Macula Densa Cell-Targeting Gene Therapy for Kidney Tissue Remodeling

Background: Intravital imaging and multiomics studies recently identified the neuronal differentiation, novel key sensory and regulatory functions, and an endogenous kidney tissue remodeling and regenerative program of the understudied renal cell type of the macula densa (MD). The high, MD-specific expression and secretion of several angiogenic, cell growth, patterning and extracellular matrix remodeling factors including cell communication network (CCN1), and the tissue size and IGF signaling regulator pregnancy-associated plasma protease A2 (PAPPA2) in the mouse and human kidney suggested the therapeutic potential of targeting MD CCN1/PAPPA2 to enhance endogenous kidney tissue regeneration. The present study aimed to develop iv injectable MD targeting gene therapies and to characterize their specificity, efficacy, and the resulting renal phenotype. Methods: AAV9 with GFP/CCN1/PAPPA2 (sh)RNA was injected iv/ip (10^9 GC/animal) in WT and Cdh5-Confetti mice followed by tissue harvest and classic renal histology and RNAscope one month later. Results: Single-dose iv injection of AAV9-GFP resulted in highly MD-specific (76.6±2.7%) and efficient (80.2±2.8%) GFP expression in the mouse kidney (n=4). Treatment with AAV9-mCcn1 RNA in healthy C57BL6 mice resulted in >5-fold MD-specific Ccn1 overexpression as confirmed by RNAscope, and increased glomerular capillary density, endothelial and vascular remodeling resulting in enlarged afferent arterioles. Treatment with AAV9-mPappa2 shRNA resulted in >90% silencing (knockdown) of MD-specific Pappa2 expression based on RNAscope, and approx. 50% smaller glomeruli and increased JG renin expression. No signs of glomerular injury were found in these mice compared to control based on histological PLVAP, ICAM, VCAM immunolabeling and proteinuria assay. Conclusion: This study validated the feasibility of performing long-lasting, specific, MD-targeting genetic overexpression or knockdown (gene therapy) in vivo with a single ip/iv injection. The efficacy and potency of this new therapeutic approach to modulate renal tissue remodeling may open the door for the future development of MD mimetics as a new class of renoprotective therapies.