Abstract:
Podocytes are specialized epithelial cells that play an important role in maintaining the function of the blood filter (glomerulus) in the kidney. Podocytes have a unique morphology that consists of actin-based projections called foot processes that are connected by protein bridges (slit diaphragm; SD). The underlying causes of most chronic kidney diseases are due to podocyte injuries, which lead to foot process effacement and SD disruption, followed by cell detachment and loss. Our current knowledge about podocyte development and the mechanisms by which their dysfunction contributes to glomerular disease processes is inadequate. Therefore, a better understanding of the signaling pathways that control podocyte development and genes that regulate their integrity and function is necessary in order to develop improved therapies.
The zebrafish pronephros is an easily accessible model kidney system that has a high degree of genetic conservation with mammalian renal tubules. In this manuscript, I developed a transient injury and repair podoctye model in the zebrafish pronephros using protamine sulphate (PS), a positively charged compound that compromises the glomerular filtration barrier and inhibits mitochondrial function. Similar to mammalian models of PS-induced glomerular dysfunction, I found that PS induces transient glomerular leakiness with a downregulation in the expression of key podocyte genes (wt1a, wt1b, nphs1 ,nphs2, podxl, cd2ap, cfl1, and α-actn4) by 1 day post-injection (dpi). This was followed by a transcriptional rebound at 3 dpi that lasted until 9 dpi. Given prior reports that pharmacological inhibition of GSK3β can ameliorate glomerular dysfunction and stabilize podocytes foot processes, I tested 1-Azakenpaullone (1-AZKP; a selective GSK3β inhibitor) to investigate whether GSK3β plays a role in PS-induced injury. Pre-treating zebrafish with 1-AZKP before PS-induced injury was successful in rescuing blood filter leakiness and restored expression of podocyte markers. To gain insights into the processes of PS-induced injury and 1-AZKP rescue, RNA-Seq analysis was performed on purified podocytes (PS treated, 1-AZKP treated and PS+1-AZKP treated) at 1 dpi and 3 dpi. The results of the RNA-Seq analysis suggest that PS induces global transcriptional changes that affect the expression of podocyte as well as more general genes, mRNA processing, protein translation, and potentially the biogenesis of
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mitochondrial and ribosomes. Many of these changes were ameliorated by 1-AZKP which I speculate, based on known roles of GSK3β, are due to protective effects on preserving mitochondria function and counteracting oxidative stress. However, I found that long-term treatment with 1-AZKP was toxic to podocytes. In conclusion, I have developed a novel zebrafish model of podocyte injury and revealed new insights into the molecular processes that occur during acute podocyte stress and recovery. I demonstrate that this model has utility for testing novel therapeutics, of which 1-AZKP may have potential as a short-term protector against acute podocyte injury caused by mitochondrial damage.