Immortalized Mouse Cortical Collecting Duct Cell Line (mCCD(cl1))

Downregulation of Hemin-Induced Nrf2 Pathway Activation Decreases Hepcidin Synthesis

The release of hemoglobin during hemolysis can lead to deposition in renal tubules, causing oxidative stress and AKI. It has been reported that urinary hepcidin levels are elevated in patients with AKI, which indicates the protective role of kidney-derived hepcidin, but the regulatory mechanism is not yet known. The Nrf2 pathway is crucial for cellular stress defense and regulates hepcidin synthesis in the liver. Iron exposure activates Nrf2 in proximal tubule cells, and hemin induces hepcidin synthesis. Diepeveen et al. explored whether Nrf2 mediates hemin-induced hepcidin expression in mCCD_cl1 cells and how Nrf2 modulation affects this synthesis, using the inhibitor trigonelline.

Treatment with hemin increased HO-1 mRNA expression as an indicator of heme catabolism and oxidative stress, which was attenuated by co-incubation with trigonelline (Fig. 1A). Nrf2 activation was shown by an increase in mRNA of target genes TXNRD1, GCLM and NQO1, and increase in NQO1 protein (Fig. 1B, C). Trigonelline caused a significant decrease in KEAP1 and GCLM mRNA, and NQO1 protein expression (Fig. 1B, C). Nuclear Nrf2 protein levels increased after incubation with hemin while the total cellular Nrf2 levels did not change (Fig. 1D). Co-incubation with trigonelline significantly reduced the Nrf2 translocation, however, this effect was also seen in control cells, so the data suggests that hemin activates the Nrf2 pathway in mCCD_cl1 cells, which can be inhibited by trigonelline. Hemin significantly increased hepcidin mRNA expression, as well as luciferase activity, both of which were decreased after treatment with trigonelline. The data presented above suggest that hepcidin synthesis in these cells is dependent on Nrf2 activation and occurs after hemin-induced injury.

Fig. 1. Hemin-induced Nrf2 pathway activation and subsequent downregulation using trigonelline in mCCD_cl1 cells (Diepeveen LE, Stegemann G, et al., 2022).

Aldosterone Increases Expression of Claudin-8 in Cultured Mouse CD Principal Cells

The aldosterone-sensitive distal nephron, a major site of transcellular Na⁺ reabsorption, tightly couples its activity to the tight-junction protein claudin-8, but the molecular mechanism is unknown. To test the hypothesis that aldosterone signaling through the mineralocorticoid receptor and the kinases GSK-3, Lyn and Abl up-regulates claudin-8 to strengthen the paracellular Na⁺ barrier.

Sassi et al. investigated how aldosterone regulates the expression of tight-junction core proteins in collecting-duct principal cells (mCCD_cl1). Aldosterone augmented transepithelial current without affecting transepithelial resistance (Fig. 2A). Blockade of ENaC activity with 10⁻⁵ M benzamil only slightly increased resistance in control and aldosterone-treated monolayers, indicating that ENaC activity only modestly contributes to resistance. Of the three major claudins expressed in collecting ducts (claudin-4, -7 and -8), only claudin-8 was regulated: aldosterone time-dependently increased claudin-8 protein and mRNA, while claudin-4, claudin-7 and the tight-junction scaffold protein ZO-1 were unchanged (Fig. 2B, D). Immunofluorescence confirmed that the increase of claudin-8 occurred along the lateral membrane (Fig. 2C). The aldosterone-induced up-regulation of the canonical target Sgk-1 served as a positive control (Fig. 2B, C).

Fig. 2. Aldosterone (Aldo) modulates the expression of claudin-8 in cultured mouse collecting duct principal cells. mCCD_cl1 cells were grown to confluence on filters and treated or not treated with 10−6 M aldosterone for 24 h (Sassi A, Wang Y B, et al., 2021).