Diabetes and SGLT2 Inhibitor Treatment Alter Podocyte Glycocalyx

Background: The glycocalyx between the podocyte foot processes and the glomerular basement membrane (GBM) has been difficult to study in vivo, therefore its function remains elusive. Recently we developed and initially characterized the Pod-Sdc1-GFP mouse model with podocyte specific genetic labeling to study the physiological role of podocyte glycocalyx. Here we aimed to visually demonstrate the alterations in podocyte glycocalyx in diabetes with and without SGLT2i treatment in vivo. Methods: New genetic mouse models were generated by the knock-in of pCAG-Lox-STOP-Lox-EGFP-Sdc1 to generate animals with Cre-dependent expression of a syndecan-1 (Sdc1) fusion protein with N-terminal, extracellular GFP. Sdc1-GFPflox animals were crossed with Podocin-Cre mice for the specific genetic labeling of the glycocalyx in podocytes. Intravital multiphoton microscopy (MPM) was used to measure changes in the depth of glycocalyx and corresponding changes in GFB function in vivo over time in streptozotocin induced diabetic kidney disease with or without SGLT2i empagliflozin treatment. Alexa-594 conjugated wheat germ agglutinin (WGA) lectin was used to label the glycosaminoglycan (GAG) component of the glycocalyx, and Alexa-680 conjugated albumin was used to visualize GFB permeability. Results: Co-localization of genetic GFP and Alexa-594 WGA confirmed podocyte glycocalyx labeling. MMP9 injection significantly reduced podocyte glycocalyx thickness and resulted in glomerular albumin leakage confirming podocyte glycocalyx functionality. In vivo MPM imaging in diabetic mice revealed a significant decrease in glycocalyx thickness on both the apical and the basal surface (0.76+/-0.01 um) compared to control (1.03+/-0.02 um) and increased GFB permeability. SGLT2i treatment restored podocyte glycocalyx thickness (0.9+/-0.01 um) and reduced albumin leakage in diabetic mice but not in healthy control animals ( 1.01+/-0.01 um). Conclusion: In conclusion, the specialized podocyte glycocalyx at the GBM may function as an important new layer of the GFB, may play an important role in the disruption of GFB functions in diabetic kidney disease, and may be involved in the pleiotropic renoprotective effects of SGLT2i treatment. Funding: NIDDK Support, Private Foundation Support