Background: Diabetic nephropathy (DN) is characterized by increased endocytosis and degradation of nephrin, a protein that comprises the molecular sieve of the glomerular filtration barrier. While nephrin internalization has been found activated in diabetes-stressed podocytes, the post-internalization trafficking steps that lead to the eventual depletion of nephrin and the development of DN are unclear. Our work on an inherited podocytopathy uncovered that dysregulated dynein could compromise nephrin trafficking, leading us to test whether and how dynein mediates the pathogenesis of DN. Methods: We analyzed the transcription of dynein components in public DN databases, using the Nephroseq platform. We verified altered dynein transcription in diabetic podocytopathy by quantitative PCR. Dynein-mediated trafficking and degradation of nephrin was investigated using an in vitro nephrin trafficking model and was demonstrated in a mouse model with streptozotocin (STZ)-induced DN, as well as in human kidney biopsy sections. Results: Our transcription analysis revealed increased expression of dynein in human DN and diabetic mouse kidney, correlated significantly with the severity of hyperglycemia and DN. In diabetic podocytopathy, we observed that dynein-mediated post-endocytic sorting of nephrin was upregulated, resulting in accelerated nephrin degradation and disrupted nephrin recycling. In hyperglycemia-stressed podocytes, Dynll1, one of the most upregulated dynein components, is required for the recruitment of dynein complex that mediates the post-endocytic sorting of nephrin. This was corroborated by observing enhanced Dynll1-nephrin colocalization in podocytes of diabetic patients, as well as dynein-mediated trafficking and degradation of nephrin in STZ-induced diabetic mice with hyperglycemia. Knockdown of Dynll1 attenuated lysosomal degradation of nephrin and promoted its recycling, suggesting the essential role of Dynll1 in dynein-mediated mistrafficking. Conclusion: Our studies show that hyperglycemia stimulates dynein-mediated trafficking of nephrin to lysosomes by inducing its expression. The decoding of dynein-driven pathogenesis of diabetic podocytopathy offers a spectrum of new dynein-related therapeutic targets for DN.
Diabetic nephropathy (DN) is characterized by increased endocytosis and degradation of nephrin, a protein that comprises the molecular sieve of the glomerular filtration barrier, but the key trafficking mechanism that connects the initial endocytic events and the homeostasis of nephrin is unknown. Our work implicates cytoplasmic dynein, a transport complex that is upregulated in DN, plays a critical role in triaging the endocytosed nephrin between recycling and proteolytic pathways. Using Nephroseq platform, our transcription analysis in public DN databases revealed dynein overexpression in human DN and diabetic mouse kidney, correlated with the severity of hyperglycemia and nephropathy. The increased expression of dynein subunits was confirmed in high glucose-treated podocytes and in glomeruli isolated from streptozotocin (STZ)-induced diabetic mice. Using live cell imaging, we illustrated that dynein-mediated post-endocytic sorting of nephrin was upregulated, resulting in accelerated nephrin degradation and disrupted nephrin recycling. In diabetic podocytopathy, Dynll1 is one of the most upregulated dynein components that was recruited to endocytosed nephrin. This was corroborated by observing enhanced Dynll1-nephrin colocalization in podocytes of diabetic patients, as well as dynein-mediated trafficking and degradation of nephrin in STZ-induced diabetic mice. Knockdown of Dynll1 attenuated lysosomal degradation of nephrin and promoted its recycling, suggesting the essential role of Dynll1 in dynein-mediated mistrafficking. Defining the role of dynein-mediated mistrafficking of nephrin in diabetes will not only fill the knowledge gap about the early events of DN, but also inspire novel therapeutics that target a broad spectrum of molecular events involved in the dynein-mediated trafficking.
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