Effects of Loop Diuretics

The observation that inhibition of NaCl transport along the loop of Henle is associated with blockade of the TGF mechanism has been of fundamental importance in understanding the initiation of the TGF signaling pathway. TGF inhibition has been rather uniformly observed in the presence of loop diuretics such as furosemide, bumetanide, piretanide, ethacrynic acid, triflocin or l-ozolinone. Concentrations causing half-maximal inhibition of transport and feedback appear to be similar, about 5×10 ⁻⁵ M for furosemide and about 10 ⁻⁶ M for bumetanide (own unpublished data). Furosemide also blocked TGF responses during retrograde perfusion, suggesting that metabolic consequences of TAL inhibition are not transmitted by convective transport to the MD cells. Since distal Na and Cl concentrations are greatly elevated during loop NaCl transport inhibition, TGF responses do not appear to be caused by luminal NaCl concentration changes per se , but by changes in cellular NaCl uptake mediated by the furosemide-inhibitable Na,K,2Cl co-transporter NKCC2. The concentration dependence of feedback responses is probably the result of concentration dependence of NaCl uptake. Studies in mice with selective deletions of the A or B isoform of NKCC2 indicate that NKCC2B mediates TGF in the low NaCl concentration range, while NKCC2A is required for responsiveness to higher NaCl concentrations. Thus, the presence of two isoforms of NKCC2 in the macula densa extends the NaCl range over which TGF operates ( Figure 23.7 ). The concept that TGF responses are generated by the successive activation of NKCC2B and NKCC2A is supported by expression studies in Xenopus oocytes that have shown a higher Cl affinity of NKCC2B than NKCC2A, 9 mM versus 45 mM. Other aspects, such as the dependence of the inhibitory potency of furosemide on Cl concentration, have also been found to hold true for the TGF response. Diuretic agents with primary actions outside the loop of Henle such as acetazolamide, chlorothiazide, and amiloride do not possess TGF-inhibitory properties.

Relationship between loop of Henle perfusion rate and the relative reduction of P _SF (±SEM) in mice lacking NKCC2B (circles: (A) only) or NKCC2A (dots: (B) only). Dashed lines indicate the positions of V _½ , the flow rates causing half-maximum reduction of P _SF ; bars indicate the flow ranges over which 90% of the total responses occur.

(Data from . )

Effect of K Channel Blockade

Retrograde application of the K channel blocker U37883A caused an almost complete inhibition of TGF responses. This effect is mediated by inhibition of ROMK type K channels, since TGF responses were largely absent in mice with targeted ROMK deletion, a finding that has been confirmed in mice in which selective breeding of surviving animals has generated ROMK-deficient mice with less compromised kidney function and well-maintained blood pressure. The observation that inhibition of NKCC2 and ROMK has similar effects on TGF responses argues against a specific “sensor” function of these transport proteins, and for a critical role of some consequence of MD NaCl transport. Since ambient distal K concentrations near the MD are close to the K affinity of the co-transporter, it is possible that variations in luminal K may in part regulate TGF response magnitude. Nevertheless, the increase in distal K concentration accompanying acute hyperkalemia was associated with attenuation, not enhancement, of TGF responses.

Ion Substitution Studies

That NaCl uptake by NKCC2 is the initial step in the feedback transmission pathway is further supported by parallels in the ionic requirements for both TGF- and NKCC2-mediated NaCl transport across TALH. During retrograde perfusion of the MD segment ( Figure 23.8a ), TGF responses were not seen during perfusion with isotonic or hypotonic solutions of Na salts such as NaHCO ₃ , NaNO ₃ , NaI, NaSCN, Na acetate, Na gluconate or Na isethionate. In contrast, isotonic solutions of Cl salts ( Figure 23.8b ) accompanied by small monovalent cations such as K, Rb, Cs or NH ₄ elicited full TGF responses, as did the bromide salts of Na and K ¹⁴⁸ . It is to be noted that some of these small cations have been found to be substrates for either the Na or the K site on the NKCC2 co-transporter.

(a) Effect of Na salts with various anionic substitutions on the response of V _EP to retrograde perfusion of the macula densa segment. (b) Effect of Cl and Br salts with various cationic substitutions on the response of V _EP to retrograde perfusion of the macula densa segment.

(Data from ref. ).

The requirement for sizable Cl or Br concentrations, and the apparent lack of dependence on Na concentration, are consistent with an involvement of Na,K,2Cl co-transport, since the apparent overall affinity of NKCC2 for Cl in both TAL and MD cells is much lower than that for Na or K. Thus, the relatively low Cl affinity would predictably create an apparent Cl dependency of transport, while the small amounts of Na or K entering initially Na- or K-free solutions are sufficient to sustain near normal NKCC2 activity. When Na was replaced with large cations such as choline or TMA, TGF responses of normal magnitude were not seen, even though Cl was present in sufficiently high concentrations. Considering the cationic selectivity of the paracellular shunt, replacing luminal Na for choline will result in a sizable lumen positive Na diffusion potential that is predicted to reduce NaCl absorption by increasing Cl backflux. This explanation is consistent with the observation that NaCl transport rates of isolated TAL were found unaltered when studied under symmetric conditions with high choline Cl on both sides of the epithelium. In this case, Na backdiffusion and voltage-dependent inhibition of NaCl absorption is not to be expected as long as low concentrations of Na and K are present.

Active Nacl Transport

Transport inhibition caused by metabolic inhibitors such as cyanide, antimycin A or uncouplers of oxidative phosphorylation has also been found to reduce TGF responsiveness. TGF responses are not affected by peritubular application of ouabain, and this is probably related to the fact that the α1 Na,K-ATPase in rodents is rather insensitive to cardiac glycoside inhibition. In fact, when α1 Na,K-ATPase was genetically engineered to become ouabain-sensitive, intravenous or luminal administration of the glycoside caused marked reductions of TGF responses. The effect of luminal ouabain may not be related to H,K-ATPase inhibition, since TGF responses were unaltered in H,K-ATPase-deficient mice. The striking effect of loop transport blockade suggests that NaCl uptake through the furosemide sensitive Na,K,2Cl co-transporter, and Na extrusion through an energy-dependent pathway are critical steps in generating feedback responses.

Glomerular Capillary Pressure (P _GC )

Direct measurements in superficial glomeruli of Munich-Wistar rats have shown that saturating flow increments cause a significant fall in P _GC , with fractional decreases ranging between 15 and 22%. The slope of the P _GC change in the most sensitive flow range was 1.3 mmHg min/nl. P _GC measured directly in an in vitro preparation of juxtamedullary nephrons also fell during TGF activation. P _GC in nephrons without superficial glomeruli can be estimated from measurements of stop flow pressure (P _SF ). In response to a saturating increase in loop flow, mean P _SF of 23 studies fell by 22%, from 39.0±0.8 to 30.3±0.8 mmHg. A reduction in P _SF was also observed in the dog when loop flow was increased from zero to normal and supranormal values. In the mouse, TGF responses of P _SF are similar in magnitude as those seen in rats, but the sensitivity range is shifted to lower flows. Since multiple determinations of P _SF can be made in the same nephron with small perfusion flow increments, the nonlinear relationship between loop of Henle flow and P _SF was apparent long before a similar feedback function for SNGFR was defined. In 15 experimental series, the maximum P _SF decrease averaged 7.9±0.6 mmHg, with a mean V _½ of 20.1±1.1 nl/min. The maximum sensitivity varied substantially between different studies, but in general was between 1 and 2 mmHg min/nl. Two laboratories have reported that the TGF-induced change in P _SF was identical to the TGF-induced change in P _GC .

The main uncertainty in the determination of feedback-induced changes in the glomerular arteriolar pressure drop results from the evidence that, at least in the rat, a portion of the preglomerular resistance resides in the cortical radial arteries rather than in the afferent arterioles. As a consequence, afferent arteriolar resistance, calculated from the artery-to-glomerulus pressure difference, overestimates true afferent resistance, while the relative change caused by the TGF mechanism is underestimated. If preglomerular resistance is equally apportioned between interlobular artery and afferent arteriole, the TGF-induced resistance change along the afferent arteriole would be about 15% greater.

Preglomerular Resistance

The micropuncture studies agree that increasing loop of Henle flow produces a 30–40% increase in preglomerular resistance. TGF-induced reductions in afferent arteriolar diameter have been directly observed in the blood-perfused juxtamedullary nephron preparation, and in an isolated perfused tubule/vessel preparation. TGF-induced resistance changes estimated from such diameter observations are consistently much larger than those measured with the micropuncture approach. As suggested above, the location of a sizable resistance along large intrarenal arteries would lead to an underestimation of the TGF-induced resistance change. It is also possible that in the small preglomerular arterioles resistance estimates may deviate from Poiseuille’s law. Finally, cTGF, the vasodilator effect at the level of the connecting tubule, may blunt TGF in vivo , but would be absent in the perfused tubule.

The ultimate cause for TGF-induced vasoconstriction is a rise in intracellular calcium that, at least to a large extent, is mediated by activation of voltage-dependent calcium channels. Depolarization of afferent arteriolar smooth muscle cells during TGF activation has recently been demonstrated using voltage-sensitive dyes. A role for the resulting activation of L-type Ca channels in TGF is supported by the TGF inhibitory effects of intravenous or peritubular application of Ca channel blockers. A decrease in protein kinase A activation may be an additional component of TGF-mediated vasoconstriction of the afferent arteriole, since Db-cAMP in the presence of 50 µM IBMX or the luminal application of forskolin, a stimulator of adenyl cyclase, significantly reduced the TGF response magnitude.

Observation of the TGF response indicates that the afferent arteriole immediately adjacent to the glomerulum is the main direct target of the TGF mediator. The glomerular entrance segment of the vessel is the part of the afferent arteriole in which agents affecting TGF response magnitude, such as adenosine and angiotensin II, exert their largest constrictor action. TGF-induced local constriction may elicit a vascular conducted response that spreads to proximal portions of the arteriole by electrotonic coupling or myogenic excitation. Spreading of contractile responses during local application of KCl has been observed in juxtamedullary nephrons, indicating electrotonic coupling of afferent arteriolar smooth muscle cells. Upstream propagation of TGF-induced vasoconstriction is also responsible for the functional coupling of nephrons that are supplied by a common interlobular artery. It is probable that conducted vasoconstriction relies on the presence of various connexins in endothelial and smooth muscle cells. In view of the functional connection between afferent arteriolar smooth muscle cells, one may conclude that the total vasoconstrictor response to a NaCl step-change is composed of a local MD-generated effect and an upstream myogenic constrictor component.

Postglomerular Resistance

Evidence for a concomitant constriction of efferent arterioles during TGF activation has been obtained by micropuncture studies showing a reduction of GFR with unaltered glomerular capillary pressure, therefore suggesting proportional increases in the tone of afferent and efferent vessels. Furthermore, an increase of flow within the low-to-ambient flow range is associated with a greater change of SNGFR than of P _SF , suggesting that either there was balanced afferent and efferent vasoconstriction, with the SNGFR fall being a consequence of the reduced plasma flow or that total resistance did not change and the SNGFR change was a consequence of a reduced K _f . Direct observations of juxtamedullary efferent arterioles during TGF activation did not reveal any vasoactivity. In contrast to these observations, luminal NaCl has been observed to dilate efferent vessels in the double-perfused nephron preparation of the rabbit, an effect that was prevented by antagonists of A2 adenosine receptors. There is some experimental evidence in support of the possibility that a reduction in the filtration coefficient may contribute to TGF-induced reduction of GFR.

Adenosine

Adenosine was originally proposed as a mediator of the TGF response, since it provided a conceptual link between the energy expended for NaCl transport and the generation of a vasoactive ATP metabolite. Stimulation of Na transport in the proximal tubule is in fact associated with a decrease in cellular ATP, and increased NaCl secretion in the shark rectal gland causes a decrease in ATP, as well as an increase in adenosine release.

The vasomotor action of adenosine in most organs and vascular beds consists of vasorelaxation that reflects the wide distribution of the two types of A2 adenosine receptors, A2aAR and A2bAR. Although the kidney is usually considered an exception to this rule, several studies have shown that the steady-state response to an intravenous administration of adenosine is a clear reduction of renal vascular resistance, whereas the initial constrictor response is only short-lasting. In contrast, persistent vasoconstriction of afferent arterioles by adenosine and A1 adenosine (A ₁ ) analogs was observed in the hydronephrotic kidney and in isolated perfused afferent arterioles when adenosine receptors were activated from the interstitial aspect of the vessel. The constrictor effect was absent in arterioles from A1 adenosine receptor (A1AR)-deficient mice. Thus, adenosine causes a lasting vasoconstriction only when the nucleoside is generated in a restricted interstitial region so that A1AR can be accessed without general activation of the more dominant A2 receptors. Expression data, as well as functional observations, indicate that the terminal afferent arteriole is a vessel with high representation of A1AR. A1AR-mediated vasoconstriction of afferent arterioles is initiated by Gi-dependent activation of phospholipase C, release of Ca from intracellular stores, and subsequent Ca entry through L-type Ca channels. The A1AR-mediated vasoconstrictor effect of adenosine in afferent arterioles was stable for extended periods of time, indicating absence of rapid receptor desensitization. Tubular administration of A1AR agonists augments the vasoconstrictor response to increased loop flow rates. This effect does not appear to be mediated through apical A1AR, but rather to reflect a direct interaction with A1AR on afferent arterioles, and it thereby demonstrates the vasoconstrictor potency of A1AR activation and its effect on glomerular capillary pressure in vivo .

Two laboratories have independently generated mouse strains with targeted deletion of A1AR, and both groups observed a complete absence of TGF responses in A1AR ^−/− animals using micropuncture measurements of stop flow pressure or single nephron GFR ( Figure 23.10 ). Furthermore, specific A1AR antagonists such as 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or PSB-36 inhibit TGF responses when added to either the tubular lumen or the peritubular blood. A similar effect has been seen earlier with non-specific blockers such as theophylline or 3-isobutyl-1-methylxanthine, IBMX. Addition of the nonxanthine A ₁ receptor antagonist FK838 to the perfusate or bath also eliminated TGF responses in an isolated tubule preparation. There is evidence that adenosine generated during TGF activation may also interact with A2 adenosine receptors, but the site of action is unclear. Enhanced TGF responses have been observed during A2AR blockade, suggesting that activation of A2AR tonically diminishes A1AR-mediated vasoconstriction of afferent arterioles, perhaps through the release of nitric oxide. On the other hand, adenosine is able to vasodilate preconstricted efferent arterioles through A2AR, and A2AR inhibition has been found to block TGF-mediated vasodilatation of efferent arterioles in the isolated perfused JGA preparation. The latter findings are difficult to reconcile with the absence of in vivo evidence for a reduction of efferent arteriolar resistance during TGF activation.

(a) Mean responses of stop flow pressure to an increase in loop of Henle perfusion rate from 0 to 30 nl/min in wild-type (A1AR ^+/+ ) and in adenosine 1 receptor-deficient mice (A1AR ^−/− ). Gene targeting and phenotypic studies were performed by two independent groups of investigators (Closed symbols: data from ; Open symbols: data from ). (b) Recordings of stop-flow pressure (P _SF ) and arterial blood pressure (AP) in a wild-type mouse (A1R ^+/+ ) and in an adenosine 1 receptor-deficient mouse (A1R ^−/− ). During periods indicated by black bars, loop of Henle perfusion rate was increased to activate the TGF mechanism. Note absence of perfusion-related changes of P _SF in the nephron of the A1 receptor-deficient animal.

ATP

The preponderance of current evidence indicates that extracellular ATP serves as the major source for the generation of adenosine in the JG interstitium. Strong support for this notion is furnished by the demonstration that basolateral membranes of MD cells appear to have an ATP release pathway that is modulated by changes in luminal NaCl. Release of ATP by MD cells is suggested by the observation that PC12 or mesangial cells placed near the basolateral MD cell membrane responded to changes in luminal NaCl over the 20–60 mM range with an increase in cytosolic calcium. This Ca response of the biosensor cells reflects P2 receptor activation, since it was inhibited by suramin. Patch-clamp studies in cell-attached and excised patches of the basolateral membrane of MD cells showed the presence of an anion channel of 380 pS whose activity was dependent on the presence of extracellular NaCl, and that was active in the presence of ATP as the only anion. Channel activity was blocked by gadolinium, but not by the Cl channel inhibitors DPC or NPPB. The molecular nature of this large conductance anion channel has not been identified, but it may be identical to a functionally described maxi-anion channel that is widely expressed throughout the body. Release of ATP across connexin hemichannels would be another possibility, but the evidence for the presence of connexins in the basolateral membrane of MD cells is weak.

It is likely that ATP released into the JGA interstitium serves as substrate for membrane-bound ecto-ATPases and nucleotidases, ultimately resulting in the formation of the vascular mediator adenosine. In fact, TGF responses have been found to be greatly reduced in mice with deletion of NTPDase1/CD39, the major renal ecto-ATPase that dephosphorylates both ATP and ADP to AMP. Furthermore, evidence has been obtained suggesting that adenosine formation by e-5′NT/CD73 may be critical in the generation of adenosine during the TGF response. Blocking adenosine formation with the e-5′NT-inhibitor α,β-methylene adenosine 5′-diphosphate (MADP) significantly reduced the compensatory TGF efficiency and the slope of the TGF relationship. When the level of TGF activation was fixed by saturating concentrations of CHA, TGF efficiency was further reduced, and retrograde administration of MADP in combination with CHA caused vasoconstriction and abolition of TGF response. Consistent with these data are observations in mice with targeted deletion of e-5′NT/CD73 in which TGF responses were found to be markedly compromised. Finally, in the double-perfused JGA preparation, bath addition of the ecto-ATPase apyrase enhanced, and MADP abolished, TGF responses, suggesting that extracellular generation of adenosine from ATP is critical for JGA signaling. Exogenous e-5′NT was also found to improve the defective TGF component of renal autoregulation in Thy-1 nephritic rats. Dephosphorylation of ATP and AMP is likely to occur through enzymes anchored to the surface of mesangial cells. Whether adenosine levels in the JG interstitium are regulated by adenosine release across equlibrative nucleoside transporters (ENT) is unclear, since evidence based on the effect of ENT inhibitors such as dipyridamole or dilazep on TGF responses is inconclusive.

As an alternative to a role of ATP as precursor for adenosine, it has been proposed that ATP may directly elicit TGF responses, most likely through activation of P2 receptors on extraglomerular mesangial cells and gap junctional transmission of the resulting increase of cytosolic Ca to the afferent arteriole. ATP may also directly activate arteriolar P2 receptors, with P2X1 receptors being the most likely receptor subtype since its presence in afferent arterioles has been well-documented. In fact, ATP causes rapid and reversible constriction of afferent arterioles, an effect that may be more pronounced in juxtamedullary than superficial nephrons. A similar effect is seen with the P2X receptor-specific ligand α,ß-methylene ATP, suggesting mediation of vasoconstriction by P2X purinoceptors. Nevertheless, unequivocal evidence in support of a P2 receptor-mediated action of ATP in TGF in vivo is not available. TGF responses in superficial nephrons of P2X1 knockout mice were not statistically distinguishable from wild-type animals. Likewise, TGF responses were not significantly affected by the infusion of the broad-spectrum inhibitors of P2 receptors PPADS or suramin, although these agents clearly diminished the blood pressure response to the P2 agonist α,ß-methylene ATP. Results from the double-perfused JGA preparation are controversial. In an earlier study on rabbit tissue, suramin had been without effect on the response of afferent arteriolar diameter to an increase of luminal NaCl. Since acceleration of ATP breakdown enhanced, and inhibition of 5′-ecto nucleotidase and of A1 adenosine receptors blocked, the arteriolar diameter response, these results were consistent with the ATP dephosphorylation/adenosine paradigm. However, in similar preparations from rabbit or mouse, suramin was recently found to abolish afferent arteriolar Ca and constrictor responses, while the A1AR antagonist DPCPX was without effect. The reasons for this divergence are presently unclear, but it is to be noted that a novel approach was used in at least part of the latter study, in that only the macula densa cells of the perfused nephron were maintained, while all thick ascending limb cells were removed by microdissection. While it is conceivable that the presence of thick ascending limb cells is required for the “conventional” TGF response that is flow-independent, NaCl-dependent, and affects vascular tone through A1AR activation, the possibility of technical reasons for the obvious discrepancies cannot be excluded.

Link between Nacl Transport and TGF

Exactly how an increase of NaCl transport by MD cells is coupled to the release of ATP or the subsequent activation of the purinergic signaling pathway is one of the major open questions in TGF physiology. Cell swelling is a very common cause for eliciting ATP release, and this mechanism may be the critical factor for linking NaCl transport to ATP egress in MD cells. Nevertheless, as discussed above, the relationship between the compositional changes associated with elevated loop flow rates and MD cell volume is not entirely clear. ATP release could also be related to any of the changes caused by stimulation of NaCl transport acrosss apical or basal membranes of MD cells. NKCC2 activation is followed by increases in cytosolic Na and Cl concentrations, increases in cytosolic pH, and membrane depolarization. MD depolarization by about 30 mV following an increase of luminal [NaCl] to 150 mM occurs over the 20–60 mM range, and it therefore parallels the TGF response curve. Depolarization may be directly involved in TGF responses, since the cation ionophore nystatin elicited afferent arteriolar vasoconstriction in the presence of low luminal NaCl and furosemide. TGF responses were also seen when MD cells were depolarized by luminal application of the K ionophore valinomycin, together with 50 mM KCl. The valinomycin-induced vascular response was not affected by the Cl channel inhibitor NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), whereas the normal NaCl-induced response was fully blocked by NPPB. Thus, it appears that TGF responses depend upon MD cell depolarization independent of the specific mechanism underlying the PD change, but evidence is needed to establish that the depolarization by valinomycin or nystatin is restricted to MD cells, and does not affect arteriolar smooth muscle cells.

MD cell depolarization may be linked to TGF responsiveness through its effect on MD cytosolic calcium [Ca] _i . Transport-induced changes in MD [Ca] _i have been suggested to play a specific role in coupling the luminal electrolyte signal to the vascular response. The Ca ionophore A 23187 in the presence of luminal Ca enhanced TGF responsiveness, whereas blocking Ca release from intracellular stores with 8-(N,N-diethyl-amino)-octyl-3,4,5-trimethoxybenzoate (TMB 8) and chelation of Ca with BAPTA-AM diminished it. Ca entry may occur through a voltage-activated and nifedipine-sensitive Ca channel in the basolateral membrane, but increased flux across a 20 pS non-selective cation channel with finite Ca permeability reduced Ca extrusion by Na/Ca exchange, and utilization of a conductive Ca entry pathway across the apical cell membrane is an alternative possibility. Despite the evidence supporting an important role of [Ca] _i it is far from certain that the [Ca] _i changes occurring in response to fluctuations of luminal NaCl between 20 and 60 mM in vivo are large and consistent enough to establish a systematic and causal association between MD [Ca] _i and the TGF response. In fact, the relationship between luminal NaCl and MD cytosolic calcium has been described as positive, negative or entirely absent, perhaps indicating that the changes of [Ca] ⁱ in MD cells may be too small to be safely distinguishable from experimental noise in this technically demanding experimental approach.

A novel role for MD cell depolarization may be activation of NADPH oxidase and generation of superoxide that may directly or by scavenging NO enhance TGF. An increase in NaCl delivery to the MD in the isolated perfused JGA preparation caused about a three-fold increase in superoxide production that was largely inhibited by tempol or apocynin. Activation of NADPH oxidase appears to be the direct result of depolarization, since depolarization with valinomycin increased superoxide production and the hyperpolarization following furosemide-induced inhibition of NaCl uptake reduced it. The enhanced activity of the NOX2 isoform of NADPHase oxidase following membrane depolarization may be associated with translocation of the small GTP-binding protein Rac to the plasma membrane.

Hypertonic Nacl

Administration of hypertonic NaCl causes vasodilatation in most vascular beds, but in the kidney it results in an anomalous vasoconstriction. This response may be a whole kidney equivalent of the response of SNGFR to increased loop flow rate. Both TGF- and NaCl-induced vasoconstriction are enhanced by salt depletion and inhibited by furosemide, theophylline or DOCA-salt treatment. Furthermore, hypertonic non-chloride containing solutions usually do not produce vasoconstriction. A micropuncture study in the rat revealed that an infusion of hypertonic NaCl reduced proximal tubular fluid absorption and increased loop flow rate, and that SNGFR fell as a result of these changes.

Protein Feeding

The vasodilatation caused by acute and chronic protein feeding may include a TGF-dependent component. In conscious dogs, furosemide and ethacrynic acid blocked the acute rise in GFR following a meat meal, suggesting that the postprandial vasodilatation may be TGF-dependent. It has been proposed that the rise in filtered amino acids causes an increase in proximal Na and fluid absorption, and a decrease in MD Na delivery.

Chronic consumption of a high-protein diet induced a rightward resetting in the feedback curve of normal and Goldblatt hypertensive rats, so that higher flows were necessary to suppress GFR than in control or low-protein fed animals. In normotensive animals the response amplitude and the slope of the feedback function were unaltered. These effects appear to be due to alterations in transport along the loop of Henle, since NaCl concentrations in distal tubular fluid during loop perfusion were 30% lower in rats on a high-protein diet than in rats on a low-protein diet. Consistent with this notion is the observation that TGF responses were the same in the two groups of rats when loops of Henle were perfused in a retrograde fashion, indicating that functional changes in the loop of Henle rather than at the level of the JGA were responsible for the protein-induced changes in TGF characteristics. The increased rate of NaCl transport along the loop of Henle caused by high-protein feeding may result from structural adaptations.

Inhibition of Proximal Transport

Diuretics that inhibit predominantly proximal tubular fluid absorption may cause a TGF-dependent decrease in GFR as a result of increased distal NaCl delivery. In support of this hypothesis, several studies using carbonic anhydrase inhibitors, as well as chlorothiazide, suggest that these agents cause SNGFR to fall more with the TGF loop intact than with the TGF loop interrupted. In view of the Cl dependency of TGF, however, the acute TGF activation by carbonic anhydrase inhibition is unexpected since these drugs, while causing an increase of early distal [Na], do not elevate distal Cl concentrations. There is no evidence to show that HCO ₃ can substitute for Cl in initiating TGF responses. In fact, recent studies have shown that the effect of benzolamide to reduce GFR and RBF was maintained in A1AR ^−/− mice which are unable to generate a TGF response. Thus, the mechanism of the renal vasoconstriction caused by CA inhibitors is not entirely clear, but a rise in tubular pressure may be an important contributor. In addition, studies in rats and mice indicate that major increases in urine flow, including those caused by furosemide, may lead to increases in total renal vascular resistance even when participation of TGF is a priori unlikely.

SNGFR of mice with targeted deletion of AQP1 was found to be significantly reduced compared to wild-type when it was determined in distal nephron segments with the TGF pathway intact. The fall in GFR was dependent upon distal fluid delivery, since it was not observed when SNGFR was measured in the proximal tubule with the TGF loop interrupted. Mice deficient in both AQP1 and A1AR which combine a proximal transport defect with absent TGF responsiveness, have been found to have a normal distal SNGFR , supporting the notion that the fall of GFR in AQP1 ^−/− mice is TGF-mediated.

Very similar results were obtained in mice with a knockout mutation in the apical Na/H exchanger NHE3, another model of established proximal tubular NaCl and fluid malabsorption. Considering that distal flow rates were not different between wild-type and AQP1 or NHE3 knockout mice, it seems unlikely that TGF activation is due to an increased NaCl concentration at the MD. Resetting of the TGF function curve (discussed below) subsequent to extracellular volume depletion seems to be more likely as causation for enhanced TGF engagement.

Extracellular Fluid Volume

Adaptations in the TGF function occur whenever MD NaCl concentrations deviate from normal for an extended period of time. Typically, such deviations result from alterations in body Na content, with volume expansion associated with persistently increased, and volume depletion associated with decreased, MD NaCl concentrations. Formally, two types of adaptation in the TGF relationship can be distinguished. TGF resetting refers to a shift in the range over which the system is operating, either a shift to the right, to higher flows or concentrations or a shift to the left, to lower flows or concentrations. A change in TGF response sensitivity refers to an altered response, either altered slope and/or maximum response magnitude. By and large, volume depletion is associated with a left shift and an increase in response magnitude, and volume expansion with the opposite, but the actual adaptation observed has varied with the protocol used.

A number of studies have established that acute expansion of the extracellular space by infusion of isotonic saline or plasma reduces the TGF response magnitude and slope, and increases V _½ in both superficial and juxtamedullary nephrons. Impaired TGF responses have been observed during chronic volume expansion caused by the administration of DOCA together with isotonic saline as drinking fluid. The combination of a right shift and a reduced response magnitude is not invariant. Short-term volume expansion by a bolus injection of dilute rat plasma shifted the responsive range to higher flows, although this protocol produced an increase rather than a decrease in the maximum response magnitude ; SNGFR at zero loop flow rose 60%, whereas kidney GFR and free flow SNGFR rose only moderately with the position of the operating point and a large proximal–distal SNGFR difference indicating that the suppressing effect of the TGF mechanism was greater than normal. Thus, in these studies, the TGF mechanism appeared to counteract TGF-independent vasodilator influences on the renal vasculature. These data are consistent with closed-loop studies in which acute volume expansion with plasma did not reduce the maximum homeostatic efficiency, but shifted it away from ambient flows to lower flows, enhancing its dilatory and reducing its constrictor potency.

The effects of an acute decrease of ECV have been studied in rats after acute hypotensive hemorrhage, and in dehydrated rats. In general, these interventions are accompanied by an increase in TGF response magnitude. Hypotensive hemorrhage induced a shift of the feedback curve to the left. Since proximal absorption increased at the same time, the operating point tended to move toward the shoulder of the reset feedback function. Thus, in this circumstance, the resetting results in reduced dilatory and enhanced constrictor capacity.

Studies have been performed to determine the time course of TGF adaptation. When single nephrons were perfused for extended periods of time, resetting developed over the initial 30–40 minutes of hyperperfusion, whereas changes in response magnitude were slower, requiring 40–60 minutes. Similarly, acute volume depletion by furosemide restored TGF responses in chronically volume-expanded rats within 60–120 minutes.

Ureteral and Nephron Obstruction

In the first few hours following complete unilateral ureteral obstruction (UUO), TGF reactivity appears to be completely abolished. Elimination of the restraining effect of TGF is reflected by increased renal and glomerular plasma flows and elevated glomerular capillary pressures. After persistence of ureteral obstruction for 24 hours, TGF activity is restored and possibly slightly enhanced, as indicated by a reduced V _½ . Augmentation of TGF responses in hydronephrotic kidneys appears to be caused by increased superoxide formation and NO deficiency, since nNOS blockade was without effect and tempol administration normalized TGF responsiveness in hydronephrotic animals. The dominant effect of prolonged ureteral obstruction is a marked reduction of glomerular capillary pressure and glomerular plasma flow, changes which result in a dramatic reduction of GFR. The apparent absence of a luminal signal suggests that vasoconstriction is not equivalent to the standard TGF response discussed so far. On the other hand, obstruction of a single nephron for 4 hours also causes marked vasoconstriction, indicating that persistent interruption of distal delivery produces a local constrictor signal of unknown nature.

Following release of short-lasting ureteral obstruction TGF reactivity is increased, and it is maintained in its somewhat activated state after release of an obstruction of 24 hours duration. It is possible that the activated TGF mechanism is in part responsible for the continued vasoconstriction following release of both ureteral and single nephron obstruction. However, additional mechanisms appear to contribute to the reduction in filtration after release of obstruction, since SNGFR at zero flow increased only slightly above distal values. Furthermore, nephron and kidney filtration rates were also markedly suppressed after release of bilateral ureteral obstruction, even though in this experimental condition TGF responses were blunted rather than enhanced.

During partial unilateral ureteral obstruction for 3–6 weeks, TGF reactivity appears to be in the normal range. During volume expansion, on the other hand, TGF reactivity increased in the hydronephrotic kidney whereas it was strongly inhibited in the non-obstructed contralateral kidney. The enhanced TGF reactivity seen in hydronephrotic kidneys during volume expansion could be prevented by thromboxane synthase inhibition. A similar paradoxical enhancement of TGF reactivity by volume expansion was less pronounced during chronic bilateral ureteral obstruction.

Loss of Renal Mass

In the first hours following unilateral nephrectomy, TGF responses in the residual kidney may be enhanced and ambient distal flows may exert a GFR-depressing effect as judged from an increase in the proximal–distal SNGFR difference, without a change in the proximal value. However, at later time points uninephrectomy or 5/6 ablation has been shown to shift the TGF function to the right, with or without increasing the maximum response. A similar response to nephrectomy was noted in transplanted kidneys. On the other hand, a striking variability in SNGFR responses to loop of Henle perfusion was noted in rats with subtotal nephrectomy, in which the average TGF response was zero. Unexpectedly, AT1 blockade restored TGF responses for reasons that are not clear. To the extent that the most striking change is a TGF-independent increase in GFR (Y-intercept of the TGF function or proximal SNGFR ) these results are reminiscent of the findings during growth-related increases in renal weight, and suggest an adaptation of the TGF function curve to a primary increase in GFR.

Hyperglycemia

Moderate hyperglycemia produced by either acute glucose infusion or streptozotocin-induced diabetes mellitus reduced the amplitude of the TGF response and shifted V _½ to higher flow rates. Type 2 diabetic rats of the OLETF strain have significantly diminished TGF responses accompanied by reduced autoregulatory efficiency even in the pre-diabetic stage. Reduced TGF responses have also been observed in the Akita mouse model of type 1 diabetes mellitus, as well as in hydrated db/db mice, a model of type 2 diabetes. This reduced capacity to compensate for experimentally-induced perturbations was also demonstrable under closed-loop conditions. The reduction in the TGF response magnitude may in part be caused by a reduction in NaCl concentration, and the presence of glucose in tubular perfusion fluid.

It has been suggested that the hyperfiltration of early diabetes is caused by TGF, as a result of excessive salt reabsorption in nephron segments upstream from the MD and the resulting reduction in MD NaCl concentration. This hypothesis is supported by the demonstration that tubular transport proximal to the macula densa is in fact enhanced in diabetic animals. Structural adaptations participate in tubular hyper-reabsorption; inhibition of the enzyme ornithine decarboxylase blocked the renal hypertrophy in a rat model of diabetes mellitus, and attenuated both the enhanced proximal reabsorption and the increase in GFR. On the other hand, it has been argued that TGF may actually prevent excessive hyperfiltration in diabetes. A protective action of TGF is supported by the finding that diabetic mice of the Akita strain display an exaggerated hyperfiltration when TGF is rendered inoperative by A1AR deletion. Hyperfiltration in alloxan-diabetic mice deficient in A1AR also suggests that TGF is not the primary cause of hyperfiltration in this diabetic model.

Both in streptozotocin-treated rats and in diabetic humans, a paradoxical relationship between salt intake and GFR or renal plasma flow has been observed, with high salt intake producing a decrease instead of the expected rise of GFR, an effect which was absent when kidney growth was suppressed by inhibition of ornithine decarboxylase. These observations are consistent with the notion that the TGF adaptation during changes in extracellular fluid volume may be defective in diabetic animals. Whereas TGF desensitization normally prevents a non-homeostatic reduction of GFR during volume expansion, absent or incomplete resetting of TGF in diabetes appears to permit persistent GFR deviations. Altered resetting in diabetic animals could be a consequence of abnormal RAS activation or dysregulation of NO generation, factors thought to be involved in TGF adaptation. Nevertheless, the paradoxical low salt-induced increase and high salt-induced decrease of GFR has not been found in all studies of diabetic patients and animals. Differences in the dietary background, aside from NaCl content, may be an important modifying factor.

Renal Sympathetic Nerve Activity

Whereas some experiments did not detect an effect of acute denervation or renal nerve stimulation on the TGF function curve in normotensive animals, other studies have reported that denervation causes a time-dependent resetting of TGF to higher values of V _½ without changes in the maximum response. Changes in TGF function persisted for at least one week, and were associated with increased GFR and Na excretion.

Renin–Angiotensin System

The local activity of the renin–angiotensin system appears to be the most consistent determinant of TGF sensitivity. Converting enzyme inhibitors or angiotensin-receptor blockers in relatively high doses cause a reduction of the TGF response magnitude by about 50–60%. An essentially complete inhibition of TGF responsiveness was seen in mice with a null mutation in the AT1A receptor, the major renal receptor for angiotensin I ¹⁸⁵ . Similarly, TGF responses were essentially absent in ACE knockout mice, an effect that was in part reversible with infusion of subpressor doses of angiotensin II. Studies in mice with deletion of tissue ACE or with selective expression of ACE in either blood vessels or proximal tubules suggest that the angiotensin II that is required for full TGF responsiveness is derived both from the action of membrane-associated ACE in endothelial cells, and from systemic ACE, while exclusive expression of ACE in proximal tubules is unable to sustain normal TGF responses. This conclusion is consistent with earlier observations that angiotensin infusion partly restored feedback responsiveness during captopril-induced TGF inhibition. Intravenous or peritubular infusion of angiotensin II enhanced TGF responses in untreated control rats, a property not shared by other vasoconstrictors such as vasopressin and norepinephrine. Conversely, the arteriolar constrictor response to angiotensin II was greater during simultaneous TGF activation in both the isolated afferent arteriole/MD double-perfusion approach, and in the blood-perfused juxtamedullary nephron preparation. Since local adenosine levels are thought to increase during TGF activation, this augmentation is possibly due to the effect of adenosine in preventing angiotensin II desensitization. In accordance with the role of TGF in autoregulation, angiotensin II has been noted to enhance the TGF component of dynamic autoregulation. The suppression of TGF responsiveness caused by acute volume expansion could be fully overcome by the infusion of angiotensin II at doses that restored normal plasma angiotensin II levels. Taken together, these results indicate that an AT1A receptor-mediated effect of angiotensin II is a required constituent of the TGF pathway. The requirement for angiotensin II may result from the well-described synergistic interaction between the vascular effects of angiotensin II and of the TGF mediator adenosine. The mechanism of this interaction has been suggested to result from an intracellular action of adenosine that enhances the calcium sensitivity of myosin light chain phosphorylation or prevents desensitization of angiotensin II receptors. Contributing factors may be upregulation of MD NaCl transport by angiotensin II or enhanced production of feedback-enhancing superoxide radicals.

The TGF-modifying effect of angiotensin II gains special importance in view of the fact that changes in NaCl in the tubular fluid in the MD region not only affect vascular tone, but also regulate renin secretion (see below). This dual effect of MD NaCl has the potential to automatically adjust TGF sensitivity to the NaCl status of the organism. When the combined external forces determining GFR and proximal and loop of Henle absorption cause deflections in MD NaCl concentration which exceed the range over which TGF operates effectively, persistent changes in MD NaCl occur which will cause an inverse change in renin secretion. The change in angiotensin II concentration resulting from the altered rate of renin secretion, in turn, is predicted to alter TGF sensitivity. For example, an increase in MD NaCl resulting from extracellular volume expansion will decrease renin secretion, and the decrease of angiotensin II concentration expected to gradually develop is then predicted to uncouple GFR from MD control.

Eicosanoids

The presence of both isoforms of cyclooxygenase (COX) in the juxtaglomerular region raises the possibility of a participation of prostaglandins in JGA cell-to-cell signaling. COX-1 is expressed in mesangial cells and in endothelial cells of afferent arterioles, whereas COX-2 activity has been demonstrated in epithelial cells of thick ascending limb and MD. Maximum TGF responses have been found to be inhibited by the intravenous or luminal application of high concentrations of non-specific COX inhibitors, as well as specific inhibitors of both COX-2 and COX-1. The conclusion that the net effect of PGs on TGF is enhanced vasoconstriction is supported by the finding that arachidonic acid elicits a constrictor rather than a dilator response when administered by retrograde luminal infusion. Thromboxane (TP) is a vasoconstrictor prostaglandin that has been implicated in TGF on the basis of the finding that the intravenous administration of inhibitors of TP receptors or of TP synthesis reduced the magnitude of the TGF-induced vasoconstrictor response. TP receptor blockade also interfered with the TGF reducing effects of COX-2 and COX-1 inhibitors, indicating that these effects were TP-dependent. Conversely, activation of TP receptors by U-46,619 or by the isoprostane 8-isoprostaglandin F2α enhanced TGF responses, and COX-1 generated prostaglandins, presumably TP, may contribute to the TGF-enhancing effects of angiotensin II. Low levels of glomerular thromboxane synthase may limit thromboxane formation under basal and low-salt conditions ; in fact, attenuation of TGF responses by systemic or luminal application of blockers of TP receptors or of TP synthesis has not been found in all laboratories, and normal TGF responses have been observed in TP receptor knockout mice. Administration of a high-salt diet, on the other hand, caused a 20-fold increase of thromboxane synthase expression, as well as a stimulation of TP receptor levels. These observations provide an explanation for the finding that the TGF response in the presence of the TP mimetic U-46,619 is augmented in high-salt fed animals. TP receptor activation may also contribute to the exaggerated TGF responses observed in young spontaneously hypertensive rats (SHR). In contrast to these conclusions, studies in the blood-perfused juxtamedullary nephron preparation indicate that TGF activation is accompanied by nNOS-dependent enhancement of COX-2 activity and subsequent generation of vasodilatory PG metabolites which counteract TGF-mediated vasoconstriction.

There is some evidence that 20-hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid metabolite endogenously generated in afferent arterioles by the 4A family of cytochrome P450 enzymes, may be involved in the TGF response. Two inhibitors of cytochrome P450 enzymes, 17-octadecynoic acid (ODYA) and clotrimazole, caused a marked attenuation of the TGF response when added to the luminal perfusate for an extended period of time, and this inhibition could be overcome by the administration of exogenous 20-HETE. The presence of the mRNAs for cytochrome P450 4A2, 4A3, and 4A8 has recently been demonstrated by RT-PCR in glomeruli and most segments of the tubule, while preglomerular arterioles appear to express only the 4A2 isoform. Immunocytochemistry with a polyclonal non-specific P450 4A antibody showed cortical presence of P450 4A protein in proximal tubules, thick ascending limbs, glomeruli, and preglomerular arterioles. Exactly how locally formed 20-HETE affects the TGF pathway is unclear. In addition to its vasoconstrictor properties, 20-HETE has been shown to inhibit TAL NaCl transport by blocking Na,K,2Cl co-transport, Na,K-ATPase, and by closing K channels. This combination of effects should result in a powerful inhibition of NaCl transport in thick ascending limb and presumably MD cells, so that increments in 20-HETE production would be expected to attenuate, not enhance, TGF responses. It is conceivable, therefore, that ODYA and clotrimazole inhibit the TGF response in a non-specific way by causing a reduction in baseline afferent arteriolar tone. It is also possible that 20-HETE acts as an intracellular second messenger for the TGF-mediating agent.

Nitric Oxide

Luminal application of non-specific and nNOS-selective inhibitors of NO synthases has been shown to enhance MD-mediated vasoconstrictor responses both in vivo and in vitro . These findings demonstrate tonic attenuation of TGF responsiveness by NO generated by nNOS in MD cells, an effect that is cGMP-dependent. TGF responses of stop flow pressure in situ and afferent arteriolar diameter reductions in vitro in response to elevated MD [NaCl] were similar in nNOS-deficient and wild-type mice, but the specific mutation in these mice does not exclude the possibility of maintained expression of β and γ splice variants of nNOS. Nevertheless, the proximal–distal SNGFR difference was significantly higher in the nNOS ^−/− animals, and luminal administration of a non-specific NOS inhibitor (NLA 10 ⁻³ M) caused an augmentation of TGF responses only in wild-type, but not in nNOS knockout, mice. Luminal and systemic administration of NOS inhibitors reversed the attenuation of TGF responses caused by an acute saline infusion, suggesting that NO contributes to the TGF resetting caused by volume expansion.

NO may affect TGF by altering the function of MD cells. In the isolated double-perfused JGA preparation, inhibition of soluble guanylate cyclase or cGMP-dependent protein kinase mimicked the TGF-potentiating effect of a nNOS inhibitor when administered from the tubular side, whereas there was no effect when inhibitors were added to the vascular perfusate. The cGMP-dependent mechanism may be inhibition of NaCl uptake, since inhibition of Cl fluxes by NO is mediated by soluble guanylate cyclase in TAL cells. Formation of NO may occur in MD cells, but a contribution of NO produced by eNOS in TAL cells appears to also affect MD cell function.

The relationship between luminal NaCl concentration and NO formation in MD cells has been addressed by using the NO-binding agents DAF-AM DA and DAF-AM. Two independent studies agree that an increase in luminal NaCl causes an increase in fluorescence in MD cells, as well as in their surroundings, and that this increase was prevented by an inhibitor of nNOS. Since the concentration steps causing increased NO formation were between 35 and 135 mM in one study and between 60 and 150 mM in the other, it has been concluded that supraphysiological NaCl changes are necessary to stimulate NOS. That the stimulation of nNOS activity by high luminal NaCl is Ca-dependent is unlikely, since NaCl does not consistently elevate [Ca] _i , and since an activation of NOS was seen in Ca-free medium. Another explanation for the increased formation of NO is based on the fact that the pH optimum of NOS is in the slightly alkaline range. Since an increase in luminal NaCl caused cell alkanization, it is conceivable that pH-dependent disinhibition of nNOS is responsible for the enhanced NO generation. This notion is supported by the observation that dimethyl amiloride increased TGF responses, and that an nNOS-specific inhibitor in the presence of the NHE blocker did not further enhance the TGF reaction. Furthermore, amiloride as well as 7-nitroindazole blunted the increase in NO formation caused by elevated luminal NaCl. Stimulation of NO formation by high-luminal NaCl is consistent with the earlier observations that the effect of NOS blockade on TGF-dependent vasoconstriction is greater at high than at low flows. Increases in luminal flow rate also stimulate NO formation by TAL cells, but in contrast to MD cells stimulation is strictly shear stress-dependent, and independent of NaCl transport. NaCl independence is difficult to reconcile with direct NO measurements performed with carbon fiber electrodes in the distal tubule which have shown an increase in the amount of NO and in NO concentration during perfusion of loops of Henle with furosemide, suggesting that transport inhibition along the entire loop of Henle stimulates emission and downstream convection of NO. These results seem consistent with observations in an MD cell line showing increased NO generation during exposure to low chloride or furosemide.

In the absence of hemoglobin, the biological half-life of NO in relation to the diffusion distance across the JGA would seem long enough to permit a direct interaction of NO released by MD cells with smooth muscle cells of the afferent arterioles. Nevertheless, NO inactivation may be an important modulator of the effect of NO on TGF. Reactive oxygen species have been identified as a factor that can markedly reduce bioactive NO levels. MD cells express NADPH oxidase isoforms NOX2 and NOX4, together with the required constituents of the active enzyme complex p47phox, p67phox, p22phox, and Rac. A high-luminal NaCl appears to activate NADPH oxidase, and this activation is pH-dependent since blocking Na/H exchange with dimethyl amiloride or acidification of the luminal perfusate diminished superoxide production. Production of the NO scavenger may limit the impact of NO that is stimulated in MD cells at the same time by the same mechanism. The membrane-permeant superoxide dismutase mimetic tempol can diminish TGF responses in vivo and in the perfused JGA preparation, but this effect is usually not very pronounced under control conditions. Since tempol had no effect in the presence of a NOS inhibitor, it appears that any effect is due to an increase in NO bioavailability. In spontaneously hypertensive rats (SHR) the expression of NADPH oxidase subunits is elevated, and the TGF-inhibiting effect of tempol is enhanced compared to normotensive controls, suggesting increased oxidative stress in SHR and reduced bioavailability of NO. This observation is in accordance with the earlier finding that inhibition of nNOS did not enhance TGF responses in SHR. Angiotensin II may be in part responsible for the activation of NADPH oxidase in SHR, since candesartan restored normal TGF responses to NOS inhibition. A high salt intake may be another situation in which the generation of superoxide is increased as indicated by increased expression of NADPH oxidase subunits, and increased excretion of isoprostanes. However, the TGF-enhancing effect of NOS inhibition was augmented rather than reduced in rats on a high-salt diet. NO availability may also be regulated by ADMA (asymmetric dimethylarginine), an endogenous NOS inhibitor that also inhibits cellular uptake of the NOS substrate arginine. In fact, ADMA in the luminal perfusate enhanced TGF responses, and this effect appears to be the result of both inhibition of arginine uptake and of NOS activity. Finally, NO may reduce TGF responses by inhibition of ecto-5′-nucleotidase, an intervention that would be predicted to reduce extracellular adenosine levels.

Other Vasoactive Factors

A reduction in TGF reactivity has been observed subsequent to the systemic administration of renal vasodilators such as atrial natriuretic factor, histamine, dopamine, high concentrations of PGI ₂ , bradykinin, uroguanylin, and a number of vasodilating drugs. Inhibition of heme oxygenase with stannous mesoporphyrin enhanced TGF-induced vasoconstriction in the isolated tubule/vessel preparation, and the administration of the CO-releasing agent CORM-3 (tricarbonylchloro[glycinato]rutheniumII) inhibited it, suggesting that carbon monoxide reduces TGF. Since the presence of heme oxygenases in the MD has not been established, it is likely that carbon monoxide affects TGF by its vasodilatory properties. Furthermore, TGF reactivity is decreased by an acute reduction of arterial blood pressure, probably as a consequence of non-TGF-mediated autoregulatory vasodilatation. It seems unlikely that all these agents specifically interact with the TGF mechanism at the JGA level to cause a reduction of renal vascular resistance. Rather, the adjustment of TGF responsiveness may be the non-specific consequence of vasodilatation, reflecting a dependency of vascular resistance changes on the initial wall thickness–radius ratio. Since the predominant effector of the TGF response is the afferent arteriole, a resistance change at this site would appear to be most likely to modulate TGF sensitivity.

Interstitial Pressure

TGF reactivity has been shown to increase during peritubular capillary perfusion with a hyperoncotic solution, whereas perfusion with protein-free solutions reduced TGF responses. Changes in response were seen after a time delay of about 20 minutes. Based on these observations, the concept was developed that net interstitial pressure (the difference between interstitial hydrostatic pressure and interstitial oncotic pressure) may be an important determinant of TGF reactivity in general. It was subsequently shown that TGF reactivity correlated inversely with net interstitial pressure during acute NaCl infusion, during short-lasting ureteral obstruction, and after contralateral nephrectomy. In all these conditions, V _½ increased and the TGF response amplitude decreased. Conversely, net interstitial pressure was found to be reduced by 24 hours of dehydration, and before and after release of 24 hour unilateral ureteral occlusion. TGF reactivity was increased in these experimental situations. Exactly how net interstitial pressure affects TGF reactivity is unclear.

Resetting by Luminal Factors

There are a number of circumstances in which unidentified factors in tubular fluid have been shown to modify responses. During chronic dietary salt-loading, TGF control was inhibited when native tubular fluid was used as perfusate, whereas responses were only slightly blunted during perfusion with an artificial fluid. A luminal factor has also been reported to modify TGF during the infusion of atrial natriuretic peptide, but this factor appears to enhance TGF responses compared to the blunting observed with artificial solutions. Loop of Henle perfusion with electrolyte-free plasma dialysates from patients with acute renal failure and liver dysfunction produced exaggerated TGF responses which could not be blocked by furosemide, suggesting that the plasma in certain disease states contains a factor which can elicit NaCl-independent vasoconstriction when present in the tubular lumen.

No in MD-Dependent Renin Release

In view of the dual effects of NO on renin secretion and the ambiguity about the directional changes of juxtaglomerular NO with changes in loop of Henle flow rates, it is not surprising that the precise role of NO in MD control of renin release has remained equivocal. In the isolated perfused JGA preparation during perfusion with a low NaCl concentration, the luminal addition of l-arginine stimulated renin secretion and this stimulation was abolished by NOS blockade, suggesting that in this setting NO is renin-stimulatory. Consistent with this conclusion is the observation that the NaCl dependency of renin secretion was essentially abolished in the presence of an NOS blocker in the tubular lumen, a change that was due entirely to prevention of the rise of renin secretion caused by a low luminal NaCl. The conclusion that a low NaCl concentration at the MD stimulates renin secretion in an NO-dependent fashion is also supported by findings showing that the increased renin secretion caused by a reduction in arterial or perfusion pressure in kidneys of conscious dogs and in isolated rat kidneys was markedly and consistently blunted by NOS inhibition. In other studies, the administration of a loop diuretic has been used to simulate a reduction in MD NaCl concentration. In dissected rat renal microvessels, NOS inhibition abolished the increase in renin release caused by furosemide pretreatment. Similarly, the stimulation of renin secretion by furosemide in vivo was inhibited by the administration of NOS inhibitors. Plasma renin activity in nNOS knockout mice and basal renin secretion in isolated perfused kidneys from nNOS ^−/− or eNOS ^−/− mice were found to be consistently lower than in wild-type animals, suggesting that tonic release of NO enhances renin release in mice. The relative increases of renin secretion by furosemide were essentially normal in nNOS ^−/− or eNOS ^−/− mice, but were markedly reduced by general NOS inhibition. Furthermore, the administration of the NO donor SNAP in kidneys in which endogenous NO production was blocked by L-NAME completely restored the stimulatory effect of loop diuretics. According to this recent evidence, it would appear that exposition of JG cells to NO regardless of its exact cellular source is necessary for the MD pathway to operate normally. The nature of this permissive effect of NO may be to inhibit PDEIII, and thereby to sensitize the renin secretory mechanism to the renin mediator that we assume to act through activation of the cAMP/PKA pathway ( Figure 23.12 ).

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