| Objective:Renal stones is one of the most common diseases in urology department.At present,removal of stones through surgery is the first choice and the most common way,but its high recurrence rate and medical economic burden have brought great troubles to doctors and patients.In order to reduce the incidence and recurrence rate of kidney stones and reduce the burden of disease,it is very important to study the prevention measures.60%to 90%of renal stones contain calcium oxalate,so it is particularly important to prevent the formation of calcium oxalate stones.At present,the prevention of kidney stones is still at a relatively superficial level,and the main prevention means is only to reduce the intake of kidney stone components such as oxalic acid and calcium in food or drink more water every day.These preventive measures mainly belong to the adjustment of dietary structure and the effect is not obvious.With the deepening of research on gut microbiota in recent years,it has found many links between gut bacteria and adiseases in humans.The relationship between metabolic diseases,such as metabolic syndrome,and the gut microbiota is the focus of much attention.The disorder of gut microbiota can induce metabolic syndrome by mediating inflammatory response,metabolic disorder,insulin resistance,etc.Metabolic disorder further aggravates the disorder of gut microbiota.The use of probiotics can improve gut microbiota.In recent years,many scholars believe that kidney stones belong to a kind of metabolic disease,its occurrence and development are closely related to human metabolism,including various metabolic diseases of human body.Exploring the relationship between kidney stones and gut microbiota and metabolic disorders will help us to further explore the formation mechanism of stones.This will help us to find a new direction for renal stones and other metabolic diseases.Diabetes mellitus is one of the clinical symptoms of metabolic syndrome.In the present study,it was also found to be closely related to gut microbiota.On the other hand,some large cohort studies have shown that the incidence of kidney stones in diabetic patients is higher and some studies have found that the incidence of diabetes in patients with kidney stones is also increased.This suggests that diabetes may increase the risk of developing kidney stones,and kidney stones may also affect the course of diabetes.The correlation between diabetes and gut microbiota also suggests that we can look for the association between these two diseases from the perspective of microorganisms.This study will be carried out from the following aspects:1.To clarify the relationship between diabetes and renal calcium oxalate stones:(1)To explore whether diabetes is an independent risk factor for the occurrence of renal stones from the epidemiological perspective;(2)To analyze the association of gut microbiota between clinical type 2 diabetes mellitus and renal calcium oxalate stones in fecal samples;2.Further explore the relationship between diabetes and kidney calcium oxalate stones from the perspective of gut microbiota.Materials and Methods:This study is divided into four parts:Part I:Explore the relationship between diabetes and kidney stones.A retrospective study was conducted on people aged 20 years or above from the NHANES database from 2007 to 2018.Clinical data were collected.Statistical methods including t test,chi-square test and multivariate analysis were used to explore whether diabetes mellitus was associated with the incidence of kidney stones.Part Ⅱ:Explore the role of gut microbiota in renal calcium oxalate stones by detecting the composition of intestinal microflora in diabetic patients with or without renal calcium oxalate stones and healthy people.(1)Fecal specimen collection:During specimen collection,the principle of aseptic operation was strictly observed.A disposable sterile feces collector was used to collect the feces of the participants,and the surface feces were removed with a sterile cotton swab.In exchange for fresh cotton swabs,an appropriate amount of specimens was taken from the inner layer of feces and put into a cryopreservation tube with a capacity of 2ml.A total of 3 tubes of fecal samples of each participant were kept.The samples were stored at-80℃.(2)Detection of gut microbiota:Based on Illumine HiSeq 2000 sequencing platform,the variable region of 16S rRNA gene of different gut microbiota was sequenced and compared with the existing bacterial 16S rRNA gene database.Part Ⅲ:Through animal experiments verify the vinegar can prevent the happening of the diabetic rat kidney stones,and explore the related mechanism:6 Sprague Dawley(SD)rats and 24 Goto-Kakizaki(GK)diabetes rats were divided into five groups.Rats were molded with ethylene glycol and fed with vinegar.GK rats in diabetes control group had free access to sterile water and 2 mL/kg ddH2O by gavage.GK rats in diabetes model group rats were supplied with drinking water with 1%(v/v)ethylene glycol(EG)and 2 mL/kg ddH2O by gavage.In diabetes vinegar group,GK rats had free access to sterile water and were given 2 mL/kg vinegar by gavage every day.In diabetes vinegar model group,GK rats were given 2 mL/kg vinegar by gavage and 1%(v/v)EG in drinking water every day.In SD model group,SD rats were supplied with drinking water with 1%(v/v)ethylene glycol(EG)and 2 mL/kg ddH2O by gavage.Intestinal contents and kidneys were stored at-80℃.The crystal formation of kidney,the change of gut microbiota and the expression of tight junction protein in kidney tissue were detected.Part Ⅳ:Cell experiments were conducted to verify whether high glucose induced human renal tubular epithelial cells increased the adhesion ability of calcium oxalate crystals and whether acetate could reduce the adhesion ability.HK-2 cells were grown in normal or high glucose medium and managed by sodium acetate and/or CaOx crystals.Cells were divided into groups,named control group,acetate group,CaOx crystals group,acetate with CaOx crystals group,high glucose group,high glucose acetate group,high glucose CaOx crystals group and high glucose acetate with CaOx crystals group.When the density of HK-2 cells in 6-well plates was about 60%,20 mM sodium acetate and/or 100μg/mL CaOx crystals were added into the culture medium for 24 h.Then medium containing 100μg/mL ponceau-stained CaOx crystals was added into wells.HK-2 cells were washed again using PBS thoroughly after 10 min and observed under microscope at the magnification of 100-fold(CARL ZEISS,Germany).PCR and WB were used for determining the content of Occludin.Results:Part Ⅰ:The follow-up population information from 2007 to 2018 was included in the NHANES database.A total of 25163 people who did not answer the question of"whether they have kidney stones" in the NHANES database were excluded,and a total of 8218 people who lacked important clinical data were excluded.26,461 people were finally included.The calculi group and the non-calculi group average age was 55 years old and 46 years old,the proportion of females was 45.4%and 53%,the average BMI was 30.38±6.68kg/m2 and 29.11±6.84kg/m2,the proportion of diabetic patients was 18.7%and 8.7%,the proportion of smokers(calculated as having smoked more than 100 cigarettes in life)was 47.7%and 43.1%,and the proportion of hypertensive patients was 46.9%and 30%,respectively.The dietary intake of the two groups was 75.6gm of protein,232.5gm of carbohydrate and 96.3gm of sugar in the stone group.The non-stone group consumed 76.5gm of protein,232.4gm of carbohydrate and 96.1gm of sugar.Multivariate logistic regression analysis was conducted,the OR value of patients with diabetes was 1.61,the OR value of women was 0.70,the OR value of patients without hypertension was 0.76,the OR value of age was 1.018,and the OR value of BMI was 1.022.Diabetes,gender,hypertension,age,BMI,HOMA-IR and Glycohemoglobin were all independent risk factors(P<0.05).Then to set diabetes as the dependent variable,we found that kidney stones,gender,smoking,hypertension,age,BMI and sugar intake were all independent risk factors for diabetes(P<0.05).Part Ⅱ:The bacteria richness in the four groups of samples is high and the testing depth was sufficient.The results of species difference showed that Bacteroides,Faecalibacterium,Prevotella and Roseburia were abundant in the intestinal tract of healthy control group,while the number was significantly decreased in the intestinal tract of patients with renal calcium oxalate stones and patients with type 2 diabetes mellitus(P<0.05).Faecalibacterium,Prevotella,Rossiella and Akkermansia were further significantly decreased in the intestinal tract of patients with renal calcium oxalate stones complicated with diabetes mellitus(P<0.05).The expression of Lachnoclostridium in the intestines of healthy control group was low,while the expression of Lachnoclostridium in the intestines of patients with simple diabetes and patients with simple renal stones was increased(P<0.05).Part Ⅲ:Vinegar treatment significantly reduced calcium oxalate crystal deposition in renal tissue of diabetic rats modeled with glycol stones.He staining score and VK staining value(crystal area)in vinegar group were lower(P<0.05).Ruminococcus torques and Faecalibaculum in the intestines of GK diabetic rats were significantly increased after the intervention of vinegar(P<0.05),while Ruminococcus gauvreauii and Lachnospiraceae in the intestines of GK diabetic rats were significantly decreased after the treatment of ethylene glycol(P<0.05).Compared with the gut microbiota of GK diabetic rats and SD rats after the same glycol model,the abundance of Romboutsia and Turicibacter in the intestinal tract of GK diabetic rats decreased,while the abundance of Blautia and other bacteria increased(P<0.05).Occludin expression in renal tissue of rats decreased after ethylene glycol modeling,but increased after vinegar intervention.Part Ⅳ:After calcium oxalate crystal treatment,the mRNA and protein expression of Occludin in HK-2 cells decreased significantly.After sodium acetate treatment,the mRNA and protein expression of Occludin in HK-2 cells increased significantly.After calcium oxalate crystal treatment,the number of crystal adhesion in HK-2 cells increased significantly.After sodium acetate treatment,the number of crystal adhesion in HK-2 cells decreased significantly.These changes persisted in the high-glucose environment.Conclusion:1.Diabetes mellitus is an independent risk factor for the formation of kidney stones,and having kidney stones is an independent risk factor for the occurrence of diabetes;2.There are differences in the composition of intestinal flora between patients with diabetes and kidney stones and healthy people;The abundance of short-chain fatty acid producing bacteria such as Bacteroides,Faecalibacterium,Prevotella and Roseburia decreased in the gut microbiota of patients with diabetes and kidney stones,and decreased more in patients with diabetes.3.Diabetic rats produce more renal calcium oxalate crystals after the formation of ethylene glycol kidney stones.After intragastric administration of vinegar,the crystal deposition and tight junction protein Occludin expression were significantly decreased in renal calcium oxalate stone model and increased in renal tissue.4.In vitro,high glucose environment can increase the crystal adhesion of HK-2 cells;acetate could increase the Occludin expression of HK-2 cells and reduce the cell crystal adhesion ability. |
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