studies have shown that CKD is associated with gut microbiota dysbiosis, highlighting the importance of the gut-kidney axis as a potential direction for disease or treatment strategies. Regulating gut microbiota through non-pharmacological adjuvant therapies, such as probiotics and prebiotics, offers a promising approach for CKD treatment. With advenced genomic sequencing technology, we can better understand the relationship between diseases and gut microbiota, identify key species involved in disease progression, and thereby improve disease outcomes. This study aims to establish a screening method for next-generation probiotics. Using third-generation sequencing technology, we identified candidate strains from healthy and CKD feline and validated them using absolute quantification techniques, real-time quantitative polymerame chain reaction (PCR). Furthermore, we evaluated the potential of the probiotics to improve CKD in animal model and investigated the underlying mechanisms. Microbiota analysis revealed that the gut microbiota composition in CKD feline is severely imbalanced, with significantly lower richness, evenness, and total observed species count compared to healthy feline. Additionally, the relative abundance of several butyrate-producing bacteria was significantly lower in CKD feline, and butyrate plays a crucial role in regulating intestinal integrity and stability. This suggests that the reduction of butyrate-producing bacteria is significant for disease progression. We selected Faecalibacterium prausnitzii as a candidate strain and confirmed through absolute quantification that its abundance is significantly higher in healthy feline than in CKD feline. Using an adenine-induced CKD mouse model, we investigated its potential as a next-generation probiotic. The results showed that F. prausnitzii could restore the gut microbiota imbalance caused by CKD, increase the abundance of butyrate-producing bacteria, elevate butyrate levels in the gut, and improve intestinal permeability associated with CKD, thereby enhancing the mice's diet, water intake, and body weight. Although F. prausnitzii could not effectively improve adenine-induced kidney damage, it reduced the concentrations of renal injury markers, fibrosis precursors, inflammation-related factors, and oxidative stress indicators, demonstrating its potential to mitigate the disease. Importantly, administering F. prausnitzii to mice did not cause any adverse health effects, further highlighting its potential as a probiotic. In summary, F. prausnitzii, identified through genomic analysis, can improve gut microbiota imbalance and CKD, providing a direction for developing microbiota-regulating therapeutic strategies and promoting the development of new adjuvant therapies for CKD.