converting enzyme 2 and angiotensin-converting enzyme

Glomerular Localization and Expression of Angiotensin-Converting Enzyme 2 and Angiotensin-Converting Enzyme:Implications for Albuminuria in Diabetes

Minghao Ye, Jan Wysocki, Josette William, Maria Jose Soler, Ivan Cokic, and Daniel BatlleDepartment of Medicine, Division of Nephrology and Hypertension, Feinberg School of Medicine, NorthwesternUniversity, Chicago, Illinois

Angiotensin-converting enzyme 2 (ACE2) expression has been shown to be altered in renal tubules from diabetic mice. Thisstudy examined the localization of ACE and ACE2 within the glomerulus of kidneys from control (db/m) and diabetic (db/db)mice and the effect of chronic pharmacologic ACE2 inhibition. ACE2 co-localized with glomerular epithelial cell (podocyte)markers, and its localization within the podocyte was confirmed by immunogold labeling. ACE, by contrast, was seen onlyin glomerular endothelial cells. By immunohistochemistry, in glomeruli from db/db mice, strong ACE staining was foundmore frequently than in control mice (db/db 64.6 � 6.3 versus db/m 17.8 � 3.4%; P < 0.005). By contrast, strong ACE2 stainingin glomeruli from diabetic mice was less frequently seen than in controls (db/db 4.3 � 2.4 versus db/m 30.6 � 13.6%; P < 0.05).For investigation of the significance of reduced glomerular ACE2 expression, db/db mice were treated for 16 wk with a specificACE2 inhibitor (MLN-4760) alone or combined with telmisartan, a specific angiotensin II type 1 receptor blocker. At the endof the study, glomerular staining for fibronectin, an extracellular matrix protein, was increased in both db/db and db/m micethat were treated with MLN-4760. Urinary albumin excretion (UAE) increased significantly in MLN-4760–treated as comparedwith vehicle-treated db/db mice (743 � 200 versus 247 � 53.9 �g albumin/mg creatinine, respectively; P < 0.05), and theconcomitant administration of telmisartan completely prevented the increase in UAE associated with the ACE2 inhibitor(161 � 56; P < 0.05). It is concluded that ACE2 is localized in the podocyte and that in db/db mice glomerular expression ofACE2 is reduced whereas glomerular ACE expression is increased. The finding that chronic ACE2 inhibition increases UAEsuggests that ACE2, likely by modulating the levels of glomerular angiotensin II via its degradation, may be a target fortherapeutic interventions that aim to reduce albuminuria and glomerular injury.

J Am Soc Nephrol ●● : ●●● –●●● , ●●●● . doi: 10.1681/ASN.2006050423

A ngiotensin-converting enzyme (ACE) is a monomeric,membrane-bound, zinc- and chloride-dependentpeptidyl dipeptidase that catalyzes the conversion of

the decapeptide angiotensin I (AngI) to the octapeptide AngIIby removing a carboxy-terminal dipeptide (1). ACE2 is a car-boxypeptidase that preferentially removes carboxy-terminalhydrophobic or basic amino acids (2,3). ACE2 is the onlyknown and enzymatically active homologue of ACE in thehuman genome (1). AngI and AngII, as well as numerous otherbiologically active peptides, are substrates for ACE2, but bra-dykinin is not (1). ACE2 cleaves AngI and AngII into inactiveAng 1-9 and the vasodilator and antiproliferative Ang 1-7 (4,5),respectively; therefore, ACE2 has the potential to counterbal-ance the effects of ACE (2,3). Whereas ACE is ubiquitouslydistributed, ACE2 is more tissue restricted. Initially, it wasfound to be restricted to heart, kidney, and testis (1), but morerecently, it has been found also in lung and other tissues (6,7).

Studies in both rat and mouse kidney have shown the presenceof ACE2 in renal tubules and glomeruli (8–10). In samples thatwere obtained from human kidney biopsies, ACE2 was foundin glomerular epithelium and vascular smooth muscle cells ofinterlobular arteries (11).

There is growing interest in a possible role of ACE2 indiabetic kidney disease (9,10,12). Activation of the renin-angiotensin system (RAS) is widely believed to contribute tokidney injury in diabetes (13). We have suggested that ACE2,acting as a negative regulator of the RAS, may exert a reno-protective action (10). The pattern of ACE and ACE2 expres-sion in renal cortical tubules of young db/db mice was char-acterized by low ACE but increased ACE2 protein (10). Morerecently, we found that these alterations in ACE2 protein inrenal tubules from diabetic mice are accompanied by corre-sponding changes in enzymatic activity (12). In this study,we examined the localization of ACE and ACE2 in the glo-merulus of control and diabetic mice. The glomerulus is thesite of the nephron where the lesions of diabetic nephropathyappear earlier, and an increase in glomerular permeability isan early manifestation of diabetic kidney disease as reflectedby the presence of albuminuria. Accordingly, we wanted toexamine the localization of ACE2 within the glomerulus andalso to test the hypothesis that downregulation of ACE2

Received May 1, 2006. Accepted August 12, 2006.

Published online ahead of print. Publication date available at www.jasn.org.

Address correspondence to: Dr. Daniel Batlle, Northwestern University, FeinbergSchool of Medicine, Department of Medicine, Division of Nephrology & Hyper-tension, 303 East Chicago Ave., SRL 10-475, Chicago, IL 60611-3008. Phone:312-908-8342; Fax: 312-503-0622; E-mail: [email protected]

Copyright © ●●●● by the American Society of Nephrology ISSN: 1046-6673/●●●● -0001

. Published on October 4, 2006 as doi: 10.1681/ASN.2006050423JASN Express

activity may lead to worsening of albuminuria in diabeticmice.

Materials and MethodsAnimals

Obese db/db mice (C57BLKS/JLepr) were used as a model of type 2diabetes, and their lean littermates (db/m) served as nondiabetic con-trols (Jackson Laboratory, Bar Harbor, ME). The db/db mouse is one ofthe best characterized and most extensively studied rodent models oftype 2 diabetes (14). Heterozygous db/m littermates are lean and arespared from the induction of type 2 diabetes and its secondary com-plications (14). For this study, we used young (8 wk of age) female db/dbmice to study an early phase of diabetes (3 to 4 wk of onset) withoutrenal pathology by light microscopy (14). The Institutional Animal Careand Use Committee approved all procedures.

Tissue PreparationPentobarbital sodium was administered intraperitoneally, and kid-

neys were perfused with ice-cold PBS at a constant flow rate of 20ml/min by using an infusion pump to flush out blood. Kidneys wereremoved quickly, cut longitudinally, and fixed with 10% bufferedformalin phosphate (Fisher Scientific, Hanover Park, IL) for overnight.After paraffin embedding, tissue sections (4 �m) were deparaffinized inxylene and rehydrated through graded ethanol series.

ImmunohistochemistryKidney sections (4 �m) were deparaffinized and rehydrated. Antigen

retrieval was performed with a pressure cooker at 120°C in targetretrieval solution (DAKO, Carpinteria, CA). Endogenous peroxidaseactivity was blocked with 3% hydrogen peroxide (Fisher Scientific). Theprimary antibodies and dilutions that were used in this study are asfollows: Anti-ACE (1:2000; 5C4 gift of Dr. Sergei Danilov), anti-ACE2(1:100; rabbit antibody [15]; a gift from Drs. M. Chapell and C.M.Ferrario, Wake Forest University, Winston-Salem, NC), and anti-fi-bronectin (1:400; Sigma-Aldrich, St. Louis, MO). Sections for ACE stain-ing were washed with Tris-buffered saline with Tween-20 (DAKO) andincubated with biotinylated rabbit anti-rat IgG (Vector Laboratories,Burlingame, CA) followed by peroxidase-labeled streptavidin (DAKO).Sections for ACE2 and fibronectin staining were washed and incubatedwith goat anti-rabbit IgG conjugated with peroxidase-labeled polymer(DAKO). Peroxidase labeling was revealed using a liquid diaminoben-zidine substrate-chromagen system (DAKO). Sections were counter-stained with hematoxylin (Sigma) and dehydrated, mounted with Per-mount (Fisher Scientific), and coverslipped. Sections were examinedand photographed with a Zeiss microscope. Nonimmune serum wasused as control for specificity.

For assessment of the degree of ACE, ACE2, and fibronectin stainingin glomerular tuft, a semiquantitative analysis of the immunoperoxi-dase stained sections was done as based on a pathologist-establishedscore as follows, as described previously (16): 1 � no staining; 2 � weakstaining; 3 � strong staining. Sections were examined blindly by threedifferent observers, who assessed staining intensity of 100 glomerulifrom each slide.

Immunofluorescence and Confocal MicroscopyThe paraffin-embedded kidney sections (4 �m) were deparaffinized

and rehydrated. After antigen retrieval, sections were permeabilizedwith 0.5% Triton-X100 for 5 min and blocked with 5% normal donkeyserum in PBS for 1 h at room temperature. The sections were incubatedwith primary antibodies diluted in 2.5% donkey serum in PBS over-night at 4°C. Primary antibodies that were used for the immunofluo-

rescence immunostaining were rat monoclonal ACE antibody (5C4;1:100), our affinity-purified polyclonal ACE2 antibody (1:200), and oneof the specific cell type markers. As podocyte markers, we used anti-nephrin (1:100; Santa Cruz Biotechnologies), which localizes specifi-cally in the slit diaphragm (17); an antibody against synaptopodin(1:100; Santa Cruz Biotechnologies), which is an actin-associated pro-tein in the podocyte foot process (18); and anti-podocin (diluted 1:100;Santa Cruz Biotechnologies), which is specific for the basal pole ofpodocyte along the glomerular basement membrane (19). Platelet-endothelial cell adhesion molecule (PECAM-1; CD31) antibody (1:100;Santa Cruz Biotechnologies) was used as an endothelial cell marker.Anti–�-smooth muscle actin antibody (1:200; Sigma) was used to stainmesangial cells. Sections were washed with Tris-buffered saline withTween-20 three times and then incubated for 45 min with one of therespective secondary antibodies (Alexa Fluor 488 donkey anti-rat, Al-exa Fluor 555 donkey anti-rabbit, Alexa Fluor 647 donkey anti-goat, andAlexa Fluor 647 donkey anti-mouse IgG; Molecular Probes, Eugene,OR) diluted 1:200 in PBS with 2.5% donkey serum. Sections werewashed three times, coverslipped with Prolong Gold antifade reagent(Molecular Probes), and sealed with nail polish. Sections were visual-ized with a Zeiss LSM 510 confocal microscope (Carl Zeiss Microscopy,Jena, Germany). Negative controls for immunofluorescence stainingwere performed by substitution of nonimmune serum for the primaryantibodies.

Immunogold Electron MicroscopyKidney cortex was cut into 1-mm3 blocks and fixed in 2.5% glutar-

aldehyde in 0.1 M sodium cacodylate buffer at 4°C for overnight. Tissueblocks were postfixed with 1% osmium tetroxide in 0.1 M sodiumcacodylate buffer for 1 h and dehydrated in graded ethanols. Blockswere infiltrated in LR white resin, transferred into gelatin capsules withresin, and polymerized at 60°C for 24 h. Immunolabeling was per-formed on ultrathin sections that were cut with an ultramicrotome andpicked up on nickel grids. Sections were rinsed with PBS and incubatedin blocking solution (Electron Microscopy Sciences, Hatfield, PA) for 30min, rinsed, and incubated overnight at 4°C with rat anti-ACE (1:100)or rabbit anti-ACE2 antibody (1:200) diluted in PBS with 0.1% normalgoat serum. After washing in PBS, sections were incubated for 1 h withgoat anti-rat or goat anti-rabbit IgG coupled with 10 and 15 nm of goldparticles (Electron Microscopy Science), respectively. The sections werepostfixed with 2.5% glutaraldehyde, stained with uranyl acetate andlead citrate, and examined using a JEOL 1220 electron microscope.Images were captured with a Gatan digital camera.

Pharmacologic ACE2 Inhibition in db/m and db/db MiceA specific ACE2 inhibitor, MLN-4760 (gift from Millennium Phar-

maceuticals, Cambridge, MA), was injected into db/m and db/db micesubcutaneously (40 to 80 mg/kg body wt, every other day), starting at8 wk of age until the mice reached the age of 24 wk. Vehicle controlmice received injections of sterile PBS in the same volume. A group ofdb/db mice received both the AT1 receptor antagonist telmisartan(Boehringer Ingelheim, Ingelheim, Germany), in drinking water in adose of 2 mg/kg body wt per d, and the subcutaneous injections ofMLN-4760.

Urinary Albumin/Creatinine RatioTo measure albumin/creatinine ratio in urine samples, ELISA kit for

murine urinary albumin and creatinine companion kit from Exocell(Philadelphia, PA) were used according to the manufacturer’s instruc-tions. Spot urine samples were collected at 8 wk of age, before initiation

2 Journal of the American Society of Nephrology J Am Soc Nephrol ●● : ●●● –●●● , ●●●●

of the administration of the ACE2 inhibitor and the AT1 blocker (at 8wk of age), and after 12 and 16 wk of administration of these agents.

Statistical AnalysesStatistical analysis was performed using unpaired t test or ANOVA

when appropriate. Significance was defined as P � 0.05. Data areexpressed as mean � SEM.

ResultsLocalization of ACE and ACE2 Using Immunofluorescenceand Confocal Microscopy

ACE and ACE2 co-localized strongly in the apical brushborder of the proximal tubule (Figure 1A). ACE seemed to berestricted to the apical border, whereas ACE2 also was present,albeit weakly, in the cytoplasm (Figure 1A). Both ACE andACE2 were present in the glomerulus, but in contrast to prox-imal tubules, there was no co-localization of ACE and ACE2 inthe glomeruli (Figure 1B).

To localize further ACE and ACE2 within the glomerularstructures, we used markers for epithelial, mesangial, and en-dothelial cells. ACE co-localized with PECAM-1 (an endothelialcell marker; Figure 2, top), whereas ACE2 did not (Figure 2,bottom). ACE did not co-localize with either nephrin (Figure 3,top), synaptopodin (Figure 4) or podocin. In contrast, ACE2co-localized with nephrin (Figure 3, bottom), synaptopodin(Figure 4), and podocin (data not shown).

ACE2 also co-localized with �-smooth muscle actin, a markerof mesangial cells, whereas ACE did not (Figure 5). In sum-mary, glomerular ACE2 co-localized with both podocyte andmesangial cell markers, whereas ACE did not. ACE co-local-

ized with an endothelial marker, whereas ACE2 did not. Thepattern of cell-specific distribution of glomerular ACE andACE2 was similar in kidneys from db/m and db/db mice.

Glomerular ACE2 and ACE Localization by ImmunogoldElectron Microscopy

Immunogold electron microscopy labeling was used to studythe ultrastructural localization of ACE2 and ACE in glomeruli

Figure 1. (A) Immunofluorescence staining of angiotensin-con-verting enzyme (ACE; green; left) and ACE2 (red; middle) inproximal tubules. Merge of both images (yellow; right) showsco-localization of ACE and ACE2 at the apical site of proximaltubules. (B) Immunofluorescence staining of ACE (green; left)and ACE2 (red; middle) in a glomerulus from mouse kidney.Merging of both images shows essentially no co-localization ofACE and ACE2 in the glomerulus (right).

Figure 2. Triple immunofluorescence staining of ACE (green;A), ACE2 (red; D), and the endothelial cell marker platelet-endothelial cell adhesion molecule (PECAM-1; blue; B and E).Merged images show that ACE strongly co-localizes with PE-CAM-1 (light blue, arrows; C), whereas ACE2 does not co-localize with PECAM-1 (F).

Figure 3. Triple immunofluorescence staining of ACE (green;A), ACE2 (red; D), and the podocyte slit diaphragm markernephrin (blue; B and E). Merged images show that ACE doesnot co-localize with nephrin (C), whereas ACE2 clearly co-localizes with nephrin (pink, arrows; F).

J Am Soc Nephrol ●● : ●●● –●●● , ●●●● ACE2 and ACE and Albuminuria in Diabetic Mice 3

from db/m and db/db mice. ACE2 labeled with gold particles waspredominantly localized in podocyte foot processes (Figure6A). ACE2 also was found in the body and slit diaphragm ofthe podocyte and, to a smaller extent, in mesangial cells (datanot shown). In contrast to ACE2, ACE labeled with gold parti-cles was not found in podocytes or mesangial cells but wasexpressed abundantly in glomerular endothelial cells (Figure6B). Neither ACE nor ACE2 was found in the glomerular basalmembrane.

ACE and ACE2 Expression in Control and Diabetic MiceKidneys

In glomeruli from 8-wk-old db/db mice, ACE staining wasincreased as compared with db/m (Figure 7, compare B with A).ACE2, by contrast, was decreased in db/db as compared withdb/m (Figure 7, compare D with C). Strong staining was used tosemiquantify the observed changes on the basis of data fromkidneys from six animals in each group (see the Materials andMethods section). In diabetic mice, the percentage of glomeruliwith strong ACE staining was increased as compared withglomeruli from control mice (db/db 64.6 � 6.3 versus db/m 17.8 �

3.4%; P � 0.005; Figure 7). In contrast to these findings withACE, the percentage of glomeruli with strong ACE2 stainingwas reduced in diabetic mice in comparison with controls(db/db 4.3 � 2.4 versus db/m 30.6 � 13.6%; P � 0.05; Figure 7).

In kidneys from db/db mice, proximal tubular staining forACE was less intense than in tubules from the db/m mice(Figure 7, compare B with A). By contrast, ACE2 staining intubules from the db/db mice was increased as compared withthe control db/m mice (Figure 7, compare D with C). Thisfinding confirms our previous studies in tubules from the db/dbmice (10). Like in proximal tubules, glomerular parietal epithe-lial ACE2 staining was increased in the db/db mice (Figure 7D).There was no ACE staining in parietal glomerular epitheliumfrom either db/db or db/m mice.

Effect of Chronic ACE2 Inhibition on Albumin Excretionand Glomerular Fibronectin Deposition

To determine whether chronic ACE2 inhibition results inincreased albuminuria in the db/db mice, we administered aspecific ACE2 inhibitor, MLN-4760, for 16 consecutive weeksstarting at 8 wk of age. At 8 wk of age, before starting MLN orvehicle administration, db/db mice from both groups had virtu-ally indistinguishable levels of urinary albumin excretion(UAE; 69.5 � 18 versus 81 � 15 �g albumin/mg creatinine,respectively). As previously reported by others (14), we foundthat at this age, albumin excretion is already significantlyhigher in the db/db than in the db/m mice (81 � 15 versus 45 �

5 �g albumin/mg creatinine, respectively). In db/db mice thatreceived MLN-4760, albumin/creatinine ratio was significantlyhigher than in their vehicle-treated counterparts already after12 wk of treatment (474 � 166 versus 124 � 23 �g/mg, respec-tively; P � 0.05). After 16 wk of treatment, at the age of 24 wk,MLN-treated db/db mice had approximately three-fold in-creased UAE in comparison with vehicle db/db controls (743 �

200 versus 247 � 53.9 �g/mg, respectively; P � 0.05; Figure 8).In db/m mice that were treated with MLN-4760, UAE washigher than in the vehicle-treated db/m controls, but the differ-ence was small and not statistically significant (55 � 24 versus32 � 3, �g/mg, respectively; NS).

To study whether the effect of ACE2 inhibition is mediatedvia AngII and, more specific, the AT1 receptor, we gave bothMLN-4760 and telmisartan to db/db mice. The administrationof telmisartan to diabetic mice completely prevented theincrease in urinary albumin that was associated with admin-istration of the ACE2 inhibitor (Figure 8). This shows that theeffect of ACE2 inhibition on albuminuria requires stimula-

Figure 4. Triple immunofluorescence staining of ACE (green;A), ACE2 (red; D), and synaptopodin (blue; B and E), a podo-cyte foot process marker. ACE2 weakly co-localizes with syn-aptopodin (pink, arrows; F), whereas ACE does not co-localizewith it (C).

Figure 5. Triple immunofluorescence staining of ACE (green;A), ACE2 (red; D), and mesangial cell marker �-smooth muscleactin (�-SMA; blue; B and E). ACE and �-SMA do not co-localize in the glomeruli (C). ACE2 shows co-localization with�-SMA in some areas of the glomerular tuft (pink, arrows; F).


tion of the AT1 receptor presumably by increased levels ofAngII.

Chronic ACE2 inhibition also was associated with in-creased glomerular deposition of fibronectin, an extracellularmatrix protein (Figure 9). In glomeruli from db/m mice thatreceived MLN-4760, fibronectin staining was increased ascompared with their vehicle-treated db/m counterparts (Fig-ure 9, compare B with A). In db/db mice, the MLN-4760administration also was associated with an exaggeration offibronectin staining (Figure 9, compare D with C). The num-ber of glomeruli with strong fibronectin staining was used tosemiquantify the observed changes in kidneys from 12 to 16mice in each group (see the Materials and Methods section).

In db/m mice that received MLN-4760, the percentage ofglomeruli with strong fibronectin staining was increased ascompared with glomeruli from vehicle-treated db/m controls(41.1 � 4.1 versus 17.3 � 5.2%, respectively; P � 0.005; Figure9). Similar to the findings in db/m mice, the percentage ofglomeruli with strong fibronectin staining was increased indiabetic mice that were treated with MLN-4760 in compari-son with the db/db mice that received vehicle (54.8 � 4.6versus 28.5 � 6.4%, respectively; P � 0.005; Figure 9).

DiscussionThis study shows that ACE and ACE2 strongly co-localize on

the apical surface of the proximal tubules, whereas in glomer-

Figure 6. ACE2 and ACE immunogold labeling in glomeruli. ACE2 labeled with 15 nm of gold particles is distributed in podocytefoot processes and slit diaphragm (A, arrows). ACE labeled with 10 nm gold particles is localized in endothelial cells (B, arrows).The glomerular basement membrane (GBM) does not have either ACE or ACE2 immunogold particles. Magnification, �30,000(JEOL 1220 transmission electron microscope).

Figure 7. (Left) Immunohistochemistry of ACE (A and B) and ACE2 (C and D) in kidney sections from db/m (A and C) and db/dbmice (B and D) showing an example of glomerular ACE and ACE2 staining. In db/db mice, ACE staining within glomerular tuft(B, wide arrow) is more intense than in nondiabetic db/m controls (A, wide arrow). Unlike glomeruli, proximal tubules from db/dbmice have less staining for ACE (B, narrow arrow) than proximal tubules from db/m controls (A, narrow arrow). The reversepattern is seen for ACE2 staining within glomeruli, where ACE2 staining is less pronounced in diabetic mice (D, wide arrow) thanin controls (C, wide arrow), whereas proximal tubular staining is stronger in db/db than in db/m mice (C and D, single narrowarrow). ACE2 staining in glomerular parietal epithelium also is shown (D, double arrows). (Right) The percentage of stronglystained glomeruli for ACE and ACE2 in 8-wk-old db/m (�; n � 6) and db/db mice (f; n � 6). Strong ACE staining is markedlyincreased in glomeruli from diabetic mice in comparison with controls, whereas ACE2 is significantly decreased.


uli, ACE and ACE2 are present but do not co-localize. Toidentify ACE and ACE2 within distinct glomerular structures,we used subcellular and cell type–specific markers for immu-nofluorescence staining with confocal microscopy as well asimmunogold labeling. ACE2 was expressed mainly in visceralepithelial cells (podocytes) and in parietal epithelial cells of theBowman’s capsule. Within the glomerular tuft, ACE2 co-local-ized with nephrin (a slit diaphragm protein), podocin (a markerof the basal pole of the podocyte), and synaptopodin (a markerof the podocyte foot process), indicating that ACE2 is present inthe podocyte/slit diaphragm complex. ACE2 also co-localized,albeit not as strongly, with �-smooth muscle actin, which indi-cates its presence in mesangial cells. ACE, by contrast, did notco-localize with podocyte or mesangial cell markers. ACE co-localized with PECAM-1, reflecting its presence in glomerularendothelial cells. Immunogold studies further showed thatACE2 is present in podocyte foot processes and ACE in endo-thelial cells (Figure 6). ACE2 also was found in the body and slitdiaphragm of the podocyte and, to a smaller extent, in mesan-gial cells. The glomerular localization of ACE and ACE2 byimmunofluorescence and immunogold electron microscopywas similar in db/m and db/db mice. By immunohistochemistry,however, the pattern of strong glomerular ACE and ACE2protein staining differed strikingly between db/db and their leancounterpart, the db/m mice, of the same age (8 wk). That is,strong ACE2 protein staining in glomeruli from diabetic micewas decreased, whereas strong ACE protein staining, by con-trast, was increased (Figure 7).

On the basis of our findings of glomerular cell specificity ofACE and ACE2, we can infer the glomerular sites that areaccountable for the differences between db/m and db/db mice interms of ACE and ACE2 expression that were observed byimmunohistochemistry (Figure 7). Because ACE was expressed

in endothelial cells of db/db and db/m mice but not in podocytesor mesangial cells, it is reasonable to conclude that the strongglomerular staining of ACE that was seen in the db/db micereflects an increase at the level of glomerular endothelial cells.This is supported further by the fact that we found no evidenceof aberrant expression of ACE (i.e., ACE outside endothelialcells) in the immunolocalization studies that were performedin the db/db mice. Conversely, because ACE2 was not present inendothelial cells of either db/db or db/m mice, the reduction inglomerular staining of ACE2 in the db/db mice likely reflects adecrease in protein expression at the level of the podocyte andpossibly mesangial cells (or both).

The compound MLN-4760 is a specific ACE2 inhibitor thatexerts its inhibitory action by binding to two metallopeptidasecatalytic subdomains of the ACE2 enzyme (20). To examine thepotential role of ACE2 enzyme in the development of albumin-uria, we administered MLN-4760 for several weeks. This re-sulted in a significant increase in albumin excretion in the db/dbmice (Figure 8). By 24 wk of age, albumin excretion was ap-proximately three-fold higher in db/db mice that were treatedwith MLN-4760 as compared with vehicle-treated db/db con-trols. The specific AT1 blocker, telmisartan, prevented the in-crease in UAE that was associated with MLN-4760, suggestingthat the effect of ACE2 inhibition is mediated by AngII viastimulation of the AT1 receptor (Figure 8). ACE2 inhibition alsowas associated with increased glomerular expression of fi-bronectin in both db/m and db/db mice (Figure 9). In normalkidney, fibronectin is present along the basement membranes.During glomerular injury, fibronectin deposition increases, andthis increase is considered a marker of extracellular matrixaccumulation (21). Glomerular fibronectin accumulation occursas early as 7 d after AngII infusion (22). We think that MLN-4760, by inhibiting ACE2, leads to increased extracellular ma-trix deposition by promoting AngII accumulation within theglomerulus. Although we did not measure AngII levels afterACE2 inhibition, others have shown that in Ace2 knockout,AngII is either endogenously elevated (15,23) or increasedabove the levels of wild-type mice after infusion of exogenousAngII (24).

Our finding that ACE2 inhibition did not increase albuminexcretion significantly in nondiabetic female mice is in keepingwith the work of Oudit et al. (23) using an Ace2 knockout. Theseauthors found that deletion of the Ace2 gene was associatedwith the development of albuminuria over time (12 mo of age)in male but not in female mice. In general, it is more difficult toproduce albuminuria in female than in male mice (14,25). Inthis respect, it is noteworthy that in this study we used femaledb/db mice, because we were not aware of the findings of Ouditet al., which were published just recently (23). Our finding thatin female mice ACE2 inhibition resulted in worsening of albu-minuria further shows the importance of this enzyme in thecontrol of the glomerular permeability. We surmise that in malemice, ACE2 inhibition would promote albuminuria to a furtherdegree and that this would affect nondiabetic mice as well asdiabetic mice, but this will await further studies. It also is ofinterest that ACE2 inhibition in female db/m mice did not resultin significant albuminuria despite a significant increase in glo-

Figure 8. Urinary albumin/creatinine ratio (UAR) in diabeticdb/db mice at 24 wk of age. Starting at 8 wk of age, mice receivedvehicle or ACE2 inhibitor alone (MLN-4760) or in combinationwith the AT1 receptor blocker telmisartan (MLN-4760�TELM)for a period of 16 wk. UAR was significantly higher in db/dbmice that received ACE2 inhibitor when compared with db/dbvehicle controls (P � 0.05). The increase in urinary albumin inACE2 inhibitor–treated mice was prevented by the administra-tion of telmisartan (P � 0.05 versus db/db mice that were givenMLN-4760 alone). *P � 0.05.


merular fibronectin staining, a marker of mesangial matrixdeposition.

AngII impairs the function of glomerular barrier, leading toincreased protein excretion, and agents that interfere withAngII activity, such as ACE inhibitors and AT1 blockers, reducefiltration of macromolecules across the glomerular barrier(13,26–28). A recent study further showed that the abnormalprotein efflux across the glomerular membrane could be medi-ated by AngII-induced actin cytoskeleton rearrangement inglomerular epithelial cells (29). We propose that the presence ofACE2 in the podocyte/mesangial compartment of the glomer-ulus could have an important counterregulatory role by pre-venting glomerular AngII accumulation (Figure 10). In thisrespect, the reduction in glomerular ACE2 that was observed inthe young db/db mice could be deleterious because AngII deg-radation via ACE2 is apt to be decreased, particularly whencoupled with increased AngII formation that is driven by aug-mented ACE activity in endothelial cells. It should be noted thatthe db/db mice at the age of 8 wk have no evidence of glomer-ular lesions by light microscopy, but at this early age, albuminexcretion was already significantly higher in the db/db than inthe db/m mice, as previously reported by Sharma et al. (14). Thisincrease in albumin excretion reflects an increase in glomerularpermeability related to changes in glomerular hemodynamics,subtle podocyte injury, or both (30). We suggest that down-regulation of ACE2 may play a role by reducing AngII degra-dation, whereas the increase in endothelial ACE activity furtherresults in excess AngII. A cross-talk between podocyte andendothelial cells was proposed recently to explain the effect ofvascular endothelial growth factor that is produced in thepodocytes on glomerular endothelial permeability (31). It ispossible that the effect of AngII on augmenting glomerularpermeability involves increased vascular endothelial growthfactor mRNA translation, as recently suggested (32,33).

It is known that there are AngII receptors (AT1) in glomer-ular epithelial cells (podocytes) and that AngII activates signaltransduction pathways in these cells. The glomerular podocytehas a local RAS (34–36), and a recent study showed that me-chanical stress increases AngII production in conditionally im-mortalized podocytes (37). Our study shows that ACE is notpresent in the podocyte, which is consistent with in vitro studiesby Druvasula et al. (37) on immortalized podocytes showing noACE-dependent AngII formation. We propose that whether thesource of Ang peptides is systemic, from paracrine sources or

Figure 9. (Left) Immunohistochemistry of kidney sections from db/m (A and B) and db/db mice (C and D) showing an example ofglomerular fibronectin staining after vehicle (A and C) or MLN-4760 administration (B and D). In vehicle-treated db/m mouse, theglomerulus shows slight fibronectin staining (reddish-brown; A), which is increased in a db/m mouse that received MLN-4760 (B).In an MLN-treated db/db mouse, there is a marked increase in glomerular fibronectin staining (D) as compared with a glomerulusfrom a vehicle-treated db/db mouse (C). (Right) The percentage of strongly stained glomeruli for fibronectin in vehicle-treated (�)and MLN-4760–treated mice (f).

Figure 10. A proposed scheme whereby excess glomerularangiotensin II (AngII) accumulation in diabetic nephropathyresults from increased ACE and decreased ACE2 in theglomeruli.


locally generated within the podocyte, ACE2 could be critical indetermining the levels of Ang peptides by promoting AngIIdegradation to Ang 1-7 and AngI degradation to Ang 1-9. Anydirect role, if any, of these peptides in affecting glomerularpermeability needs to be examined. Regardless of any potentialeffect of these peptides on glomerular permeability, a de-creased expression of ACE2 protein and an increase in ACEfavors AngII accumulation, which, in turn, would lead to in-creased glomerular permeability (Figure 10).

Glomerular ACE2 and, most specific, its presence within thepodocyte/slit diaphragm complex normally could be protec-tive against AngII-mediated increases in glomerular permeabil-ity. We suggest that ACE2 activity within the glomerulus exertsa renoprotective effect by favoring the rapid degradation ofAng peptides and thereby preventing exposure to high levels ofAngII. This may be particularly relevant at the level of thepodocyte, a cell that may not be programmed to tolerate AngII,which would be in keeping with the lack of ACE expression.

Our findings in the glomerulus are in sharp contrast with thefindings in renal cortical tubules from db/db mice, where ACEstaining is decreased but ACE2 is increased (Figure 7). Thelatter finding confirms our previous report (10). A decrease intubular ACE was described originally by Anderson et al. (38) instreptozotocin-treated rats. Moreover, these authors suggestedalso that glomerular ACE was increased in this model of dia-betes (38). There also have been reports of an increase in ACEin the glomerulus of patients with diabetes and nephropathy(16). An increase in ACE expression in glomerular endothelialcells from animals and humans with diabetes may be the resultof generalized endothelial dysfunction, which is recognizedincreasingly in early stages of diabetes. Hyperfiltration, whichis present already at an early age in the db/db mice, could playan additional role at the level of the glomerular endothelium.We speculate that excessive ACE activation could be an impor-tant event in the activation of the RAS in diabetes and thereforeplay a more proximate role than generally appreciated. A pri-mary role of ACE overactivity on diabetes-related renal injurycan be inferred from studies in transgenic mice with threecopies of the Ace gene (39). Transgenic mice with one, two, orthree copies of ACE were studied after induction of diabeteswith streptozotocin (39). After induction of diabetes, there wasa moderate but significant increase in UAE in one- and two-copy mice but a much larger increase in the three-copy ACEmice (39). The overexpression of endothelial ACE coupled withthe underexpression of ACE2 in podocytes and mesangial cellsis a combination that is apt to increase AngII within the glo-merulus (Figure 10).

ConclusionOur study shows that ACE2 is present in podocytes and, to a

lesser extent, in glomerular mesangial cells, whereas ACE, bycontrast, is present only in endothelial cells. We propose thatACE2, by regulating the degradation of Ang peptides, preventsAngII accumulation in the glomerulus. Reduced glomerularACE2 in the db/db mice likely is deleterious by favoring AngIIaccumulation, leading to an increase in glomerular permeabil-ity early, and may foster progressive glomerular injury over

time. Our finding of increased albuminuria in the db/db micethat were treated with an ACE2 inhibitor suggests a role of thisenzyme in the regulation of Ang peptides and thus glomerularpermeability. The possibility of therapies that are targeted toamplify glomerular ACE2 expression as a way to reduce pro-teinuria and confer renoprotection early in the course of dia-betic nephropathy and possibly other kidney diseases needs tobe investigated.

AcknowledgmentsThis work was supported by a grant from the American Diabetes

Association (D.B.), and during the conduction of these studies, D.B. wassupported by the National Institute of Diabetes and Digestive andKidney Diseases.

Portions of this work were presented at the American Society ofNephrology Renal Week; November 8 through 13, 2005; Philadelphia,PA.

We thank Dr. Yashpal Kanwar for expert and generous advice on theimmunogold studies, and Dr. Rudolph Seidler for providing telmisar-tan.

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