Aim In vivo, renal medullary interstitial cells (RMICs) and collecting duct principal cells (mpkCCD cells) are subjected to inflammatory, oxidative and mechanical stress as a result of unilateral ureteral obstruction (UUO). Because heat‐shock protein (HSP) 27 and HSP70 are induced by cellular stresses and play a role in cytoprotection, we hypothesized that HSP27 and HSP70 are increased in rats subjected to acute UUO and in RMICs and mpkCCD cells exposed to inflammatory, oxidative or mechanical stress. Methods Rats were subjected to acute UUO for 6 h and 12 h. To examine the expression of HSP27, phosphorylated HSP27 (pHSP27) and HSP70 in response to inflammatory, oxidative and mechanical stress in vitro, we exposed RMICs and mpkCCD cells to interleukin 1β (IL‐1β), hydrogen peroxide (H2O2), and stretch stimulation over time. Results The phosphorylated form of HSP27 (pHSP27) was increased in the renal inner medulla (IM) after 6‐h and 12‐h UUO, while HSP27 and HSP70 were unchanged. Furthermore, after 6 h and 12 h of UUO, the expression of inflammatory (IL‐1β) and oxidative [haem oxygenase 1 (HO‐1)] markers was induced. Exposure to inflammatory, oxidative and mechanical stress changed HSP27 and pHSP27 expression in RMICs but not in mpkCCD cells, while HSP70 was not affected by any of the stress conditions. Exposure of RMICs to oxidative and mechanical stress induced HSP27 phosphorylation via a p38‐dependent mechanism. Conclusion These data demonstrate that, in response to acute UUO, different forms of cellular stresses modulate HSP27 expression and phosphorylation in RMICs. This may affect the ability of renal cells to mount an effective cytoprotective response.