Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan

Correspondence: Sadayoshi Ito, M.D., Second Department of Internal Medicine, Tohoku University School of Medicine, 1 to 1, Seiryo-cho, Aoba-ku, Sendai, Japan. E-mail: db554@mail.cc.tohoku.ac.jp

Abstract

Characteristics of the isolated perfused juxtaglomerular apparatus. Tubuloglomerular feedback (TGF), which operates between the tubule and the parent glomerulus, is important to renal autoregulation and homeostasis of body fluid and electrolytes. The juxtaglomerular apparatus (JGA) has long been suggested as the anatomical site of TGF. To study the function of the JGA directly, we developed an in vitro preparation in which both the afferent arteriole (Af-Art) and macula densa (MD) of a microdissected rabbit JGA are microperfused simultaneously. We see that increasing the [NaCl] of the MD perfusate constricts the afferent arteriole in the segment close to the glomerulus. This constriction is blocked by furosemide, a loop diuretic known to inhibit TGF. On the other hand, microperfusion of Af-Arts alone showed the myogenic response to exist in the more proximal segments. Such an anatomical relationship between the myogenic response and TGF may enable the kidney to achieve its extremely efficient autoregulation.

Tubuloglomerular feedback (TGF), which operates between the tubule and the parent glomerulus, is important to renal autoregulation and homeostasis of body fluid and electrolytes^1,2. Because the juxtaglomerular apparatus (JGA) has an intimate anatomical relationship between the specialized tubular epithelial cells (macula densa, MD) and the vasculature (afferent and efferent arterioles), it has long been suggested as the anatomical site of TGF³. The first evidence of a functional link between the distal tubule and glomerulus was provided by Thurau and Schnermann⁴, who observed collapse of the proximal tubule on injecting solutions with a high [NaCl] into the early distal tubule of the same nephron. Numerous micropuncture studies have now established that each nephron has a feedbeck mechanism that operates between the distal tubule and glomerulus to control glomerular filtration rate (GFR).

To study the function of the JGA more directly, we developed an in vitro preparation in which both the afferent arteriole (Af-Art) and MD of a microdissected rabbit JGA are microperfused simultaneously Figure 1 and 2⁵. This preparation has the advantage of allowing control of both pressure in the Af-Art and luminal fluid composition at the MD. Real-time images of the Af-Art can be obtained. Unlike other preparations, few structures interfere with direct observation of the vascular pole, permitting accurate measurement of the luminal diameter even at the most distal segment, which is overlapped by the glomerulus. Finally, there are no systemic hemodynamic or hormonal influences.

Figure 1.

Schematic representation of the pipette arrangement used for perfusion of an afferent arteriole (Af-Art) and attached macula densa (MD). Abbreviations are: TALH, thick ascending limb of Henle's loop; GL, glomerulus; DCT, distal convoluted tubule; Ef-Art, efferent arteriole; Hold-Pip, holding pipette; Perf-Pip, perfusion pipette; Exch-Pip, exchange pipette; Pre-Pip, pressure pipette.

Full figure and legend (65K)

Figure 2.

Left: simultaneous perfusion of an afferent arteriole (Af-Art) and attached macula densa (MD). Right: after perfusion has been established, both the MD and Af-Art can be visualized. Abbreviations are: DCT, distal convoluted tubule; TAL, thick ascending limb of the loop of Henle.

Full figure and legend (217K)

Using this preparation, we have observed that increasing the [NaCl] of the MD perfusate constricts the afferent arteriole in the segment close to the glomerulus Figure 3⁵. This constriction is blocked by furosemide, a loop diuretic known to inhibit TGF, suggesting that tubular transport is a critical step for the initiation of this response. On the other hand, microperfusing Af-Arts alone showed that these constrict significantly when intraluminal pressure is elevated^6,7, demonstrating the myogenic response. Of note is the fact that the myogenic response and the TGF exist in series along the Af-Art, with the former being in the more proximal and the latter in the terminal segment. Thus, the myogenic response is the first to respond to changes in renal perfusion pressure to keep glomerular capillary pressure constant, whereas any changes in GFR that are not prevented by the myogenic response are reflected as changes in [NaCl] at the MD, with subsequent tuning of the distal segment by the TGF. Such interactions of the myogenic response and TGF enable the kidney to achieve an extremely efficient autoregulation. Indeed, when the distal [Cl^-] and the proximal tubular pressure, an index of single nephron GFR, are measured simultaneously, they oscillate synchronously, with an increase in the [Cl^-] being associated with a decrease in pressure⁸. These studies indicate that the TGF is an exquisitely intricate mechanism for maintaining a constant GFR at the single nephron level.

Figure 3.

Constriction of the afferent arteriole induced by a high [NaCl] at the macula densa (MD). With the afferent arteriole in focus, the MD cannot be seen clearly.

Full figure and legend (187K)

There are several advantages in our preparation. First, we can control the perfusion pressure in the Af-Art, eliminating the hemodynamic influence of the interlobular artery, which also expresses a myogenic response⁹. Second, because it is feasible to microdissect the midcortical and juxtamedullary JGA¹⁰, our preparation can be used to study the heterogeneity of MD-mediated glomerular hemodynamics among different nephron populations. Third, because our preparation is an isolated JGA, we will be able to manipulate various paracrine hormones much more effectively than we can in vivo. Therefore, this preparation will be useful in studying mediator(s) and/or modulator(s) of the mechanism by which the MD controls glomerular hemodynamics. Indeed, we observed that selective inhibition of nitric oxide (NO) synthase within the MD causes constriction of the Af-Art when the MD was perfused with a high [NaCl] but not with a low [NaCl]¹¹, demonstrating the modulatory role of the MD NO pathway in the TGF. Finally, it is feasible to perfuse both the efferent arteriole and the MD¹². Such preparation would be useful to study a possible role of the efferent arteriole in the MD control of glomerular hemodynamics.