Abstract
ABSTRACT. It has been shown that injection of combinations of anti-aminopeptidase A (APA) monoclonal antibodies (mAb) that inhibit the enzyme activity induces an acute albuminuria in mice. This albuminuria is not dependent on inflammatory cells, complement, or the coagulation system. APA is an important regulator of the renin-angiotensin system because it is involved in the degradation of angiotensin II (Ang II). This study examined the potential role of glomerular Ang II in the induction of albuminuria. The relation among renal Ang II, glomerular APAX enzyme activity, and albuminuria was examined first. Injection of the nephritogenic combinations ASD-3/37 and ASD-37/41 in BALB/c mice induced albuminuria, whereas the non-nephritogenic combination ASD-3/41 had no effect. There was no clear relation between the inhibition of glomerular APA activity and albuminuria, yet it was evident that intrarenal Ang II levels were significantly increased in albuminuric mice and not in nonalbuminuric mice. As a next step, anti-APA mAb were administered to angiotensinogen-deficient mice that do not produce Ang II, and kidney morphology and albuminuria were determined. Angiotensinogen-deficient mice also developed albuminuria upon ASD-37/41 administration. Altogether, these findings clearly demonstrate that Ang II is not required for the induction of albuminuria upon injection of enzyme-inhibiting anti-APA mAb.
Aminopeptidase A (APA; EC 3.4.11.7) is a homodimeric type II membrane-bound protease, which specifically hydrolyses N-terminal glutamyl and aspartyl residues from peptides (1,2). APA has a widespread organ distribution, but in mouse kidney it is predominantly expressed on podocytes and brush borders of proximal tubular epithelial cells (3–5). APA is an important regulator of the renin-angiotensin system, because it inactivates its most vasoactive component angiotensin II (Ang II) by cleaving off the N-terminal aspartate (6,7).
In an effort to develop a mouse model of membranous nephropathy, we generated monoclonal antibodies (mAb) against APA. Injection of some of these mAb in BALB/c mice induces an acute albuminuria that is not dependent on systemic mediators, such as inflammatory cells, complement, or the coagulation system (3). Recently, we showed that injection of combinations of the anti-APA mAb, in doses that do not cause albuminuria when given alone, induces a massive acute albuminuria. This albuminuria is observed only when using combinations of mAb that reduce the APA enzyme activity in vivo considerably (8).
The latter finding suggested that Ang II might play a crucial role in the induction of the acute albuminuria in our model. It is widely known that the APA substrate Ang II can increase urinary protein excretion in various experimental settings (9–11). Clearly, there is a role for Ang II in the regulation of glomerular hemodynamics (12), but Ang II can also induce expression of several factors that may contribute to proteinuria (13,14). Because we never observed changes in mean arterial pressure (MAP) upon administration of combinations of anti-APA mAb in BALB/c mice (8), a systemic effect of Ang II seemed unlikely. Moreover, our previous study showed that the albuminuria is lowered to a similar degree by treatment with either a combination of an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin type 1 (AT1) receptor blocker or a combination of a β-blocker, a vasodilator, and a diuretic (8). We concluded from these findings that the acute reduction of the proteinuria observed with the ACE inhibitor-AT1 receptor blocker combination was the result of a fall in systemic BP rather than a specific effect of Ang II. However, a role for glomerular Ang II in the induction of albuminuria in our mouse model cannot be excluded entirely.
In the present study, therefore, we wanted to clarify the role of glomerular Ang II in our model more specifically, and we started by determining albuminuria, glomerular APA enzyme activity, and intrarenal Ang II levels after injection of different (combinations of) anti-APA mAb in BALB/c mice. As we found significantly increased intrarenal Ang II levels in albuminuric mice only, we wanted to examine whether the presence of Ang II is absolutely essential for the occurrence of albuminuria or whether Ang II is produced as a consequence of albuminuria upon blocking APA. In the next set of experiments, we therefore injected a nephritogenic (ASD-37/41) and a non-nephritogenic (ASD-3/41) combination of anti-APA mAb in angiotensinogen-deficient (Agt −/−) mice that do not produce Ang II as a result of a targeted deletion in the endogenous angiotensinogen gene. From the albumin excretion measurements in these mice, we conclude that Ang II is not involved in the induction of albuminuria in our anti-APA mAb mouse model.
Materials and Methods
Animals
BALB/c mice were obtained from Charles River (Sulzfeld, Germany). Heterozygous angiotensinogen-deficient mice (Agt +/−) that were backcrossed seven generations into the C57BL/6J background were a gift of O. Smithies and J. Krege (University of North Carolina, Chapel Hill, NC) (15). These mice were further intercrossed to obtain animals that were homozygous for the targeted mutation (Agt −/−). Homozygous APA-deficient (APA −/−) mice held on a mixed 129/C57BL/6 background were provided by M. Cooper (University of Alabama, Birmingham, AL) (16). These mice were further intercrossed with C57BL/6 mice to obtain a breeding colony. All procedures involving mice were approved by the Animal Care Committee of the University of Nijmegen and conformed to the Dutch Council for Animal Care and National Institutes of Health guidelines.
Genotyping of Transgenic Mice
DNA was prepared from tail biopsies using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI). For routine genotyping of mutant angiotensinogen mice, PCR was performed with sense primer 148 (5′-GTATACATCCACCCCTTCCA-3′, located in exon 2), antisense primer 149 (5′-GGAAGTGAACGTAGGTGTTGAA-3′, located in exon 2), and sense primer 162 (5′-TGCTCCTGCCGAGAAAGTAT-3′, located in the neomycin cassette). Genotyping of mutant APA mice was performed using APA gene-specific sense primer 231 (5′-ACACAACCCCAGCTCCTTCC-3′, located in exon 1) and antisense primer 232 (5′-TCTTCTGCAGCCTGGATCAC-3′, located in exon 1) and neomycin-specific sense primer 233 (5′-ACTGGGCACAACAGACAATCG-3′) and antisense primer 234 (5′-CAAGCTCTTCAGCAATATCACG-3′). Genomic DNA (200 ng) was PCR-amplified in a mixture containing 100 ng of the corresponding primers and 0.5 U of Thermostable DNA polymerase (Integro, Dieren, The Netherlands). Amplification was carried out under the following conditions: an initial 5-min denaturation at 94°C, followed by 30 cycles of 94°C for 1 min, 58°C for 1 min, and 72°C for 2 min, with a final extension for 10 min at 72°C. Amplified products were electrophoretically resolved on agarose gels. The presence of the angiotensinogen wild-type and/or mutated alleles was demonstrated by amplification of a 746-bp fragment with primers 148 and 149 and/or a 1364-bp fragment with primers 149 and 162, respectively. For APA genotyping, the presence of wild-type and/or mutated alleles was demonstrated by a 367-bp amplicon with primers 231 and 232 and a 630-bp amplicon with primers 233 and 234, respectively.
Experimental Design
The characteristics of the three rat mAb against mouse APA used in this study (ASD-3, ASD-37, and ASD-41) have been described previously (8). Only ASD-3 and ASD-37 are able to inhibit the APA enzyme activity considerably in vitro and in vivo. When used in combination, ASD-3/37 and ASD-37/41 almost completely inhibited the enzyme activity in vivo, whereas ASD-3/41 did not (8). Injection of the combinations ASD-3/37 and ASD-37/41 into BALB/c mice induced a massive acute albuminuria at day 1, whereas the combination ASD-3/41 had no effect (8). The anti-APA mAb have been propagated in vitro by hollow fiber culture (Nematology Department, Agriculture University Wageningen, The Netherlands). After purification by Protein A column affinity chromatography, protein concentrations of the batches were measured by means of Lowry. Experiments were performed in different mouse strains. APA-deficient 6-wk-old mice received an intravenous injection of a total dose of 4 mg of the nephritogenic combination ASD-37/41 (1:1 weight ratio) to control the specificity of the anti-APA mAb used in this study. Six- to 8-wk-old BALB/c mice received an intravenous injection of a total dose of 4 mg of the nephritogenic combinations ASD-3/37 and ASD-37/41 or of the non-nephritogenic combination ASD-3/41 (1:1 weight ratio) or of 8 mg ASD-37 alone. Next, 8- to 16-wk-old angiotensinogen-deficient and wild-type (Agt +/+) mice received an intravenous injection of 4 mg of the combinations ASD-37/41 or ASD-3/41 (1:1 weight ratio). Urine samples were collected from 6 to 24 h after injection of the anti-APA mAb. Subsequently, mice were killed and kidneys were removed to be processed for histology and/or measurements of intrarenal Ang II levels (vide infra). Urinary albumin was measured by radial immunodiffusion (17). Serum creatinine was measured in an autoanalyzer using standard techniques (18). The MAP of untreated Agt −/− and Agt +/+ mice was measured intra-arterially after anesthetization with 0.8% isoflurane (Cyprane, Keighley, UK) in combination with 400 cc O2/min as described (8).
Determination of Intrarenal Ang II
Liquid nitrogen-frozen kidneys (0.3 to 0.45 g) were rapidly homogenized (1:10, wt/vol) using a Polytron PT10/35 (Kinematica, Luzern, Switzerland) in an iced solution of 0.02 mol/L HCl in ethanol (19). A known amount of 125I-angiotensin I (Ang I) was added as an internal standard before the extraction procedure. Homogenates were prepared for reversed-phase HPLC as described previously (20,21). The fractions that contained Ang II were neutralized with 50 μl of 0.16 mol/L sodium hydroxide and vacuum dried, and concentrations of Ang II were measured by RIA (20). Concentration of intact 125I-Ang I in the HPLC fractions was measured in a γ counter, and the recovery of 125I-Ang I after HPLC separation was used to correct for losses (maximally 20% to 30%) that occurred during extraction and separation (21).
Light Microscopy, Immunofluorescence, and Electron Microscopy
For light microscopy, kidney fragments were fixed in Bouin’s solution, dehydrated, and embedded in paraplast (Amstelstad, Amsterdam, The Netherlands), and 2-μm sections were stained with periodic acid-Schiff and methenamine silver (22). For immunofluorescence, kidney fragments were snap-frozen in liquid nitrogen, and 2-μm cryostat sections were stained with FITC-labeled goat anti-mouse C3, FITC-labeled rabbit anti-mouse fibrinogen (Nordic, Tilburg, The Netherlands), and Alexa 488-labeled goat anti-rat Ig (Molecular Probes, Eugene, OR) to examine the presence of the injected anti-APA mAb. APA expression was analyzed by immunofluorescence using the anti-APA mAb ASD-38 followed by incubation with Alexa 488-labeled goat anti-rat Ig. Tissue sections were examined using a fluorescence microscope (Leica GmbH, Wetzlar, Germany) (3). For electron microscopy, small pieces of cortex were fixed overnight at 4°C in 2.5% glutaraldehyde dissolved in 0.1 M sodium cacodylate buffer (pH 7.4) and washed in the same buffer. Tissue fragments were postfixed in cacodylate-buffered 1% OsO4 for 1 h, dehydrated, and embedded in Epon 812 (Merck, Darmstadt, Germany). Ultrathin sections were prepared using an ultratome (Leica; Reichert Ultracuts, Wien, Austria) and stained with 4% uranyl acetate for 45 min and subsequently with lead citrate for 4 min at room temperature. Sections were examined in a JEOL 1200 EX2 electron microscope (JEOL, Tokyo, Japan).
Enzyme Histochemistry
The enzyme activity of APA in the kidneys was visualized by enzyme histochemistry according to Lojda and Gossrau (23) with the APA-specific substrate l-glutamic acid-4-methoxy-β-naphtylamide (Bachem, Bubendorf, Switzerland) (4). The intensity of the developed color was recorded semiquantitatively on a scale from 0 to 4 (8).
Statistical Analyses
For multiple group comparisons, ANOVA was used and post hoc analyses were done with Tukey’s. One-sided unpaired t tests were performed to compare the effect of ASD-37/41 with ASD-3/41 in the Agt −/− and Agt +/+ mice. P < 0.05 was regarded as significant. All values are expressed as means ± SEM.
Results
Injection of Anti-APA mAb in BALB/c Mice
Injection of various (combinations of) in vitro produced anti-APA mAb in BALB/c mice resulted in a varying reduction of glomerular APA enzyme activity. A dose of 4 mg of the combination ASD-3/37 completely inhibited the enzyme activity, and the combination ASD-37/41 or 8 mg of ASD-37 alone reduced the enzyme activity almost completely. The combination of ASD-3/41 had considerably less effect. Only the combinations ASD-3/37 and ASD-37/41 affected urinary albumin excretion. These findings are in line with our previous results and do not demonstrate a clear relation between the inhibition of glomerular APA enzyme activity as measured by enzyme histochemistry and the level of albuminuria (Table 1). In previous studies, we always used mAb propagated as ascites, but for obvious reasons we now use in vitro produced mAb. The histology observed after injection of the anti-APA mAb in BALB/c mice and the binding of the mAb was the same as for ascites-propagated mAb (8). Upon injection of the nephritogenic anti-APA mAb combination in APA-deficient mice (generated by the group of Cooper (16)), the normal binding pattern of the anti-APA mAb along the capillary wall (Figure 1B) was not observed. In fact, the antibody-binding pattern analyzed by immunofluorescence was identical to the negative APA expression pattern in APA-deficient kidneys, as shown in Figure 2E. As a result of the lack of anti-APA mAb binding, injection of the nephritogenic combination ASD-37/41 in APA-deficient mice did not lead to albumin excretion. These findings demonstrate that our mAb are highly specific for APA and underscores the significance of epitopes on the APA molecule in the induction of proteinuria.
Effect of anti-aminopeptidase A monoclonal antibodies administered to BALB/c mice
Figure 1. Histology of Agt −/− and Agt +/+ mice that received an injection of 4 mg of the nephritogenic combination ASD-37/41. The intensity of the binding of the injected rat mAb to the glomerular capillary wall was studied by immunofluorescence. The glomerular binding was comparable between Agt −/− (A) and Agt +/+ (B) mice. Both figures demonstrate binding of the mAb to the brush border and basolateral membranes (arrows). In Agt −/− mice (A), the intensity of mAb binding to the brush borders is slightly less, compatible with an increased shedding (arrowheads) of APA as a result of the higher proteinuria. In addition, in the Agt −/− mice, we observed more intracytoplasmic resorption vesicles. Electron micrograph of a glomerulus of Agt −/− mice (C) showing podocytes with partial retraction of foot processes (arrow). Agt +/+ mice (D) displayed similar but less retraction of foot processes (arrow). The swelling of endothelial cells observed in the treated Agt −/− mice was similar to untreated Agt −/− mice. C, capillary lumen; U, urinary space. Magnifications: ×400 in A and B; ×8500 in C and D.
Figure 2. Aminopeptidase A (APA) expression and enzyme histochemistry. Immunofluorescence, using the anti-APA monoclonal antibody (mAb) ASD-38, of Agt −/− (A) and Agt +/+ (C) mice showed normal APA expression in podocytes of glomeruli and brush borders of proximal tubular epithelial cells. In addition, APA enzyme activity was comparable in kidney sections of Agt −/− (B) and Agt +/+ (D) mice. Note that in kidney sections of APA knockout mice, APA expression (E) and APA enzyme activity (F) were completely absent. G, glomerulus. Magnification, ×400.
Because Ang II is the only known substrate of APA in the kidney, we determined whether inhibition of APA enzyme activity causes a rise in intrarenal Ang II levels. We observed significantly higher Ang II levels in mice that received the nephritogenic combinations ASD-3/37 or ASD-37/41. Intrarenal Ang II levels in mice that received non-nephritogenic mAb, either the single enzyme-inhibiting ASD-37 or the non-enzyme-inhibiting combination ASD-3/41, were not different from values in control mice (Table 1). These findings show that albuminuria is associated with raised Ang II levels in this experimental model.
Characteristics of Angiotensinogen-Deficient Mice
To determine whether Ang II is required for the induction of the acute albuminuria in our model, we used angiotensinogen-deficient mice (Agt −/−). Light microscopy of kidneys of Agt −/− mice aged 6 to 14 wk showed many distinctive pathologic lesions, as described previously (15,24,25). These lesions include a prominent but variable atrophy of the medulla causing a wider pelvic space, a pronounced wall thickening of particularly the intralobular arteries and afferent arterioles (Figure 3A, arrow), and areas of interstitial fibrosis with a mild to moderate influx of mononuclear cells (Figure 3A, *) and atrophic tubules. In addition, small immature and sometimes sclerosing glomeruli were found, which are situated closely together. Other glomeruli sometimes showed an increase in mesangial matrix and cells, whereas the podocytes looked normal. We observed in some glomeruli additional proliferation of parietal epithelial cells (Figure 3B, arrows) that appeared as small crescents, a finding that has not been mentioned before. Agt +/+ mice of 4 to 12 wk of age displayed normal kidney histology (Figure 3, C and D). Also, kidneys of Agt +/− mice aged 4 to 10 wk revealed no abnormalities (data not shown). In addition to traces of complement factor C3 in the mesangium of some glomeruli in both Agt +/+ and Agt −/− mice, a few sclerosing segments of the immature glomeruli in Agt −/− mice showed minor C3 deposits; however, the presence of traces C3 in the mesangium and in sclerotic lesions is a normal finding in mice. The presence of glomerular abnormalities in Agt −/− mice was confirmed by electron microscopy. In mature glomeruli of both Agt −/− and Agt +/+ mice, podocytes were normal and showed no signs of retraction of foot processes (Figure 4, A and B), whereas cuboid podocytes without developed foot processes were observed in immature and sclerosing glomeruli in Agt −/− mice. Some capillary loops of Agt −/− mice showed some swelling of endothelial cells, which was seen less in Agt +/+ mice. The glomerular basement membrane (GBM) was normal in all glomeruli. Renal APA expression was normal in Agt −/− mice compared with wild-type littermates (Agt +/+) as demonstrated by immunofluorescence using the anti-APA mAb ASD-38 (Figure 2, A and C) and enzyme histochemistry (Figure 2, B and D). As expected, both immunofluorescence and enzyme histochemistry were completely negative in kidney sections of APA-deficient mice (Figure 2, E and F). Renal APA expression and enzyme histochemistry also were normal in Agt +/− mice (data not shown). Normal APA presence in Agt mutant mice is of course a prerequisite to achieve the anti-APA mAb model in these knockout mice.
Figure 3. Histology of Agt −/− and Agt +/+ mice. Light microscopy of Agt −/− mice revealed a pronounced arterial wall thickening (arrow), areas of interstitial fibrosis with a mild to moderate influx of mononuclear cells (*), and the presence of mature glomeruli but also of small immature and sclerosing glomeruli (A). Some glomeruli of Agt −/− mice showed proliferation of parietal epithelial cells (B, arrows). Proliferation of parietal epithelial cells was also observed in areas where the interstitium is normal. Agt +/+ mice showed no abnormalities of the glomeruli, the interstitium, or the blood vessels (C and D). Magnifications: ×250 in A and C; ×400 in B and D.
Figure 4. Analysis of mature glomeruli of nontreated Agt −/− and Agt +/+ mice. Electron micrographs showing normal podocytes without signs of retraction of foot processes in Agt −/− (A) and Agt +/+ mice (B). C, capillary lumen; U, urinary space. Magnification, ×8500.
Angiotensinogen-knockout mice showed significantly lower MAP compared with wild-type mice (58 ± 0.5 versus 95 ± 1 mmHg, respectively; P < 0.001), which is consistent with earlier findings (15,25,26). As expected, intrarenal Ang II could not be detected in Agt −/− mice, whereas the Ang II levels in Agt +/+ mice amounted to 155 fmol/g wet weight (median). Plasma creatinine was normal in Agt −/− mice (41 ± 3 versus 44 ± 1 μmol/L in Agt +/+ mice) as was urinary albumin excretion (measured in 4- to 15-wk-old mice).
Injection of Anti-APA mAb in Angiotensinogen-Deficient Mice
To examine whether Ang II is a prerequisite for the induction of a massive acute albuminuria, we injected 4 mg of the nephritogenic (ASD-37/41) or non-nephritogenic (ASD-3/41) combination into Agt −/− and Agt +/+ mice. If indeed Ang II is involved in the induction of albuminuria in this model, then one would expect no increase in albumin excretion in Agt −/− mice after injection of the mAb combination ASD-37/41. Surprising, injection of ASD-37/41 into Agt −/− mice caused albuminuria (8262 ± 2066 μg/18 h; n = 5), which was even numerically higher than that observed in Agt +/+ mice (3860 ± 1292 μg/18 h; n = 5; Figure 5). The mAb combination ASD-37/41 also induced an acute albuminuria in heterozygous mice (3231 ± 759 μg/18 h; n = 10). As anticipated, the non-nephritogenic combination ASD-3/41 did not result in an albuminuria in Agt +/+ (90 ± 38 μg/18 h; n = 3), Agt +/− (92 ± 2 μg/18 h; n = 3), or Agt −/− mice (127 ± 34 μg/18 h; n = 4; Figure 5). It is of note that the albumin excretion upon administration of the nephritogenic combination is strikingly lower in the Agt homozygous, heterozygous, and wild-type mice that are held on a C57BL/6J background than in BALB/c mice (Table 1). On the basis of our observations in the anti-GBM model, we anticipate that the differences in albuminuria most likely result from different susceptibilities of the mouse strains.
Figure 5. Albuminuria in Agt −/− and Agt +/+ mice at 1 d after intravenous injection of 4 mg of the nephritogenic combination ASD-37/41 (▪) or 4 mg of the non-nephritogenic combination ASD-3/41 (◼). *P < 0.05, **P < 0.01 versus ASD-3/41. Values are given as means ± SEM, n = 3–5. Note that the albuminuria in Agt −/− mice upon administration of the nephritogenic combination was even numerically higher than in wild-type and Agt +/− mice.
Light microscopy of all treated mice revealed no morphologic differences compared with their untreated counterparts. In particular, there was no evidence of influx of inflammatory cells or thrombosis. The intensity of glomerular binding of the injected mAb was checked with immunofluorescence, and no differences were observed among Agt −/−, Agt +/+ (Figure 1, A and B), and Agt +/− mice (data not shown). We observed, however, subtle differences in the mAb-binding pattern between the two mAb combinations used in this study. In both knockout and wild-type mice, injection of the combination ASD-37/41 resulted in a granular pattern in the glomeruli compared with the more homogeneous binding pattern observed after administration of the combination ASD-3/41. Injection of ASD-37/41 resulted in a decreased presence of APA in the brush border and an increase in intensity of basolateral membrane APA as described previously (27). When comparing the Agt −/− and Agt +/+ mice, there was more heterogeneity in the Agt −/− mice, but overall the intensity of the binding of the mAb to the brush border was slightly decreased. This is consistent with the increased shedding of APA observed in Agt −/− mice, which is due to the higher proteinuria. In addition, the number of intracytoplasmic vesicles was increased in Agt −/− mice, reflecting a higher degree of resorption. These resorption vesicles are not observed in mice that received an injection of non-nephritogenic anti-APA mAb such as ASD-41 alone (27) or the mAb combination ASD-3/41. By electron microscopy, we observed a partial retraction of the foot processes in Agt −/− mice (Figure 1C) and to a lesser extent in Agt +/+ mice that received an injection of ASD-37/41 (Figure 1D), whereas the nonalbuminuric mice that were treated with ASD-3/41 showed no differences compared with nontreated mice.
Discussion
Our study clearly demonstrates that the presence of Ang II is not required for the induction of albuminuria in the anti-APA mAb mouse model. We previously showed that injection of some combinations of antibodies directed against APA in mice induces a massive acute albuminuria that is independent of inflammatory cell influx, activation of complement, or glomerular thrombosis (8,28). Recent findings of our group concerning MAP measurements and effects of different antihypertensive therapies on the acute albuminuria in our model indicated already that a systemic effect of Ang II was unlikely (8). However, we could not exclude a specific role for glomerular Ang II in the induction of albuminuria. In the current set of experiments, we showed that the nephritogenic effect of anti-APA mAb was also present in mice that do not have Ang II, because the nephritogenic combination of anti-APA mAb still produced albuminuria in these mice. Our data certainly prove that Ang II is not responsible for inducing the albuminuria associated with reduced APA enzyme activity, yet albuminuria is induced by specific combinations of anti-APA mAb. Upon injection of the nephritogenic combination ASD-37/41 in APA-deficient mice, we observed no binding, and these mice did not develop albuminuria. These findings demonstrate that our mAb are highly specific for APA and also validates the importance of APA-specific epitopes in our model. In this respect, recent findings of Chugh et al. (29) are of relevance. These investigators have studied nephrotoxic serum nephritis in the rat induced by injection of sheep anti-rat antibodies. This model is also characterized by the absence of an inflammatory response. The data indicate that the induction of proteinuria upon administration of this antiserum is related to the presence of antibodies directed against APA.
To prove unambiguously that Ang II has no role in the induction of albuminuria in our model, we performed experiments in angiotensinogen-deficient mice that lack the ability to produce Ang II. The APA expression in the glomeruli and brush borders of Agt −/− mice was comparable to that in Agt +/+ mice, which validates the use of our model in these knockout mice. Evidently, injection of the mAb combination ASD-37/41 induced a massive acute albuminuria even in these knockout animals. There was no evidence of cell influx or complement activation, indicating that the model in the angiotensinogen-deficient mice is not different from the model in BALB/c mice. From these experiments, it is clear that Ang II is not required for the induction of an acute albuminuria in our model. It is interesting that despite the lower MAP in the Agt −/− mice, the albuminuria in these knockouts was higher than in Agt +/+ mice. This finding suggests that the glomerular filter in Agt −/− mice may be more susceptible to injury. Indeed, there is an argument for preexisting damage in the Agt −/− mice as indicated by the presence of more sclerosing and immature glomeruli with subtle changes in the podocytic foot processes. In addition, Agt −/− mice have increased mRNA levels of neuronal nitric oxide synthase (30), renin (26), ACE, and AT1 (31,32). Effects of these alterations on the induced albuminuria in Agt −/− mice cannot be ignored completely.
Although Ang II is not needed for the induction of albuminuria in our anti-APA mAb model, we observed greatly elevated renal Ang II levels in albuminuric mice. Determination of albuminuria and renal Ang II levels after injection of the anti-APA mAb in BALB/c mice revealed that albuminuria was observed in mice that received the enzyme-inhibiting combinations ASD-3/37 or ASD-37/41, whereas neither the non-enzyme-inhibiting combination ASD-3/41 nor a single enzyme-inhibiting mAb (ASD-37) had any effect. Although it is possible that minor differences in APA enzyme activity may not be detected with the enzyme-histochemical procedure used, we did not find a simple relation between reduction of APA enzyme activity and the induction of albuminuria. However, intrarenal Ang II levels were significantly increased in mice that received the nephritogenic combinations (ASD-3/37 or ASD-37/41) compared with non-nephritogenic mice, providing evidence that albuminuria is associated with elevated Ang II levels. We hypothesize that the increased intrarenal Ang II levels may be the consequence of albuminuria. Evidence for such an increased production of Ang II has also been provided in studies of other experimental models of glomerular disease, such as renal ablation (33), passive Heymann nephritis (34), anti-Thy1 glomerulonephritis (35), anti-GBM nephritis (36,37), and also glomerulosclerosis (38). Initially elevated Ang II levels might contribute to late development of glomerular injury and proteinuria, as suggested by Pagtalunan et al. (39).
If Ang II is not required for the induction of albuminuria in our model, then other explanations must be sought. Theoretically, albuminuria might be the result of inhibition of tubular albumin resorption upon binding of the antibodies to brush border APA. However, in immunofluorescence, more resorption vesicles were observed in the proximal tubules of mice that were treated with the nephritogenic combination ASD-37/41 compared with nonproteinuric mice that were treated with ASD-3/41 or ASD-41 (27). These findings provide evidence that albuminuria, at least partly, must result from increased glomerular leakage. If Ang II is not involved in the induction of albuminuria, then other substrates may be, although we are unaware of other known APA substrates in the kidney. Therefore we suggest that the binding of the anti-APA mAb may result in podocytic alterations such as changes in the cytoskeleton or production of mediators such as collagenases or oxygen radicals. Future research in this area should help elucidate the pathogenic mechanisms involved in the induction of albuminuria.
Acknowledgments
This work was supported by a grant from the Dutch Kidney Foundation (C 96.1534). We thank Dr. Max D. Cooper from the University of Alabama (Birmingham, AL) for providing us with the APA-deficient mice.
- © 2001 American Society of Nephrology
