Farnesoid X receptor (FXR), a ligand-activated transcription factor, plays an important role in maintaining water homeostasis by up-regulating aquaporin 2 (AQP2) expression in renal medullary collecting ducts; however, its role in the survival of renal medullary interstitial cells (RMICs) under hypertonic conditions remains unclear. We cultured primary mouse RMICs and found that the FXR was expressed constitutively in RMICs, and that its expression was significantly up-regulated at both mRNA and protein levels by hypertonic stress. Using luciferase and ChIP assays, we found a potential binding site of nuclear factor kappa-B (NF-κB) located in the FXR gene promoter which can be bound and activated by NF-κB. Moreover, hypertonic stress-induced cell death in RMICs was significantly attenuated by FXR activation but worsened by FXR inhibition. Furthermore, FXR increased the expression and nuclear translocation of hypertonicity-induced tonicity-responsive enhance-binding protein (TonEBP), the expressions of its downstream target gene sodium myo-inositol transporter (SMIT), and heat shock protein 70 (HSP70). The present study demonstrates that the NF-κB/FXR/TonEBP pathway protects RMICs against hypertonic stress.

Unilateral ureteral obstruction (UUO) is associated with increased hydrostatic pressure, inflammation, and oxidative stress in the renal parenchyma. Previous studies have demonstrated marked cyclooxygenase (COX)-2 induction in renal medullary interstitial cells (RMICs) in response to UUO. The aim of the present study was to evaluate the effect of quercetin, a naturally occurring antioxidant, on COX-2 induction in vivo and in vitro. Rats subjected to 24 h of UUO were treated intraperitoneally with quercetin (50 mg·kg(-1)·day(-1)). Quercetin partly prevented COX-2 induction in the renal inner medulla in response to UUO. Moreover, RMICs exposed to conditions associated with obstruction, inflammation (produced by IL-1β), oxidative stress (produced by H2O2), and mechanical stress (produced by stretch) showed increased COX-2 expression. Interestingly, quercetin reduced COX-2 induction in RMICs subjected to stretched. Similarly, PGE2 production was markedly increased in RMICs exposed to stretch and was reversed to control levels by quercetin treatment. Furthermore, stretch-induced phosphorylation of ERK1/2 was blocked by quercetin, and inhibition of ERK1/2 attenuated stretch-induced COX-2 induction in RMICs. These results indicate that quercetin attenuated the induction of COX-2 expression and activity in RMICs exposed to mechanical stress as a consequence of acute UUO and that the MAPK ERK1/2 pathway might be involved in this quercetin-mediated reduction in COX-2.

OBJECTIVE: Metformin is a first-line antidiabetic drug for type 2 diabetes (T2D) with a relatively good safety profile. Metformin activates AMP-activated protein kinase (AMPK), which is crucial in maintaining renal medullary function, with inappropriate AMPK activation facilitating renal medullary interstitial cells (RMICs) apoptosis under hypertonic challenge. The present study was to determine the effects of metformin on RMIC survival in both normal and T2D mice.
METHODS: Mice (C57BL/6, db/m, and db/db) were treated with 450 mg/kg metformin for 7 days and subjected to 24-h water restriction (=dehydration) before being killed. Cell apoptosis in the renal medulla was determined by the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay. Cultured RMIC were treated with 10 mmol/L metformin in the presence or absence of hypertonic stress. Cell viability was determined and the underlying mechanisms were investigated.
RESULTS: Metformin induced significant apoptosis of RMIC in dehydrated normal mice and both hydrated and dehydrated T2D mice. Hypertonicity increased ATP production and inhibited AMPK phosphorylation in RMIC, which was attenuated by metformin. Metformin augmented hypertonicity-induced apoptosis of RMIC, suppressed the nuclear factor-κB/cyclo-oxygenase-2 pathway, reduced reactive oxygen species production and inhibited transcriptional activation of tonicity-responsive enhancer binding protein (TonEBP) and its downstream osmoprotective gene expression.
CONCLUSIONS: Metformin treatment is associated with increased RMIC apoptosis in both normally hydrated and dehydrated T2D mice. The results confirm AMPK as a critical factor involved in the maintenance of RMIC viability in T2D and raise safety concerns for metformin and other AMPK-activating antidiabetic drugs in dehydrated diabetic patients.

OBJECTIVE: 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 (H2 O2 ), 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.
CONCLUSIONS: 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.