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【关键词】 nephron
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Department of Pathology, Emory University, Atlanta, Georgia
ABSTRACT
To determine the role of the local renin-angiotensin system in renal function, micropuncture was performed on two lines of mice in which genetic changes to the angiotensin-converting enzyme (ACE) gene markedly reduced or eliminated the expression of renal tissue ACE. Whereas blood pressure is low in one line (ACE 2/2), it is normal in the other (ACE 1/3) due to ectopic hepatic ACE expression. When normalized for renal size, levels of glomerular filtration rate [GFR; μl?min1?g kidney wt1 (KW)] and single-nephron GFR (SNGFR; nl?min1?g KW1) were similar between wild-type (WT) and ACE 1/3 mice, while both measures were significantly reduced in ACE 2/2 mice (WT: 500 ± 63 and 41.7 ± 3.5; ACE 1/3: 515.8 ± 71 and 44.3 ± 3.3; ACE 2/2: 131.4 ± 23 and 30.3 ± 3.5). Proximal fractional reabsorption was not significantly different between WT and ACE 1/3 mice (51 ± 3.5 and 49 ± 2.3%), and it was increased significantly in ACE 2/2 mice (74 ± 3.5%). Infusion of ANG II (50 ng?kg1?min1) increased mean arterial pressure by 7 mmHg in all groups of mice and reduced SNGFR in WT and ACE 1/3 mice (to 30.9 ± 2.8 and 31.9 ± 2.5 nl?min1?g KW1) while increasing it in ACE 2/2 mice (to 55.3 ± 5.3 nl?min1?g KW1) despite an increase in total renal vascular resistance. The tubuloglomerular feedback (TGF) response was markedly reduced in ACE 1/3 mice (stop-flow pressure change 2.5 ± 0.9 mmHg) compared with WT despite similar blood pressures (8.3 ± 0.6 mmHg). In ACE 2/2 mice, TGF was absent (0.7 ± 0.2 mmHg). We conclude that the chronic lack of ACE, and presumably ANG II generation, in the proximal tubule was not associated with sustained proximal fluid transport defects. However, renal tissue ACE is an important contributor to TGF.
nephron filtration rate; proximal fluid reabsorption; tubuloglomerular feedback; angiotensin II kidney weight
THE RENIN-ANGIOTENSIN SYSTEM has been established to play a critical role in extracellular volume and blood pressure homeostasis. Renin is a proteolytic enzyme that is secreted by the juxtaglomerular granular cells of the kidney and that catalyzes the release of angiotensin I from the renin substrate angiotensinogen (28, 29). Angiotensin I is cleaved by angiotensin-converting enzyme (ACE) to form bioactive angiotensin II (28, 29). There can be little doubt that systemically formed angiotensin II acting in an endocrine fashion exerts important effects throughout the organism. Nevertheless, it has become clear that in addition to the so-called systemic renin-angiotensin system, production of angiotensin II can also occur by enzyme-substrate interactions within certain tissues (11). The most consistent of these tissue renin-angiotensin systems are found in the brain and in the kidney, but such local angiotensin generation appears to be a characteristic of many other tissues as well (9, 12, 22).
Discriminating between the actions of angiotensin II formed systemically or formed locally has been difficult because the standard intervention of administering ACE inhibitors or angiotensin receptor blockers does not distinguish between the endocrine or paracrine pathways. Nonconditional gene deletions have been helpful in delineating the role of the renin-angiotensin system in developmental and global functional aspects but have been unable to address the question of specific, local roles of angiotensin II depending on its place of origin (17, 18). Definitive progress has been made with mouse models in which a nonnative renin substrate has been expressed in proximal tubules exclusively and in which mice with this transgene have been found to develop arterial hypertension (8, 10). Since plasma angiotensin II levels were found to be normal, and high levels of the imported substrate were retrieved in the urine, it appeared as if local generation of angiotensin II in the proximal tubule was responsible for the blood pressure aberration (8, 10).
These observations are in agreement with the evidence that proximal tubules can generate renin substrate and, catalyzed by locally produced or filtered renin, can produce angiotensin I (23). Angiotensin I can be converted to angiotensin II by ACE that is abundantly expressed in the brush border of proximal tubules (6, 35, 37). It has been suggested therefore that proximal tubular reabsorption may be under the control of angiotensin II formed locally in the lumen of the proximal tubule and that such a local action may therefore be responsible for some of the hypertensinogenic actions of the peptide (19, 26).
The present experiments were performed to further study the role of the local renin-angiotensin system in the kidney in controlling proximal tubule function and tubuloglomerular feedback (TGF) responsiveness. Previous studies have shown that deletion of the COOH terminus of ACE generates mice with plasma ACE but without any tissue-associated ACE (ACE 2/2 mice) (14). Thus ACE 2/2 mice lack renal brush border-associated ACE and therefore are incapable of forming angiotensin II in the proximal tubule. Since most of total body ACE activity is represented by enzyme anchored in the membrane of endothelial and epithelial cells, complete elimination of tissue ACE was associated with a marked reduction of blood pressure and a deficiency in overall renin-angiotensin system function (14). The blood pressure abnormality has been corrected in a second line of genetically modified mice in which the ACE gene was ectopically expressed in the liver under the control of the albumin promoter (ACE 3/3). These mice have normal blood pressure despite the lack of all endothelial ACE (6). In the present study, we used a compound heterozygote variant of this strain in which one ACE allele is null and the other ACE allele targets ACE expression to the liver (ACE 1/3) (7). These mice have normal blood pressure and, like the ACE 2/2 and ACE 3/3 mice, lack expression of ACE in the endothelium. The ACE 1/3 mice have only 67% the levels of renal ACE found in wild-type (WT) animals. These very low levels of ACE are detectable throughout the proximal tubule, as opposed to WT mice in which ACE expression increases toward the S3 segment of the proximal tubule (6).
The main goal of the present study was to determine rates of nephron filtration and proximal reabsorption in WT mice and in mice without tissue ACE. Using the micropuncture technique in WT, ACE 2/2, and ACE 1/3 mice, we found that the absence of tissue ACE, and therefore of ACE in the proximal tubule brush border, did not significantly affect the relationship between glomerular filtration rate (GFR) and reabsorption. Thus these observations do not yield evidence that the chronic lack of local production of angiotensin II is associated with major changes in proximal reabsorption under basal conditions.
METHODS
Animals. All animal studies were performed according to protocols examined and approved by the Animal Use and Care Committee of the National Institute of Diabetes and Digestive and Kidney Diseases. Experiments were performed in three strains of mice: homozygous ACE 2/2, ACE 1/3, and WT mice. For studies with ACE 2/2 mice, animals were generated from the mating of ACE.2 heterozygous mice. This mating also produced WT littermate controls. ACE 1/3 mice were produced by mating heterozygous ACE.1 mice with homozygous mutant ACE.3 mice (13). All genotype determinations were made as previously described. All mice used in this study were male. They varied in age from 8 to 24 wk.
The animals were maintained on standard rodent chow and tap water. Mice were anesthetized with Inactin (100 mg/kg ip) and ketamine (100 mg/kg im). Body temperature was maintained at 38°C by placing the animals on an operating table with a servo-controlled heating plate. The trachea was cannulated, and a stream of 100% O2 was blown toward the tracheal tube throughout the experiment. The jugular vein was cannulated with two hand-drawn polyethylene catheters, one for an intravenous maintenance infusion of 2.25 g/100 ml bovine serum albumin in saline at a rate of 0.5 ml/h and the other for substance injections. The femoral artery was catheterized for measurement of arterial blood pressure and blood withdrawal. Another catheter was inserted in the urinary bladder for continuous urine collection. The left kidney was approached from a flank incision, freed of adherent fat and connective tissue, and placed in a lucite cup adapted for the size of the mouse kidney. The kidney was then covered with mineral oil.
To determine kidney GFR and nephron filtration and reabsorption rates, mice were infused with [125I]iothalamate (Glofil, Questcor Pharmaceuticals, Hayward, CA) at 40 μCi/h. The first blood samples were obtained after 45 min of equilibration. Free-flow micropuncture was performed according to techniques used previously in rats. Briefly, end-proximal segments were identified by injecting a bolus of artificial tubular fluid stained with FD&C green from a 3- to 4-μm tip pipette connected to a pressure manometer. This pipette remained in place during the collections to permit control of intratubular pressure. All proximal collections were done in the last surface segment, and collection times were between 2 and 4 min. Fluid volume was determined from column length in a constant-bore capillary. The sample was then transferred into a counting vial, and radioactivity was determined in a gamma counter. Blood samples were collected in heparinized 5-μl microcaps at the beginning of micropuncture, after 4560 min, and after 110120 min. [125I]iothalamate radioactivity for determination of kidney GFR was measured in duplicate 0.5-μl samples of plasma and urine. Following a control period of 4560 min, angiotensin II was infused at the rate of 2 ng/min (50 ng?kg1?min1) for the second experimental period of about equal duration. Experiments did not extend beyond 2 h.
Measurements of stop-flow pressure (PSF) during loop of Henle perfusion were done by identifying a late proximal tubule segment from the staining pattern following microperfusion of a randomly selected proximal segment with the FD&C-stained perfusate. The tubule was blocked with wax, the pump was inserted into the last superficial proximal segment, and the pressure pipette was inserted into an early proximal segment recognizable from the widening of the tubular lumen. When PSF had stabilized, the loop of Henle perfusion rate was increased to 30 nl/min and PSF responses were determined. The perfusion fluid contained (in mM) 136 NaCl, 4 NaHCO3, 4 KCl, 2 CaCl2, and 7.5 urea as well as 100 mg/100 ml FD&C green (Keystone).
Renal blood flow. For measurement of renal blood flow (RBF), mice were anesthetized with Inactin (100 mg/kg ip) and ketamine (100 mg/kg im). The left renal artery was approached from a flank incision and carefully dissected free to permit placement of a 0.5PSB nanoprobe connected to a T402-PB flowmeter (Transonic Systems, Ithaca, NY). The probe was held in place with a micromanipulator. The flow signal was digitized and analyzed using PowerLab software (ADInstruments, Colorado Springs, CO). RBF was determined for 10 min, and control values represent the 10-min average. Following baseline RBF measurements, the response of RBF, heart rate, and blood pressure to intravenous bolus injections of angiotensin I (13 and 65 ng) or angiotensin II (10 and 50 ng) was determined in the same animals. In another group of animals, measurements of baseline RBF were followed by a 10-min infusion of angiotensin II at 3 ng/min.
Statistics. Data are given as arithmetic means and variations as SE. Significance comparisons were done with ANOVA in combination with the Bonferroni post hoc test or with the t-test as appropriate.
RESULTS
Body and kidney weights. Mean body weights were 39.7 ± 1.1 g in WT (n = 9), 41.3 ± 3 g in ACE 2/2 (n = 7; not significant), and 27.8 ± 0.5 g in ACE 1/3 mice (n = 11; P < 0.001 compared with WT). Kidney weights were 584 ± 17.5 mg in WT and 392 ± 30.5 and 378 ± 11.5 mg in ACE 2/2 (P < 0.001) and ACE 1/3 (P < 0.001) mice, respectively. Thus, while body weights of WT and ACE 2/2 mice were the same, kidney weights were significantly lower in ACE 2/2 than control mice. The relationship between body weight and kidney weight is shown in Fig. 1. It can be seen that body and kidney weights of ACE 1/3 mice were lower than of WT animals but that the relationship between body and kidney weights was the same in the two strains of mice. In contrast, the kidney weights of ACE 2/2 mice were consistently lower for any given body weight than in either WT or ACE 1/3 mice.
Hemodynamic response to angiotensin. Responses of arterial blood pressure and renal vascular resistance to bolus injections of angiotensin II (10 and 50 ng) and angiotensin I (13 and 65 ng) were determined in five WT, five ACE 2/2, and five ACE 1/3 mice. Mean arterial blood pressure in response to angiotensin II increased in these three groups of mice with comparable sensitivity, whereas blood pressure changes in response to angiotensin I were virtually absent in ACE 2/2 and diminished in ACE 1/3 mice (Fig. 2). The reduced blood pressure response to angiotensin I in the ACE 1/3 mice is consistent with the original publication on these animals (7). This probably reflects the high circulating angiotensin I levels found in these mice and the reduced total levels of ACE (7). These determinations, combined with measurements of RBF by using ultrasonic flowmeters, permitted calculation of the response of renal vascular resistance to angiotensins. Renal resistance increased significantly in all groups of mice in response to angiotensin II although the increase was less in ACE 1/3 mice. As expected, angiotensin I had markedly lower effects on renal vascular resistance in ACE 2/2 and ACE 1/3 than WT mice (Fig. 2).
GFR and RBF. RBF (1 kidney), as determined with an ultrasonic flowmeter, averaged 1.14 ± 0.08 ml/min in WT (n = 5), 0.77 ± 0.1 ml/min in ACE 2/2 (n = 5), and 1.27 ± 0.13 ml/min in ACE 1/3 mice (n = 5; Fig. 3). Mean arterial blood pressures in these anesthetized and laparatomized animals were 97 ± 7.4, 48 ± 5.5, and 84 ± 4 in WT, ACE 2/2, and ACE 1/3 mice, respectively. Renal vascular resistance tended to be lower in ACE 2/2 mice, but differences between genotypes were not significant. The effect of an intravenous infusion of angiotensin II at 3 ng?kg1?min1 on renal hemodynamics is included in Fig. 3. It can be seen that angiotensin II decreased RBF and increased renal vascular resistance in WT mice as well as in ACE 2/2 and ACE 1/3 mice.
GFR expressed both in absolute terms and normalized for kidney weight is summarized in Fig. 4. As can be seen, GFR was not significantly different between WT (500 ± 63 μl?min1?g kidney wt1 and 294.6 ± 39.6 μl/min; n = 9) and ACE 1/3 mice (515.8 ± 71 μl?min1?g kidney wt1 and 184.3 ± 24.2 μl/min; n = 6) when tested by ANOVA with a Bonferroni correction. The tendency for the lower absolute GFR in the ACE 1/3 mice probably reflects their lower kidney weight. In contrast, the GFR in ACE 2/2 mice was significantly reduced compared with WT or ACE 1/3 when expressed both in absolute and kidney weight-corrected terms (131.4 ± 23 μl?min1?g kidney wt1 and 53.6 ± 13.6 μl/min; n = 7). Mean arterial blood pressure in this group of animals again was significantly lower in ACE 2/2 mice than in either WT or ACE 1/3 animals (63 ± 4 compared with 95 ± 4.5 and 85 ± 4 mmHg). As also shown in Fig. 4, infusion of angiotensin II reduced GFR in WT mice to 385 ± 99 μl?min1?g kidney wt1 (P = 0.03 vs. control) and in ACE 1/3 mice to 360 ± 47 μl?min1?g kidney wt1 (P = 0.02 vs. control), whereas it increased GFR in ACE 2/2 mice to 179 ± 31 μl?min1?g kidney wt1 (P = 0.007 vs. control). Mean arterial blood pressure during the infusion of angiotensin II increased by about 67 mmHg in all groups.
Single-nephron GFR and tubular reabsorption. Single-nephron GFR (SNGFR) and rates of proximal tubular reabsorption were determined in five WT, four ACE 2/2, and five ACE 1/3 transgenic animals using standard free-flow micropuncture. Mean body weight of the mice used in the micropuncture studies was 42 ± 1 g in WT mice, 37.2 ± 1.6 g in ACE 2/2, and 27.8 ± 1.07 g in ACE 1/3 mice (P < 0.001 compared with either WT or ACE 2/2, ANOVA). Left kidney weights averaged 306 ± 23.1 mg in WT, 185 ± 11 mg in ACE 2/2 (P < 0.001 compared with WT), and 177 ± 4 mg in ACE 1/3 (P < 0.001 compared with WT). SNGFR (Fig. 5) averaged 11.7 ± 0.9 nl/min in WT (n = 14), 4.9 ± 0.6 nl/min in ACE 2/2 (n = 12; P < 0.001 compared with WT, ANOVA), and 7.8 ± 0.6 nl/min in ACE 1/3 mice (n = 23; P < 0.001 compared with WT, P < 0.05 compared with ACE 2/2, ANOVA). As also shown in Fig. 5, the difference in SNGFR between WT and ACE 1/3 mice was abolished by kidney weight correction (41.7 ± 3.5 nl?min1?g kidney wt1 vs. 44.3 ± 3.4 nl?min1?g kidney wt1), whereas SNGFR remained lower in ACE 2/2 mice (30.3 ± 4 nl?min1?g kidney wt1; P < 0.05 compared with WT, ANOVA).
The response of SNGFR to an infusion of angiotensin II consisted of a significant reduction in both WT and ACE 1/3 mice to 8.6 ± 0.7 and 5.7 ± 0.4 nl/min, respectively (P = 0.01 and P = 0.008 compared with control). In contrast, angiotensin II increased SNGFR in ACE 2/2 mice to 9.1 ± 0.8 nl/min (P = 0.001 compared with control). Kidney weight correction did not alter the conclusion that angiotensin II significantly reduced SNGFR in WT and ACE 1/3 mice and increased it in ACE 2/2 mice (Fig. 5). Mean arterial blood pressure during the time of micropuncture increased with angiotensin II from 102.5 ± 3 to 109.6 ± 3.3 mmHg in WT (P = 0.02), from 68.5 ± 1.7 to 75 ± 2.4 mmHg in ACE 2/2 (P = 0.0004), and from 83 ± 2 to 90 ± 2 mmHg in ACE 1/3 mice (P < 0.00001). Blood pressure was significantly higher in WT than in ACE 2/2 mice (P < 0.01).
Proximal fractional reabsorption averaged 51.3 ± 3% in WT and 49 ± 2.3% in ACE 1/3 mice (not significant). In contrast, fractional reabsorption in ACE 2/2 mice was significantly higher, averaging 74.4 ± 3.5%. Rates of proximal tubular fluid reabsorption without and with correction for kidney weight are summarized in Fig. 6. While rates of absorption were significantly lower in both ACE 2/2 and ACE 1/3 than in WT mice when expressed in absolute terms, there were no differences in normalized reabsorption rates between the different genotypes. The relationship between SNGFR and proximal reabsorption showing identical slopes for WT and ACE 1/3 mice indicates that differences in absolute reabsorption reflect differences in kidney weight and not in intrinsic reabsorptive capacity (Fig. 7, top). In contrast, the glomerulotubular balance function was shifted upward in ACE 2/2 mice, reflecting the increased fractional reabsorption. Angiotensin II infusion increased fractional reabsorption to 60.4 ± 3.2% in WT (P = 0.05 compared with control) and decreased it in both ACE 2/2 and ACE 1/3 mice to 55.6 ± 2.3 and 40.8 ± 2.5%, respectively (P = 0.0004 and P = 0.02 compared with control). During the infusion of angiotensin II, the reabsorption rate increased significantly in ACE 2/2 mice compared with control, probably as a result of the markedly increased filtration rate. Reabsorption rates tended to decrease in WT mice, and they fell significantly in ACE 1/3 mice. The relationship between SNGFR and proximal reabsorption rate in individual tubules of WT, ACE 2/2, and ACE 1/3 mice suggests that the infusion of angiotensin II was associated in ACE 1/3 mice with a slight reduction of reabsorption at each level of SNGFR compared with either WT or ACE 2/2 mice (Fig. 7, bottom).
TGF. TGF was assessed as the reduction of PSF caused by a saturating increase in the loop of Henle perfusion rate (30 nl/min). The findings are summarized in Fig. 8. PSF fell from 38.8 ± 1.3 to 30.5 ± 1.4 mmHg in WT (n = 13; P < 0.0001) and from 31.8 ± 1.5 to 29.3 ± 1.3 mmHg in ACE 1/3 mice (n = 10; P = 0.02), whereas it did not significantly change in ACE 2/2 mice (28.3 ± 0.6 vs. 27.7 ± 0.6; n = 9; P = 0.07). The decrease in PSF by 8.3 ± 0.6 mmHg in WT mice was significantly greater than that of 0.7 ± 0.22 mmHg in ACE 2/2 (P < 0.001 compared with WT) and of 2.5 ± 0.9 mmHg in ACE 1/3 mice (P < 0.001 compared with WT). Mean arterial blood pressure in these studies was 107 ± 2.6 mmHg in WT, 70.1 ± 1.7 mmHg in ACE 2/2, and 85 ± 5.9 mmHg in ACE 1/3 mice.
DISCUSSION
The renin-angiotensin system influences numerous aspects of renal function related to the control of renal vascular resistance, net salt and water reabsorption, and the renal concentrating mechanism (15). While most of the actions of angiotensin II are mediated through the AT1 receptor, the peptide can also bind to the AT2 receptor, eliciting effects that widen the spectrum of its renal actions (3). One of the most vexing problems in understanding the consequences of changes in the activity of the renin-angiotensin system is related to the fact that angiotensin II can be generated systemically, with plasma renin being the rate-limiting step in most cases, but that it can also be formed by substrate-enzyme interactions entirely located in a number of tissues including in the kidney (1). The concept of intrarenal formation of angiotensin II is based on the presence of renin substrate, renin enzyme, and ACE in cellular and extracellular compartments within the kidney. As a consequence of local enzymatic processes, proximal tubular angiotensin II levels have been found markedly elevated compared with those in plasma (2, 36). Furthermore, renal angiotensin II levels do not always mirror plasma peptide concentrations (25).
The main goals of the present studies were to assess the effect of a chronically deficient formation of angiotensin II by proximal tubules on proximal fluid reabsorption and to determine the effect of the absence of tissue ACE on TGF. Progress in this area has been made possible by the generation of mice with deficiencies in specific components of the renin-angiotensin system through genetic interventions in embryonic stem cells. In the present study, we used two strains of mice with selected mutations of ACE: ACE 2/2 and ACE 1/3 (7, 14). ACE 2/2 mice, in which the membrane anchoring COOH-terminal domain has been deleted, lack membrane-bound ACE in the lung, kidney, and other tissues. Although some plasma ACE activity is retained, ACE 2/2 mice have reduced plasma angiotensin II levels and a significant reduction in arterial blood pressure (5, 14). ACE 1/3 mice, on the other hand, have been engineered to express ACE in the liver while still lacking all ACE expression in the endothelium (7). These animals have been found to compensate for the genetic modification, and they are able to maintain largely normal blood pressure levels (7).
Our results show that proximal tubular fluid reabsorption of these genetically altered mice was comparable to that observed in WT mice despite the essentially complete absence of tissue ACE. As is well known, proximal fluid reabsorption varied with SNGFR, but the relationship between SNGFR and proximal tubular reabsorption of fluid was not different between WT and ACE 1/3 mice, and it even shifted upward in ACE 2/2 mice as a reflection of an increase in fractional fluid reabsorption (Fig. 6, top). The mechanism for this increase in fractional fluid reabsorption in proximal tubules of ACE 2/2 mice was not explored in this study, but it is likely that it is related in some way to the reduced SNGFR. Our observations are unexpected in view of previous demonstrations that the intratubular administration of converting enzyme inhibitors caused a marked reduction in proximal fluid reabsorption (30) and that the same effect was seen with angiotensin II receptor blockers (31). These findings clearly show that an acute reduction in local angiotensin II formation and action can exert a profound inhibitory effect on fluid reabsorption. We assume therefore that the difference between these previous results and the present data lies in the chronicity of angiotensin II blockade. Chronic ACE deficiency is apparently associated with compensatory events that normalize fluid reabsorption along the proximal tubule. On the basis of these findings, one would not expect that chronic interference with local angiotensin II formation in the proximal tubule plays a major role in the maintenance of reductions in extracellular fluid volume. In fact, careful previous measurements have been unable to show measurable plasma volume contraction in either ACE 2/2 or ACE 1/3 mice (5, 7). A similar discrepancy between the effects of acute and chronic blockade of a transport-regulatory pathway has also been observed during inhibition of adenosine 1 receptors (A1R). While acute A1R blockade with CVT-124 caused a marked inhibition of proximal fluid reabsorption, proximal transport was unaltered in A1R-deficient mice (40, 41). Given the multifactorial nature of the regulation of proximal fluid transport, it is perhaps not unexpected that the chronic elimination of one regulatory input appears to be relatively inconsequential. It would be important to examine the related question of whether extracellular volume expansion can result from chronic overproduction of angiotensin II or other stimulatory transport regulators in the proximal tubule.
The infusion of angiotensin II at doses that caused a mild elevation of blood pressure did not significantly alter the rate of fluid reabsorption in WT mice, although there was a decrease in SNGFR. Thus angiotensin II infusion caused a mild stimulation of NaCl transport for a given SNGFR, shifting the reabsorption-SNGFR function curve slightly upward. Stimulation of proximal reabsorption during systemic infusion of angiotensin II has been described earlier (20). In contrast, fluid reabsorption fell in near proportion to SNGFR in angiotensin-infused ACE 1/3 mice. Since plasma angiotensin II levels in these animals have been found to be three times WT levels (7), it is conceivable that angiotensin II-dependent proximal reabsorption had reached its maximum before the angiotensin II infusion was started and that further increases in plasma angiotensin II were therefore not associated with further stimulation of transport (16). It is also conceivable that the elevated angiotensin II level caused downregulation of AT1 receptors throughout the organism.
In ACE 2/2 mice, angiotensin II infusion produced a marked increase in proximal fluid reabsorption (Fig. 6) that was paralleled by an increase in nephron as well as kidney GFR (Fig. 5) and may thus result from flow dependence of proximal fluid reabsorption, reflecting the well-known phenomenon of glomerulotubular balance. In addition, a direct effect of angiotensin II may contribute to transport stimulation. The cause for the increase in GFR is probably at least partly related to the increase in arterial blood pressure. However, angiotensin II elevated blood pressure to the same extent in WT and ACE 1/3 mice while lowering instead of increasing GFR. It is especially noteworthy that angiotensin II increased GFR in ACE 2/2 mice while at the same time doubling total renal vascular resistance (Fig. 3). Since a preglomerular vasoconstriction would result in a decrease in GFR, one is forced to assume that angiotensin II caused a predominantly postglomerular resistance increase in the ACE 2/2 mouse strain. Modeling suggests that an increase in postglomerular resistance causes an increase in GFR only at very low initial values of efferent resistance (24). Because of the low angiotensin II levels, low blood pressure, and the absence of TGF, it is likely that the glomerular arterioles of ACE 2/2 mice have a rather low resistance under ambient conditions (5). The notion that angiotensin II infused into the low-angiotensin II state of the ACE 2/2 mice may cause a predominantly efferent constriction is feasible in view of the evidence that efferent arterioles are more sensitive to angiotensin II than afferent arterioles when starting from zero angiotensin II, particularly at low pressures (42). The low initial filtration fraction (7%) and its increase with angiotensin II (to 12%) is consistent with this assumption, although it is in itself no proof for a predominantly efferent constriction (4).
The present studies show that mice without tissue ACE have a markedly reduced capacity to transform changes in luminal NaCl concentration into changes in glomerular capillary pressure. This TGF response deficit is reminiscent of earlier studies in AT1AR and ACE knockout mice from our laboratory (34, 38). Furthermore, pharmacological inhibition of AT1AR and ACE causes a significant reduction in TGF responsiveness (32). Conversely, the administration of angiotensin II by either systemic or peritubular infusion enhances TGF responses (21). The reduction in TGF responses in ACE 2/2 mice may be aided by the markedly reduced blood pressure since blood pressure has been shown earlier to be a determinant of the response magnitude (33). However, our observations in ACE 1/3 mice in which blood pressure was only marginally altered indicate that lack of angiotensin II availability contributes importantly to the attenuation of TGF responsiveness. Since plasma angiotensin II levels in ACE 1/3 mice have been found to be clearly higher than normal, the presence of angiotensin II in the systemic circulation appears to be insufficient to sustain normal TGF responses. It would appear rather that angiotensin II generation by tissue ACE is required to support this function. It is possible that tissue ACE in the vascular wall of afferent arterioles normally provides TGF-relevant angiotensin II. On the other hand, it is also conceivable that angiotensin II generated in the tubular lumen affects NaCl transport across macula densa cells and that this increase in transport enhances the basal sensitivity of the TGF system.
The present studies show that kidneys of ACE 2/2 mice are significantly hypoplastic compared with age-matched WT animals. The reasons for the reduced growth rate are unclear and need to be evaluated further. Reduced kidney weights compared with WT have also been reported in mice with combined AT1AR and AT1BR deficiency, but in this case there was also a proportional difference in total body weight (27). Pharmacological blockade of angiotensin II receptors in neonatal rats also caused reduced somatic and renal growth (39). One of the functional consequences of the reduced kidney mass is that tissue-specific RBF in ACE 2/2 mice was not significantly different from WT, although absolute values were significantly lower in ACE 2/2 than in WT mice. In addition, our data show that the reduction in kidney GFR in ACE 2/2 mice compared with WT was more marked (81%) than that in superficial nephron GFR (58%). One explanation for this observation is that fewer nephrons contribute to total GFR in ACE 2/2 than WT mice. Calculation of the "effective" number of glomeruli from kidney and SNGFR yields values of 24,000 in WT and ACE 1/3 but only 9,000 in ACE 2/2 mice. While this calculation assumes equal rates of filtration in all nephrons of the kidneys and obviously only provides an estimate of the number of glomeruli, the difference appears to be clear and beyond all possible sources of error. A reduced nephron number has also been reported to be associated with neonatal administration of losartan (39). Thus it appears that structural differences are partly responsible for the reduced renal function in ACE 2/2 mice.
In conclusion, our results show that the relationship between proximal tubule fluid reabsorption and filtration rate is comparable between WT mice and two strains of mice that lack tissue ACE activity. Thus the absence of angiotensin II generation in the proximal tubule does not appear to be associated with sustained inhibition of proximal fluid transport that could be directly responsible for volume depletion and reductions of arterial blood pressure. Generation of angiotensin II by tissue ACE contributes importantly to the magnitude of TGF responses.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-39777, DK-51445, and DK-55503 (K. E. Bernstein) and by intramural funds from the NIDDK. S. Hashimoto was the recipient of a Visiting Fellowship of the NIDDK.
ACKNOWLEDGMENTS
Present address of S. Hashimoto: Department of Medicine II, Hokkaido Univ., Sapporo, Hokkaido, Japan 060 8638.
FOOTNOTES
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.
REFERENCES
Bader M, Peters J, Baltatu O, Muller DN, Luft FC, and Ganten D. Tissue renin-angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med 79: 76102, 2001.
Braam B, Mitchell KD, Fox J, and Navar LG. Proximal tubular secretion of angiotensin II in rats. Am J Physiol Renal Fluid Electrolyte Physiol 264: F891F898, 1993.
Carey RM and Siragy HM. Newly recognized components of the renin-angiotensin system: potential roles in cardiovascular and renal regulation. Endocr Rev 24: 261271, 2003.
Carmines PK, Perry MD, Hazelrig JB, and Navar LG. Effects of preglomerular and postglomerular vascular resistance alterations on filtration fraction. Kidney Int Suppl 20: S229S232, 1987.
Cole J, Ertoy D, Lin H, Sutliff RL, Ezan E, Guyene TT, Capecchi M, Corvol P, and Bernstein KE. Lack of angiotensin II-facilitated erythropoiesis causes anemia in angiotensin-converting enzyme-deficient mice. J Clin Invest 106: 13911398, 2000.
Cole J, Quach du L, Sundaram K, Corvol P, Capecchi MR, and Bernstein KE. Mice lacking endothelial angiotensin-converting enzyme have a normal blood pressure. Circ Res 90: 8792, 2002.
Cole JM, Khokhlova N, Sutliff RL, Adams JW, Disher KM, Zhao H, Capecchi MR, Corvol P, and Bernstein KE. Mice lacking endothelial ACE: normal blood pressure with elevated angiotensin II. Hypertension 41: 313321, 2003.
Davisson RL, Ding Y, Stec DE, Catterall JF, and Sigmund CD. Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice. Physiol Genomics 1: 39, 1999.
Diet F, Pratt RE, Berry GJ, Momose N, Gibbons GH, and Dzau VJ. Increased accumulation of tissue ACE in human atherosclerotic coronary artery disease. Circulation 94: 27562767, 1996.
Ding Y, Davisson RL, Hardy DO, Zhu LJ, Merrill DC, Catterall JF, and Sigmund CD. The kidney androgen-regulated protein promoter confers renal proximal tubule cell-specific and highly androgen-responsive expression on the human angiotensinogen gene in transgenic mice. J Biol Chem 272: 2814228148, 1997.
Dzau VJ. Evolving concepts of the renin-angiotensin system. Focus on renal and vascular mechanisms. Am J Hypertens 1: 334S-337S, 1988.
Dzau VJ. Tissue angiotensin and pathobiology of vascular disease. Hypertension 37: 10471052, 2001.
Esther CR, Howard TE, Marino EM, Goddard JM, Capecchi MR, and Bernstein KE. Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab Invest 74: 953965, 1996.
Esther CR, Marino EM, Howard TE, Machaud A, Corvol P, Capecchi MR, and Bernstein KE. The critical role of tissue angiotensin-converting enzyme as revealed by gene targeting in mice. J Clin Invest 99: 23752385, 1997.
Hall JE and Brands MW. The renin-angiotensin-aldosterone systems: renal mechanisms and circulatory homeostasis. In: The Kidney: Physiology and Pathophysiology (3rd ed.), edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p.10091045.
Harris PJ and Young JA. Dose-dependent stimulation and inhibition of proximal tubular sodium reabsorption by angiotensin II in the rat kidney. Pflügers Arch 367: 295297, 1977.
Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, and Coffman TM. Regulation of blood pressure by the type 1A angiotensin II receptor gene. Proc Natl Acad Sci USA 92: 35213525, 1995.
Krege JH, Kim HS, Moyer JS, Jennette JC, Peng L, Hiller SK, and Smithies O. Angiotensin-converting enzyme gene mutations, blood pressures, and cardiovascular homeostasis. Hypertension 29: 150157, 1997.
Lavoie JL, Lake-Bruse KD, and Sigmund CD. Increased blood pressure in transgenic mice expressing both human renin and angiotensinogen in the renal proximal tubule. Am J Physiol Renal Physiol 286: F965F971, 2004.
Liu FY and Cogan MG. Angiotensin II: a potent regulator of acidification in the rat early proximal convoluted tubule. J Clin Invest 80: 272275, 1987.
Mitchell KD and Navar LG. Enhanced tubuloglomerular feedback during peritubular infusions of angiotensins I and II. Am J Physiol Renal Fluid Electrolyte Physiol 255: F383F390, 1988.
Muller DN, Bohlender J, Hilgers KF, Dragun D, Costerousse O, Menard J, and Luft FC. Vascular angiotensin-converting enzyme expression regulates local angiotensin II. Hypertension 29: 98104, 1997.
Navar LG. The intrarenal renin-angiotensin system in hypertension. Kidney Int 65: 15221532, 2004.
Navar LG. The regulation of glomerular filtration rate in mammalian kidneys. In: Physiology of Membrane Disorders, edited by Andreoli TE, Hoffman JF and Fanestil DD. New York: Plenum, 1978, p. 593627.
Navar LG and Harrison-Bernard LM. Intrarenal angiotensin II augmentation in angiotensin II dependent hypertension. Hypertens Res 23: 291301, 2000.
Navar LG, Harrison-Bernard LM, Wang CT, Cervenka L, and Mitchell KD. Concentrations and actions of intraluminal angiotensin II. J Am Soc Nephrol 10, Suppl 11: S189S195, 1999.
Oliverio MI, Kim HS, Ito M, Le T, Audoly L, Best CF, Hiller S, Kluckman K, Maeda N, Smithies O, and Coffman TM. Reduced growth, abnormal kidney structure, and type 2 (AT2) angiotensin receptor-mediated blood pressure regulation in mice lacking both AT1A and AT1B receptors for angiotensin II. Proc Natl Acad Sci USA 95: 1549615501, 1998.
Peach MJ. Renin-angiotensin system: biochemistry and mechanisms of action. Physiol Rev 57: 313370, 1977.
Peart WS. The renin-angiotensin system. Pharmacol Rev 17: 143182, 1965.
Quan A and Baum M. Endogenous production of angiotensin II modulates rat proximal tubule transport. J Clin Invest 97: 28782882, 1996.
Quan A and Baum M. Regulation of proximal tubule transport by angiotensin II. Semin Nephrol 17: 423430, 1997.
Schnermann J and Briggs JP. Function of the juxtaglomerular apparatus: control of glomerular hemodynamics and renin secretion. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 945980.
Schnermann J and Briggs JP. Interaction between loop of Henle flow and arterial pressure as determinants of glomerular pressure. Am J Physiol Renal Fluid Electrolyte Physiol 256: F421F429, 1989.
Schnermann JB, Traynor T, Yang T, Huang YG, Oliverio MI, Coffman T, and Briggs JP. Absence of tubuloglomerular feedback responses in AT1A receptor-deficient mice. Am J Physiol Renal Physiol 273: F315F320, 1997.
Schulz WW, Hagler HK, Buja LM, and Erdos EG. Ultrastructural localization of angiotensin I-converting enzyme (EC 3.4151) and neutral metalloendopeptidase (EC 342411) in the proximal tubule of the human kidney. Lab Invest 59: 789797, 1988.
Seikaly MG, Arant BS Jr, and Seney FD Jr. Endogenous angiotensin concentrations in specific intrarenal fluid compartments of the rat. J Clin Invest 86: 13521357, 1990.
Sibony M, Gasc JM, Soubrier F, Alhenc-Gelas F, and Corvol P. Gene expression and tissue localization of the two isoforms of angiotensin I converting enzyme. Hypertension 21: 827835, 1993.
Traynor T, Yang T, Huang YG, Krege JH, Briggs JP, Smithies O, and Schnermann J. Tubuloglomerular feedback in ACE-deficient mice. Am J Physiol Renal Physiol 276: F751F757, 1999.
Tufro-McReddie A, Romano LM, Harris JM, Ferder L, and Gomez RA. Angiotensin II regulates nephrogenesis and renal vascular development. Am J Physiol Renal Fluid Electrolyte Physiol 269: F110F115, 1995.
Vallon V, Richter K, Huang DY, Rieg T, and Schnermann J. Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice. Pflügers Arch 448: 214221, 2004.
Wilcox CS, Welch WJ, Schreiner GF, and Belardinelli L. Natriuretic and diuretic actions of a highly selective adenosine A1 receptor antagonist. J Am Soc Nephrol 10: 714720, 1999.
Yuan BH, Robinette JB, and Conger JD. Effect of angiotensin II and norepinephrine on isolated rat afferent and efferent arterioles. Am J Physiol Renal Fluid Electrolyte Physiol 258: F741F750, 1990.