RanBP3L is a NFAT5 target gene and its deficiency in principal cells promotes epithelial mesenchymal transition through MAPK signaling

Abstract

Inner medullary kidney cells are exposed to an increasing hyperosmolality altering several intracellular processes. As a key regulator involved in adaption to hyperosmotic environment the transcription factor nuclear factor of activated T cells‐5 (NFAT5) was identified. Its nuclear translocation results in activation of different genes to restore homeostasis. By using primarily cultured rat renal inner medullary collecting duct cells (IMCD) and microarray analysis we identified hundreds of genes that showed differences in expression level in a time‐ and osmolality‐dependent manner. The kidney specific Ran binding protein 3 like (RanBP3L) showed the highest changes in expression level and is located in IMCDs. Within the collecting duct its expression is specific to the principal cells. Since studies describing the renal function are missing, we further characterized the cellular and physiological role of RanBP3L in a renal mouse cell line.We used the murine kidney collecting duct cell line mpKCCD creating a RanBP3L deficient cell clone by using CRISPR/Cas9. To investigate its physiological role the gene expression profiles were analyzed by Next Generation Sequencing. We further analyzed the migration and colony forming capability of the cells and used immunofluorescence to study differences in cell morphology and specific signaling pathways. These experiments were performed under isoosmolar (300 mosmol/kg) and hyperosmolar (600 mosmol/kg) conditions. Since RanBP3L expression is induced by hyperosmolality, the NFAT5 dependent expression of RanBP3L was examined through CRISPR/Cas9 mediated NFAT5 knock out.Loss of RanBP3L under 300 mosmol/kg showed no significant changes in the morphology while under 600 mosmol/kg massive changes were induced resulting in a loss of epithelial structures. This correlates with our functional analysis showing a higher migration and invasiveness capacity. Furthermore RanBP3L deficiency is associated with a massive change in gene expression, indicating a dysregulation of stress associated pathways. Immunofluorescence showed that TGF‐β stimulation in wild type induced a similar phenotype to RanBP3L knock out cells. Downstream analysis of MAPK pathway showed that the key proteins JNK, c‐JUN and Smad2 are deregulated in RanBP3L deficient cells resulting in an epithelial mesenchymal transition (EMT) like phenotype, which is a hallmark in cancer development. This matches with the data from the cancer genome atlas from patients with renal clear cell carcinoma (RCC) where low RanBP3L expression level is associated with shorter overall survival. It seems as if the expression level of RanBP3L in kidney cells is crucial for cell protection. Here we could show that RanBP3L expression is reduced in NFAT5 deficient cells, indicating that RanBP3L is a NFAT5 target gene.Taken together we have shown that RanBP3L is expressed NFAT5 dependent and prevents a MAPK associated EMT in renal cells and should be used as a prognostic marker for patients with RCC. Further experiments, like in‐vivo studies of a conditional RanBP3L knock out mouse will give us more insights to resolve the underlying pathway.Support or Funding Informationgerman research foundation