Temporal adjustment of the juxtaglomerular apparatus during sustained inhibition of proximal reabsorption (original) (raw)
The existence and functional significance of the TGF system have been established in publications from many laboratories. Most of this research has involved examining adjustments in glomerular filtration brought about within 2–3 minutes in order to compensate for abrupt changes in tubular flow. In such studies, the overall efficiency of the TGF system has been quantified, and the range of tubular flow rates over which TGF is maximally efficient has been shown to surround the natural tubular flow rate (1).
For the purposes of most micropuncture experiments, the behavior of TGF is considered to be static. However, it is obvious that TGF cannot be a static process. For example, the immediate action of TGF is to confer an inverse dependence of SNGFR on VLP. Therefore, any physiologic circumstance that permits or requires SNGFR and VLP to change in the same direction also requires a change in the behavior of TGF. Trivial examples of this include normal growth and development of the kidney, pregnancy, the renal response to changes in extracellular volume, changes in cardiovascular hemodynamics, and the single-nephron response to reduced renal mass. Furthermore, TGF adaptation to changing physiologic circumstances cannot be accounted for merely by increases or decreases in the maximum range of the TGF response or by changes in the TGF slope, because such changes would not account for the preservation of TGF efficiency that usually occurs (1).
Whereas it is clear that TGF must be capable of resetting, current understanding of how this occurs is minimal. In the past, we documented rightward TGF resetting within 1 hour when proximal flow was supplemented (2) or proximal reabsorption was inhibited (12). Therefore, it is possible to link TGF resetting to changes in the TGF signal that persist for 1 hour. The present studies were performed to examine the nature of TGF resetting over a period of 24 hours, a model arguably more analogous to certain real-life situations, such as the need for the kidney to grow or to respond to changes in salt intake. To induce TGF resetting, animals were repeatedly administered the proximal tubular diuretic BNZ. Rather than test for evidence of resetting under the continued influence of BNZ, we elected to look for evidence of resetting in the form of rebound hyperfiltration after the effects of BNZ on proximal reabsorption had subsided. The justification for this approach is as follows: Under the continuous influence of BNZ, SNGFR would likely remain somewhat less than baseline and VLP would likely remain somewhat greater than baseline, regardless of whether or not TGF has reset. When SNGFR and VLP change in opposite directions, single measurements of SNGFR and VLP obtained before and after these changes cannot reveal whether the nephron has shifted onto a new TGF curve or has moved along an original TGF curve. In contrast, SNGFR and VLP cannot change in the same direction unless there has been resetting of the TGF curve. Therefore, a simultaneous increase in both SNGFR and VLP to values above baseline would constitute positive proof of TGF resetting. We predicted that such proof might be obtained after the primary effect of BNZ had mostly subsided, but would not be revealed by data collected under the full ongoing influence of BNZ. Furthermore, the specificity of the results obtained with the chosen protocol would not be affected by tachyphylaxis to BNZ, inconstant tissue levels of BNZ, or reduced extracellular volume resulting from BNZ. In fact, the only assumptions required by the present approach regarding the pharmacokinetics or pharmacodynamics of BNZ are that BNZ did reduce proximal reabsorption and that it was less than maximally active at reducing proximal reabsorption during the time that physiologic data were being collected. A pictorial presentation of this argument is contained in Figure 6.
Graphic representation of the hypothesis to account for the observed effects of BNZ on ambient SNGFR and VLP, and to justify performing measurements after BNZ rather than during BNZ. The sigmoid curves refer to various TGF curves. Solid lines refer to the dependence of VLP on SNGFR owing to proximal GTB. Points A–E refer to the operating points where the various TGF and GTB curves intersect. Arrows refer to the PDD at each operating point. The control nephron operates at point A. The immediate effect of BNZ is to reduce proximal reabsorption, thereby moving the nephron to point B, where the TGF curve is relatively flat. Under the continuous influence of BNZ, the TGF curve resets rightward and the nephron moves to point C, where TGF efficiency is restored. If, as we hypothesize, it is the persistent activation of TGF that provides the stimulus for TGF resetting, then it follows that SNGFR (C) ≤ SNGFR (A) and VLP (C) > VLP (A). However, establishing these 2 inequalities cannot verify TGF resetting, because the same inequalities apply to the relationship between point A and point B. Furthermore, administration of the diuretic BNZ might activate sodium-conserving mechanisms extrinsic to TGF, which would buffer the effect of BNZ on proximal reabsorption and exert a downward pressure on GFR through mechanisms that operate independently of TGF. Therefore, comparing point A with point C was not considered an appropriate means to test for TGF resetting during BNZ treatment. On the other hand, allowing proximal tubular reabsorption to increase by withdrawing BNZ would move the operating point to point D, where SNGFR (D) > SNGFR (A) and VLP (D) > VLP (A). These 2 inequalities cannot both be satisfied unless there is rightward and/or upward resetting of TGF. Furthermore, increases in SNGFR are contrary to any expected outcome mediated through the effects of BNZ on extracellular volume. Therefore, establishing that SNGFR (D) > SNGFR (A) and VLP (D) > VLP (A) is sufficient to validate that TGF has reset. However, it cannot be determined from the coordinates of the operating point whether the resetting is primarily rightward (point D) or rightward and upward (point E). This latter issue can be addressed by the testing for changes in the PDD and by measuring SNGFR from the proximal tubule during minimum and maximum stimulation of TGF. If TGF resets purely rightward during BNZ, then the increase in ambient SNGFR after BNZ is withdrawn will be associated with a subnormal PDD (point D). If the PDD is not subnormal after withdrawal of BNZ, then there must be an upward component to the resetting of TGF (point E). In the present experiments, the PDD was unaffected by previous treatment with BNZ, inferring that the TGF curve had shifted both upward and rightward.
In this study, it was considered that TGF resetting in response to reduced proximal reabsorption might require increased NO formation by the macula densa. The basis for this consideration derives from a previous micro-puncture study in which we demonstrated that the TGF curve shifted downward, leftward, and became more steep when macula densa NOS was blocked acutely by infusing the nonselective NOS blocker L-NMMA into the lumen of the free-flowing late proximal tubule (5). Those observations implied that NO formed by the macula densa of the euvolemic rat exerts a tonic upward pressure on SNGFR and a rightward pressure on the TGF curve, and suggested that a rightward and upward shift in the TGF curve would be the expected result if endogenous macula densa NO activity were to increase. Furthermore, others have suggested that the tonic influence of macula densa NOS over nephron function and TGF is enhanced when rats are fed a high-salt diet (13, 14), even though the high-salt diet reduces immunoreactive NOS in the macula densa (15, 16). There are few parallels to draw between rats receiving the diuretic BNZ and rats receiving a high-salt diet. However, these disparate treatments each cause flow to increase downstream from the early proximal tubule; each involves resetting of TGF to accommodate higher tubular flow; and each is associated with enhanced sensitivity of GFR to reduction by NOS inhibitors.
In the present study, NOS-I blockers were either administered systemically, along with each dose of BNZ, or added to the proximal tubular fluid of individual nephrons at the time of micropuncture. Both of these approaches yielded data consistent with idea that normal TGF resetting can be prevented by blocking macula densa NOS. As a caveat, NOS-I outside of the kidney performs a counterregulatory function in the control of renal sympathetic nerve traffic at the level of the central nervous system (17) and spinal cord (18). Because treatment with the diuretic could predispose to increased renal sympathetic nerve traffic, this role of NOS-I may have contributed to the subnormal GFR observed in rats treated with both 7-NI and BNZ. However, the heightened sensitivity of the post-BNZ nephron to microperfusion with SMTC should be free of any such confounding influence.
Adding SMTC to ATF in control rats reduced SNGFR by similar amounts, regardless of whether TGF was being maximally or minimally activated at the time. The effects of SMTC on SNGFR are remarkably similar to effects that we reported previously for microperfusion with L-NMMA (5). When delivered into nephrons of post-BNZ rats, the effects of SMTC on SNGFR were similar in character, but of significantly greater magnitude than the effects of SMTC on control nephrons. As a result, the difference in SNGFR between control and BNZ groups was substantially reduced when each was perfused with SMTC (Figure 3). This confirms that macula densa NO exerts a greater tonic influence over SNGFR after BNZ, as would be expected if increased NO were responsible for resetting of TGF during BNZ treatment. To our knowledge, this experimental model is the first setting in which changes in macula densa NOS immunoreactivity and the magnitude of an associated physiologic response to NOS inhibition have not been paradoxical (13–16).
TGF is defined traditionally as a series of processes that occur in the JGA. However, when examined by micropuncture, it is most common to manipulate the TGF system from a site in the late proximal tubule. When this approach is taken, changes in loop of Henle function that alter the relationship between VLP and salt concentration at the macula densa may falsely suggest changes in the behavior of TGF. In this study, separate experiments were performed to test for the possibility that the apparent resetting of TGF could be due to a compensatory increase in loop of Henle transport. Indeed, previous treatment with BNZ was associated with increased loop of Henle reabsorption of both water and solute. However, the early distal flow rate remained elevated in the post-BNZ animal, and neither fractional reabsorption nor the ionic content of early distal tubular fluid was changed by previous treatment with BNZ. For a primary increase in loop reabsorption to account for the observed adaptation of TGF, the NaCl concentration at the macula densa during hyperfiltration must assume a value below baseline. If this criterion is not met, then increases in reabsorption noted during hyperfiltration are consistent with GTB or flow-dependent increases in transport, but not with a primary increase in transport sufficient to mimic the apparent resetting of TGF.
To normalize for differences in delivered load, changes in tubular function can be evaluated by examining fractional reabsorption. Free-flow micropuncture data alone do not yield a complete picture of the effects of previous BNZ administration on the loop of Henle. Therefore, we performed microperfusion studies that revealed that the volume of fluid reabsorbed from the loop of Henle was increased in post-BNZ animals, but only during perfusion with supraphysiologic concentrations of bicarbonate. This suggests an increased capacity for sodium-proton exchange in the nephron downstream from the perfusion site that is not brought to bear under natural conditions. One limitation of perfusing individual nephrons to study loop of Henle reabsorption is that the increment in volume reabsorbed per increment in applied VLP will underestimate the change that occurs when an equivalent increase in VLP affects a whole population of nephrons. This underestimation occurs because the hydro-osmotic force that governs water flux from the descending limb is influenced by the sum of the solute that is reabsorbed into the interstitium from the ascending limbs of many nearby nephrons, not just the index nephron. This accounts for the fact that the volume absorbed from the loop of Henle showed little dependence on VLP during microperfusion. However, using changes in early distal ionic content during loop perfusion as evidence for true changes in ascending limb function should not be encumbered by this problem, except when delivery to the ascending limb is affected. Given these caveats, the microperfusion data did not suggest a major primary increase in loop reabsorption after BNZ administration sufficient to account for an increase in SNGFR to the levels observed. This point was reinforced by the lack of any increase in the main thick ascending limb transporter, BSC-1. Therefore, whereas ambient reabsorption of both salt and water from the loop of Henle was increased after prolonged inhibition of proximal reabsorption, the events responsible for TGF resetting in this model must take place within the JGA rather than within the loop of Henle.
These studies demonstrate that during a sustained reduction in proximal reabsorption, events occur within the JGA that alter the behavior of TGF in such a way that both SNGFR and VLP become supranormal when proximal reabsorption returns toward normal. When TGF is depicted as a curve in the plane defined by VLP and SNGFR, this TGF adaptation is represented by a shift in the TGF curve that is upward and to the right (Figure 6). At the same time, there is only a small reduction in the maximum range of the TGF response, and the magnitude of the tonic influence exerted by TGF over ambient SNGFR is minimally affected. Thus, the steepest portion of the TGF relationship remains aligned with the actual tubular flow for optimal TGF efficiency. This is not to say that TGF efficiency is unaffected by previous treatment with BNZ. TGF efficiency, or the degree to which a TGF response succeeds in offsetting a particular disturbance in tubular flow, depends on the actual slope of the TGF curve, not merely on where the steep portion of the curve sits relative to the tubular flow (1). It is possible that the maximum slope of the TGF curve was affected by previous treatment with BNZ, but the current studies were not designed to test this hypothesis.
The type of TGF resetting observed after BNZ treatment resembles the TGF adaptation that accompanies normal growth, in the sense that SNGFR and VLP both increase, whereas the macula densa signal, as indexed by the ionic content of the luminal fluid, remains normal. These events correlate with increased expression of NOS-I activity in the macula densa and can be largely prevented by inhibition of NOS-I. Why is such adaptation important? The ability of TGF to reset is essential to maintaining the effectiveness of TGF as a mediator of short-term or dynamic autoregulation of nephron function. Also, and perhaps more importantly, the loss of normal TGF adaptation would curtail the ability to effect long-term control of the extracellular volume through changes in proximal tubular reabsorption. This is illustrated by the following example. When a step increase in salt intake is imposed on an animal that is salt-balanced, salt excretion will increase progressively until a new equilibrium is achieved a few days later. The amount of salt accrued in the course of reaching the new equilibrium will be proportional to the time required to reach that equilibrium (19). The natriuretic response to salt loading normally includes a shift in proximal GTB, i.e., reduced proximal reabsorption at any given SNGFR (20). This shift occurs because the proximal tubule is sensitive to neurohumoral signals that are linked to changes in total body salt. However, the natriuretic effect of any given shift in proximal GTB depends on whether there is simultaneous resetting of TGF. Whereas the nature of TGF is to buffer any short-term disturbance in GTB, thereby stabilizing VLP, a rightward resetting of TGF will reduce the increment in total body salt required to bring about a given increment in VLP through reduced proximal reabsorption. Thus, rightward resetting of TGF will shorten the time required to reach equilibrium after an increment in salt intake, and will lessen the long-term impact of salt intake on steady-state salt content. Conversely, impaired TGF resetting would prolong the time required to achieve equilibrium salt balance in response to increased salt intake, thus increasing the effect of salt intake on steady-state total body salt content. Thus, the phenomenon of TGF resetting has obvious consequences for the long-term control of blood pressure.
Most research concerning TGF in pathophysiologic states has focused on assessing the acute TGF response. Such studies have demonstrated that TGF efficiency or the maximum TGF response can be suppressed or enhanced in certain conditions. This study confirms the previous observation that TGF normally resets rightward during a sustained reduction in proximal reabsorption (12), and demonstrates that this resetting can persist for many hours after the stimulus for resetting is removed. Furthermore, this form of TGF resetting appears to require generation of NO in the macula densa. Finally, these results suggest that time be viewed as a relevant dimension when considering the potential pathophysiologic consequences of altered interactions between the tubule and glomerulus.