CONCEPT OF TUBULOGLOMERULAR FEEDBACK

The vascular response

Goormaghtigh, Harsing, and Thurau clearly recognized that the existence of a tubulovascular connection at the site of the macula densa (MD) provides an ideal pathway along which changes in the composition of the urine at that point can affect afferent arteriolar tone and thereby glomerular filtration rate (GFR)^1,2,3. The site of the MD is particularly suited for the location of a chemoreceptor because [NaCl] at this site is hypotonic, variable, and determined almost exclusively by loop of Henle flow rate. Beginning with Thurau's microinjection experiments, numerous investigators have now established firmly that GFR is in fact inversely related to [NaCl] at the MD⁴. The vascular response to a change in [NaCl] occurs over a limited range of concentrations extending, in the hydropenic rat, from about 15 to 60 mM⁵. The response is nonlinear with sensitivity greatest in the range of ambient [NaCl], about 20 to 30 mM in the hydropenic rat. Because an increase in [NaCl] decreases single nephron GFR (SNGFR), the relationship between epithelial and vascular cells represents a negative feedback loop, usually called the tubuloglomerular feedback (TGF) mechanism.

The renin secretory response

It is now clear that changing MD [NaCl] has a second effect: it regulates the secretion of renin from the granular cells of the juxtaglomerular apparatus (JGA) and thereby is a determinant of extracellular angiotensin II (Ang II) concentrations. As originally suggested by Vander, a decrease in MD [NaCl] stimulates, and an increase inhibits renin secretion⁶. This response, like the TGF response, has been shown in vitro to occur over the range of about 10 to 60 mM. The difference between minimal and maximal rates of secretion in this preparation is large, about sixfold⁷. Concurrent operation of the two MD-dependent mechanisms might be predicted to generate two rigidly coupled functional triads: states of high MD [NaCl] associated with vasoconstriction and an inhibited renin-angiotensin system (RAS) or, conversely, states of low MD [NaCl] combined with vasodilatation and activation of the RAS, yet these triads are almost never observed. In all states of volume depletion, for example, MD [NaCl] and transport are almost certainly decreased, but these are states of vasoconstriction, not vasodilatation. It is likely that this paradox can be resolved by the very different temporal characteristics of the two responses.

Co-ordination of vascular and renin secretory responses

The TGF response, which takes about 10 seconds to become maximal, is the faster by far of the two responses. The renin secretory response in the single perfused JGA increases over 5 to 15 minutes; in the whole animal, the time course is not quite clear. Maximal changes in plasma levels of Ang II produced by a persistently changed MD [NaCl] probably require 30 to 60 minutes. These temporal characteristics make it likely that TGF contributes substantially to the control of afferent arteriolar tone when distal [NaCl] is changed by rapid and randomly oscillating factors. Such factors may impact on GFR without being related to body salt balance. An example would be step changes in arterial pressure, changes that, in the physiological context, are typified by the random and fast changes in blood pressure found during long-term pressure recordings⁸. These random fluctuations in MD NaCl probably are dampened effectively by operation of TGF but, given their high frequency, do not cause changes in renin secretion, and certainly not systemic plasma renin and Ang II levels Figure 1.

Figure 1.

Function of the juxtaglomerular apparatus (JGA). (A) Short-term function. With random high-frequency perturbations, macula densa (MD) [NaCl] and glomerular filtration rate (GFR) oscillate around a set point, whereas plasma renin does not change. (B) Long-term function of the JGA. With prolonged perturbations exceeding the operating range of the tubuloglomerular feedback (TGF) mechanism, plasma renin changes concomitantly with resetting of the TGF function curve. Resetting permits stabilization of MD [NaCl] and GFR at a new operating point.

Full figure and legend (31K)

On the other hand, renin secretion and extracellular Ang II levels will change when MD [NaCl] is dislodged from its set point for a prolonged time. This will occur when the combined external forces determining GFR and proximal and loop absorption cause deflections of MD [NaCl] that exceed the range of flows over which TGF operates effectively. Deflections of MD [NaCl] will then inevitably change both renin secretion and extracellular [Ang II]. The time frame within which MD [NaCl] may be expected to produce this effect is not quite certain but can be estimated to be in the order of 30 to 60 minutes. Thus, tight control of distal NaCl delivery by TGF is the function of the JGA only in the short term. In the long term, the JGA is primarily responsible for the secretion of renin at a rate that is optimal for the maintenance of Na balance.

It is important to realize Figure 1 that the TGF system in the new steady state, after MD [NaCl] and renin secretion have changed, does not remain inoperative but regains homeostatic efficiency by the phenomenon of resetting, an observation anticipated and first made by Thurau^4,9. In volume expansion, for example, inhibition of proximal or loop of Henle transport causes MD [NaCl] and transport to increase. This change, when it persists, causes renin secretion to decrease but also causes TGF to adapt in the sense that it becomes less sensitive. Thus, when [NaCl] at the MD is increased for a prolonged time, the decrease in GFR predicted from the acute TGF function does not occur. Rather, steady-state GFR stays the same or may even increase. The advantage is twofold: adaptation keeps TGF in the regulatory range so that it can continue to function as a fast controller, and it prevents changes in GFR when these would be maladaptive.

The mechanism of resetting is presumably multifactorial, but the most important single factor is extracellular Ang II, with increasing Ang II causing a left shift and decreasing Ang II causing a right shift of the TGF function curve. Because Ang II is controlled by chronic changes in MD NaCl, the phenomena of MD-controlled renin secretion and TGF resetting are mechanistically coupled. The mechanism by which Ang II affects the TGF function curve is presumably a reflection of its specific interaction with adenosine, which is an important mediator of the TGF response.

Should the TGF system, in fact, act only as a fast control system, its regulatory importance under chronic conditions may have to be reconsidered. When it is observed experimentally that MD [NaCl] is increased or decreased, and one may assume that this change has persisted for some time, it would seem a priori unlikely that TGF has been responsible for any inverse changes in GFR that may be found at the same time. For example, an increased MD [NaCl] following prolonged inhibition of proximal transport (as occurs with a carbonic anhydrase blocker) would be unlikely to be the cause of the observed fall of GFR. It is more likely that the predicted fall of renin secretion in this condition (provided volume depletion is prevented), or some other consequence of increased distal flow, will cause TGF adaptation, thereby preventing a decrease of GFR. If GFR decreases anyway, this is probably due to some other, non—JGA-related causes. Furthermore, observing that the TGF function, as reflected in the relationship between late proximal flow (V_LP) or MD [NaCl] and SNGFR, is altered or reset, the conclusion that the change in the TGF function is a primary event permitting MD [NaCl], and with it GFR, to change may not be justified. For example, the desensitization of TGF in volume expansion has been assumed to be causal in the elevated GFR often found in this condition. However, it may be more likely that the relationship is just the reverse, that is, that a change in [NaCl] at the MD elicited by factors extrinsic to the JGA causes the feedback function to adapt or reset. This notion has recently also been expressed by Thomson et al, and we fully share their sentiment that "TGF resetting is more likely a result, rather than a cause, of any sustained increase in (SN)GFR"¹⁰.

NEW DEVELOPMENTS IN TGF REGULATION

Transgenic mice as experimental models

The problem of the identity of humoral or other factors participating in mediation of the NaCl-induced vasomotor response has been difficult to study. Progress in this area has depended largely on studies of the effect of pharmacological interventions designed to interfere with the operation of potential mediator pathways. Despite the important insights yielded by the administration of exogenous agonists and antagonists, the validity of conclusions reached depends on assumptions that cannot always be proven, particularly not in complex in vivo studies. In general, neither the specificity of the pharmacological agent used nor the completeness of blockade can be definitively established, mainly because the concentration at the site of action is usually unknown. Furthermore, experimental approaches may be too demanding to permit the construction of full dose-response relationships, and conclusions are often based on the use of single drug doses.

Advances in transgenic and gene-targeting approaches provide alternative or complementary approaches to studying complex cell-cell signaling pathways. Random insertion of gene constructs by either retroviral gene delivery or injection of pronuclei has generated animals, usually mice, that overexpress certain genes and are therefore an alternative method to minipump or other chronic infusion studies for examining the long-term effect of endogenous bioactive agents^11,12. Homologous recombination, in contrast, has permitted the generation of transgenic mice with targeted deletions of specific genes^13,14. Thus, these animals can be used as the genetic equivalent of pharmacological blocker or surgical ablation studies. Definitive, and often unexpected, information on the developmental contributions of certain genes has been obtained. These animals also offer a new and independent means of addressing some of the questions that have been studied previously with pharmacological tools, and they are, therefore, increasingly used in studies of the effect of gene deletions on specific tissue and organ functions.

The use of gene-knockout models offers the unique advantage that, with an optimal targeting construct, a gene product of interest simply is not expressed in the animal; thus, a change in function implies a direct or indirect role of this gene in the phenomenon under study. On the other hand, if no effect is seen, one may conclude either that the targeted gene is of no, or only minor, importance in the studied system or that compensatory mechanisms have been up-regulated to take the place of the deleted product. Studies of such compensatory mechanisms acting in mice with null mutations would seem to be promising in their own right. When the gene deletion is lethal either before or shortly after birth, the use of transgenic mice is limited to heterozygous animals¹⁵. In general, expression of the targeted gene in heterozygotes is intermediate between the normal and the homozygous mutant animal, and the change in the level of the gene product may be sufficiently large to cause detectable functional alterations. Functional approaches in null mutant animals may also be problematic if the gene deletion causes severe structural alterations in the organ under study¹⁶.

Studies of renal function in mice

Due no doubt to the size of the animal, about one tenth of that of a laboratory rat, measurements of baseline physiological parameters in the mouse are not abundant but are needed to permit comparison with animals carrying single gene mutations. The acquisition of a wide array of such data will certainly require miniaturization of existing methods, a process that is hoped to be driven eventually by increasing demand. However, in some instances, methods that have been used for studies in rats can be directly transferred to mouse studies.

We have explored the feasibility of utilizing standard micropuncture techniques for studying nephron function in both normal and transgenic mice and have found that this approach is applicable to mice with relatively minor modifications¹⁷. A combination of thiobutabarbital (100 mg/kg i.p.) and ketamine (50 mg/kg i.m.) reliably induces a controllable level of anesthesia. Not surprisingly, preparation of mice for renal micropuncture is substantially more time consuming than in rats, mainly due to more difficult blood vessel cannulation requiring exceptional care and patience. Cannulation of the femoral vein and artery with fine, hand-drawn polyethylene catheters is felt to be the best method of vascular access for infusion (2.25 g% bovine serum albumin in saline at 0.5 ml/hr) and blood pressure (BP) measurement. Although it is possible to cannulate the ureter close to the kidney, we have found that these catheters have a tendency to obstruct with small urinary crystals so that urine is collected usually with a bladder catheter.

Baseline renal function in C57BL/6 mice (body wt 30 to 40 g) is summarized in Table 1. Mean arterial BP (MAP) in 13 anesthetized and laparatomized animals was 87 6 mm Hg. Mean renal blood flow determined with a Transonic Systems ultrasonic flowmeter and a 0.5-mm V-series probe averaged 1.1 ml/min per kidney (N = 9). GFR determined as ¹²⁵Iothalamate clearance was 0.18 0.02 ml/min/kidney. Nephron number, determined as the average of 20 glomerular counts in aliquots of macerated kidney suspensions, was 13,769 1,028. SNGFR determined with ¹²⁵Iothalamate (bolus injection of 0.5 C/100 g followed by an infusion of 10 C/100 g/h) was 15.8 0.83 nl/min when measured by complete collection from the late proximal tubule and 13.7 0.64 when determined by quantitative collections in the distal tubule. The difference (2.07 0.52 nl/min) is significant (P < 0.02) in these paired measurements. Based on these measurements, normal late proximal flow rate can be estimated to be 6 to 8 nl/min, values in agreement with earlier work¹⁸.

Table 1 - Renal and nephron function in anesthetized and laparatomized mice of the C57BL/6 strain.

Full table (25K)

Using the open-loop microperfusion approach, TGF responses of stop-flow pressure (P_SF) and SNGFR were determined in five mice. Mean P_SF at zero loop flow was 36.9 2.2 mm Hg falling to 33.8 1.5 mm Hg at 5 nl/min and to 31.5 1.3 mm Hg at 10 nl/min. Responses were saturated by flows greater than 25 nl/min, with the maximum P_SF decrease averaging 12.5 1.4 mm Hg. The flow producing a half-maximum response (V_1/2) was 9 1.2 mm Hg. Thus, commensurate with lower ambient flow rates, the TGF function is shifted to the left compared with the rat. As a consequence, as in the rat, steady-state SNGFR is situated in the most sensitive region of the TGF curve. Like P_SF, SNGFR decreases with increasing loop flow, and this decrease also occurs for the most part at flows less than 15 nl/min. TGF responses were attenuated in mice infused with 2.4 ml/h isotonic saline, an expansion of extracellular volume causing urine flow to increase from 1.5 to 8 l/min, and urine osmolarity to fall from 1050 to 365 liter/min. In these volume-expanded mice, increasing loop perfusion rate from 0 to 40 nl/min caused P_SF to decrease by only 3 mm Hg, from 40.4 0.9 mm Hg to 37.4 0.8 mm Hg.

Studies of JGA function in transgenic mice

Angiotensin 1A-receptor (AT_1A)-knockout mice

Studies were undertaken in AT_1A receptor-knockout mice using the strain generated by Ito et al¹⁹. Mean body weights and mean kidney weights were not different between wild-type, heterozygous, and homozygous animals (BW: 27.2 1.2, 26.5 0.65, and 26.4 1.3 g, respectively). MAP was 91.8 2.2 mm Hg in wild type (range 83.5 to 96) and 97.2 3 mm Hg in heterozygotes (91-104). MAP in homozygotes was significantly lower, with a mean of 80.7 3.2 mm Hg (range 71.5 to 91; P = 0.045 vs. AT_1A +/+, P = 0.007 vs. AT_1A +/-). BP responses to exogenous Ang II were similar in AT_1A +/+ and AT_1A +/- mice but were markedly blunted in AT_1A -/- animals. In addition to polymerase chain reaction genotyping, this difference in the BP response to Ang II was used as independent corroboration of complete AT_1A receptor null mutation. In wild-type littermates, mean P_SF at zero flow was 37.2 1.6 mm Hg. Increments in loop flow caused a decrease in P_SF with a V_1/2 of 8.7 0.4 nl/min. Minimum P_SF (about 29 mm Hg) was established at 15 nl/min. In AT_1A +/- mice, P_SF at zero flow was 39.9 2.4 mm Hg, falling to 35.7 2.4 mm Hg at 15 nl/min. V_1/2 was similar to that in normal mice, 8.6 1 nl/min. In contrast, in AT_1A -/- mice, P_SF did not change significantly with increases in loop perfusion rate. Overall, P_SF fell by a maximum of about 9 mm Hg in AT_1A +/+, 5.1 0.4 mm Hg in AT_1A +/- and only 0.64 0.3 mm Hg in AT_1A -/- animals Figure 2.

Figure 2.

Maximum change in stop-flow pressure (response to a loop of Henle flow change from 0 to 40 nl/min) in heterozygous and homozygous AT_1A angiotensin receptor, angiotensin converting enzyme (ACE), and nitric oxide synthase (NOS)-I knockout mice, and in wild type littermates. NOS-I heterozygotes were not studied. Vertical bars are SEM. Symbols are: () wild-type +/+; () heterozygous +/-; () homozygous -/-.

Full figure and legend (43K)

Angiotensin converting enzyme (ACE)-knockout mice. Experiments were performed on mice from a colony of the ACE mutants generated by Krege et al²⁰. MAP in four wild-type mice of this strain prepared for micropuncture was 94.4 2.65 mm Hg and 85.9 1.6 and 85.5 4.2 mm Hg in five ACE +/- and two ACE -/- mice. BP responses to angiotensin I (Ang I) were greatly attenuated in the ACE -/- mice, whereas BP responses to Ang II were not significantly different between the three groups of mice. These results are similar to a recent report by Tian et al²¹.

As in the AT mutants, TGF responses were severely blunted in the ACE-knockout animals. In ACE +/+ mice, P_SF fell by 9.2 0.9 mm Hg, from 42.1 1.3 to 32.9 1.55 mm Hg (N = 16) on increasing loop perfusion rate from 0 to 40 nl/min. In ACE -/- null mutants, P_SF was 39.5 1.7 mm Hg at zero loop flow and 39.3 1.6 mm Hg at 40 nl/min (NS; N = 10). Interestingly, TGF responses were also blunted in ACE +/- mice, with P_SF falling from 41.2 2.5 to 38.9 2.29 mm Hg (N = 12). The mean decrease (2.25 0.5 mm Hg) was significantly lower than in ACE +/+ animals Figure 2. Ang II infused at 100 ng/kg per min, a rate that does not increase MAP significantly in these mice, partly restored TGF responsiveness.

Nitric oxide synthase I (NOS I)-knockout mice

Anesthetized and laparatomized mice with a null mutation in the NOS I gene²² had a MAP of 99.6 1.7 (N = 7) versus 96.6 3.8 mm Hg in NOS I +/+ animals (N = 6). TGF responses of P_SF to changes in loop perfusion rate were not different between wild type and NOS I -/-. In NOS I +/+ mice (13 tubules), mean P_SF at zero loop flow was 40.3 1.8 mm Hg and 31.9 1.8 mm Hg at 45 nl/min (8.5 0.7 mm Hg). V_1/2 was about 12 nl/min. In NOS I -/- animals (18 tubules), mean P_SF at zero flow was 40.7 1.7 mm Hg, with a steady-state value of 31.9 2 mm Hg at 45 nl/min (9.1 0.7 mm Hg). V_1/2 was about 13 nl/min. Acute NOS inhibition augments TGF responsiveness, a finding suggesting a modifying effect of MD-derived NO in juxtaglomerular signal transmission^23,24. In contrast, the current observations show that chronic and selective absence of NO generated by NOS I in MD cells is not associated with augmented TGF responses Figure 2.

Expression of renin, determined by in situ hybridization, was reduced in NOS I -/- mice, with only 34% of vascular poles showing a renin signal (56% in control). These results coincide with a recent study reporting a renal renin content about 50% lower in NOS I -/- mice compared with mice of the two progenitor strains²⁵. It is possible therefore that reduced local activity of the RAS in NOS I-deficient mice may compensate for TGF enhancement in NO deficiency. A recent study by Brenman et al has revealed the existence of a shorter isoform of NOS I mRNA in which the second exon is spliced out²⁶. Because the second exon was targeted as the site of the gene disruption, this short isoform may be expressed in the NOS I-knockout mice and is in fact found at a low level in the brain of these animals²⁶. Its presence in MD cells and the functional importance of any remaining NOS activity in these cells are currently unknown.

SUMMARY AND PERSPECTIVES

TGF acts as a minute-to-minute stabilizer of distal salt delivery and is therefore an important mechanism controlling NaCl excretion during fast and random perturbations in filtration and absorption forces that are unrelated to body salt balance. Changes in distal salt delivery too large to be fully compensated by TGF, and therefore persisting for a prolonged time, are followed by changes in renin secretion and extracellular [Ang II], the second effect initiated by a change in MD [NaCl]. One consequence of this change in Ang II availability is adaptation or resetting of the TGF system. Restoration of TGF responsiveness at a different operating point permits the TGF mechanism to continue to function as a high frequency regulator of salt excretion.

JGA studies in genetically modified animals have confirmed that RAS integrity is critical for the operation of TGF. The response-modifying effect of Ang II appears to be mediated exclusively by AT_1A angiotensin receptors. In contrast, the presence of NOS I in MD cells, and therefore presumably of NO released by these cells, does not appear to be an important modulator under chronic conditions. Initial studies using an isolated perfused JGA preparation confirm, however, that the presence of NOS I is necessary for NaCl-dependent renin secretion.

The use of genetically altered mice already offers unique opportunities for studying regulatory problems that have been difficult to answer unequivocally. It is to be expected that advances in gene modification techniques and in the physiological methodology applicable to mice will augment the experimental options further. Chimeric mice with regional gene deletions may provide an opportunity for comparing cellular functions between mutated and normal cells in the same animal²⁷. Gene duplication will complement null mutations by permitting comparisons of the functional effect of a gene over a fourfold range in the number of gene copies²⁸. The generation of double knockout animals by interbreeding will expand the analysis of the consequences of gene deletions and maybe particularly useful for the analysis of compensatory mechanisms. The use of tetracycline-responsive targeting constructs may provide temporal control over gene activation as well as inactivation²⁹. Finally, tissue-specific gene inactivation may become feasible by the use of Cre-mediated homologous recombination^30,31. In summary, the combination of genetic manipulation with detailed and precise physiological analysis is expected to provide an array of approaches with almost unlimited potential for improving our understanding of integrated body functions.

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Acknowledgments

Work from our laboratory was supported by National Institutes of Health Grants DK 37448, DK 39255, and DK 40042. We are indebted and grateful to the following for providing breeder pairs of transgenic animals: Drs. T.M. Coffman and M.I. Oliverio, Duke University (AT_1A mutant mice); Drs. J.H. Krege and O. Smithies, University of North Carolina (ACE mutant mice); Drs. P.L. Huang and M.C. Fishman, Massachusetts General Hospital and Harvard University; and Dr. O.A. Carretero, Henry Ford Hospital (NOS I mutant mice).