Department of Medicine, Division of Nephrology-Hypertension, University of California and Veterans Affairs San Diego Health Care System, San Diego, California 92021
Submitted 24 September 2003 ; accepted in final form 2 June 2004
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ABSTRACT |
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glomerular filtration; macula densa; micropuncture; nitric oxide; tubuloglomerular feedback
In contrast, subtype-specific NOS blockers should be less encumbered by confounding effects. Two groups have used a selective NOS II antagonist, L-NIL, to determine whether the so-called "inducible" NOS II contributes to diabetic hyperfiltration. In neither case was a role demonstrated for the NOS II isoform in diabetic hyperfiltration (18, 31).
Given its prominence as a vasodilator, one might consider endothelial NOS III as a candidate mediator for diabetic hyperfiltration. Because there are no antagonists specific to NOS III, this issue can only be addressed indirectly. Reports on the expression of NOS III in diabetic kidneys or glomeruli are equally divided as to whether expression is increased (4, 31) or not increased (6, 18).
In the present study, we tested for abnormalities of renal hemodynamics related to the NOS I isoform in rats with early streptozotocin diabetes. Experiments were designed based on what is already known about NOS I in normal kidney physiology. For example, we know that NOS I is featured in the macula densa (MD) where it performs certain functions (32). One of these functions is to buffer the acute tubuloglomerular feedback (TGF) response (30). Another is to participate in the normal rightward resetting of the TGF response that occurs within 3060 min when TGF is continuously stimulated (5, 23, 25, 26). Regarding the signals that regulate MD NOS I, it has been shown that MD NO production can increase rapidly when the apical MD is perfused with salt (9). Regarding tissue content of the enzyme itself, MD NOS I generally varies inversely with what the kidney sees as the effective volume state of the animal, although the amount of NOS I enzyme and the magnitude of its functional effect are often dissociated (reviewed in Ref. 22).
With these points borne in mind, experiments were performed to evaluate whether dysregulation of NOS I in the diabetic kidney contributes to glomerular hyperfiltration, reduced TGF efficiency (29), or abnormal resetting of TGF. Findings suggest that the tonic influence of NOS I over glomerular filtration is enhanced in diabetes and that the normal dependence of NOS I activity on MD salt is lost such that the diabetic kidney generates excess, and functionally significant NO, even when not stimulated to do so by variations in MD salt.
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METHODS |
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Effects of NOS I Blockers on Diabetic Hyperfiltration
GFR was measured in control and diabetic rats pretreated with placebo, with the water-insoluble NOS I blocker 7-nitroindazole (7-NI), or with the water-soluble NOS I blocker S-methyl thiocitrulline (L-SMTC), n = 57 for each group. 7-NI was given as a single dose after anesthesia, 25 mg/kg ip in 150 µl DMSO. This dose was chosen to inhibit NOS I in the juxtaglomerular apparatus (JGA) without affecting blood pressure (2, 27). Controls for 7-NI were injected with DMSO vehicle. SMTC was administered in Ringer saline at 5.1 µmol·kg1·h1 by continuous infusion to avoid sudden changes in blood pressure. This dose of SMTC inhibits NOS I in the kidney while minimally affecting the blood pressure (20).
GFR was determined in two serial 15-min collection periods. Subsequently, infusion was begun of a nonselective NOS blocker, NG-monomethyl-L-arginine (L-NNMA; 0.5 mg·kg1·min1 iv). After 30 min, GFR was determined twice more from serial 15-min timed urine collections. The point of infusing L-NMMA in NOS I-blocked rats was to test for any residual NO, presuming that this would be due to NOS III.
Effect of NOS I Blockade and Diabetes on Resetting of TGF
The JGA normally calibrates the TGF response to be steepest near the ambient tubular flow. If a prolonged TGF stimulus is imposed, then the system normally adapts by shifting the TGF response rightward within 60 min (23, 25). At the same time, RBF normally increases back toward baseline by a mechanism that requires NOS I (5). To investigate whether this function of MD NOS I is preserved in diabetes, we monitored RBF with a perivascular ultrasonic transit time flow probe (Transonics T206, Ithaca, NY) while infusing a proximal tubular diuretic, benzolamide (5 mg/kg bolus followed by infusion at 5 mg·kg1·h1), as described (5, 25). Rats were equilibrated for 2 h and baseline RBF was established during the final 20 min. Then, benzolamide was begun and continued for 1 h. In one group, RBF was stabilized during SMTC infusion before benzolamide was started.
MD NOS I and TGF Efficiency
Micropuncture in free-flowing nephrons. When flow in the proximal tubule is disturbed, a fraction of the disturbance is buffered by TGF and this fraction constitutes an index of TGF efficiency (21). TGF efficiency is reduced in rats with 56 wk of diabetes (29). Nonspecific NOS blocker delivered to the MD by tubular microperfusion increases TGF efficiency in normal nephrons (30). Therefore, decreased TGF efficiency in diabetes might result from excess NOS I. As an index of TGF efficiency, the fractional compensation for small perturbations in late proximal tubule flow was measured before and during addition of SMTC to the free-flowing late proximal nephrons according to an established protocol (21, 23, 26, 29, 30). Perturbations were applied with a Hampel nanoliter pump (University of Tuebingen, Dept. of Pharmacology) filled with standard artificial tubular fluid (ATF) (21). For diabetic rats, ATF also contained 10 mM glucose. Flow was monitored upstream from the perturbation by a noninvasive optical technique (videometric flow velocitometry) as described (21). Perturbations ranged from 8 to +8 nl/min in 4-nl/min increments and were done in variable order. SMTC was added (2 nM at 4 nl/min) using a second pump positioned slightly downstream from the perturbation pump. The perturbation pump was adjusted, in turn, to compensate for the additional flow of the drug pump. Fractional compensation was calculated by linear regression of the changes in measured flow against the applied perturbations.
MD Salt as Determinant of NOS I Activity
Micropuncture in wax-blocked nephrons. In addition to initiating the TGF response, MD salt may activate NOS I to buffer that response (9). If this is true, then blocking NOS I should reduce single-nephron GFR (SNGFR) to a greater extent when there is more salt being delivered to the MD. To test this theory, we performed micropuncture to manipulate MD salt while blocking NOS I. Two microperfusion pipettes were placed in the most downstream visible segment of the proximal tubule. One pipette contained ATF and the other contained ATF + SMTC (1 µM). A wax block was inserted immediately upstream from the perfusion pipettes. A series of four timed collections was made upstream from the wax block to determine SNGFR by [3H]inulin clearance. The first collection was made during perfusion with ATF at 38 nl/min, the second during ATF at 8 nl/min, the third during ATF at 30 nl/min + SMTC at 8 nl/min, and the fourth during SMTC at 8 nl/min. Collections were made for 3 min. Outcome measures included the range of the TGF response (difference in SNGFR during high- and low-microperfusion rates) and the influence of TGF stimulation on the effect of SMTC.
Statistics
Effects were tested by ANOVA with a design for repeated measures and Tukeys test for post hoc intergroup comparisons where appropriate. Significance is defined as P < 0.05.
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RESULTS |
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Response to NOS I inhibitors (7-NI or SMTC) was measured after 2 wk of diabetes (Table 1 and Figs. 1 and 2). General characteristics are shown in Table 1, confirming that diabetic kidneys were larger and that there was no important confounding by body weight, blood pressure, or glycemic control.
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L-NMMA significantly reduced GFR in those diabetics in which NOS I had not previously been blocked. L-NMMA had no significant effect on GFR in any other group (Figs. 1 and 2). Because increased blood pressure confounds the direct effects of L-NMMA on renal function (reviewed in Ref. 22), the present data do not imply absence of NOS III activity in the normal kidney.
RBF Recovery During Activation of TGF With Benzolamide
Hemodynamic evidence for TGF resetting (5) was sought during a 1-h infusion of benzolamide, with or without SMTC, in 26 animals. In these experiments, SMTC caused arterial blood pressure to increase by 10 mmHg in nondiabetics (P < 0.05) and
5 mmHg in diabetics (P = not significant). Over the subsequent 60-min period of benzolamide infusion, blood pressure gradually declined by
5 mmHg. There was no apparent effect of diabetes or SMTC on the minor drift in blood pressure during benzolamide (Fig. 3). By multiway ANOVA, RBF was greater among diabetic rats (P = 0.001); initial activation of TGF reduced RBF (P < 0.0005); SMTC reduced basal RBF (P < 0.0005) by similar amounts in diabetics and controls. SMTC tended to enhance the initial response to benzolamide and this effect appeared greater in diabetes, but this effect of diabetes was not statistically significant (P
0.15). Those diabetic rats that went on to receive SMTC began with greater RBF than those diabetic rats that did not receive SMTC. This was unintentional but did result in similar RBF among both groups of diabetic rats at the time benzolamide was started. After initially declining, RBF gradually increased toward normal during continuous infusion of benzolamide in controls. This restoration of RBF was prevented with SMTC, as previously reported (5). In diabetic rats, RBF remained depressed throughout 1 h of benzolamide and was unaffected by SMTC (Fig. 4).
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TGF efficiency was measured by perturbation analysis before and during SMTC microperfusion in 14 control nephrons, 14 nephrons from 1-wk-old diabetic rats, and 12 nephrons from 2-wk-old diabetic rats (Fig. 5). The 2-wk data were obtained after the discovery that TGF efficiency was not reduced as much as expected in 1-wk diabetic rats. That expectation was based on past experience with rats after 56 wk of diabetes (29). Consistent with prior experience, neither diabetes nor NOS I blockade affected ambient late proximal flow. In 2-wk diabetic rats, fractional compensation was 50% less than in control (P = 0.005). The addition of SMTC to nephrons caused TGF efficiency to increase (P = 0.007 by repeated-measures ANOVA applied to all groups). The effect of SMTC appeared to be greatest in 2-wk diabetic rats. However, this difference could not be made statistically significant without an untenable increase in sample size.
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Serial micropuncture data were obtained from 45 nephrons in 2-wk diabetic rats and 25 nephrons from nondiabetic controls (Table 2 and Figs. 6 and 7). Diabetic hyperfiltration was confirmed, and there was a TGF response in each nephron. We refer to SNGFR during minimal TGF stimulation as SNGFRmax and SNGFR during maximal stimulation of TGF as SNGFRmin. The addition of SMTC to nondiabetic nephrons reduced SNGFRmax by 9% (P = 0.07) and SNGFRmin by 24% (P < 0.0005). In diabetic rats, adding SMTC reduced SNGFRmax by 24% (P < 0.0005) and SNGFRmin by 31% (P < 0.0005). SNGFRmax was more sensitive to NOS I blockade in diabetic rats than in nondiabetic rats (P = 0.006). SNGFRmin was similarly sensitive to NOS I blockade in diabetic and nondiabetic rats. The range of the TGF response (SNGFRmax-SNGFRmin) was increased by NOS I blockade in controls (P < 0.05) but not diabetic rats (P = not significant). In other words, imposing a large salt signal on the MD increased the sensitivity of SNGFR to NOS I blockade in normal rats. In diabetic rats, SNGFR was highly sensitive to NOS I blockade regardless of the amount of salt being delivered to the MD.
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DISCUSSION |
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The first point mentioned above is that the diabetic kidney is under increased influence from NOS I. This confirms the recent report of another group (8) who observed remarkably similar effects of NOS blockers in whole kidney clearance studies. The most obvious utility of such data might be to supplement the list of putative causes of diabetic hyperfiltration (14). However, these data may be equally useful for considering the role of MD NOS I in normal physiology. This is because there are unlikely to be any regulatory pathways that are unique to diabetes or uniquely absent in diabetes. Therefore, if the diabetic kidney responds differently to NOS I inhibition, this should be due to quantitative differences in the activities of mechanisms that exist in nondiabetic and diabetic subjects alike. In this way, nuances in the diabetic response to NOS I blockade could reveal details of a mechanism that happens to be confounded or concealed under normal conditions.
The second point to be made from the present data relates to salt delivery as a determinant of NOS I activity in the MD. So far, apical salt entry is the only proximate stimulus that has been directly observed to activate NOS I in MD cells (9). The present data from control rats confirm that this mechanism can be physiologically relevant. Apical entry of salt is also the first step toward eliciting a vasoconstrictor TGF response (17) and it is well known that NO made in the MD normally offsets the TGF response (30). Based on this situation, one might imagine that the osmotic effect of glucose in the proximal tubule leads to increased distal delivery of salt, continuous overproduction of NO by the MD, and glomerular hyperfiltration. This would make NO the link between hyperglycemia and glomerular hyperfiltration, which is a cardinal feature of early diabetes. Such a simple scheme may be appealing, but it is fatally flawed for at least two reasons. First, increased distal salt delivery is not a prerequisite for diabetic hyperfiltration. In fact, due to a marked increase in reabsorptive capacity of the proximal tubule, delivery of salt to the MD falls well below normal in diabetes, notwithstanding the increased reabsorptive burden imposed by hyperfiltration (16, 19, 29). Therefore, the initial requirement for increased salt to stimulate NOS I is not met. Second, the present data reveal that the diabetic JGA can produce abundant NO independently of MD salt. The relevance of this finding should extend beyond diabetes because, again, it is unlikely that any regulatory pathway is unique to diabetes. Henceforth, any theory to explain the regulation of MD NOS I must allow for abundant NO formation that is independent of MD salt. Once it is accepted that NOS I blockers can affect glomerular hemodynamics independently of flow past the MD, then it is no longer necessary to conclude that all of the relevant NOS I resides within MD cells, notwithstanding that the NOS I enzyme is concentrated in the MD (32).
The third point to be made from the present data pertains to the site where NO acts to influence glomerular filtration. NO from the MD is thought to impact glomerular hemodynamics in an autocrine manner by inhibiting salt entry into MD cells (13), thereby blunting the sensory limb of the TGF system. NO from the MD might also impact glomerular hemodynamics by a paracrine vasodilatory effect on the glomerular microcirculation. However, demonstrating that NO is capable of these autocrine and paracrine effects does not reveal to what degree each of these impacts kidney function. The present data help to sort this out. For there to be an autocrine effect of MD NO on the TGF response, there must be NO available to inhibit salt transport and there must be salt transport available for NO to inhibit. Our main clue regarding how much NO is present comes from the decrement in SNGFR when NOS I is acutely blocked with SMTC. However, the amount of NO may not be the only determinant of the response to SMTC. For example, if MD NO primarily affects SNGFR by blocking MD transport then, for a given amount of NO, there will be a lesser effect of SMTC when there is less transport to inhibit. There are two opportunities to observe this among the present findings. The first, and most obvious, is that MD transport is eliminated as a target for NO when SNGFR is measured without flow through Henle's loop. The finding that SMTC reduced SNGFR in diabetic nephrons under this circumstance eliminates MD salt transport as the sole target of MD NO in diabetes. Therefore, NO made in the JGA must be able to act on the glomerular microvessels. Although it is unlikely that the mechanism involved is unique to diabetes, this conclusion could only be reached by considering the results in diabetic rats, because flow dependence of the SMTC effect in nondiabetic rats makes it impossible to rule out MD transport as the sole target of NO in those animals. Thus a physiological mechanism has been confirmed by studying pathophysiology. Second, MD salt content is less in diabetes (13, 16, 19, 29). Therefore, if MD salt were both the main originator and the main target of MD NO in diabetes, there would be a lesser effect of NOS I blockade on GFR. Because this is contrary to the present finding, the role of NOS I as a modulator of TGF cannot be limited to inhibiting MD transport.
The fourth point to be made from the present data pertains to the role of MD NO in the temporal adaptation, or resetting, of TGF. Most research on TGF assumes there to be a static relationship between SNGFR and MD salt. However, it is obvious that the TGF relationship cannot be static because SNGFR and MD salt could never change in the same direction unless there is a change in the behavior of TGF itself. In fact, TGF can be made to reset by a wide variety of events in the systemic milieu or by events confined to the JGA, with major implications for the body's internal environment. TGF resetting within the JGA is the subject of several papers (5, 20, 23, 25, 26) that can be summarized for present purposes. Briefly, ambient tubular flow in a nephron tends to reside within a narrow domain where the TGF curve is steep. This alignment is maintained because each nephron gradually adjusts its TGF curve in response to sustained changes in tubular flow. This adjustment can occur on the time scale of 3060 min and is mediated within the JGA. There are also hemodynamic events that correspond to TGF resetting, although different measurements are necessary to record these events than to observe resetting of the TGF curve at the single-nephron level. The hemodynamic events corresponding to TGF resetting were recently studied (5) using the same benzolamide infusion protocol that was originally employed to analyze migration of the TGF curve (25). When TGF is initially activated with benzolamide, both RBF and GFR rapidly decline. Over the next 60 min, RBF increases back to baseline but GFR does not (5). Therefore, TGF resetting cannot be mediated by a simple tachyphylaxis of the TGF response elements. This has certain implications for the vascular response elements and renal metabolism that are discussed elsewhere (5).
NO is a prime suspect to mediate TGF resetting by the JGA because MD NOS I can be activated by MD salt (9) and because endogenous NO modulates TGF in the same rightward direction as MD salt (23, 25, 30). When MD NOS I is active, there will be increased distal salt delivery for any given GFR and increased GFR for any given distal salt delivery. The importance of MD NOS I as a long-term modulator of TGF is revealed by positive salt balance and increased blood pressure that gradually ensue when the TGF curve is forced to shift leftward by chronically inhibiting MD NOS I (12).
We recently confirmed that NO must be present for normal resetting in the 1-h benzolamide model (5). The present data tell us more about the role of NO by revealing that the mere presence of NO is not sufficient for normal TGF resetting. In other words, resetting is abnormal when NOS I is suppressed with NOS blockers or when NOS I is active but unresponsive to MD salt (e.g., in diabetes). These findings suggest that normal TGF resetting requires modulation of MD NO by distal salt delivery. This clue may guide future investigations into the mechanism of TGF resetting where difficult issues remain, such as explaining the prominence of the efferent arteriole and the 30- to 60-min time frame for resetting (5).
As a final caveat, overactive NOS has received attention as a potential cause of diabetic hyperfiltration. From the present data, it is clear that ambient NO excess does not fully explain diabetic hyperfiltration. This is not surprising given that diabetic kidneys are large and that, all else being equal, GFR should vary in proportion to kidney mass (24). Even had acute NOS I blockade completely normalized GFR in diabetes, it would not have been appropriate to invoke this as proof that NO "caused" hyperfiltration in the first place. Whether continuous exposure to excess NO is necessary for the diabetic kidney to become large is a subject distinct and separate from the present study.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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