Role of Long Noncoding RNA H19 and its Target microRNA/mRNA Pathways in Acute Kidney Injury: A Comparative Study in...

Hypoxia is a key regulator of renal epithelial cell function and contributes to the pathogenesis of kidney diseases. Although two-dimensional cell cultures are commonly used to study hypoxic responses, three -dimensional spheroid cultures better represent the in vivo microenvironment, including cell -cell and cell -matrix interactions. In this study, we compared hypoxia -induced gene expression in two - and three -dimensional cultures of HK2 cells.

We induced hypoxia at 12 hours in two -dimensional cultures and at 24 hours in three -dimensional spheroids, with confirmation of hypoxia by hypoxia -inducible factor 1α protein expression and vascular endothelial growth factor mRNA upregulation. With quantitative real -time polymerase chain reaction analysis, we noted that long noncoding RNA H19 was significantly upregulated in both culture models, accompanied by modulation of NLRP3, Wnt1, β -catenin, interleukin 1 β, interleukin 6, tumor necrosis factor α, interleukin 10, microRNA -30a -5p, and microRNA -196a.

Tumor necrosis factor α and microRNA -30a-5p expression were decreased, with strong interleukin 10 upregulation, in two -dimensional cultures. In three -dimensional cultures, expression levels of both microRNA -30a -5p and microRNA -196a were further upregulated, with less pronounced reduction of tumor necrosis factor α and limited increase of interleukin 10 expression.

Our findings suggested that microRNA -30a -5p and microRNA -196a may play a role in modulating hypoxia -induced cytokine responses, contributing to a more balanced cellular adaptation in three -dimensional cultures. Three-dimensional HK2 spheroids may provide a physiologically relevant model for studying hypoxic stress and may reveal the potential involvement of long noncoding RNA H19 and specific microRNAs in regulation of inflammatory and signaling pathways. Of note, this study represents one of the first investigations of hypoxia -induced gene expression in three -dimensional HK2 cultures, offering a novel contribution to understanding renal epithelial responses under low oxygen conditions.