The Role And Mechanism Of Exosomal MiR-148b-5p Regulating Calcitonin Receptor In Kidney Stone Formation

【Background】The prevalence of kidney stones in China is as high as 5.8%.Calcium-containing stones are the most common type of stone.Due to their unknown pathogenesis,there is a lack of effective prevention and treatment measures in clinical practice.The recurrence rate has remained high,which has not only imposed a heavy economic burden on individuals and families but also a significant burden on society.Therefore,the in-depth elucidation of the pathogenesis of calcium-containing stones and the development of new effective preventive measures are important clinical issues that need urgent attention.Exosomes are tiny disc-shaped membrane vesicles that can be secreted by almost all cells.They can,in part,reflect the pathophysiological state of the cell that secretes the exosome,and in the kidney,they also play a key role in many of its pathophysiological processes.The miRNAs in exosomes are more stable and can indicate the miRNA levels in their parent cells.Exosomal miRNAs are considered potential molecular diagnostic markers in many areas of research.In addition,exosomal miRNAs are increasingly being explored in the context of targeted therapies.However,studies related to urinary exosomes in patients with kidney stones are still in the preliminary stage,and no in-depth studies have been reported.The most important role of calcitonin is to regulate calcium metabolism in humans,and its corresponding regulatory effect is exerted through its binding to the calcitonin receptor.The main location of the calcitonin receptor in the kidney is at the brush border of the renal tubules,and its effect on urinary calcium and renal papillary calcification after binding to calcitonin is not well established.【Objective】This study aims to reveal the molecular mechanism of exosomal miR-148b-5p in promoting kidney stone formation through in vivo and ex vivo experiments,multiple cellular and animal models,and to elucidate the role of calcitonin receptors in the formation of calcium-containing kidney stones.The ultimate goal is to provide a new experimental basis and intervention strategy for the pathogenesis and clinical prevention of kidney stones.【Methods】In vitro experiments:Mi R-148b-5p mimics and inhibitors,circ RNA-83536overexpression,and sh RNA plasmids were transfected into HK-2,MDCT,and m IMCD-3 cells to detect changes in miR-148b-5p and circ RNA-83536 expression levels,as well as calcitonin receptor protein levels.RNA pull-down assay was used to screen for downstream miR-24-3p that could bind to circ RNA-83536.Related mimics,inhibitors,and sh RNA plasmids were transfected into HK-2,MDCT,and m IMCD-3cells to detect changes in the level of calcitonin receptor protein.The direct interaction of miR-24-3p with the 3’UTR of the calcitonin receptor gene was confirmed by luciferase reporter gene assay.In vivo experiments:（1）A rat model of high-calcium urinary kidney stones was created by administering high-calcium drinking water and intramuscular injections of Vit D₃.The experimental group received weekly intraperitoneal injections of miR-148b-5p antagomir,further confirming the in vitro experiments that miR-148b-5p plays a role in regulating urinary calcium and stone formation by regulating circ RNA-83536.This experiment investigated the role of miR-148b-5p in regulating urinary calcium and stone formation.（2）Renal tubular-specific calcitonin receptor knockout mice were bred and analyzed for changes in urinary calcium and renal papillary calcification in both wild-type and mutant mice.A mouse model of hypercalciuria was constructed by providing high-calcium water and intramuscular Vit D₃ to observe the effect of renal tubular calcitonin receptor knockout mice on hypercalcemic load.【Results】1.In the three cell lines,overexpression or inhibition of miR-148b-5p resulted in a corresponding decrease or increase in the expression level of circ RNA-83536 and the level of calcitonin receptor protein.Overexpression or inhibition of circ RNA-83536 resulted in a corresponding decrease or increase in the expression level of downstream miR-24-3p and a corresponding decrease or increase in the level of calcitonin receptor protein.The level of calcitonin receptor protein was correspondingly increased or decreased upon overexpression or inhibition of miR-24-3p.Luciferase reporter gene assays showed that miR-24-3p directly interacts with the 3’UTR of the calcitonin receptor gene.2.In a rat model of hypercalciuric kidney stones,urinary calcium was significantly higher in the experimental group of rats compared to the control group.Rats in the experimental group injected with miR-148b-5p antagomir showed decreased urinary calcium compared to pre-injection,and immunofluorescence and FISH revealed elevated levels of circ RNA-83536 expression and calcitonin receptor protein in the kidney.In renal tubule-specific calcitonin receptor knockout mice, there was a significant increase in urinary calcium in the mutant mice.CT scans showed a distinct high-density stone shadow in the kidneys of the experimental group of rats,and calcium oxalate crystals were visible in the knockout mice when stained for calcium oxalate stones.【Conclusion】Exosomal miR-148b-5p increases urinary calcium excretion and promotes the formation of kidney stones via the circ RNA-83536/miRNAs/calcitonin receptor signaling pathway.Blocking this pathway is expected to be used in the treatment of hypercalciuria and the prevention of kidney stones.