Introduction:
Diabetic kidney disease (DKD) is the leading cause of end-stage renal failure in North America. The transforming growth factor beta 1 family (TGFβ1) has been shown to promote kidney proximal tubular cell apoptosis while contributing to the pathogenesis of DKD by promoting interstitial fibrosis. The mechanism involves the activation of SMAD pathways which release cytokines and growth factors that promote interstitial inflammation and myofibroblast activation, which leads to the increased production and accumulation of extracellular matrix proteins in the interstitium. Given that clinical inhibition of TGFβ1 has posed various challenges due to its pleiotropic roles, newer therapies and strategies are needed to ameliorate its effect in enhancing DKD.
Previously, we have identified a significant increase in miRNA(miR)-299a-5p expression in primary mesangial cells treated with TGFβ1, as well as in the tubules and glomeruli of mice with chronic kidney disease. We also showed that this miR attenuated expression of the antifibrotic protein follistatin. However, the role of miR-299a-5p in DKD is as yet unexplored. Here we studied the role of this miR in regulating tubular apoptosis and promoting fibrosis in DKD.
Methods:
Immortalized human kidney proximal tubular cells (HK2) were treated with high glucose (HG) at 30mM. miR299a-5p was detected by qPCR in HK2 cells and in-situ hybridization in mouse and human kidneys. HK2 cells were transfected with miR overexpression and inhibition plasmids using Transfectamine reagent. Apoptosis was detected using the Annexin V kit. Immunoblotting and immunohistochemistry were used to assess protein expression. miR-299a-5p was inhibited in type 1 diabetic mice using lock-nuclei acid technology to investigate the role of miR-299a-5p in promoting tubular cell apoptosis and interstitial fibrosis.
Results:
HG increased the expression of miR299a-5p in HK2 cells. HG and TGFβ1 combined had an additive effect. miR299a-5p expression was also significantly increased in type-1 diabetic Akita mice overexpressing TGFβ1 (HH-Akita), a model which accelerates DKD, as well as in human diabetic kidney biopsies. Furthermore, bioinformatics screening of four databases showed that miR299a-5p targets MAP3K2, a protein kinase primarily expressed in the tubules. MAP3K2 is a major activator of extracellular signal-regulated kinase 5 (ERK 5), a known anti-apoptotic and anti-inflammatory kinase. miR299a-5p overexpression reduced the protein expression of MAP3K2 and phosphorylation of ERK5, while augmenting endoplasmic reticulum (ER) stress and the expression of apoptotic markers in response to HG. Conversely, miR299a-5p inhibition restored MAP3K2 expression and ERK5 phosphorylation to basal levels, while attenuating HG-induced apoptosis. In vivo inhibition of miR-299a-5p in HH-Akita mice reduced tubular cell apoptosis, fibrosis, urine TGFβ1 and ER stress markers, while upregulating phosphorylation of ERK5.
Conclusions:
These data support an important role for miR299a-5p in regulating apoptosis and fibrosis in DKD through regulation of MAP3K2/ERK5. Future studies will explore the potential role of regulating ERK5 activity as a potential therapeutic for DKD.
I have no potential conflict of interest to disclose.
I did not use generative AI and AI-assisted technologies in the writing process.