Nephron Function in Transgenic Mice with Selective Vascular or Tubular Expression of Angiotensin-Converting Enzyme
Sean P. Kessler*,
Seiji Hashimoto,
Preenie S. Senanayake,
Christina Gaughan*,
Ganes C. Sen* and
Jurgen Schnermann
* Department of Molecular Genetics, Lerner Research Institute, and Department of Ophthalmic Research, Cleveland Clinic Foundation, Cleveland, Ohio; and National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Address correspondence to: Dr. Sean P. Kessler, Department of Molecular Genetics NE20, Lerner Research Institute, 9500 Euclid Avenue, Cleveland, OH 44195. Phone: 216-444-0884; Fax: 216-444-0512; kessles{at}ccf.org
Received for publication February 8, 2005.
Accepted for publication August 24, 2005.
Angiotensin-converting enzyme (ACE) null mice display aberrantrenal pathology. Inadequate formation of angiotensin II (AngII) results in hypotension, loss of fluid homeostasis, lackof urine concentration, and failure to regulate GFR throughthe tubuloglomerular feedback (TGF) mechanism. For examiningthe tissue-specific role of ACE in renal structure and regulationof renal filtrate formation, single-nephron GFR, proximal tubularfluid reabsorption, and TGF responsiveness were determined inmice that expressed ACE in only one tissue. Maximum TGF responsesin mice that expressed somatic ACE (sACE) in proximal tubulecells (Gs strain) or germinal ACE in the serum (Pg strain) werereduced significantly compared with wild-type (WT) mice. Incontrast, TGF responses in mice that expressed sACE in vascularendothelial cells (Ts strain) were not different from control.Single-nephron GFR was reduced in Ts compared with WT mice,but fractional reabsorption and therefore glomerulotubular balancewere not distinguishable. BP responses to exogenous Ang I werediminished in Ts, Gs, and Pg mice, whereas those to Ang II werethe same in the different strains. Plasma and renal tissue AngI of all transgenic mouse strains was significantly higher thanWT, whereas Ang II levels were generally lower; aldosteronelevels were significantly lower than WT in Ts mice but not inthe two other transgenic strains. Our results demonstrate thatvascular expression of sACE can largely but not completely restoreTGF regulation of GFR. Proximal fluid reabsorption in the chronicabsence of proximal tubule ACE is normal.
Control of fluid homeostasis by the kidneys requires the participationof numerous paracrine agents that act in concert to regulatethe formation of a large glomerular ultrafiltrate and the subsequentretrieval of essential constituents along the tubular system.It has been recognized that among these regulatory pathwaysthe renin-angiotensin-aldosterone system (RAAS) seems to playan important and perhaps dominant role (1). Both angiotensinII (Ang II) as the end product of the enzymatic actions of reninand angiotensin-converting enzyme (ACE) and aldosterone, whichis secreted in response to Ang II, have profound effects onthe absorption of NaCl and water in most segments of the renalnephron. In addition, Ang II is a vasoactive agent that affectsGFR by vasoconstrictor actions in glomerular arterioles (2).Tubular absorption and glomerular filtration are linked by thetubuloglomerular feedback (TGF) mechanism. As NaCl concentrationrises in the tubular fluid passing the macula densa, for exampleas a result of a reduction in proximal fluid absorption, vasoconstrictionof afferent arterioles occurs, and the resultant change of GFRlimits the delivery of fluid to the late nephron (3). It isthought that this tubulovascular cross-talk assists in the preservationof electrolyte balance by limiting sodium excretion. Whereasadenosine acting through A1 adenosine receptors seems to bethe direct mediator of this response, a substantial body ofevidence indicates that the formation of Ang II is necessaryfor adenosine to exert its actions (4).
Studies of the effects of pharmacologic inhibitors of the RAAShave been used widely to assess its role in physiology and pathophysiology.This approach was complemented recently by the development ofmutant mouse strains defective in the expression of renin, reninsubstrate, ACE, or angiotensin receptors (5–9). The operationof the RAAS as a single functional unit is supported by thefindings that all of these strains have essentially identicalabnormalities, including arterial hypotension, renal corticaland papillary atrophy, and hyperplasia of the renal vasculature.Specifically, ACE null mice that are generated in our laboratoryin the FVB background have identical renal defects: Hypotensionand male sterility (10). In addition, Ace–/– miceare runty and have a life expectancy of only 6 to 8 mo as aresult of the severe renal defects. Although the occurrenceof these abnormalities is highly informative in itself, theseverity of the RAAS null phenotype limits the usefulness ofthese animals for physiologic studies.
In addition, it has become clear that the RAAS fulfills bothendocrine and paracrine roles through the operation of systemicgeneration of Ang II on the one hand and the local formationof Ang II in certain tissues on the other hand (11). Both theuse of pharmacologic inhibitors and the generation of null mutantsare unable to distinguish between these two modes of RAAS operation.To address this question, we produced several strains of micewith tissue-specific expression of ACE with the expectationthat such animals would facilitate the distinction between systemicand local RAAS actions. The general strategy was to use tissue-specificpromoters to drive the expression of a single ACE isoform inmice with an ACE null FVB background (10,12,13).
The main goal of this study was to assess the effect of selectiveexpression of somatic ACE (sACE) in vascular endothelial cellson TGF responsiveness and proximal tubule function in an otherwiseAce–/– animal. The Tie-1 promoter was used to drivethe expression of sACE in vascular endothelial cells (Ts strain).For comparison, TGF responses were also determined in mice withexpression of sACE in renal proximal tubule cells using the-glutamyl transpeptidase promoter (Gs strain) and in a strainof mice that expresses germinal ACE (gACE) in sperm using thehuman PGK2 promoter (Pg strain). Mice of the Ts and Gs strainshave sACE in their serum as a result of the natural cleavage-secretionprocessing of membrane-anchored ACE (13), whereas Pg mice ectopicallyexpress gACE in the serum (10). Our observations in the Ts strainshow that TGF regulation was largely but not entirely normalizedby expression of ACE in vascular endothelial cells. Furthermore,the absence of ACE in renal proximal tubule cells does not causemeasurable changes of fluid reabsorption. These results furthersupport the notion that specific physiologic functions of ACEare determined by the location of ACE expression. Moreover,the expression of ACE in proximal tubules is redundant in thesense that its long-term absence does not compromise proximalNaCl and fluid reabsorption.
Mice
The FVB strains of mice that were used in this study have beencharacterized previously (10,13) and have the following genotypes:Ts (Ace–/–, Ts+/+), Gs (Ace–/–, Gs+/–)and Pg (Ace–/–, Pg+/–). Animals were maintainedon a standard rodent diet and sterile tap water, and studieswere performed at the age of 4 mo.
Aldosterone and Ang I and II Levels
Plasma aldosterone levels were determined as described previously(14) in four age-matched adult mice of each genotype using theCoat-A-Count method according to the manufacturer’s instructions(Diagnostic Products Corp., Los Angeles, CA). The genotype averagesthat were obtained by duplicate assays from each mouse are reported±95% confidence interval (CI). Plasma and renal Ang Iand Ang II levels both were measured as described previously(13,15). Duplicate assays on a minimum of three plasma pools,each pool obtained from four to five age-matched mice, wereaveraged and reported ±95% CI. Duplicate assays on thekidneys from five mice of each genotype were averaged and reported±95% CI.
GFR and Renal Blood Flow
For determining GFR, mice were infused with 125I iothalamate(Glofil, Questcor Pharmaceutical, Hayward, CA) at approximately5 µCi/h. After 30 to 45 min of equilibration, a bloodsample of approximately 4 µl was collected into heparinized5-µl microcaps (Drummond, Broomall, PA), and a urine collectionwas made for 30 min followed by a second blood collection. 125Iiothalamate radioactivity was measured in duplicate 0.5-µlaliquots of plasma and urine in a Riastar -counter (PackardInstrument Co., Downers Grove, IL). Urine volume was determinedgravimetrically. Measurements of renal blood flow (RBF) wereperformed in a separate group of wild-type (WT) and Ts mice.The left renal artery was approached from a flank incision andcarefully dissected free to permit placement of a 0.5PSB nanoprobeconnected to a T402-PB flow meter (Transonic Systems, Ithaca,NY). The probe was held in place with a micromanipulator. Theflow signal was digitized and analyzed using PowerLab software(ADInstruments, Colorado Springs, CO). RBF was determined for20 min and values given represent the average for the observationperiod.
Micropuncture Studies
The preparation of mice and the techniques that were used toassess proximal tubule function and TGF responses of stop flowpressure have been described in detail (16). In short, the leftkidneys of mice under inactin/ketamine anesthesia were approachedfrom a flank incision and placed in a Lucite cup adapted forthe size of the mouse kidney. Nephron filtration and absorptionrates were determined from timed end-proximal fluid collectionsusing 125I iothalamate as a volume marker. TGF responses wereassessed as the change of stop flow pressure (PSF) in responseto an increase of loop of Henle perfusion rate from 0 to 30nl/min. The perfusion fluid contained (in mM/L) 136 NaCl, 4NaHCO3, 4KCl, 2CaCl2, and 7.5 urea and 100 mg/100 ml FD&Cgreen (Keystone).
Immunohistochemistry
Adult male mice were killed by inhalation of 100% CO2. Histochoice-preserved,paraffin-embedded kidney sections were prepared as describedpreviously (13). The monoclonal mouse anti-rabbit ACE antibody3C5 (Mono-ACE, River Forest, IL), which recognizes rabbit ACEbut not mouse ACE, was applied at 1:25 in PBST blocking solutionfor 16 h at 4°C (17). After washes with PBS at 25°C,goat anti-mouse AlexaFluor 594 (Molecular Probes, Eugene, OR)was applied at a 1:1500 dilution in blocking buffer for 2 hat 25°C. Sections were washed in PBS before mounting withVectashield + DAPI. Slides were visualized with a Leica fluorescencemicroscope at x40 total magnification.
BP Responses to Angiotensin
BP was measured through a catheter in the femoral artery, andpressure signals were digitized, recorded, and analyzed usingPowerLab software. Responses to Ang I and II were assessed insix WT, seven Ts, five Gs, and four Pg mice by giving bolusinjections of freshly prepared peptide solutions through a jugularvein catheter. Peptides were prepared as 1-ng/µl solutions,and volumes of 5, 10, 20, 50, and 100 µl were given todeliver 5, 10, 20, 50, or 100 ng. The same mice were used toestablish a full dose-response relationship. After each injection,BP was allowed to return to baseline before a new injectionwas made.
Statistical Analyses
Data are given as arithmetic means and variations as SEM. Significancecomparisons were done with ANOVA in combination with the Bonferronipost hoc test or with the t test as appropriate.
Phenotypic Profile of Experimental Mice
The transgenic mice that were used in this study have been previouslycharacterized (10,13), and their phenotypes, compared with WTor ACE null mice, are summarized in Table 1. In those studies,only the Ts strain that expressed ACE in vascular endothelialcells was found to have WT BP levels, despite the absence ofproximal tubule ACE expression. Circulating ACE of either germinalor somatic isoform was sufficient to restore gross renal structure,function, health, and blood vessel wall architecture (arteriolethickness). Male fertility was restored in the Pg strain, inwhich germinal ACE is expressed on the surface of sperm (10,18).Renal ACE levels in all strains were intermediate between WTand ACE null mice. Despite WT serum ACE levels in both Gs andPg strains, BP was not fully restored. Male fertility was restoredonly in Pg mice that expressed germinal ACE in sperm.
Immunohistochemistry
ACE expression in the kidney was determined by immunohistochemicalstaining using the 3C5 mAb that binds only to transgene-expressedrabbit ACE (Figure 1, B through D) but not WT murine ACE expressedin the vascular endothelium and proximal tubules (Figure 1A).sACE staining in the Gs strain was observed in the S1 regionof the proximal tubules nearest the Bowman’s capsule ofthe glomerulus (Figure 1B). Staining of gACE in the Pg strainwas localized to the S1 region of a very small number of renalproximal tubules but was observed primarily in serum flowingthrough glomerular capillaries and in the blood of larger vessels(Figure 1C). In the Ts strain, sACE expression is restrictedto the vascular endothelial cells of blood vessels and in theserum (Figure 1D).
Figure 1. Transgenic angiotensin-converting enzyme (ACE) expression in the kidney. Slices from age-matched, adult kidneys from wild-type (WT; Ace+/+; A), Gs strain (Ace –/–, Gs +/–; B), Pg strain (Ace –/–, Pg +/–; C), and Ts strain (Ace –/–, Ts +/+; D) FVB mice were prepared and stained with anti-rabbit ACE mAb 3C5 as described in Materials and Methods. Slides were mounted with VectaShield + DAPI and viewed with a Leica fluorescence microscope at x40 magnification. G, glomerulus; V, blood vessel.)
Endogenous Hormone Levels
We determined the endogenous level of Ang I and Ang II in thekidney and plasma of WT and transgenic strain mice. Figure 2shows that the level of Ang II was decreased in the kidneysof all transgenic strains (pg Ang II/g kidney), significantlyso in the Ts strain (WT = 621 ± 100, Ts = 369 ±37, Gs = 437 ± 30, Pg = 539 ± 39). A significantreduction in the plasma Ang II (pg Ang II/ml) in the Ts andPg strains (WT = 85.2 ± 11.3, Ts = 54.5 ± 7.4,Gs = 77.5 ± 7.0, Pg = 55.8 ± 9.6) was also observed.Conversely, the level of Ang I was significantly elevated inthe kidney (pg Ang I/g kidney; WT = 280 ± 34, Ts = 655± 27, Gs = 395 ± 46, Pg = 386 ± 39) andthe plasma (pg Ang I/ml; WT = 222 ± 53, Ts = 969 ±85, Gs = 572 ± 79, Pg = 1221 ± 119) of all transgenicstrains. This result illustrates the partial restoration ofAng II production as a result of tissue-restricted expressionof ACE in each transgenic strain. Because Ang II stimulatesaldosterone release by the adrenal cortex, we also measuredthe plasma aldosterone levels in all experimental groups. Asshown in Figure 3, the aldosterone level in the Ts and Pg strainsbut not the Gs strain were significantly different from WT strainmice (ng aldosterone/ml plasma; WT = 2.7 ± 0.2, Ts =2.3 ± 0.1, Gs = 2.6 ± 0.2, Pg = 3.1 ± 0.1).
Figure 2. Mean plasma and renal angiotensin I (Ang I) and Ang II levels in WT mice and in three transgenic ACE-expressing lines (n = 5 for all genotypes). Error bars indicate 95% confidence interval (CI), and significances are given in comparison with WT mice by unpaired t test (*P 0.01, **P 0.001, ***P 0.0001, ****P 0.00001, *****P 0.0000001).
Figure 3. Mean plasma aldosterone levels in WT and in three transgenic ACE-expressing lines (n = 4 for all genotypes). Error bars indicate 95% CI, and significances are given in comparison with WT mice by unpaired t test (*P 0.01, **P 0.05).
BP Response to Angiotensin
To functionally assess ACE activity in the vascular system,we determined the acute BP response to intravenous bolus injectionsof Ang I. We also determined BP responses to Ang II to comparethe effect of angiotensin at the receptor and postreceptor level.Data are summarized in Figure 4. It can be seen that the responseto Ang I was reduced in all transgenic mice, particularly atlower doses of injected Ang I. For example, at a dose of 10ng, Ang I increased BP by 22 ± 2.7 mmHg in WT but onlyby 3.2 ± 0.7 mmHg in Ts, by 5.4 ± 2.5 mmHg inPg, and by 7.7 ± 0.8 mmHg in Gs mice. Responses weresignificantly lower in all transgenic mice compared with WTbut were not different between transgenic animals. In contrast,responses to Ang II were comparable and not different betweenall animals tested.
Figure 4. Average change of mean arterial BP (mmHg) in response to intravenous bolus injections of Ang I (left) and Ang II (right) in WT mice (n = 6) and in three transgenic strains of mice with vascular (TS+/+; n = 7), proximal tubular (PG+/–±; n = 5), or serum ACE (GS+/–; n = 4) expression. Values are means, and vertical bars indicate SEM (n = 4).
TGF Response
TGF responses were determined in five WT, five Ts, five Pg,and four Gs mice. The gender of all animals was male. Therewere no significant differences in body weight among WT, Ts,Pg, and Gs mice (31 ± 1.0, 30 ± 0.8, 28 ±1.7, and 30 ± 0.9 g, respectively). Similarly, kidneyweights of the micropunctured left kidneys were not differentbetween genotypes (194 ± 4.7, 239 ± 11, 229 ±18, and 217 ± 12 mg). PSF without perfusion of the loopof Henle averaged 39.4 ± 0.9 mmHg in WT (n = 17), 42.6± 3.1 mmHg in Ts (n = 17), 42.1 ± 1.3 mmHg inPg (n = 17), and 39 ± 1.5 mmHg in Gs (n = 17). Differenceswere not statistically different. In response to an increasein loop of Henle perfusion to 30 nl/min, a flow rate that isknown to saturate the TGF response in mice, PSF fell significantlyin all genotypes, by 8.1 ± 0.9 mmHg in WT, by 7.1 ±0.9 mmHg in Ts, by 4.5 ± 0.8 mmHg in Pg, and by 5.0 ±0.43 mmHg in Gs mice (Figure 5). Changes in PSF were significantlysmaller than control in Pg and Gs strains when tested by ANOVAwith Bonferroni post hoc test (P < 0.01 and P < 0.05,respectively). Mean arterial BP of anesthetized mice for theperiod of the micropuncture procedure averaged 113 ±1.2 mmHg in WT, 93 ± 2.5 mmHg in Ts, 98 ± 1.4mmHg in Pg, and 89 ± 2.0 mmHg in Gs mice. Mean BP ofall genetically variant mice under anesthesia were significantlylower than WT mice at P < 0.001 (ANOVA). Among the transgenicmice, BP in Gs mice was significantly lower than in animalsof the Pg strain (P < 0.01).
Figure 5. Individual values of stop flow pressure changes (mmHg) induced by a saturating increase of loop of Henle perfusion rate in WT mice and in the three transgenic mouse strains studied in this work. Horizontal bars indicate mean values. Significances are given for comparisons with WT mice (ANOVA with Bonferroni post hoc test).
Kidney GFR and RBF
Kidney GFR averaged 476 ± 47 µl/min in WT mice(n = 4) and 427 ± 77 µl/min (n = 4) in Ts mice,a filtration rate that is not significantly different from WT.RBF was 1.09 ± ml/min in WT and 0.83 ± 0.21 ml/minin Ts mice at mean arterial pressures of 86 ± 11 and87 ± 5.7 mmHg, respectively.
Single-Nephron GFR and Tubular Absorption
Single nephron GFR (SNGFR) and rates of proximal tubular reabsorptionwere determined in four WT (FVB strain; Taconic) and four Tstransgenic animals using standard free-flow micropuncture. SNGFRaveraged 9.4 ± 0.9 nl/min in WT (n = 16) and 7.7 ±0.38 nl/min in Ts mice (n = 34; P = 0.043 by unpaired t test).Individual measurements of SNGFR in the two groups of mice aredisplayed in Figure 6. The reduction of SNGFR is probably relatedto the lower mean arterial BP in this group of Ts mice (77.5± 3.7 versus 98 ± 2.0 mmHg). Proximal fractionalabsorption of 47.1 ± 3% in WT and 50.3 ± 2.2%in Ts were not significantly different (P = 0.41; Figure 7).Similarly, absolute rates of proximal reabsorption were notdifferent between WT and Ts animals (4.6 ± 0.6 versus3.9 ± 0.3 nl/min; P = 0.21). As shown in Figure 8, glomerulotubularbalance, the relationship between SNGFR and tubular fluid reabsorptionin the proximal tubule, was not different between WT and Tsmice.
Figure 6. Individual measurements of single-nephron filtration rate (SNGFR) in WT and TS+/+ mice. Horizontal bars indicate mean values. Significance of difference was tested by unpaired t test.
Figure 7. Individual measurements of fractional proximal fluid absorption in WT and TS+/+ mice. Horizontal bars indicate mean values. Significance of difference was tested by unpaired t test.
ACE, as well as all other components of the renin-angiotensinsystem (RAS), is expressed in a variety of organs, includingthe kidney (16,19,20). The generation of Ang II by the tissueRAS suggests a role of the peptide as a paracrine modulatorof organ function in addition to its function as a circulatinghormone. Distinguishing between the importance of systemic andlocally produced Ang II is not trivial because pharmacologicinhibitors as well as knockout approaches affect both modesof angiotensin action. Our study used transgenic mice that hadbeen genetically engineered to express a single ACE isoformin a cell type–specific location on the background ofan ACE null genotype (10,13). This approach seems well suitedto assess the notion of a location-specific role of ACE andto determine the contribution of this restricted expressionpattern to the overall health and organ function of an animal.
Previous studies in AT1A and ACE knockout mice as well as earlierexperiments with ACE inhibitors and angiotensin receptor blockershave shown that an intact RAS is necessary for the regulatoryresponse of GFR to changes in distal NaCl concentration, theso-called TGF response (3,21). The main focus of this reportwas to evaluate whether selective expression of ACE in vascularcells is capable of maintaining TGF activity and to comparethe role of vascular ACE with that of proximal tubule ACE. Inaddition, we examined proximal tubular fluid reabsorption inmice that express only vascular ACE but lack ACE activity inproximal tubules. A strain of transgenic mice in which the Tie-1promoter was used to drive vascular ACE expression in mice witha null mutation of the native ACE gene was generated and previouslydescribed (13). In these experiments, we used Ts mice to investigatethe specific role of vascular ACE on TGF and proximal tubulefunction. Functional studies in these mice are greatly facilitatedby the lack of the renal deformities that characterize micewith null mutations in any of the RAS components, includingthe ACE-deficient strains studied to date (6,7,12,13,18).
In confirmation of previous observations in conscious animals,our data show that BP was somewhat reduced in anesthetized micewith selective expression of ACE in the proximal tubule andin mice that expressed only germinal ACE. In addition, anesthetizedTs mice had BP that in two of the three experimental serieswere slightly but significantly lower than in WT, a findingthat is in contrast to previous measurements in the consciousstate (13). It is possible that this is a consequence of theanesthesia, and one could speculate that the absence of a functionalbrain RAS in these Ts strain mice reduces an ACE-dependent componentthat in WT maintains normal BP during anesthesia. It is to benoted that BP varied considerably between experimental seriesin a given strain, suggesting that BP during anesthesia andsurgery depends more on the response of individual animals tothe procedure than on pre-existing pressure levels. Overall,however, BP reductions were not as limiting as they are in ACEand AT1 null mutants. This is also reflected in the fact thatPSF at zero loop flow, a variable that is generally dependenton BP, did not vary significantly between WT and transgenicmice.
Our data show that TGF responses were essentially normal inmice that express ACE in the vasculature but that are devoidof membrane-associated ACE expression at all other natural ACEexpression sites. Thus, it seems that endothelial ACE is animportant contributor to the Ang II required for full TGF responsiveness.The same conclusion was reached in a recent study in which TGFresponses were found to be significantly reduced in ACE nullmice that do not possess tissue-bound ACE in either vascularor proximal tubules locations and in which a normal BP was achievedby ectopic expression of ACE in the liver (22). In contrast,significant impairment of TGF responsiveness was found in theGs and Pg strains, presumably as a result of lack of endothelialACE expression.
Nevertheless, TGF responses in both Pg and Gs strain mice werenot abolished, indicating that maintenance of TGF responsiveness,albeit at a reduced level, can be achieved by Ang II generatedby plasma ACE. Some contribution of systemically generated AngII to TGF responsiveness is also supported by previous findingsshowing that intravenous or peritubular administration of AngII augmented TGF responses (23,24). Thus, the amount of AngII that is required for full TGF responsiveness seems to bederived mainly from the action of membrane-associated ACE inendothelial cells, but ACE in the circulating blood stream cancontribute to this Ang II pool to some lesser extent. An openquestion is that of the actual activity of circulating ACE.Previous data have shown that plasma ACE activity determinedin vitro by cleavage of a small synthetic substrate was essentiallynormal in Gs and Pg animals (10,13). However, our experimentsshowed that BP responses to Ang I were reduced and that theAng I/Ang II ratio in both kidney and plasma of all transgenicmice was increased, observations consistent with some degreeof functional ACE deficiency. We have no satisfactory explanationfor this discrepancy other than suggesting that either plasmaACE activity is in fact not the main determinant of acute AngI conversion or that the use of the synthetic substrate doesnot reflect Ang I conversion in vivo. It is important to pointout the underlying assumption of the above arguments that theoverall vascular responsiveness of the vasculature is not alteredin the transgenic models used in our study. This assumptionis supported by the normal responses of BP to systemically appliedAng II.
Another goal of this study was to assess proximal tubular absorptivefunction in mice that lack ACE and, therefore, Ang II productionin the proximal tubules. Using Ts mice as a model of proximaltubule ACE deficiency, we observed that although the rate ofsingle-nephron filtration was significantly reduced, the relationshipbetween proximal tubular reabsorption and GFR was not significantlydifferent from WT animals. Thus, we conclude that the chronicabsence of proximal tubular Ang II production does not exertlong-term effects on proximal fluid and NaCl transport. It ispossible that Ang II is delivered to the proximal tubule lumenby filtration. However, because proximal tubule Ang II concentrationshave been found, at least in the rat, to exceed plasma levelsby a factor of approximately 50, it is unlikely that sufficientlyhigh luminal concentrations can be reached by ultrafiltrationalone (25,26). The absence of a measurable inhibition of proximalfluid absorption in Ts mice is distinct from the clear reductionof proximal fluid transport that results from acute and localadministration of ACE inhibitors or Ang II receptor blockers(27,28). Thus, one has to conclude that changes of Ang II formationin the proximal tubule can exert acute effects on salt transportbut that other factors can substitute for Ang II when its generationis absent for prolonged periods of time.
We conclude that ACE in vascular tissue is responsible for mostof the Ang II generation required for full expression of TGFresponsiveness, whereas plasma ACE plays a minor role in contributingto the Ang II pool that affects TGF. Absence of local Ang IIgeneration in the proximal tubule is not associated with long-termeffects on proximal tubular fluid absorption.
Acknowledgments
This work was supported by National Institutes of Health GrantHL-48258 (to G.C.S.) and by the Intramural Research Programof the National Institute of Diabetes and Digestive and KidneyDiseases (S.H. and J.S.).
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[Abstract][Full Text][PDF]
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Abstract
Introduction
-glutamyl transpeptidase promoter (Gs strain) and in a strain of mice that expresses germinal ACE (gACE) in sperm using the human PGK2 promoter (Pg strain). Mice of the Ts and Gs strains have sACE in their serum as a result of the natural cleavage-secretion processing of membrane-anchored ACE (13), whereas Pg mice ectopically express gACE in the serum (10). Our observations in the Ts strain show that TGF regulation was largely but not entirely normalized by expression of ACE in vascular endothelial cells. Furthermore, the absence of ACE in renal proximal tubule cells does not cause measurable changes of fluid reabsorption. These results further support the notion that specific physiologic functions of ACE are determined by the location of ACE expression. Moreover, the expression of ACE in proximal tubules is redundant in the sense that its long-term absence does not compromise proximal NaCl and fluid reabsorption. 
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