| ObjectiveKidney is a complex organ composed of multiple structures and more than twenty one types of cells.It plays a major role in maintaining the hemostasis of our body.The kidney has limited repair capability after injury.However,it is still unclear on the cell and molecular mechanism involved in the kidney repair process because of the multiple cell types and complicated structures in the kidney.Recent studies have showed that progenitor cells possibly exist in adult kidney.However,it is still controversial whether these cells are involved in the kidney repair/regeneration process.Therefore,it is the dream of many clinician and research in nephrology to identify new types of adult stem cells/progenitor cells,and use adult stem cells/precursor cells to treat kidney diseases in clinic.Lgr5 has been reported as an adult stem cell marker in other organs.In kidney,Lgr5 is only expressed in the embryonic kidney,and the expression of Lgr5 is silenced normally one week after birth.Could Lgr5 gene be reactivated in the disease state and take part in kidney repair/regeneration after injury,like in other organs?It is still unknown whether the descendants of embryonic Lgr5+cells exsit in adult kidney or not.In this study,we will use the cell lineage tracing technique to explore whether Lgr5+cells are a subset of adult renal stem cells/progenitors and involved in kidney repair/regeneration after injury.We also explored the role of Lgr5+ renal embryonic progenitor cells in renal development and repair after injury.MethodsIn the first part,we breed Lgr5-EGFP-ires-creERT2 and ROSA26-LSL-RFP mice to generate Lgr5-EGFP-ires-creERT2/+;ROSA26-LSL-RFP/+double positive mice.Cre activity was induced with 2.5mg/tetrahydroxy tamoxifen one day before surgery.Bilateral ischemia-reperfusion(AKI model)and unilateral ischemia-reperfusion nephrectomy(CKD model)procedure were performed on 8 weeks old mice the following day.After the operation,the expression of GFP and RFP were directly detected using immunofluorescence microscopy,which indicate the activation of Lgr5 gene.In the second part,we used Lgr5-EGFP-ires-creERT2;RFP+mouse to perform fate-mapping analysis of Lgr5+cells during kidney development.Pregnant females were injected intraperitoneally with a single dose of O.lmg/g 4-hydroxytamoxifen at E13.5,E15.5 andE 18.5 gestational days once.The kidneys of Lgr5-EGFP-IRES-CreERT2/+;RFP/+mouse were collected at 8 weeks to analysis the fate of the RFP labelled cells in kidney.ResultsIn the first part,we did not observe the activation of Lgr5 gene in the kidney from AKI and CKD mice models we used in this study.In the second part,we found that descendants of Lgr5+ embryonic progenitors did exist in adult kidney.The numer of RFP+labelled cells were different at different time point induction.The descendants of Lgr5+embryonic progenitors were mainly localized at the junction of cortex and medulla.It also located in the glomerular and tubular structures with different appearance.The immunostaining results showed that RFP+labelled cells were not co-localized with any known renal tubular markers,indicating that these cells are a novel cell population in the kidney.Compared to the sham group,the number of RFP+labelled cells did not dramatically change 11 days after UIRI.Further investigation need to be done to confirm these results.ConclusionIn summary,our study showed that Lgr5 is not a renal adult stem cell/progenitor cell and was not involved in the kidney repair/regeneration in our AKI or CKD model.Our study also showed that the descendants of Lgr5+embryonic progenitors still exist in adult kidney.It’s still unclear about the structure and function of these cells in the adult kidney. |
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