Fluid Flow Shear Stress-Induced Podocyte Glycosylation Changes: Implications for Glomerular Function

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Background: We have demonstrated that hyperfiltration-induced increase in fluid flow shear stress (FFSS) causes podocyte injury and loss of glomerular function following unilateral nephrectomy (UNx) in rodent models. To explain how glycosaminoglycans (GAGs) on cell surfaces respond to FFSS, Tarbell (J Intern Med. 2006:339) introduced the 'wind in the trees' model. Here, GAGs, akin to branches of proteoglycans in the glycocalyx, detect fluid flow (wind) and convey the force to the membrane/actin cytoskeleton network (ground) through the core protein (tree trunk). To address the chain of events and mechanism of FFSS-induced podocyte injury, we hypothesize that increased FFSS affects the membrane glycoproteins with extensive network of glycans exposed to the environment. Methods: Differentiated immortalized murine podocytes were treated with FFSS (2 dynes/cm2) for 2 h (37°C, 5% CO2) and studied at the end of FFSS treatment, and 2 h and 24 h post-FFSS were termed End-FFSS, Post-2h, Post-24h and Control (untreated). Western blot analysis of podocyte glycoproteins was performed using lectins specific for high-mannose glycans (GNL), N-glycans with 3 and 4 antennas (PHA), alpha2,3-linked sialic acid (MAA), alpha2,6-linked sialic acid (SNA), and core 1 O-glycans (jacalin). RT-qPCR using Qiagen PCR arrays was performed to determine changes in gene expression of selected glycosylation enzymes. Results: Lectins GNL, PHA, and SNA reacted with more glycoproteins of a wide range of molecular mass compared to MAA and jacalin. Podocytes at End-FFSS and Post-2h showed increased alpha2,6- and alpha2,3-linked sialic acid and more tri- and tetra-antennary N-glycans compared to Control. Post-24h samples showed results comparable to Control. Gene expression of specific glycosyltransferases, such as sialyltransferases and GlcNAc-transferases was upregulated at End-FFSS or Post-2h compared to Control and reverted to Control levels. Conclusion: Brief FFSS caused immediate changes in podocyte glycosylation including N-glycosylation consistent with increased branching and sialylation. These transient but substantial changes were paralleled by changes in the expression of specific glycosylation enzymes. Persistent hyperfiltration-induced increase in FFSS may result in lasting changes in glycan metabolism and thus alter the membrane interface leading to functional changes. Funding: NIDDK Support

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