Effect Of Lactobacillus Rhamnosus GG On CKD Rat Vascular Calcification And The Mechanisms Exploration

BackgroundVascular calcification is the commonest pathological phenomenon in patients with chronic kidney disease(CKD).Existing evidence suggests that vascular calcification is significantly associated with the risk of cardiovascular events,and is an independent risk factor for cardiovascular complications in CKD patients.However,there is currently no effective way to prevent and treat CKD-related vascular calcification.In recent years,there has been increasing evidence that the gut microbiota plays a crucial role in various diseases,including vascular calcification.Gut dysbiosis,an alteration in composition and function of the gut microbiome,are a striking feature of CKD patients.On the one hand,the imbalance of intestinal flora causes intestinal barrier damage,which leads to endotoxemia and systemic inflammatory state,promoting the progress of vascular calcification in CKD patients.On the other hand,changes in intestinal flora affect the metabolism of amino acids and choline to regulate the production of uremic toxins such as trimethylamine oxide(TMAO)and p-cresol,and increase the risk of vascular calcification in CKD patients.Modifying the intestinal flora,protection of intestinal barrier,anti-inflammatory are new directions to prevent and treat CKD-related vascular calcification.As the most simple and effective way to regulate intestinal flora,protect intestinal barrier,and anti-inflammatory,probiotics are currently a potential effective strategy for disease prevention and treatment.However,in recent years,although various researches have been finished on exogenous supplementation of specific probiotics at the animal models and in humans,our knowledge about the applicable conditions and side effects of these microorganisms is limited.Nowadays,probiotics are widely used to try to protect kidney function and reduce uremic toxins production in CKD patients.However,several studies have confirmed that multiple strains of probiotics have significant bone formation-promoting effects.As we all know,vascular calcification is an active regulatory process with similar histopathological features and initiation mechanisms to bone formation.Meanwhile,vascular calcification and bone formation share many of the same signaling pathways.Targeting bone formation may affect the progression of CKD-related vascular calcification.Unfortunately,there are no reports of probiotics affecting vascular calcification in CKD patients.Lactobacillus rhamnosus GG(LGG),one of the most extensively studied and clinically promising probiotics,was isolated by Gorbach and Goldin from a fecal sample of a healthy volunteer in 1985,With inflammation,anti-oxidative stress,protection of intestinal barrier and other functions.However,an in vivo study demonstrated that LGG was able to stimulate bone formation through Treg cell-mediated WNT10B expression.Whether LGG can affect the vascular calcification process in CKD patients remains unclear.ObjectiveThe purpose of this study was to explore the effect of probiotic LGG supplementation on the process of CKD-related vascular calcification and its underlying mechanism,providing reference for the correct use of probiotic therapy in CKD patients.Methods and results1.Firstly,5/6 nephrectomy model was used to induce renal failure in rats.After 2 weeks,serum creatinine level of rats was measured to determine whether the CKD model was established successfully.Then,high phosphorus diet combined with VitD3 was used to induce vascular calcification in RATS with CKD,while LGG was supplemented by gavage to explore the effect of LGG on vascular calcification in rats with CKD.The perfusion dose of rats in LGG group was 1*109 CFU/day.Our results revealed that LGG supplementation significantly promoted the weight loss of CKD rats compared with the Model group.Aortic alizarin red staining,Micro-CT and vascular sections results indicated that LGG supplementation did not alleviate but significantly promoted vascular calcification in CKD rats.Western blot results confirmed that LGG supplementation increased the expression of the osteogenic transcription factor Runx2.These results suggest that probiotic LGG can significantly promote vascular calcification in CKD rats.2.16sRNA sequencing was used to analyze the effect of LGG supplementation on the intestinal flora of CKD rats.Analysis of gut microbiota composition showed that at the family level,LGG supplementation significantly promoted the enrichment of Lactobaculaceae in CKD rats.At the genus level,LGG supplementation significantly promoted the enrichment of Lactobacillus.LEFSE analysis indicated that LGG supplementation significantly altered the gut microbiota and the abundance of Lactobacillus was significantly different among the three groups.Spearman correlation analysis showed that there was a significant positive correlation between the expression of Runx2 and the level of Lactobacillus.3.EVs secreted by LGG were extracted by ultracentrifugation to explore its role in vascular calcification.We used EVs at three concentrations of 4?g/ml,8?g/ml and 16?g/ml to treat rat vascular smooth muscle cells(VSMCs)in the presence of calcifying medium.Alizarin red staining and calcium quantification suggestd that LGG-derived EVs promoted the calcification of rat VSMCs in a concentration-dependent manner.q-PCR and western blot results showed that EVs significantly up-regulated the expressions of Runx2 and BMP2 in VSMCs,promoting Osteoblast-like phenotypic transformation.Similarly,we found that EVs can promote the calcification of isolated aortic vascular rings in vitro in a high phosphorus and calcium environment.In vivo experiments showed that EVs also promoted vascular calcification in CKD rats.These results suggest that LGG promotes vascular calcification by secreting EVs.4.Our results indicated that LGG and EVs supplementation activated the PI3K/AKT signaling pathway in CKD rats.In vitro experiments also showed that LGG-derived EVs activated the PI3K/AKT signaling pathway in rat VSMCs in a concentration-dependent manner.ConclusionsIn this study,we demonstrated for the first time that Lactobacillus rhamnosus GG(LGG)and its EVs can promote vascular calcification in rats with CKD,and this effect is related to the activation of PI3K/AKT signaling pathway.Patients with CKD may be cautious about using specific probiotic strains,such as LGG.