Modified HEK cells simulate DCT cells in their sensitivity and response to changes in extracellular K

Abstract

A potassium (K+) rich diet is known to have an antihypertensive effect that has been embodied by the NHLBI in the DASH diet. However, the molecular basis for this blood pressure‐lowering effect has been unclear, until a recent study proposed a model in which the DCT cells of the kidney regulate their salt transport in response to variations in intracellular chloride ([Cl−]i), which are directly regulated by serum K+. With the knowledge that WNK proteins are Cl− sensors, and are a part of the WNK/SPAK/NCC signaling cascade which regulates the NCC, the main salt transporter in the distal nephron, we examined the effect of serum K+ on the ([Cl−]i) and, in turn its effect on the WNK4 signaling pathway in a “modified HEK 293T” cell line. Using a fluorescence‐based approach in this cell line, we have shown that the membrane potential of the cell membrane is sensitive to the small changes in external KCl within the physiological range (2–5 mM), thus functioning as a K+ electrode. When the extracellular K+ was progressively increased (2–5 mM), the membrane depolarization lead to a subsequent increase in [Cl−]i measured by fluorescence quenching of an intracellular chloride sensor. Increase in extracellular [K] resulted in a decrease in the phosphorylation of the WNK4 protein and its downstream targets, SPAK and NCC. This confirms that small changes in serum K can affect WNK4/SPAK/NCC signaling and transcellular Na+ flux through the DCT and provide a possible mechanism by which a K‐rich DASH diet could reduce blood pressure.

Mammalian Cell Models of the Distal Convoluted Tubule: A Systematic Review of Cell Lines, Culture Condition and Gene Expression

“…HEK293 lines lack the native capability to mimic the DCT's specific responses to changes in extracellular potassium concentrations. While this process presents considerable challenges, the advancements signal a move towards more representative models for studying renal potassium regulation and DCT cell function (43). This discrepancy underscores the need for transfection to introduce key DCT functionalities into HEK293 cell-based models, pointing to their limitations in mimicking native DCT gene expression profiles (44).…”

Serum aldosterone and urine electrolytes dynamics in response to DASH diet intervention – An inpatient mechanistic study

“…A change in ion channel composition in the tubule epithelium or activation may mediate the effects of the DASH diet. Previous studies tried to identify the relevant ion channel, based on cell cultures [43] and animal models [44], yet none had conclusive results.…”

Physiological Processes Modulated by the Chloride-Sensitive WNK-SPAK/OSR1 Kinase Signaling Pathway and the Cation-Coupled Chloride Cotransporters

“…This affects the driving force for Cl − movement through ClC-Kb channels, as suggested by the reduced basolateral Cl − conductance observed in Kir4.1 −/− mice (Zhang et al, 2014). Therefore, low [K + ] e promotes hyperpolarization, which increases Cl − efflux and decreases [Cl − ] i , whereas, increased [K + ] e promotes depolarization, lowers Cl − efflux, and increases [Cl − ] i (Terker et al, 2015b;Murthy and O'Shaughnessy, 2019). As explained previously in detail, [Cl − ] i is an important regulator of the WNK4-SPAK/OSR1 signaling pathway, which ultimately regulates NCC activity.…”