NFAT5 upregulates osmolyte transporters to protect IMCD3 cells against hyperosmolarity

Abstract

The kidney inner medulla is one of the only places in the body exposed to extremely high osmolarity, reaching up to 1300 mOsm, while the iso-osmotic cortex and plasma are ≍300m mOsm. Renal epithelial cells need a mechanism to address the stress that comes with hyperosmolarity. One possible mechanism is regulation of osmolyte transporters to increase intracellular osmolyte concentration and reduce osmotic stress. NFAT5 is an important factor in regulating renal epithelial cell responses to high osmolarity. We hypothesize that NFAT5 is protective in inner medullary collecting duct cells (IMCD3) via upregulation of key transporters. To test this hypothesis, we used CRISPR to delete NFAT5 in IMCD3 cells. IMCD3 -NFAT5 null (NFAT5 null) and control (Ctl, IMCD3) cells were used and cell death assessed. Cells were plated at the same concentrations, in media (320mmol) with additional osmolarities of: urea (160mmol), mannitol (160mmol) and salt (40 and 80 mmol). Cell death was measured over 5 days using Janus Green B, which permeates living cell membranes. Cells were incubated with Janus Green B, lysed and then absorbance measured with a spectrophotometer to determine concentration. Data were analyzed as mean ± SEM; cell confluence, two-way ANOVA, n=3-5. Total mRNA was also isolated for qPCR analysis and data expressed as a fold change (DCT) over housekeeping (PPIA). At iso-osmolarity (»300mOsm), no differences were observed between NFAT5 null and Ctl cells. However, by day 3, NFAT5 null cells had reduced cell growth in all hyperosmolar conditions: urea 160 mmol (1.192 ± 0.225 vs. 1.953 ± 0.167 Wt ****p<0.0001, n=4), mannitol 160 mmol (0.852 ± 0.135 vs. 1.683 ± 0.283 Wt ****p<0.0001, n=3-4), NaCl 40 mmol (1.745 ± 0.210 vs. 2.020 ± 0.252 Wt ****p<0.0001, n=4-5), NaCl 80 mmol (0.385 ± 0.059 vs. 1.089 ± 0.191 Wt ****p<0.0001, n=4-5). Interestingly, NFAT5 null cells recovered growth patterns similar to Ctl cells by day 5. This suggests that although NFAT5 is essential in the cellular response hyperosmolarity, other mechanisms are present that allow for adaptation over time. To address a possible mechanism, we performed qPCR investigating common osmo-protective genes. We hypothesized that expression is lower in the NFAT5 null cells as compared to Wt. When cultured with 40 mmol of NaCl (n=2), we found that the NFAT5 null cells showed a 3.31 fold reduction for SMIT1, a 2924.98 fold reduction for TauT, and a 4.91 fold reduction for BGT1 as compared to Wt, each of which are osmolyte transporters. Additionally, we saw a 161.46 fold reduction for AQP1, a water channel and a 4.91 fold reduction for Akr1b3, which influences water homeostasis, as compared to Wt. Together, these results suggest that NFAT5 is a critical regulator of both osmolyte and transporters. Here, we show that loss of NFAT5 reduces IMCD cell viability in the face of hyperosmotic stress, possibly through a reduction in key osmolyte transporters. These data suggest that NFAT5 is key in the renal epithelial cell response to, and survival in, the hyperosmolar environment of the inner medulla. Undergraduate Research Opportunity Program grant NIH NIDDK DK115660-01A1 University of Utah DOIM, UCares. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.