Viewed As a Sole Terbalance APA Deﬁciency

BASIC RESEARCH www.jasn.org

Deﬁciency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury

† † ‡ Juan Carlos Q. Velez,* Ehtesham Arif, Jessalyn Rodgers, Megan P. Hicks, † | † John M. Arthur,§ Deepak Nihalani, Evelyn T. Bruner, Milos N. Budisavljevic, † † Carl Atkinson,¶ Wayne R. Fitzgibbon, and Michael G. Janech

*Department of Nephrology, Ochsner Clinic Foundation, New Orleans, Louisiana; †Division of Nephrology, Department of Medicine and Departments of |Pathology and ¶Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina; ‡Institute of Public and Preventative Health, Augusta University, Augusta, Georgia; and §Division of Nephrology, Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

ABSTRACT Aminopeptidase A (APA) is expressed in glomerular podocytes and tubular epithelia and metabolizes angiotensin II (AngII), a peptide known to promote glomerulosclerosis. In this study, we tested whether APA expression changes in response to progressive nephron loss or whether APA exerts a protective role against glomerular damage and during AngII-mediated hypertensive kidney injury. At advanced stages of FSGS, fawn-hooded hypertensive rat kidneys exhibited distinctly increased APA staining in areas of intact glomerular capillary loops. Moreover, BALB/c APA-knockout (KO) mice injected with a nephrotoxic serum showed persistent glomerular hyalinosis and albuminuria 96 hours after injection, whereas wild-type controls achieved virtually full recovery. We then tested the effect of 4-week infusion of AngII (400 ng/kg per minute) in APA-KO and wild-type mice. Although we observed no significant difference in achieved systolic BP, AngII-treated APA-KO mice developed a significant rise in albuminuria not observed in AngII-treated wild-type mice along with increased segmental and global sclerosis and/or collapse of juxtamedullary glomeruli, microcystic tubular dilation, and tubulointerstitial fibrosis. In parallel, AngII treatment significantly increased the kidney AngII content and attenuated the expression of podocyte nephrin in APA-KO mice but not in wild-type controls. These data show that deficiency of APA increases susceptibility to glomerular injury in BALB/c mice. The augmented AngII-mediated kidney injury observed in association with increased intrarenal AngII accumulation in the absence of APA suggests a protective metabolizing role of APA in AngII-mediated glomerular diseases.

J Am Soc Nephrol 28: 2119–2132, 2017. doi: https://doi.org/10.1681/ASN.2016111166

Aminopeptidase A (APA; glutamyl aminopeptidase; whereas neprilysin (NEP) converts AngII into two EC 3.4.11.7) is a homodimeric membrane–bound tetrapeptides, Ang1–4andAng5–8.3–8 Although zinc metallopeptidase capable of hydrolyzing an- these peptidases are ubiquitously expressed through- giotensin (Ang) peptides. Through cleavage at the out the body, differences in their cellular localization amino terminus, APA initiates the metabolism of the octapeptide AngII by converting it into the Received November 2, 2016. Accepted January 4, 2017. heptapeptide AngIII, which is further metabolized Published online ahead of print. Publication date available at rapidly by aminopeptidase N (APN) into AngIV.1,2 www.jasn.org. Other peptidases are capable of metabolizing AngII Correspondence: Dr. Juan Carlos Q. Velez, 1514 Jefferson at the carboxy terminus to convert it into Ang1–7 Highway, 5th Floor, Clinic Tower, Ochsner Medical Center, New (e.g., angiotensin-converting enzyme 2 [ACE2], Orleans, LA 70121. Email: [email protected] prolyl endopeptidase, and prolyl carboxypeptidase), Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 2119–2132, 2017 ISSN : 1046-6673/2807-2119 2119 BASIC RESEARCH www.jasn.org

experimental and human glomerular disease have revealed conflicting data: some reporting an increase in APA expression and others reporting a decrease.23–26 Using a robust model of spontaneous FSGS, the fawn-hooded hypertensive (FHH) rat, we sought to explore whether an adaptive change in pattern of glomerular APA expression is present during progressive nephron loss. Subsequently, we tested whether APA deficiency could influ- ence the reversibility of damage acquired in an established mouse model of glomerular injury, the sheep anti-rat glomerular lysate antiserum (nephrotoxic serum – Figure 1. Pattern of glomerular APA expression changes during progressive glo- [NTS]) injection model.27 29 Next, be- merulosclerosis. Examination of glomerular APA expression in 60-week-old FHH rat cause of its inherent property as an AngII- kidneys by (A) immunohistochemistry. In comparison with normal age-matched Wistar degrading enzyme, we examined the role rat kidneys that revealed uniform glomerular APA staining, FHH rat glomeruli showed of APA in a well established mouse model a heterogeneous pattern of expression, with a mixture of globally sclerotic glomeruli of AngII-dependent hypertensive kidney showing virtually no APA expression, segmentally sclerotic glomeruli with enhanced injury, the chronic AngII infusion APA staining in the nonsclerotic segments, and fully preserved glomeruli with overall model.30–32 We hypothesized that glomer- increased APA staining. Examination of APA expression in glomerular extracts ular APA expression changes as part of an (pooled from three rats per lane) by (B) Western blotting showed progressive loss fi adaptive response to injury. Then, we hy- of total APA over time along with a parallel progressive increase in bronectin (FN). fi The upper band for APA represents a mature transmembrane domain, whereas the pothesized that de ciency of APA could lower band corresponds to an immature endoplasmic reticulum fraction. Examination result in exacerbated glomerular injury in of APA expression in (C) human kidneys of a transplant healthy donor and an indi- response to a noxious stimuli, such as the vidual with FSGS also revealed a similar distinct patter of APA expression. Scale bars, NTS. In addition, we hypothesized that, 200 mm. during systemic exposure to exogenous AngII, full deficiency of APA may lead to reduced AngII degradation, increased in- and tissue distribution may have implications in tissue concen- trarenal accumulation of AngII, and subsequent augmented tration of AngII in health and disease. renal parenchymal injury. Studies indicate that APA, APN, NEP, ACE2, and prolyl carboxypeptidase are involved in AngII enzymatic processing in the kidney.1,9–12 However, intrarenal APA has the highest relative RESULTS tissue abundance and enzymatic activity compared with the other peptidases.13–15 Because APA is localized in glomerular Pattern of Adaptive Kidney APA Expression during podocytes,16,17 tubular epithelia,17 and medullary endothelial Progressive FSGS cells,16 it is critical for AngII metabolism in all kidney compart- Using the FHH rat model, we examined the pattern of glomer- ments. However, the notable localization of APA in podocytes is ular expression of APA during the course of the disease. In highlighted by studies showing that intravenous injection of comparison with normal age-matched Wistar rats, kidney APA mAb against APA in mice induces overt podocyte foot process expression changed at advanced stages (Figure 1A). Obsoles- effacement, glomerular granular IgG deposition, and albumin- cent glomeruli lost virtually all expression of APA. However, uria.18,19 Importantly, the kidneys possess the most robust en- coexisting surviving glomeruli exhibited prominent APA zymatic capacity to degrade AngII in the body, with up to 93% of staining. Additionally, intact areas within segmentally sclerotic AngII being degraded as it passes through the kidney compared glomeruli also showed prominent APA staining. Notwith- with 60% through systemic circulation and only 5% through standing the observed heterogeneity in glomerular APA ex- pulmonary circulation, and up to 60% of that intrarenal metab- pression, the total glomerular APA expression decreased over olism is mediated by APA.1 Despite those observations regarding time as shown by Western blotting of glomerular homoge- APA localization and function and the widely established notion nates. The upper band for APA represents a mature trans- that AngII promotes progressive glomerulosclerosis,20–22 the membrane domain protein post-Golgi processing, whereas role of APA-mediated AngII degradation in attenuating AngII- the lower band corresponds to an immature endoplasmic re- mediated kidney injury has not been elucidated. Furthermore, ticulum fraction.33 In contrast, glomerular fibronectin abun- studies examining the glomerular expression of APA in dance increased over time (Figure 1B). Furthermore, a kidney

2120 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

Renal Characterization of APA- Knockout Mice Lack of expression of APA in kidneys of BALB/c APA-knockout (KO) mice was verified by immunohistochemistry. Staining in BALB/cwild-typemice showedexpressionin podocytes and proximal tubules (Figure 3A). Immunofluorescence studies confirmed colocalization of APA and nephrin in wild-type mouse podocytes and complete absence of APA in APA-KO mice (Figure 3B). Images show APA localization at the body of the podocyte as well as colocalization of APA and nephrin at the slit diaphragms, consistent with a previous report.18 In addition, functional deficiency of intrarenal APA was verified in whole-kidney extracts of APA-KO mice by a fluorogenic substrate–based enzyme activity assay (Figure 3C) as well as a mass spectrometry–based assay (Figure 3D). The heptapeptide AngIII, a product of the cleavage of AngII by APA, was not detected in glomerular suspensions of APA-KO mice (Figure 3D). Altogether, these studies confirmed intrarenal deficiency of APA in APA-KO mice.

Renal Phenotype of Aged APA-KO Mice To determine whether APA-KO mice develop spontaneous kidney disease, kidneys from 12- month-old APA-KO and wild-type control mice were examined histologically. A few glomeruli per section were found to display mild Figure 2. Podocyte APA and GLEPP-1 expression evolve during early and advanced mesangial hypercellularity in APA-KO mice, stages of FSGS. Glomerular APA expression examined by immunohistochemistry in but this finding did not reach statistical signif- kidney sections from FHH rats in specimens obtained at 12, 24, 60, and 90 weeks of icance. In addition, no significant difference age. Sections revealed contrasting degrees of glomerular APA expression at advanced between groups was found in urine albumin- stages. Although APA staining increased in intact glomeruli as well as intact areas of to-creatinine ratio (UACR), although a trend segmentally sclerosed glomeruli, it was absent in globally sclerotic glomeruli. Staining was noted (Supplemental Figure 1). for GLEPP-1 did not show a similar expression pattern. Representative immunofluorescence images show APA staining along with faint GLEPP-1 staining in a segmentally Effect of NTS in APA-KO Mice sclerotic glomerulus at advanced stages of disease. Scale bars, 200 mminblackand green; 500 mm in yellow; 50 mm in white. Wild-type and APA-KO mice were subjected to single retro-orbital injections of NTS (50 ml). Within 24 hours, massive albumin- specimen from a subject with idiopathic FSGS showed a sim- uria was induced in both wild-type and APA-KO mice. How- ilar pattern of glomerular APA expression (Figure 1C). To ever, although wild-type mice exhibited an almost complete ascertain whether the observed pattern of expression was recovery at 96 hours postinjection, persistent albuminuria was not a consequence of a nonspecific podocyte hypertrophic observed in APA-KO mice (Figure 4A). This finding was accom- adaptation, we compared the pattern of glomerular expression panied by evidence of glomerular hyalinosis and tubular atrophy of APA with that of glomerular epithelial protein 1 (GLEPP- in APA-KO mice that was not observed in wild-type controls 1), a podocyte marker. Unlike APA, GLEPP-1 expression was (Figure 4B). Heterogeneity in response to the insult was observed. not found to be increased in either intact glomeruli or the At 12 days, in addition to glomerular hyalinosis and tubular at- nonsclerotic segments of segmentally sclerosed glomeruli dur- rophy, evidence of glomerular parietal cell hyperplasia and glo- ing advanced glomerulosclerosis (Figure 2). merular collapse was also detected in APA-KO mice (Figure 4B).

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2121 BASIC RESEARCH www.jasn.org

Figure 3. Lack of expression and activity of APA were verified in APA-KO mice. Examination of APA expression by (A) immunohistochemistry in kidney sections of wild-type (wt) and APA-KO mice showed glomerular and apical tubular distribution of APA in wt mice and negative staining in APA-KO mice. Scale bars, 200 mm. Examination by (B) immunofluorescence revealed colocalization of APA with nephrin, a podocyte marker, in wt mice and lack of glomerular APA expression in APA-KO mice. The white arrow indicates the body of the podocyte only showing APA staining (green), whereas colocalization of APA and nephrin corresponds to the podocyte slit diaphragms (yellow). DAPI, 49,6-diamidino-2-phenylindole. Fluorometry-based APA enzymatic activity (C) was measured in whole-kidney homogenates harvested from wt or APA-KO mice. Bars represent mean values, and error bars show SEMs. APA activity was measured with or without the presence of the APA inhibitor glutamate phosphonate (GluP; 10 mm). Recombinant aminopeptidase A (rAPA) was used as a positive control. (D) Conversion of AngII into AngIII was verified by LC-MS/MS, showing detection of AngIII (represented by the +3 parent ion 311.17 m/z) in glomerular suspensions of wt mice and almost complete absence of AngIII in those of APA-KO mice. The horizontal dashed line in the bar graph denotes the lower limit of detection. *P,0.001.

Effect of Chronic AngII Infusion on BP in APA-KO Mice 2, 3, and 4 weeks (Figure 5). Heterogeneity in the albuminuric Osmotic minipumps were implanted in wild-type and APA- response to AngII was observed. KO mice for a 4-week subcutaneous administration of vehicle (isotonic saline) or AngII (400 ng/kg per minute). Tail cuff Intrarenal Ang Peptide Abundance after Chronic AngII sphygmomanometry-based systolic BP (SBP) was measured Infusion in APA-KO Mice weekly throughout the experiment. The magnitude of the in- Whole-kidney homogenates were processed and analyzed by crease in SBP in AngII-treated APA-KO mice was not signif- liquid chromatography followed by tandem mass spectrometry icantly different than that of wild-type mice at 1, 2, 3, or 4 weeks (LC-MS/MS). AngI abundance was significantly greater in of infusion (Supplemental Figure 2). At 4 weeks, SBP reached vehicle-treated APA-KO mice compared with wild-type mice but 23267 and 23567 mmHg for AngII-treated wild-type and not in APA-KO mice infused with AngII (Figure 6A). However, APA-KO mice groups, respectively. The observed degree of kidney AngII abundance was not different among both vehicle- hypertension precluded testing higher doses of AngII. treated groups and AngII-treated wild-type mice. However, kidney AngII abundance significantly increased in AngII-treated APA-KO Effect of Chronic AngII Infusion on Albuminuria in APA- mice (Figure 6B). Moreover, kidney AngII content seemed to KO Mice correlate better to UACR than to SBP (Figure 6, C and D). Low levels of UACR were observed in vehicle-treated wild-type and APA-KO mice. Similarly, AngII-treated wild-type mice did Effect of Chronic AngII Infusion on Kidney Morphology not exhibit a significant increase in UACR during the 4 weeks of in APA-KO Mice AngII exposure. However, AngII-treated APA-KO mice Glomerular and vascular lesions are common morphologic fea- developed a significantly greater degree of albuminuria at 1, tures described in this model.30,34,35 Sections from wild-type and

2122 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

Figure 4. Urine albumin excretion and glomerular injury persist in APA-KO mice injected with NTS. (A) Persistence of albuminuria was observed in APA-KO mice 96 hours after injection of NTS but not in wild-type (wt) mice as shown by albumin gel electrophoresis and UACR by ELISA obtained 24 and 96 hours after NTS injections. (B) Histologic examination shows exacerbated renal parenchymal injury in APA-KO mice injected with NTS. Representative light microscopy images of kidney sections stained with periodic acid–Schiff. Tissue was harvested from wt or APA-KO mice 96 hours or 12 days after treatment with NTS injection. At 96 hours, mesangial hypercellularity was observed in both groups. However, only APA-KO mouse glomeruli showed hyalinosis, tuft adhesion to the Bowman’s capsule, and microaneurysms. Tubular atrophy with protein lakes was also seen in APA-KO mice. At 12 days, in addition to the aforementioned changes, parietal cell hyperplasia was noted in APA-KO mice. Scale bars, 200 mm in black and green; 500 mminyellow;2mminwhite.*P,0.001; #P,0.001.

APA-KO mice treated with vehicle showed no evidence of (Figure 7). Affected glomeruli were almost exclusively found glomerular damage after 4 weeks. Only a few wild-type mice at the juxtamedullary region. Because of the known effects of treated with AngII showed discrete evidence of segmental AngII in cell hypertrophy36,37 and on juxtamedullary glo- sclerosis and microaneurysms. In contrast, AngII-treated meruli volume,38 we assessed for glomerular size but did not APA-KO mice exhibited significantly more pronounced find a significant difference in glomerular volume among segmental and global sclerosis and glomerular collapse treatment groups.

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2123 BASIC RESEARCH www.jasn.org

podocyte marker, but its expression was also attenuated in AngII-treated APA-KO mouse kidneys (Figure 9). Others have reported redistribution of ZO-1 and cytoskel- etal rearrangement in response to AngII.41

Effect of Chronic AngII Infusion on Expression of Angiotensinases in APA-KO Mice We examined the intrarenal expression of key renin-angiotensin system (RAS) peptidases known to metabolize AngII by immunohistochemistry, searching for a compensatory upregulation in response to APA deﬁciency. However, no difference in expression of ACE2, NEP,or APN was found among all four treatment groups (Figure 10).

DISCUSSION

We report a distinct pattern of glomerular expression of APA during progressive glomerulosclerosis. Although the overall expression of glomerular APA decreases over time in parallel with the development of glomerulosclerosis, a heterogeneous fi Figure 5. Global de ciency of APA leads to increased albuminuria during chronic AngII population of glomeruli is recognizable at infusion. Values of urinary albumin excretion collected during a 4-week infusion of AngII advanced stages of disease. Strong APA (400 ng/kg per minute) in APA-KO mice. Urinary albumin excretion rates expressed in staining was observed in the surviving in- UACR in wild-type (wt) or APA-KO mice treated with chronic subcutaneous infusion of either vehicle control (Ctrl) or AngII (400 ng/kg per minute). Data presented in scatter dot tact glomeruli as well as the segmental plots of values obtained at (A) 1, (B) 2, (C) 3, and (D) 4 weeks showed greater magnitude of areas where the glomerular capillary loops UACR in AngII-treated APA-KO mice. #P,0.01 versus all three groups. remain intact, whereas virtually no APA staining was found in globally sclerotic glomeruli. Using enzyme histochemistry, a Sections from wild-type and APA-KO mice treated with vehicle similar adaptive pattern of APAactivity was reported in advanced control showed no evidence of tubulointerstitial damage. Mild human CKD.42 The authors labeled the glomeruli displaying patchy tubularatrophy,microcystictubulardilation,andinterstitial high APA activity as “highly resistant” because of their normal fibrosis were found in AngII-treated wild-type mice, whereas APA- morphology, despite the severe damage of the surrounding tis- KO mice infused with AngII acquired significantly worse tubuloin- sue, and implicated APA in mechanisms of cellular adaptation terstitial injury, including scattered large protein lakes (Figure 8). to progressive nephron loss.42,43 Our findings are in line These lesions were found either surrounding an affected glo- with those observations and provide a protein abundance merulus within the juxtamedullary region or at the subcapsular correlate to their functional studies. In addition, the pattern region depicting a radial pattern, perhaps after a vascular bed of APA expression did not seem to correspond to nonspecific distribution. podocyte hypertrophy, because the expression of GLEPP-1, a podocyte marker, was not enhanced in the surviving glomer- Effect of Chronic AngII Infusion on Podocyte Protein uli at advanced stages. In agreement with our findings, others Expression in APA-KO Mice reported gradual fall in GLEPP-1 expression in a rat model of Because AngII has been reported to induce podocyte injury in podocyte injury.44,45 Thus, augmentation of podocyte APA association with a decrease in nephrin expression,39,40 we ex- abundance seems to be an adaptive response to progressive amined the effect of chronic AngII infusion on nephrin. We glomerulosclerosis. found a reduction in nephrin expression in AngII-treated To further investigate the role of APA in mechanisms APA-KO mouse kidneys by Western blotting in whole-kidney of adaptation to glomerular injury, we subjected APA-KO homogenates as well as immunofluorescence in tissue sections mice to a glomerular injury model. Compared with wild- (Figure 9). Zona occludens 1 (ZO-1) was used as a colocalizing type controls, APA-KO mice exhibited persistent albuminuria

2124 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

hypertension, APA-KO mice developed marked glomerulosclerosis, glomerular collapse, and tubular damage in response to AngII treatment. This pattern of injury resembled that described in other suscep- tible mouse strains subjected to the AngII infusion model.34,35,48 Thus, APA deficiency increased the susceptibility of BALB/c mice to the pathologic actions of AngII in the kidney. Notably, affected glomeruli were almost exclusively localized at the juxtamedullary region, zonal localization that fits with previous descriptions of vulnerability of juxtamedullary glomeruli in animal models of glomerular capillary hypertension49 or FSGS50 and human FSGS.51,52 Unlike our study, APA-KO mice on a hy- brid C57BL/6J-129Sv background strain exhibited an augmented pressor response to chronic AngII. However, the authors did not report whether that pressor susceptibility was accompanied with greater kidney Figure 6. Kidney content of Ang peptides after chronic AngII infusion is altered in injury.53 Although systemic hypertension fi APA-KO mice. Quanti cation of tissue content of (A) AngI and (B) AngII performed by is a critical element driving the pathogen- LC-MS/MS in whole-kidney homogenates from wild-type (wt) or APA-KO mice treated esis of AngII-dependent glomerulosclero- with chronic infusion of either vehicle control (Ctrl) or AngII (400 ng/kg per minute) for 4 weeks (n=5 per group). *P,0.05. Correlation between kidney AngII content and (C) sis, local nonpressor effects of AngII in the UACR and (D) SBP) is shown. kidney are thought to play an independent role.54–56 Thus, the greater renal parenchymal injury in AngII-treated APA-KO mice and glomerular hyalinosis in response to NTS, suggesting that likely resulted from local intrarenal effects of AngII amplified APA participates in glomerular repair mechanisms. Because by APA deficiency. APA is an angiotensinase and AngII can promote podocyte loss A vast array of evidence indicates that AngII can directly and injury,44,46 it is plausible that the lack of recovery to the exert deleterious effects in podocyte biology, such as de- NTS injection in APA-KO mice corresponds to an exacerbated rangements of the slit diaphragm composition40,57,58 or AngII-mediated mechanism. the cytoskeleton,38,59,60 activation of profibrotic pathways,61 Our studies using a chronic AngII infusion model show that apoptosis,62–64 and podocyte detachment.44 In our studies, mice fully deficient in APA exhibit increased susceptibility to AngII infusion induced attenuation of nephrin expression AngII-mediated kidney injury as manifested by changes in in APA-KO mice but not in wild-type mice. These findings albuminuria, kidney morphology, and nephrin expression. suggest that the noxious effects of AngII on podocyte slit dia- These findings were observed in association with increased phragm integrity of BALB/c mice were enhanced in a state of intrarenal abundance of AngII in APA-KO mice infused with global APA deficiency. Because APA deficiency led to greater AngII. Thus, our data suggest that the increased susceptibility kidney AngII content, direct effects of AngII on podocytes were to AngII-mediated kidney injury may correspond to reduced likely amplified. In support of this contention, AngII has been metabolism of AngII. Intrarenal AngII content in vehicle-treated shown to reduce nephrin expression40 and mRNA57 in mouse APA-KO mice was not increased, suggesting that, under basal podocytes in vitro as well as nephrin mRNA abundance after conditions and in the absence of an amplified RAS state, other acute subcutaneous AngII injection in rats.57 The observed de- mechanisms of AngII degradation might be sufficient to coun- crease in nephrin expression should not be viewed as a sole terbalance APA deficiency. However, this equilibrium may be mediator of the albuminuric glomerular phenotype. Rather, it lost during a perturbation that leads to amplification in AngII is interpreted as a surrogate indicator for distressed glomeruli generation or enhanced AngII delivery to the kidney. caused by AngII. The lack of nephrin attenuation in wild-type Despite pronounced hypertension, only minimal renal pa- AngII-treated mice likely corresponded to the relative renal renchymal damage was observed in AngII-treated wild-type resistance of BALB/c mice to the AngII infusion model.47 mice, likely due to resistance inherent to their background Interestingly, vehicle-treated APA-KO mice were found to strain.47 In contrast, although acquiring a similar degree of have increased kidney AngI abundance. Because APA is the

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2125 BASIC RESEARCH www.jasn.org

Figure 7. Histologic examination shows exacerbated glomerular injury in APA-KO mice after chronic AngII infusion. (A) Representative light microscopy images of kidney sections stained with Masson trichrome. Tissue was harvested from wild-type (wt) or APA-KO mice treated with chronic subcutaneous infusion of either vehicle control (Ctrl) or AngII (400 ng/kg per minute) for 4 weeks. (B and C) Bar graphs show comparison of the percentages of juxtamedullary glomeruli with segmental (III, IV, and VII) or global (V and VI) sclerosis, collapse (V and VIII), or microaneurysms (IV and VIII). (D) Evidence of glomerular enlargement was compared between groups. *P,0.01. Scale bars, 200 mm. primary glomerular peptidase responsible for the conversion CONCISE METHODS of AngI to Ang2–10,65 the greater content of AngI in APA-KO mice likely corresponds to diminished AngI metabolism. In Animal Care AngII-treated APA-KO mice, the intrarenal AngI content Our protocols were approved by the Medical University of South was not increased. This may pertain to the known negative Carolina Institutional Animal Care and Use Committee and in accor- feedback mechanism by which AngII inhibits renin release dance with the procedures and practices of the National Insititutes of via stimulation of Angtype 1 receptors. Thus, AngII infusion Health Guide for the Care and Use of Laboratory Animals. All rodents may suppress renin release, thereby diminishing AngI gen- were housed at a certified facility at 22°C and in a 12:12-hour light/ eration from angiotensinogen. Altogether, our findings of dark cycle, allowed free access to tap water, fed a standard diet, and intrarenal AngI content are in accordance with the known provided environmental enrichment. All surgeries were performed RAS feedback loops. Interestingly, we did not detect any under isoflurane anesthesia (5% induction and 2% maintenance) at compensatory increase in kidney expression of ACE2, 37°C and perioperative buprenorphine. NEP, or APN in our studies. ACE2 has been implicated as a renoprotective molecule in models of diabetic kidney Rodents disease.66–68 Nonetheless, peptidomic and network model- BALB/c APA-KO (ENPEP2/2) mice were provided by Renata Pas- ing approaches have revealed dominance of APA over ACE2 qualini (University of New Mexico, Albaquerque, NM). Generation as Ang peptidase.12,69 of APA-KO mice was previously reported in detail.70 Wild-type In conclusion, deficiency of APA leads to increased suscep- BALB/c control mice were purchased from Envigo (Indianapolis, tibility to glomerular injury as shown by the NTS injection IN). Male FHH rats were an in-house colony established from ani- model and the AngII-mediated renal parenchymal injury mals obtained from Charles River Laboratories (Wilmington, MA). model. In lieu of the relative abundance and enzymatic activity Male Wistar rats were purchased from Envigo (Indianapolis, IN). of APA in the kidney and the adaptive changes in glomerular APA expression during glomerulosclerosis, our findings Human Tissue suggest a key role of APA in maintenance of intrarenal RAS Our study protocol was approved by the Medical University of South equilibrium. Carolina Institutional Review Board. Archived formalin-fixed,

2126 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

Figure 8. Histologic examination shows exacerbated tubulointerstitial injury in APA-KO mice after chronic AngII infusion. (A) Repre- sentative light microscopy images of kidney sections stained with Masson trichrome. Tissue was harvested from wild-type (wt) or APA-KO mice treated with chronic subcutaneous infusion of either vehicle (Ctrl) or AngII (400 ng/kg per minute) for 4 weeks. (B) Bar graph shows comparison of tubulointerstitial injury scores that accounted for patchy subcapsular fibrosis (III and V) and microcystic tubular dilation (III– VI). *P,0.01. Scale bars, 500 mm in yellow; 2 mm in white. paraffin-embedded kidney specimens were processed for examination albumin and creatinine were measured by ELISA (Exocell, Philadelphia, by immunohistochemistry. Five-mm sections were obtained from bi- PA) as per the manufacturer’s protocol. Samples were diluted (1:10– opsy cores from three healthy donors and five patients with diagnosis 1:100) before assay. For the NTS model, 2.5-ml urine aliquots were of idiopathic FSGS. run on a 10% SDS-PAGE, and the gels were stained with Coomassie Brilliant Blue G-250 (0.25%) prepared in 50% methanol and 10% Glomerular Injury Model acetic acid for 30 minutes as previously reported.72,73 Mice were retro-orbitally injected with 50 ml NTS (PTX-001S; Probetex Inc., San Antonio, TX) at 12 weeks of age as previously Immunohistochemistry for Kidney Morphology and reported.29,71 Pilot studies were carried out to select the dosage. Urine RAS Peptidase Expression samples were collected at preinjection and 24 and 96 hours postinjec- Parenchymal renal injury was assessed by immunohistochemistry. tion. Kidney tissues were collected at euthanasia. Kidneys harvested at euthanasia were fixed in 4% paraformaldehyde and paraffin embedded. Morphology was examined in sections AngII Infusion Model stained with hematoxylin-eosin, Jones silver, periodic acid–Schiff, An established model for AngII-mediated hypertensive kidney injury and Masson trichrome. Expression RAS peptidases and GLEPP-1 was used with some modifications.30 Eight- to 12-week-old male mice were examined using an antigen retrieval technique. Briefly, 4- to underwent osmotic minipump implantation (Alzet, Palo Alto, CA) 5-mm sections were deparaffinized in xylene, washed in ethanol gra- for delivery of AngII (400 mg/kg per minute) for 4 weeks. SBP was dient, and microwaved for 20 minutes with Cymatin Citrate (pH 6.0) measured with a tail cuff sphygmomanometer at baseline and weekly for antigen retrieval (DAKO, Carpinteria, CA) followed by washes throughout the experiment. with 3% hydrogen peroxide, PBS, and 2.5% horse serum (ImmPRESS Reagent Peroxidase Anti-Goat Ig Kit; Vector Laboratories, Burlingame, Albuminuria CA). Primary antibodies were goat anti-APA (anti–BP-1; Abcam Mice were placed in metabolic cages for 24-hour urine collection Laboratories, Cambridge, MA), 1:167 (mouse specimens) and 1:150 weekly. UACR was used as a marker of glomerular injury. Urine (rat and human specimens); mouse anti-NEP (anti-CD10; Vector

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2127 BASIC RESEARCH www.jasn.org

glomerular volume using the software AxioVi- A sion 4.6 (Dublin, CA) and standard morphom- etric equations.74 An average of 15 glomeruli per tissue section per kidney was obtained. Tubu- lointerstitial damage was determined by the presence of interstitial ﬁbrosis, tubular atrophy, and microcystic tubular dilation and rated by a standard scale: 0, 1 (,25%), 2 (25%–50%), and 3(.50%).

Immunoﬂuorescence for Glomerular Protein Expression B Procedures were performed as previously described.75 Primary antibodies for APA (1:10; Abcam Laboratories), nephrin76 (1:150), and ZO-1 (1:300; Invitrogen, Carlsbad, CA) were diluted in 5% BSA in PBS and incubated overnight at 4°C. Sections were washed, incubated with Alexa Fluor–labeled secondary antibodies at 1:1000 for 1 hour at 37°C, and mounted with antifade reagent containing 49,6-diamidino-2- phenylindole.

Western Blotting Kidney homogenates were loaded in Novex 4%– 12% Bis-Tris NuPage Gels (Invitrogen) as previously described with minor modiﬁcations.2 Primary antibodies used were goat anti-APA (Abcam), 1:500; rabbit antiﬁbronectin (Abcam), 1:1000; rabbit antinephrin (gift from Puneet Garg, University of Michigan, Ann Arbor, MI), 1:2000; and mouse anti–b-actin (Sigma-Aldrich, St. Louis, MO), 1:5000.

Figure 9. Podocyte protein expression changes observed in APA-KO mice after APA Activity by Fluorometry chronic AngII infusion. Representative images depicting the effect of chronic infusion of Mouse kidney homogenates(n=3per group)were AngII on (A) nephrin and b-actin as examined by Western blotting in whole-kidney prepared with triethanolamine sucrose buffer homogenates and (B) nephrin and ZO-1 expression as examined by immunofluores- (10 mM triethanolamine and 250 mM sucrose, m fl cence in 5 m kidney sections. Graph shown next to the Western blot re ects pH 7.6) and diluted in assay buffer (25 mM Tris, quantification of band densities from five individual mouse kidney homogenates from 50 mM CaCl2, and 0.2 M NaCl, pH 8.0) to a the wild-type (wt) control (Ctrl) group and six of each of the other three treatment 2 mg/ml concentration. Lysates were pretreated groups. *P,0.01. with or without 4-amino-4-phosphonobutyric acid (glutamate phosphonate, 10 mM final; Laboratories), 1:50; goat anti-ACE2 (Santa Cruz Biotechnology, Santa APA inhibitor77,78; gift from Robert Speth, Nova Southeastern Uni- Cruz, CA), 1:50; rabbit anti-APN (anti-CD13; Origene, Rockville, versity, Fort Lauderdale FL). The fluorogenic substrate H-Glu-AMC MD), 1:250; and mouse anti-GLEPP-1 (gift from Roger Wiggins, Uni- (Bachem, Torrance, CA) was added to 100 mg protein per well at a final versity of Michigan, Ann Arbor, MI), 1:50. The anti–GLEPP-1 anti- concentration of 100 mM. Excitation and emission were measured for body was not reactive in mouse tissue. Tissues were mounted with 30 minutes at 380 and 460 nm, respectively. Recombinant APA(0.02 mg; Cytoseal 60 (ThermoFisher Scientific, Carlsbad, CA). R&D Systems, Minneapolis, MN) was used as a positive control. Scoring of specimens were adjudicated blindly by a trained pa- thologist. Glomerular damage was rated by percentage of affected APA Activity by LC-MS/MS glomeruli and determined by the presence of segmental or global Conversion of AngII to AngIII was examined in suspensions of glo- sclerosis or hyalinosis, glomerular collapse, microaneurysms, peri- meruli isolated from wild-type and APA-KO mouse kidneys as pre- capsular fibrosis, mesangial hypercellularity, or parietal epithelial viously reported with some modifications.65 Isolation of glomeruli cell hyperplasia. Glomerulomegaly was assessed by measurement of was undertaken following a magnetic beads–based method.79

2128 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

Ang Peptide Content by LC-MS/MS Tissue abundance of Ang peptides was determined using a Triple-ToF 5600 Mass Spec- trometer (SCIEX, Framingham, MA) using previously published methods with some mod- iﬁcations.80 Frozen whole kidney was pulverized in liquid nitrogen, and 40–190 mg were trans- ferred to a glass dounce for homogenization in 60% methanol in water containing 1 mM phena- throline. Stable isotope–labeled standards for AngII, AngIII,AngIV,andAng2–10 were added to a concentration of 10.8 fmol/mg wet kidney weight.

Statistical Analyses Figures were generated using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA), and values were displayed as mean and SD. Compar- isons between groups were performed using two-way ANOVA with post hoc analysis, re- peated measures ANOVA, paired t tests, or Fischer exact tests when appropriate. Correla- tions were assessed by Spearman test. A P value ,0.05 was deemed signiﬁcant.

ACKNOWLEDGMENTS

We thank Alison Bland and Peifeng Deng for technical support. Figure 10. RAS peptidase expression does not change in APA-KO mice after chronic This work was partially funded by grants from AngII infusion. Representative images depicting the effect of chronic infusion of AngII the National Institute of Diabetes and Digestive fl on expression of NEP, ACE2, and APN as examined by immuno uorescence. Glo- and Kidney Diseases of the National Institute of merular parietal epithelial and tubular epithelial cell distribution was noted for all three Health (DK080944, J.C.Q.V.), and American enzymes, with faint podocyte localization. No difference in degree of staining was Recovery and Reinvestment Act supplement to observed across treatment groups. Ctrl, control; wt, wild type. Scale bars, 200 mm. DK080944 (W.R.F. and M.P.H.) and Dialysis Clinics Incorporated (J.C.Q.V. and M.G.J.). Two thousand glomeruli per 1 ml were incubated in Krebs buffer D.N. is supported by grant R01DK0887956-07 from the National with 1 mM AngII for 60 minutes. Buffer aliquots were serially Institute of Diabetes and Digestive and Kidney Diseases of the National obtained and analyzed by LC-MS/MS to search for generation of Institutes of Health. AngIII. Each aliquot was centrifuged at 20,0003g for 10 minutes. Part of this work was presented as a poster at American Society of Supernatant (25 ml)wasmixed1:1withstableisotope–labeled Nephrology Kidney Week in Philadelphia, Pennsylvania on November AngIII in 0.4% formic acid to a final concentration of 22.5 fmol/ml. 11–16, 2014 and in San Diego, California on November 3–8, 2015. Samples were loaded into the autosampler, and 4 mlsupernatant was injected onto an ACQUITY UPLC M-Class HSS T3 Column (1.8 mm; 75 mm3150 mm; Waters Corp., Milford, MA) at 10 ml/min DISCLOSURES with 0.2% formic acid in mass spectrometry–grade water. Peptides were J.C.Q.V. has served on advisory boards for Mallinckrodt Pharmaceuticals, separated at 10 ml/min using an M-Class UPLC (Waters Corp.) from Alexion Pharmaceuticals, and Relypsa Inc. zero to 30% mobile phase B (95% acetonitrile/0.1% formic acid) over 4 minutes. Data were acquired with a Triple Quadrupole Mass Spectrometer (TQS; Waters Corp.) using a low-flow probe at the REFERENCES source. The transitions (m/z) 311.2–419.2 and 313.3–425.3 were monitored for quantification of the native and stable isotope–labeled 1. Bauer J, Berthold H, Schaefer F, Ehmke H, Parekh N: Quantification of standard. Estimates of concentration were made against an external conversion and degradation of circulating angiotensin in rats. Am J – – Physiol 277: R412 R418, 1999 standard curve (0.5 250 fmol on column) in Targetlynx (Waters 2. Velez JC, Bland AM, Arthur JM, Raymond JR, Janech MG: Character- Corp.). Lower limit of quantification was determined as 103 SD ization of renin-angiotensin system enzyme activities in cultured mouse of five blank injections. podocytes. Am J Physiol Renal Physiol 293: F398–F407, 2007

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2129 BASIC RESEARCH www.jasn.org

3. Velez JC: Prolyl carboxypeptidase: A forgotten kidney angiotensinase. 22. Meyer TW, Anderson S, Rennke HG, Brenner BM: Reversing glomerular Focus on “Identification of prolyl carboxypeptidase as an alternative hypertension stabilizes established glomerular injury. Kidney Int 31: enzyme for processing of renal angiotensin II using mass spectrome- 752–759, 1987 try.” Am J Physiol Cell Physiol 304: C939–C940, 2013 23. Wolf G, Thaiss F, Mueller E, Disser M, Pooth R, Zahner G, Stahl RA: 4. Yang HY, Erdös EG, Chiang TS: New enzymatic route for the in- Glomerular mRNA expression of angiotensinase A after renal ablation. activation of angiotensin. Nature 218: 1224–1226, 1968 Exp Nephrol 3: 240–248, 1995 5. Allred AJ, Diz DI, Ferrario CM, Chappell MC: Pathways for angiotensin- 24. Wolf G, Thaiss F, Scherberich JE, Schoeppe W, Stahl RA: Glomerular (1—7) metabolism in pulmonary and renal tissues. Am J Physiol Renal angiotensinase A in the rat: Increase of enzyme activity following renal Physiol 279: F841–F850, 2000 ablation. Kidney Int 38: 862–868, 1990 6. Rosivall L, Narkates AJ, Oparil S, Navar LG: De novo intrarenal forma- 25. Song L, Healy DP: Kidney aminopeptidase A and hypertension, part II: tion of angiotensin II during control and enhanced renin secretion. Am J Effects of angiotensin II. Hypertension 33: 746–752, 1999 Physiol 252: F1118–F1123, 1987 26. Baelde HJ, Eikmans M, Doran PP, Lappin DW, de Heer E, Bruijn JA: 7. Li N, Zimpelmann J, Cheng K, Wilkins JA, Burns KD: The role of Gene expression profiling in glomeruli from human kidneys with di- angiotensin converting enzyme 2 in the generation of angiotensin 1-7 abetic nephropathy. Am J Kidney Dis 43: 636–650, 2004 by rat proximal tubules. Am J Physiol Renal Physiol 288: F353–F362, 27. Ophascharoensuk V, Pippin JW, Gordon KL, Shankland SJ, Couser WG, 2005 Johnson RJ: Role of intrinsic renal cells versus infiltrating cells in glo- 8. Rice GI, Thomas DA, Grant PJ, Turner AJ, Hooper NM: Evaluation of merular crescent formation. Kidney Int 54: 416–425, 1998 angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin 28. Moeller MJ, Soofi A, Hartmann I, Le Hir M, Wiggins R, Kriz W, Holzman in angiotensin peptide metabolism. Biochem J 383: 45–51, 2004 LB: Podocytes populate cellular crescents in a murine model of in- 9. Wysocki J, Ye M, Soler MJ, Gurley SB, Xiao HD, Bernstein KE, Coffman flammatory glomerulonephritis. J Am Soc Nephrol 15: 61–67, 2004 TM, Chen S, Batlle D: ACE and ACE2 activity in diabetic mice. Diabetes 29. Chen SM, Mukoyama T, Sato N, Yamagata S, Arai Y, Satoh N, Ueda S: 55: 2132–2139, 2006 Induction of nephrotoxic serum nephritis in inbred mice and suppres- 10. Grobe N, Weir NM, Leiva O, Ong FS, Bernstein KE, Schmaier AH, sive effect of colchicine on the development of this nephritis. Phar- Morris M, Elased KM: Identification of prolyl carboxypeptidase as an macol Res 45: 319–324, 2002 alternative enzyme for processing of renal angiotensin II using mass 30. Crowley SD, Frey CW, Gould SK, Griffiths R, Ruiz P, Burchette JL, spectrometry. Am J Physiol Cell Physiol 304: C945–C953, 2013 Howell DN, Makhanova N, Yan M, Kim HS, Tharaux PL, Coffman TM: 11. Velez JC, Ierardi JL, Bland AM, Morinelli TA, Arthur JM, Raymond JR, Stimulation of lymphocyte responses by angiotensin II promotes kid- Janech MG: Enzymatic processing of angiotensin peptides by human ney injury in hypertension. Am J Physiol Renal Physiol 295: F515–F524, glomerular endothelial cells. Am J Physiol Renal Physiol 302: F1583– 2008 F1594, 2012 31. Crowley SD, Gurley SB, Herrera MJ, Ruiz P, Griffiths R, Kumar AP, Kim 12. Schwacke JH, Spainhour JC, Ierardi JL, Chaves JM, Arthur JM, Janech HS, Smithies O, Le TH, Coffman TM: Angiotensin II causes hyperten- MG, Velez JC: Network modeling reveals steps in angiotensin peptide sion and cardiac hypertrophy through its receptors in the kidney. Proc processing. Hypertension 61: 690–700, 2013 Natl Acad Sci USA 103: 17985–17990, 2006 13. Troyanovskaya M, Jayaraman G, Song L, Healy DP: Aminopeptidase-A. 32. Lautrette A, Li S, Alili R, Sunnarborg SW, Burtin M, Lee DC, Friedlander I. CDNA cloning and expression and localization in rat tissues. Am J G, Terzi F: Angiotensin II and EGF receptor cross-talk in chronic kidney Physiol Regul Integr Comp Physiol 278: R413–R424, 2000 diseases: A new therapeutic approach. Nat Med 11: 867–874, 2005 14. Bioinformatics Laboratory at Wilmer Institute, Johns Hopkins University: 33. Rozenfeld R, Muller L, El Messari S, Llorens-Cortes C: The C-terminal Tissue-specific Gene Expression and Regulation (TiGER), version 1.0. domain of aminopeptidase A is an intramolecular chaperone required Available at http://bioinfo.wilmer.jhu.edu/tiger/db_gene/ENPEP-index. for the correct folding, cell surface expression, and activity of this html.” Accessed on March 2008 monozinc aminopeptidase. JBiolChem279: 43285–43295, 2004 15. Scherberich JE, Wiemer J, Herzig C, Fischer P, Schoeppe W: Isolation 34. Liu Z, Huang XR, Lan HY: Smad3 mediates ANG II-induced hyperten- and partial characterization of angiotensinase A and aminopeptidase M sive kidney disease in mice. Am J Physiol Renal Physiol 302: F986– from urine and human kidney by lectin affinity chromatography and F997, 2012 high-performance liquid chromatography. J Chromatogr A 521: 279– 35. Liao TD, Yang XP, Liu YH, Shesely EG, Cavasin MA, Kuziel WA, Pagano 289, 1990 PJ, Carretero OA: Role of inflammation in the development of renal 16. Mentzel S, Dijkman HB, Van Son JP, Koene RA, Assmann KJ: Organ damage and dysfunction in angiotensin II-induced hypertension. Hy- distribution of aminopeptidase A and dipeptidyl peptidase IV in normal pertension 52: 256–263, 2008 mice. J Histochem Cytochem 44: 445–461, 1996 36. Anderson PW, Do YS, Hsueh WA: Angiotensin II causes mesangial cell 17. Kugler P, Wolf G, Scherberich J: Histochemical demonstration of hypertrophy. Hypertension 21: 29–35, 1993 peptidases in the human kidney. Histochemistry 83: 337–341, 1985 37. Johnson RJ, Alpers CE, Yoshimura A, Lombardi D, Pritzl P, Floege J, 18. Assmann KJ, van Son JP, Dijkman HB, Koene RA: A nephritogenic rat Schwartz SM: Renal injury from angiotensin II-mediated hypertension. monoclonal antibody to mouse aminopeptidase A. Induction of mas- Hypertension 19: 464–474, 1992 sive albuminuria after a single intravenous injection. JExpMed175: 38. Hornych H, Beaufils M, Richet G: The effect of exogenous angiotensin 623–635, 1992 on superficial and deep glomeruli in the rat kidney. Kidney Int 2: 336– 19. Chugh S, Yuan H, Topham PS, Haydar SA, Mittal V, Taylor GA, Kalluri R, 343, 1972 Salant DJ: Aminopeptidase A: A nephritogenic target antigen of 39. Wang L, Chang JH, Paik SY, Tang Y, Eisner W, Spurney RF: Calcineurin nephrotoxic serum. Kidney Int 59: 601–613, 2001 (CN) activation promotes apoptosis of glomerular podocytes both in 20. Ruggenenti P, Perna A, Gherardi G, Gaspari F, Benini R, Remuzzi G: vitro and in vivo. Mol Endocrinol 25: 1376–1386, 2011 Renal function and requirement for dialysis in chronic nephropathy 40. Doublier S, Salvidio G, Lupia E, Ruotsalainen V, Verzola D, Deferrari G, patients on long-term ramipril: REIN follow-up trial. Gruppo Italiano di Camussi G: Nephrin expression is reduced in human diabetic ne- Studi Epidemiologici in Nefrologia (GISEN). Ramipril Efficacy in Ne- phropathy: Evidence for a distinct role for glycated albumin and an- phropathy. Lancet 352: 1252–1256, 1998 giotensin II. Diabetes 52: 1023–1030, 2003 21. Lewis EJ, Hunsicker LG, Bain RP, Rohde RD; The Collaborative Study 41. Macconi D, Abbate M, Morigi M, Angioletti S, Mister M, Buelli S, Group: The effect of angiotensin-converting-enzyme inhibition on di- Bonomelli M, Mundel P, Endlich K, Remuzzi A, Remuzzi G: Permse- abetic nephropathy. N Engl J Med 329: 1456–1462, 1993 lective dysfunction of podocyte-podocyte contact upon angiotensin II

2130 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 www.jasn.org BASIC RESEARCH

unravels the molecular target for renoprotective intervention. Am J contributes to podocyte injury by increasing TRPC6 expression via an Pathol 168: 1073–1085, 2006 NFAT-mediated positive feedback signaling pathway. Am J Pathol 42. Scherberich JE, Wolf G, Albers C, Nowack A, Stuckhardt C, Schoeppe 179: 1719–1732, 2011 W: Glomerular and tubular membrane antigens reflecting cellular ad- 61. Chen S, Lee JS, Iglesias-de la Cruz MC, Wang A, Izquierdo-Lahuerta A, aptation in human renal failure. Kidney Int Suppl 27: S38–S51, 1989 Gandhi NK, Danesh FR, Wolf G, Ziyadeh FN: Angiotensin II stimulates 43. Scherberich JE, Matthess A, Remelius W, Schoeppe W: Aminopepti- alpha3(IV) collagen production in mouse podocytes via TGF-beta and dase A (= angiotensinase A) in human progressive renal disease. Miner VEGF signalling: Implications for diabetic glomerulopathy. Nephrol Electrolyte Metab 18: 97–100, 1992 Dial Transplant 20: 1320–1328, 2005 44. Fukuda A, Wickman LT, Venkatareddy MP, Sato Y, Chowdhury MA, 62. Jia J, Ding G, Zhu J, Chen C, Liang W, Franki N, Singhal PC: Angiotensin Wang SQ, Shedden KA, Dysko RC, Wiggins JE, Wiggins RC: Angio- II infusion induces nephrin expression changes and podocyte apo- tensin II-dependent persistent podocyte loss from destabilized glo- ptosis. Am J Nephrol 28: 500–507, 2008 meruli causes progression of end stage kidney disease. Kidney Int 81: 63. Ding G, Reddy K, Kapasi AA, Franki N, Gibbons N, Kasinath BS, Singhal 40–55, 2012 PC: Angiotensin II induces apoptosis in rat glomerular epithelial cells. 45. Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, Am J Physiol Renal Physiol 283: F173–F180, 2002 Saunders TL, Dysko RC, Kohno K, Holzman LB, Wiggins RC: Podocyte 64. Durvasula RV, Shankland SJ: Activation of a local renin angiotensin depletion causes glomerulosclerosis: Diphtheria toxin-induced podo- system in podocytes by glucose. Am J Physiol Renal Physiol 294: F830– cyte depletion in rats expressing human diphtheria toxin receptor F839, 2008 transgene. JAmSocNephrol16: 2941–2952, 2005 65. Velez JC, Ryan KJ, Harbeson CE, Bland AM, Budisavljevic MN, Arthur 46. Hoffmann S, Podlich D, Hähnel B, Kriz W, Gretz N: Angiotensin II type 1 JM, Fitzgibbon WR, Raymond JR, Janech MG: Angiotensin I is largely receptor overexpression in podocytes induces glomerulosclerosis in converted to angiotensin (1-7) and angiotensin (2-10) by isolated rat transgenic rats. JAmSocNephrol15: 1475–1487, 2004 glomeruli. Hypertension 53: 790–797, 2009 47. Francois H, Athirakul K, Mao L, Rockman H, Coffman TM: Role for 66. Ye M, Wysocki J, William J, Soler MJ, Cokic I, Batlle D: Glomerular lo- thromboxane receptors in angiotensin-II-induced hypertension. Hy- calization and expression of angiotensin-converting enzyme 2 and pertension 43: 364–369, 2004 Angiotensin-converting enzyme: Implications for albuminuria in di- 48. Eckel J, Lavin PJ, Finch EA, Mukerji N, Burch J, Gbadegesin R, Wu G, abetes. JAmSocNephrol17: 3067–3075, 2006 Bowling B, Byrd A, Hall G, Sparks M, Zhang ZS, Homstad A, Barisoni L, 67. Nadarajah R, Milagres R, Dilauro M, Gutsol A, Xiao F, Zimpelmann J, Birbaumer L, Rosenberg P, Winn MP: TRPC6 enhances angiotensin II- Kennedy C, Wysocki J, Batlle D, Burns KD: Podocyte-specific over- induced albuminuria. JAmSocNephrol22: 526–535, 2011 expression of human angiotensin-converting enzyme 2 attenuates di- 49. Iversen BM, Amann K, Kvam FI, Wang X, Ofstad J: Increased glomerular abetic nephropathy in mice. Kidney Int 82: 292–303, 2012 capillary pressure and size mediate glomerulosclerosis in SHR juxta- 68. Wong DW, Oudit GY, Reich H, Kassiri Z, Zhou J, Liu QC, Backx PH, medullary cortex. Am J Physiol 274: F365–F373, 1998 Penninger JM, Herzenberg AM, Scholey JW: Loss of angiotensin- 50. Chen A, Sheu LF, Ho YS, Lin YF, Chou WY, Chou TC, Lee WH: Exper- converting enzyme-2 (Ace2) accelerates diabetic kidney injury. Am J imental focal segmental glomerulosclerosis in mice. Nephron 78: 440– Pathol 171: 438–451, 2007 452, 1998 69. Wysocki J, Ye M, Batlle D: Plasma and kidney angiotensin peptides: 51. Sherman RL, Susin M, Mouradian J: Focal glomerular sclerosis. Perspect Importance of the aminopeptidase A/angiotensin III axis. Am J Hy- Nephrol Hypertens 6: 175–186, 1977 pertens 28: 1418–1426, 2015 52. Danilewicz M, Wagrowska-Danilewicz M: Minimal change disease and 70. Lin Q, Taniuchi I, Kitamura D, Wang J, Kearney JF, Watanabe T, idiopathic focal segmental glomerulosclerosis in adults. A quantitative Cooper MD: T and B cell development in BP-1/6C3/aminopeptidase study. Pol J Pathol 47: 209–214, 1996 A-deficient mice. JImmunol160: 4681–4687, 1998 53. Mitsui T, Nomura S, Okada M, Ohno Y, Kobayashi H, Nakashima Y, 71. Lichtnekert J, Kulkarni OP, Mulay SR, Rupanagudi KV, Ryu M, Allam R, Murata Y, Takeuchi M, Kuno N, Nagasaka T, O-Wang J, Cooper MD, Vielhauer V, Muruve D, Lindenmeyer MT, Cohen CD, Anders HJ: Anti- Mizutani S: Hypertension and angiotensin II hypersensitivity in amino- GBM glomerulonephritis involves IL-1 but is independent of NLRP3/ peptidase A-deficient mice. Mol Med 9: 57–62, 2003 ASC inflammasome-mediated activation of caspase-1. PLoS One 6: 54. Kagami S: Involvement of glomerular renin-angiotensin system (RAS) e26778, 2011 activation in the development and progression of glomerular injury. 72. Gee HY, Sadowski CE, Aggarwal PK, Porath JD, Yakulov TA, Schueler M, Clin Exp Nephrol 16: 214–220, 2012 Lovric S, Ashraf S, Braun DA, Halbritter J, Fang H, Airik R, Vega-Warner V, 55. Urushihara M, Kinoshita Y, Kondo S, Kagami S: Involvement of the in- Cho KJ, Chan TA, Morris LG, ffrench-Constant C, Allen N, McNeill H, trarenal renin-angiotensin system in experimental models of glomer- Büscher R, Kyrieleis H, Wallot M, Gaspert A, Kistler T, Milford DV, Saleem ulonephritis. J Biomed Biotechnol 2012: 601786, 2012 MA, Keng WT, Alexander SI, Valentini RP, Licht C, Teh JC, Bogdanovic R, 56. Mahmood J, Khan F, Okada S, Kumagai N, Morioka T, Oite T: Local Koziell A, Bierzynska A, Soliman NA, Otto EA, Lifton RP, Holzman LB, delivery of angiotensin receptor blocker into the kidney ameliorates Sibinga NE, Walz G, Tufro A, Hildebrandt F: FAT1 mutations cause a progression of experimental glomerulonephritis. Kidney Int 70: 1591– glomerulotubular nephropathy. Nat Commun 7: 10822, 2016 1598, 2006 73. Ge Y, Si J, Tian L, Zhuang S, Dworkin LD, Gong R: Conditional ablation 57. Suzuki K, Han GD, Miyauchi N, Hashimoto T, Nakatsue T, Fujioka Y, of glycogen synthase kinase 3b in postnatal mouse kidney. Lab Invest Koike H, Shimizu F, Kawachi H: Angiotensin II type 1 and type 2 re- 91: 85–96, 2011 ceptors play opposite roles in regulating the barrier function of kidney 74. Haas CS, Amann K, Schittny J, Blaser B, Müller U, Hartner A: Glomerular glomerular capillary wall. Am J Pathol 170: 1841–1853, 2007 and renal vascular structural changes in alpha8 integrin-deficient mice. 58. Wennmann DO, Hsu HH, Pavenstädt H: The renin-angiotensin- JAmSocNephrol14: 2288–2296, 2003 aldosterone system in podocytes. Semin Nephrol 32: 377–384, 2012 75. Arif E, Wagner MC, Johnstone DB, Wong HN, George B, Pruthi PA, 59. Hsu HH, Hoffmann S, Endlich N, Velic A, Schwab A, Weide T, Schlatter Lazzara MJ, Nihalani D: Motor protein Myo1c is a podocyte protein that E, Pavenstädt H: Mechanisms of angiotensin II signaling on cytoskele- facilitates the transport of slit diaphragm protein Neph1 to the podo- ton of podocytes. J Mol Med (Berl) 86: 1379–1394, 2008 cyte membrane. Mol Cell Biol 31: 2134–2150, 2011 60. Nijenhuis T, Sloan AJ, Hoenderop JG, Flesche J, van Goor H, Kistler 76. Holzman LB, St John PL, Kovari IA, Verma R, Holthofer H, Abrahamson AD, Bakker M, Bindels RJ, de Boer RA, Möller CC, Hamming I, Navis G, DR: Nephrin localizes to the slit pore of the glomerular epithelial cell. Wetzels JF, Berden JH, Reiser J, Faul C, van der Vlag J: Angiotensin II Kidney Int 56: 1481–1491, 1999

J Am Soc Nephrol 28: 2119–2132, 2017 Aminopeptidase A Deﬁciency and Glomerular Injury 2131 BASIC RESEARCH www.jasn.org

77. Vazeux G, Iturrioz X, Corvol P, Llorens-Cortès C: A tyrosine residue 80. Velez JC, Janech MG, Hicks MP, Morinelli TA, Rodgers J, Self SE, essential for catalytic activity in aminopeptidase A. Biochem J 327: Arthur JM, Fitzgibbon WR: Lack of renoprotective effect of 883–889, 1997 chronic intravenous angiotensin-(1-7) or angiotensin-(2-10) in a rat 78. Karamyan VT, Gadepalli R, Rimoldi JM, Speth RC: Brain AT1 angio- model of focal segmental glomerulosclerosis. PLoS One 9: tensin receptor subtype binding: Importance of peptidase inhibition for e110083, 2014 identiﬁcation of angiotensin II as its endogenous ligand. JPharmacol Exp Ther 331: 170–177, 2009 79. Takemoto M, Asker N, Gerhardt H, Lundkvist A, Johansson BR, Saito Y, Betsholtz C: A new method for large scale isolation of kidney glomeruli This article contains supplemental material online at http://jasn.asnjournals. from mice. Am J Pathol 161: 799–805, 2002 org/lookup/suppl/doi:10.1681/ASN.2016111166/-/DCSupplemental.

2132 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2119–2132, 2017 SUPPLEMENTAL DATA

Supplemental Figure S1. Kidney histology and albuminuria examined after aging APA- KO mice. Examination of kidney sections from 12 month-old wild-type and APA-KO mice stained with periodic acid-Schiff revealed absence of significant glomerular or tubulointerstitial injury, except for sporadic mesangial hypercellularity in APA-KO mouse kidneys. Scale bars: black = 200 µM; green = 200 µM. No significant increase in urine albumin-to-creatinine ratio (UACR) was observed between groups, P = 0.07.

Supplemental Figure S2. Values of systolic blood pressure registered during a 4-week infusion of Ang II (400 ng/kg/min) in APA-KO mice. Tail-cuff systolic blood pressure readings in wild-type (wt) or APA-KO mice treated with chronic subcutaneous infusion of either vehicle control (Ctrl) or angiotensin II (Ang II) (400 ng/kg/min). Data presented in box whisker plots of values obtained at (a) 1 week, (b) 2 weeks, (c) 3 weeks and (d) 4 weeks shows large and comparable increases in SBP in wt and APA-KO mice treated with Ang II throughout the study period. *P < 0.001.