3D Biomimetic Platforms to Model Kidney Cell Biology for In Vitro Toxicity Screening

SUMMARY A large variety of chemicals require evaluation for organ specific toxicityAnimals have traditionally been used for toxicological screeninghowever this mode of evaluation is expensiveethically challenged and not always predictableIn vitro monolayer has been used as an alternativehowever cells in this format lose the specialized interactions requisite for differentiation and optimal functionThusD in vitro organotypic cellular modelsOCMthat recapitulate key in vivo features of are being soughtThe kidney poses a particularly difficult challenge due to its highly complex spatial and functional orientation that has not been replicated in vitroEmphasis is now being placed on creation of in vitro assays that mimic specific structural microenvironmentsWe have fabricated aD in vitro kidney medullary organotypic culture modelMOCMutilizing mouse ureteric budUBand metanephric mesenchymalMMprogenitor cells in co culture in sea sponge scaffolds that mimic the natural tubular vessel micro environmentThe cells in this system reciprocally induce each other to form organoids comprised of collecting duct epithelium surrounded by capillary profilesThe objective of this grant is to validate that the MOCM platform can be used for in vitro screening of toxins that affect medullary development and mature cell systemsAimwill validate theD MOCM platform as an in vitro prototype for assessment of toxic agents on medullary developmentThe UB and MM cells produce the growth factors and possess their cognate receptors for reciprocal induction of collecting duct epithelium and vasculogenesisToxic effects of known inhibitors of nephrogenesis including imatinib mesylatean inhibitor of platelet derived growth factor receptorPDGFRNVP ASTan inhibitor of glial cell derived neurotrophic factorGDNFreceptor RETand captoprilan inhibitor of angiotensin converting enzymeACEwill be evaluated for toxicity of MOCM developmentAimwill validate theD MOCM platform as in vitro prototype for the assessment of toxic agents on establishedmaturemedullary structuresOur UB cells express AQPand are responsive to vasopressinLithium is a known pharmaco chemical toxin of collecting duct principal cells via interference with vasopressin regulation of a water channel protein aquaporinAQPMature MOCM scaffolds will be treated with vasopressin to enhance AQPand the effect of lithium toxicity on this expression will be evaluatedCell toxicity in both aims will be evaluated using assessments of organoid structure and immunoreactivity of AQPand other marker proteins using immunohistochemistryimmunoblotting and ELISAData will be compared to the effects of these agents on human and rodent medullary structures in vivo as reported in the literatureThis MOCM system has the potential to provide an innovative platform to facilitate toxicant effects on the kidney medulla and will reduce animal testing Project NarrativeThere is a need for in vitroD organotypic culture platforms for toxicity testing that diminish animal experimentationWe have developed a kidney medullary organotypic culture modelMOCMcomprised of collecting duct and vascular cells in a sea sponge scaffoldThis proposal will validate the MOCM as a means to screen toxicity of compounds on medullary development and mature organ cytotoxicity in vitroSuch an in vitro kidney MOCM device will modernize the field and can be used for the study of kidney cell physiologypathologytoxicologyand drug development