Primary Culture Of Nephrolithiasis Renal Inner Medullary Collecting Duct Epithelial Cells

Background Urolithiasis is the most common benign disease in urinary system. It isreported that the incidence of stone disease in upper urinary tract is13%,12%,5-9%and10%in the USA, Canada, European countries and Japan, respectively.60%of thestones are oxalate stones. According to epidemiological data, men are affected bystone disease three times more often than women. China is one of the three countrieswhich have the highest incidence of urinary stones. The prevalence of urinary stonesis1-5%in China and5-10%in southern China. The estimated new cases of urinarystones are120-6020/100,000. The recurrence rate within7-8years of stone removal isabout50-70%, resulting a heavy burden to our society. However, the underlyingmechanism for stone formation remains largely unknown and there’s no effectiveapproaches to prevent stone formation and its recurrence. Enviromental, metabolicand genetic factors are thought to be involved in the development of urinary stones.Medullary sponge kidney (MSK) is characterized by cystic distention of innermedullary collecting duct (IMCD). Most MSK cases may also have multiple renalstones and tubular acidosis. IMCD is the last segment of kidney tubules and located inthe renal medullary pyramids. IMCD is involved in regulation of osmosis andacid-base balance. To our best knowledge, there’s no report on methods ofestablishing IMCD cell lines in China. Therefore, we aim to isolate and culture IMCDcells from kidneys affected by stones.Objective To establish a method for isolation, primary culture and identification ofIMCD cells through culturing IMCD cells from normal kidney and kidneys affectedby stones, by which laying the foundation for study on the mechanisms of renal stonesand MSK.Materials and methods IMCD tissues were obtained from renal cell carcinomapatients undergoing radical nephrectomy and renal stones and MSK patientsundergoing PCNL. Under the guidance of ultrasonography, two pieces of tissue wereobtained, put into ice-cold freezing tubes containing sterile normal saline andtransferred to the laboratory immediately. In the cell culture hood, normal saline wasremoved. The tissue was transferred into a culture plate and added with4mlDMED/F12containing collagenase IV, hyaluronidase, DNase I and antibiotics. Thetissue was minced to1mm in length and transferred into a15-ml centrifuge tube and incubated under room temperature with continuous slight shaking for8hours. Thenthe cell suspension was filtered through BD FALCON100μm cell strainer. Thenot-well-digested tissues were minced so as to pass the cell strainer. The filtrate werecentrifuged at400g for8minutes and rinsed twice with sterile PBS. The cellularsediments were added with8ml DMEM/F12containing10%FBS and antibiotics andput into2wells of a6well culture plate. Meanwhile,3drops of cell suspensions wereadded into a test tube and incubated with a drop of0.4%trypan blue for3minutes. Nostaining of the nucleus is used as indicator for good cell viability. The culture wasincubated at37°C with5%CO2. On day4, nonviable cells were washed out usingsterile PBS and the culture medium was replaced. Then the culture medium wasreplaced at a3-day interval. On day12, MEM containing D-valine,10%FBS,1:50penicillin and streptomycin was added to inhibit fibroblast cells. After theestablishment of IMCD cells stable passage, IHC was employed to detect keratinmarkers in IMCD cells.Results We found that the best digestion time under room temperature is8hours. TheIMCD cells cultured from normal kidney and kidney affected by stones are viable andmorphologically identical to IMCD cells. IHC indicates positive staining of keratin18.Therefore, it is viable to culture IMCD cells from MSK.Conclusions We have successfully established a method for culturing IMCD cellsfrom kidneys affected by stones. Our preliminary results suggest that culturing IMCDcells from MSK is possible, thus laying foundation for further studying on themechanism of renal stones and MSK.