Oscillating cortical thick ascending limb cells at the juxtaglomerular apparatus

Abstract

While studying the intracellular calcium dynamics in cells of the macula densa, the observation was made that tubular epithelial cells located near the macula densa and associated with the renal arterioles exhibit spontaneous Ca2+ oscillations. In this study, the cortical thick ascending limb-distal tubule, with attached glomerulus, was isolated and perfused. At a low luminal sodium chloride concentration, Ca2+ oscillations at a frequency of 63 mHz were observed in tubular cells that were within 100 microm of the macula densa plaque using four-dimensional multiphoton microscopy and wide-field fluorescence microscopy with fura-2. The Ca2+ oscillations were absent in the macula densa cells. Spontaneous oscillations in basolateral membrane potential suggested that Ca2+ oscillations occurred, at least in part, through depolarization-induced increases in Ca2+ entry. The amplitude of these Ca2+ oscillations was significantly enhanced by the activation of the Ca2+-sensing receptor. Increasing the luminal sodium chloride concentration or luminal flow resulted in a significant increase in both the amplitude of Ca2+ oscillations and the intracellular Ca2+ concentration in perimacular cortical thick ascending limb cells. In addition, luminal furosemide attenuated the [NaCl]L-dependent changes in intracellular Ca2+ concentration, but hydrochlorothiazide had no effect. These findings demonstrate that tubular epithelial cells at the perimeter of the macula densa exhibit spontaneous oscillations in intracellular Ca2+ concentration, enhanced by tubular flow and luminal sodium chloride. These oscillatory patterns may play a role in juxtaglomerular signaling.

Figure 1.

Representative three-dimensional segmented image demonstrating extensive region of contact between DT and afferent arteriole (AA). Portion of the cTAL has been optically removed to gain insight into the tubule. The macula densa (MD) is hidden beneath the tubular segments.

Figure 2.

Wide-field fura-2 imaging of the isolated perfused cTAL-DT preparation with attached glomerulus. (A) Bright-field photomicrograph demonstrating the cTAL, the DT, the MD plaque, and the glomerulus (G). The tubule was cannulated and perfused from the cTAL end. (B) Cumulative increases in [Ca²⁺]_i during the course of the experiment. (C) Representative [Ca²⁺]_i recording from a cell in the terminal cTAL. (D) Representative power spectrum density (PSD) plot of the [Ca²⁺]_i recordings in the frequency domain. Inset demonstrates the position of power spectrum peaks on the frequency domain.

Figure 3.

Four-dimensional multiphoton fluorescence imaging of isolated perfused tubular preparation (A) Schematic segmentation image showing the topography of the preparation in B. MD is indicated in purple. (B) Snapshots from four-dimensional reconstruction (see Supplemental Movie 1) showing spontaneous intracellular Ca²⁺ spikes (arrowheads) in the tubular epithelial cells in the perimeter of MD.

Figure 4.

Confocal imaging of membrane potential changes in the isolated perfused cTAL-DT preparation. (A and B) Three-dimensional reconstructed (A) and confocal emission ratio images (B) of the preparation loaded with the membrane potential–sensitive DiBAC₄(3) dye. Note the efferent arteriole (EA) and glomerulus (G). (C) Graph demonstrating the effect of membrane depolarization by application of KCl in the bath on the emission ratio values obtained from a preparation. (D) Pseudolinescan image along the dashed line in B demonstrating spontaneous oscillations in the membrane potential of a tubular cell in the cTAL.

Figure 5.

Effect of membrane depolarization on the [Ca²⁺]_i in the perimacular oscillatory cells. (A) Representative fura-2 ratio recording demonstrating the effect of KCl (from the lumen) on [Ca²⁺]_i in a perimacular cTAL cell located in the terminal portion of the cTAL. (B and C) Effect of bath (B) and luminal KCl (C) on [Ca²⁺]_i in perimacular tubular epithelial cells. ○, Fura-2 ratio values from individual cells; •, average values (n = 4, the values in the control and KCl groups were different from each other; P < 0.05).

Figure 6.

Effect of activation of calcium-sensing receptor on [Ca²⁺]_i in the perimacular oscillatory cells. (A) Representative fura-2 ratio recording demonstrating the effect of neomycin, an activator of calcium-sensing receptor (5 × 10⁻⁴ mol/L from the bath), on [Ca²⁺]_i in a perimacular cTAL cell located in the terminal portion of the cTAL. (B) Effect of neomycin on [Ca²⁺]_i in perimacular tubular epithelial cells. ○, Fura-2 ratio values from individual cells; •, average values (n = 8, one cell each from every preparation; the values in the control and neomycin groups were different from each other; P < 0.05).

Figure 7.

Characteristics of [NaCl]_L-dependent changes in [Ca²⁺]_i in macula densa and perimacular cTAL cells. (A and B) Wide-field fura-2 ratio (A) and pseudolinescan image (B) obtained along the line illustrated in A. Note the robust increases in [Ca²⁺]_i in the early DT upon increases in [NaCl]_L from 20 to 80 mmol/L (arrowheads).

Figure 8.

Effect of tubular flow and luminal [NaCl]_L on [Ca²⁺]_i in perimacular oscillatory cells. (A) Representative fura-2 ratio recording demonstrating the effect of elevated tubular flow from 20 to 40 nl/min (onset at the time point indicated by arrow) on the intracellular calcium dynamics in a perimacular cTAL cell located in the terminal portion of the cTAL. (B) Effect of elevations in tubular flow on the amplitude of oscillations in [Ca²⁺]_i in perimacular tubular epithelial cells. ○, Fura-2 ratio oscillation amplitude values from individual cells at different tubular flow conditions; •, average values (n = 6, one cell each from every preparation; the values in the control and high-flow groups were different from each other; P < 0.05). (C) Representative fura-2 ratio recording demonstrating the effect of an increase in [NaCl]_L and osmolality (from 20 to 80 mmol/L and from 98 to 210 mOsm/kg H₂O) on [Ca²⁺]_i in a perimacular cTAL cell located in the early portion of the DT. (D) Effect of elevations in [NaCl]_L and osmolality on [Ca²⁺]_i in perimacular tubular epithelial cells of the early DT. ○, Fura-2 ratio values from individual cells; •, average values (n = 12, one cell each from every preparation; the values in the control and high-NaCl groups were different from each other; P < 0.05).