結合多體學探討複合乳酸桿菌應用於預防及治療慢性腎臟病之研究

Abstract

慢性腎臟病 (Chronic kidney disease, CKD) 為腎臟功能不可逆的喪失而最終演變成疾病的過程，慢性腎臟病之高發生率及高盛行率使腎臟保健成為關注議題。近年來由於多體學分析技術的發展，得以進一步窺探疾病對於腸道菌相組成之影響。慢性腎臟病造成腸道菌相失衡 (gut dysbiosis) 的現象可由臨床試驗得到實證，菌相失衡的結果導致更多腸道衍生型尿毒素(gut-derived uremic toxin, GDUT) 生成，使腎臟功能持續惡化並加劇病程發展。此結果顯示，基於調整腸道菌相，進而影響其衍生代謝物之作法將可以做為減少尿毒素物質以及延緩慢性腎臟病病程發展的有效預防及治療策略。因此，本研究目的為開發具預防或延緩慢性腎臟病病程發展之益生菌，並藉由多體學分析整合慢性腎臟病動物模式及臨床試驗之成果，以進一步探究益生菌於慢性腎臟病宿主、腸道微生物及所衍生代謝物間之交互作用，以及影響慢性腎臟病病程發展之潛在作用機轉。

首先，本研究建立體外益生菌篩選平台，篩選出可降低尿毒素前趨物之潛力菌株，其中Lactiplantibacillus plantarum subsp. plantarum MFM 30−3 及 Lacticaseibacillus paracasei subsp. paracasei MFM 18之複合菌株具有最佳之清除能力，將其命名為複合乳酸桿菌 (Lactobacillus mix, Lm)，並以腺嘌呤誘發小鼠慢性腎臟病模式探討其於體內之生理功效與機制。研究結果顯示，Lm可改善慢性腎臟病所引發之腸道菌相失衡，提升腸道菌相多樣性，並回復健康個體腸道常駐菌相之豐富度。此常駐菌株大多為短鏈脂肪酸生成菌，此與Lm使腸道中丁酸含量上升以及增加醣類代謝途徑之研究成果相互呼應，短鏈脂肪酸為提供腸道上皮細胞能量的關鍵化合物，Lm可藉由調整腸道菌相組成及相關代謝物生合成進而改善腸道屏障功能。此外，Lm可降低腸道衍生型尿毒素前趨物，進而降低血液中尿毒素之濃度，透過減少毒素物質累積對腎臟組織的傷害，得以改善慢性腎臟病所引起之氧化壓力及發炎現象，降低腎臟損傷及纖維化相關蛋白質的表現，並維持腎臟組織中免疫作用的恆定，使腎臟維持較佳的功能性。研究結果顯示，Lm可作為延緩腎臟病程發展之有效預防策略，亦實證本研究開發之腎臟保健益生菌篩選平台之可行性及生理意義。

本研究更進一步與臺大醫院以及臺大動物醫院合作進行慢性腎臟病之臨床試驗，藉以評估Lm作為輔助治療策略對腎臟病指標以及降低尿毒素之效果。人體臨床試驗設計為前瞻性隨機分配開放標記盲性指標試驗 (prospective, randomized, open-labeled, blinded end-point trial, PROBE)，共招募第三期慢性腎臟病患共120位，持續給予Lm或活性竹碳三個月以評估其臨床應用功效。截至目前為止，僅49位病患完成為期三個月之試驗，其餘71位個案仍在進行試驗當中。由目前試驗結果可知，相較於對照組，慢性腎臟病患者補充三個月Lm可降低血液中腸道衍生型尿毒素之濃度，而肌酸酐 (creatinine, CRE) 及血中尿素氮 (blood urea nitrogen, BUN) 亦有降低之趨勢，且腎絲球過濾率 (estimated glomerular filtration rate, eGFR) 在補充益生菌期間每月平均提升0.17 ml/min/1.732，而對照組則每月平均下降0.67 ml/min/1.732。綜合上述，Lm可有效降低血液中氧化三甲胺 (trimethylamine-N-oxide, TMAO)、硫酸吲哚酚 (indoxyl sulfate, IS)、對硫甲酚 (p-cresyl sulfate, PCS) 及硫酸苯酯 (phenyl sulfate, PS)，推測Lm藉由改善菌相失衡而減少毒素累積，進而延緩腎臟功能惡化，以改善慢性腎臟病患腎絲球過濾率持續衰退之情況。

腎貓臨床試驗為開放標記單臂試驗設計之前導研究 (open-label single-arm pilot study)，招募第二至三期之慢性腎臟病貓共14隻，持續給予Lm八周以評估其延緩慢性腎臟病病程之功效，並結合多體學以分析腸道總菌相 (gut microbiome)及血液總代謝物組成 (serum metabolome)，以釐清Lm用以治療並延緩慢性腎臟病發展之可能機制。經過八周Lm的介入治療後，將近七成腎貓可降低或維持血中肌酸酐、尿素氮及腸道衍生型尿毒素之含量。此外，Lm顯著改善腸道菌相之多樣性，並改變腎貓特定細菌分類群、微生物功能及代謝物的比例及含量，此現象指出Lm調整腸道菌相、微生物代謝途徑及代謝物組成，而此三者之間具有密不可分的交互關係。研究中發現個別腎貓針對評估Lm延緩慢性腎臟病相關指標上存在著差異性反應，因此進一步將腎貓區分為對Lm具高反應性 (high responder, HR) 及中反應性 (moderate responder, MR)，期望可更精準釐清Lm之潛在作用機制。研究發現，高及中反應性腎貓之間對於特定微生物菌種、血清代謝物及生物代謝路徑的改變存在差異。此外，腸道中兩株益生菌株的含量更影響了Lm延緩慢性腎臟病之功效。於高反應性腎貓中可更明確指出，Lm調控腸道衍生型尿毒素及短鏈脂肪酸生合成途徑相關代謝物及微生物功能性。研究中亦發現，病程發展程度不同的腎貓具有特定的微生物菌種及代謝物標的，而其與病程發展因子亦具有不同程度之關聯性。本研究識別之特定微生物菌種及代謝物標的仍待結合臨床試驗加以驗證其生理意義，期望可確立其對於病程發展之影響，未來將有潛力作為慢性腎臟病預後及診斷之判斷依據，或開發次世代或精準益生菌用於延緩慢性腎臟病之發展。

綜觀上述，多體學分析證明Lm藉由調整腸道菌相組成及相關代謝物生成，而達到改善腸道菌相失衡及降低腸道衍生型尿毒素生合成之效，可做為慢性腎臟病有效的預防及輔助治療之益生菌療法，並可預期在長期服用之下，Lm改善上述情形之效果將可更為顯著。本研究的最終目標為提供慢性腎臟病低成本且無副作用的益生菌療法，改善腎臟功能指標及緩解患者的不適反應，在維持病患生活品質為前提之下，作為腎臟保健議題的有效解決方案。

Chronic kidney disease (CKD) is distinguished by the persistent decline of kidney function. Growing clinical evidence supports the theory that gut dysbiosis significantly contributes to deteriorating CKD progression, generating gut-derived uremic toxin (GDUT) and aggravating kidney failure. Therefore, strategies based on microbiota-based interventions could be considered preventive and therapeutic approaches to modulate gut microbiota and its metabolites to alleviate the progression of CKD. Thus, in this study, we aim to develop a probiotic mixture to prevent or alleviate CKD and further investigate its possible mechanisms via in vivo and clinical studies using multi-omics analyses.

First, we developed an in vitro probiotics screening platform based on reducing GDUT precursors. Two strains (Lactiplantibacillus plantarum subsp. plantarum MFM 30−3 and Lacticaseibacillus paracasei subsp. paracasei MFM 18) were selected due to their high clearance ability. The two strains were mixed and named Lactobacillus mix (Lm) for further animal study to verify its anti-CKD effect and the underlying mechanism through a 0.2% adenine-induced CKD mouse model. The animal study demonstrated that Lm could alleviate kidney function through reversed gut dysbiosis and further changed the abundance of commensal bacteria, especially short-chain fatty acid (SCFA) producers in the gut, leading to downregulating uremic toxins and preventing intestinal barrier disruption via modulation of metabolite production. Furthermore, Lm also significantly improved kidney function by reducing kidney injury, decreasing fibrotic-related proteins, modulating oxidative stress, downregulating proinflammatory activity, and regulating immune responses. The findings not only provided evidence that Lm could be a potential preventive approach against CKD. The novel probiotic screening platform also showed the applicable reference for selecting functional probiotics possessing the anti-CKD effect.

Next, we cooperated with National Taiwan University Hospital and National Taiwan University Veterinary Hospital to perform clinical trials to evaluate the therapeutic opportunities of Lm. A total of 120 patients with stage 3 CKD were enrolled in a prospective, randomized, open-labeled, blinded end-point (so-called PROBE) trial for 3 months to evaluate the clinical efficacy of Lm on CKD. Currently, only 49 patients have completed the 3-month study, while the remaining 71 patients are still undergoing treatments. The patients with CKD with a 3-month Lm intervention demonstrated a down-regulatory effect on GDUT compared to the control group. A decreasing trend of creatinine (CRE) and blood urea nitrogen (BUN) levels with an estimated glomerular filtration rate (eGFR) increasing rate of 0.17 per month was found after the Lm intervention. The clinical results support that Lm can effectively downregulate trimethylamine-N-oxide (TMAO), p-cresyl sulfate (PCS), indoxyl sulfate (IS), and phenyl sulfate (PS) levels in circulation, ultimately preserving renal function, as evidenced by preventing eGFR decline in patients with CKD. More analysis will be conducted after all the patients finish the study.

For the feline study, an open-label single-arm pilot study was conducted with fourteen cats with stage 2-3 CKD for 8 weeks to evaluate the effect of Lm intervention on CKD alleviation. The gut microbiome and serum metabolome were also analyzed to study the mechanisms of therapeutic impact involved in CKD alleviating effect of Lm intervention. After 8 weeks of Lm intervention, CRE and BUN of most cats with CKD were reduced or maintained. Lm also downregulated GDUTs in serum. Furthermore, Lm significantly improved intestinal diversity and changed the levels of specific bacterial taxa, microbial functions, and metabolites in cats, suggesting modulation of microbial compositions, metabolic reactions, and metabolite profiles by Lm intervention, which were strongly interconnected. We further separated the cats with CKD into Lm high responder (HR) and moderate responder (MR) to investigate the precision perspective of individual cats that showed different responses on alleviating CKD after Lm intervention. We identified the differential microbial biomarkers, serum metabolites, and KEGG level 3 pathways between HR and MR cats, and observed that the abundances of two probiotic strains in the intestinal tract affect the efficacy of Lm intervention in alleviating CKD. Furthermore, modulations of metabolites and microbial functions involved in GDUT (IS, PCS, and PS) and SCFA (acetic acid, butyric acid, and propionic acid) biosynthesis pathways were more precise among HR cats. We also discovered specific bacterial species and serum metabolites that differed between cats with stage 2 and 3 CKD, and were shown correlations with CKD progressive factors. Although the physiological significance of these specific biomarkers still requires further investigation, these biomarkers have the potential to be utilized as novel CKD prognostic/diagnostic biomarkers or developed as next-generation/precision probiotics for alleviating CKD progression.

In conclusion, multi-omics analyses demonstrated that Lm improved intestinal flora dysbiosis and downregulated harmful GDUT biosynthesis through modulation of microbial composition and metabolite production, revealing the translational potential of probiotic adjuvant therapy in CKD. A preferable alleviating effect could be expected if the patients consume a long-term Lm intervention. The final aim of this study is to provide a novel approach to improve symptoms and outcomes in patients with CKD, thereby enhancing the quality of life for this population.