Upregulation of miR‐21‐5p and VMP‐1 Precede Apical Bleb Formation in Renal Ischemia
Authors: Singh Vaishali , Paterson Mark , Chuppa Sandra , Kriegel Alison J. 
Published: November 2023

Severe acute kidney injury (AKI) can lead to residual tubular damage and chronic kidney disease. Proximal convoluted tubule cells produce apical blebs in response to ischemic stress. Blebs are shed into the tubular lumen, resulting loss of apical membrane transporters. Hypoxia regulated microRNA miR‐21 is well known to be rapidly upregulated in response to hypoxia or ischemia. The genetic sequence of miR‐21 lies within an intronic region of vacuole membrane protein 1 (Vmp1), a gene which encodes a transmembrane protein involved with autophagy. While each gene can be transcribed independently, they can also be co‐transcribed, suggesting there may be a biologically synergistic or coordinated relationship. We hypothesize that miR‐21‐5p and Vmp1 are co‐regulated in response to renal ischemia and that they may be colocalized after upregulation. The goals of this study were to establish a time course for changes in miR‐21‐5p and VMP1 protein abundance and localization, as well as proximal tubule bleb formation within the renal cortex. Acute renal ischemia or sham surgery was performed on 10 week old male Sprague Dawley rats by ligating the renal artery and vein for 15, 30, 45 and 60 minutes (n=6/time point). Kidneys were immediately collected and sectioned for subsequent histology, RNA and protein analysis. Abundance of miR‐21‐5p in the renal cortex was evaluated by qRT‐ PCR, and VMP1 protein abundance was evaluated by western blot. Trichrome staining was performed to evaluate ischemia induced bleb formation and changes in proximal tubule morphology. Combined fluorescence in situ hybridization and immunohistochemistry for miR‐21‐5p and VMP1, respectively, was performed on tissue sections from rats subjected to 15 or 30 minutes renal ischemia or sham surgeries to evaluate cellular location and colocalization. We observed a rapid increase in averaged miR‐21‐5p (~2 fold) and VMP1 protein (>2.5 fold) abundance in the renal cortex at both 15 and 30 minutes of renal ischemia, compared to sham operated controls. Histological analysis indicated blebs were not present until 30 minutes of ischemia. The relative increases in miR‐21‐5p and VMP1 were reduced by 45 of ischemia and lost by 60 minutes of ischemia. At 15 minutes miR‐21‐5p was diffusely increased in the cytoplasm of the proximal tubule epithelial cell, while VMP1 protein was observed as very small dots or clusters within the cytoplasm and and within the nucleus. Within 30 minutes, when apical blebs are observed, we see that miR‐21‐5p and VMP1 are colocalized within larger clusters. In conclusion, the rapid increase in abundance of cortical miR‐21 and VMP1 precedes the release of blebs from proximal tubules and by 30 minutes miR‐21 and VMP1 are colocalized within cytoplasmic structures. This finding of temporal expression similarities suggests that their co‐regulation is may be functionally related in renal ischemia, though it is not clear if this is directly related to autophagy. These findings support our hypothesis, however additional studies are needed to validate a functional role of VMP1 in response to ischemia.