Deficiency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury

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.

Aminopeptidase A (APA; glutamyl aminopeptidase; EC 3.4.11.7) is a homodimeric membrane–bound zinc metallopeptidase capable of hydrolyzing angiotensin (Ang) peptides. Through cleavage at the amino terminus, APA initiates the metabolism of the octapeptide AngII by converting it into the heptapeptide AngIII, which is further metabolized rapidly by aminopeptidase N (APN) into AngIV.^1,2 Other peptidases are capable of metabolizing AngII at the carboxy terminus to convert it into Ang1–7 (e.g., angiotensin-converting enzyme 2 [ACE2], prolyl endopeptidase, and prolyl carboxypeptidase), whereas neprilysin (NEP) converts AngII into two tetrapeptides, Ang1–4 and Ang5–8.^3–8 Although these peptidases are ubiquitously expressed throughout the body, differences in their cellular localization and tissue distribution may have implications in tissue concentration of AngII in health and disease.

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 tissue abundance and enzymatic activity compared with the other peptidases.^13–15 Because APA is localized in glomerular podocytes,^16,17 tubular epithelia,¹⁷ and medullary endothelial cells,¹⁶ it is critical for AngII metabolism in all kidney compartments. However, the notable localization of APA in podocytes is highlighted by studies showing that intravenous injection of mAb against APA in mice induces overt podocyte foot process effacement, glomerular granular IgG deposition, and albuminuria.^18,19 Importantly, the kidneys possess the most robust enzymatic capacity to degrade AngII in the body, with up to 93% of AngII being degraded as it passes through the kidney compared with 60% through systemic circulation and only 5% through pulmonary circulation, and up to 60% of that intrarenal metabolism is mediated by APA.¹ Despite those observations regarding APA localization and function and the widely established notion that AngII promotes progressive glomerulosclerosis,^20–22 the role of APA-mediated AngII degradation in attenuating AngII-mediated kidney injury has not been elucidated. Furthermore, studies examining the glomerular expression of APA in 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 influence the reversibility of damage acquired in an established mouse model of glomerular injury, the sheep anti-rat glomerular lysate antiserum (nephrotoxic serum [NTS]) injection model.^27–29 Next, because of its inherent property as an AngII-degrading enzyme, we examined the role of APA in a well established mouse model of AngII-dependent hypertensive kidney injury, the chronic AngII infusion model.^30–32 We hypothesized that glomerular APA expression changes as part of an adaptive response to injury. Then, we hypothesized that deficiency of APA could result in exacerbated glomerular injury in response to a noxious stimuli, such as the NTS. In addition, we hypothesized that, during systemic exposure to exogenous AngII, full deficiency of APA may lead to reduced AngII degradation, increased intrarenal accumulation of AngII, and subsequent augmented renal parenchymal injury.

Results

Pattern of Adaptive Kidney APA Expression during Progressive FSGS

Using the FHH rat model, we examined the pattern of glomerular expression of APA during the course of the disease. In comparison with normal age-matched Wistar rats, kidney APA expression changed at advanced stages (Figure 1A). Obsolescent glomeruli lost virtually all expression of APA. However, coexisting surviving glomeruli exhibited prominent APA staining. Additionally, intact areas within segmentally sclerotic glomeruli also showed prominent APA staining. Notwithstanding the observed heterogeneity in glomerular APA expression, the total glomerular APA expression decreased over time as shown by Western blotting of glomerular homogenates. The upper band for APA represents a mature transmembrane domain protein post-Golgi processing, whereas the lower band corresponds to an immature endoplasmic reticulum fraction.³³ In contrast, glomerular fibronectin abundance increased over time (Figure 1B). Furthermore, a kidney specimen from a subject with idiopathic FSGS showed a similar pattern of glomerular APA expression (Figure 1C). To ascertain whether the observed pattern of expression was not a consequence of a nonspecific podocyte hypertrophic adaptation, we compared the pattern of glomerular expression of APA with that of glomerular epithelial protein 1 (GLEPP-1), a podocyte marker. Unlike APA, GLEPP-1 expression was not found to be increased in either intact glomeruli or the nonsclerotic segments of segmentally sclerosed glomeruli during advanced glomerulosclerosis (Figure 2).

Figure 1.

Pattern of glomerular APA expression changes during progressive glomerulosclerosis. Examination of glomerular APA expression in 60-week-old FHH rat kidneys by (A) immunohistochemistry. In comparison with normal age-matched Wistar rat kidneys that revealed uniform glomerular APA staining, FHH rat glomeruli showed a heterogeneous pattern of expression, with a mixture of globally sclerotic glomeruli showing virtually no APA expression, segmentally sclerotic glomeruli with enhanced APA staining in the nonsclerotic segments, and fully preserved glomeruli with overall increased APA staining. Examination of APA expression in glomerular extracts (pooled from three rats per lane) by (B) Western blotting showed progressive loss of total APA over time along with a parallel progressive increase in fibronectin (FN). The upper band for APA represents a mature transmembrane domain, whereas the lower band corresponds to an immature endoplasmic reticulum fraction. Examination of APA expression in (C) human kidneys of a transplant healthy donor and an individual with FSGS also revealed a similar distinct patter of APA expression. Scale bars, 200 µm.

Figure 2.

Podocyte APA and GLEPP-1 expression evolve during early and advanced stages of FSGS. Glomerular APA expression examined by immunohistochemistry in kidney sections from FHH rats in specimens obtained at 12, 24, 60, and 90 weeks of age. Sections revealed contrasting degrees of glomerular APA expression at advanced stages. Although APA staining increased in intact glomeruli as well as intact areas of segmentally sclerosed glomeruli, it was absent in globally sclerotic glomeruli. Staining 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 sclerotic glomerulus at advanced stages of disease. Scale bars, 200 μm in black and green; 500 μm in yellow; 50 μm in white.

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/c wild-type mice showed expression in 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.

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 µm. 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, 4′,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 μm). 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.

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 mesangial hypercellularity in APA-KO mice, but this finding did not reach statistical significance. In addition, no significant difference between groups was found in urine albumin-to-creatinine ratio (UACR), although a trend was noted (Supplemental Figure 1).

Effect of NTS in APA-KO Mice

Wild-type and APA-KO mice were subjected to single retro-orbital injections of NTS (50 μl). Within 24 hours, massive albuminuria was induced in both wild-type and APA-KO mice. However, although wild-type mice exhibited an almost complete recovery at 96 hours postinjection, persistent albuminuria was observed in APA-KO mice (Figure 4A). This finding was accompanied by evidence of glomerular hyalinosis and tubular atrophy in APA-KO mice that was not observed in wild-type controls (Figure 4B). Heterogeneity in response to the insult was observed. At 12 days, in addition to glomerular hyalinosis and tubular atrophy, evidence of glomerular parietal cell hyperplasia and glomerular collapse was also detected in APA-KO mice (Figure 4B).

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 μm in black and green; 500 μm in yellow; 2 mm in white. *P<0.001; ^#P<0.001.

Effect of Chronic AngII Infusion on BP in APA-KO Mice

Osmotic minipumps were implanted in wild-type and APA-KO mice for a 4-week subcutaneous administration of vehicle (isotonic saline) or AngII (400 ng/kg per minute). Tail cuff sphygmomanometry-based systolic BP (SBP) was measured weekly throughout the experiment. The magnitude of the increase in SBP in AngII-treated APA-KO mice was not significantly different than that of wild-type mice at 1, 2, 3, or 4 weeks of infusion (Supplemental Figure 2). At 4 weeks, SBP reached 232±7 and 235±7 mmHg for AngII-treated wild-type and APA-KO mice groups, respectively. The observed degree of hypertension precluded testing higher doses of AngII.

Effect of Chronic AngII Infusion on Albuminuria in APA-KO Mice

Low levels of UACR were observed in vehicle-treated wild-type and APA-KO mice. Similarly, AngII-treated wild-type mice did not exhibit a significant increase in UACR during the 4 weeks of AngII exposure. However, AngII-treated APA-KO mice developed a significantly greater degree of albuminuria at 1, 2, 3, and 4 weeks (Figure 5). Heterogeneity in the albuminuric response to AngII was observed.

Figure 5.

Global deficiency of APA leads to increased albuminuria during chronic AngII infusion. Values of urinary albumin excretion collected during a 4-week infusion of AngII (400 ng/kg per minute) in APA-KO mice. Urinary albumin excretion rates expressed 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 plots of values obtained at (A) 1, (B) 2, (C) 3, and (D) 4 weeks showed greater magnitude of UACR in AngII-treated APA-KO mice. ^#P<0.01 versus all three groups.

Intrarenal Ang Peptide Abundance after Chronic AngII Infusion in APA-KO Mice

Whole-kidney homogenates were processed and analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). AngI abundance was significantly greater in vehicle-treated APA-KO mice compared with wild-type mice but not in APA-KO mice infused with AngII (Figure 6A). However, kidney AngII abundance was not different among both vehicle-treated groups and AngII-treated wild-type mice. However, kidney AngII abundance significantly increased in AngII-treated APA-KO mice (Figure 6B). Moreover, kidney AngII content seemed to correlate better to UACR than to SBP (Figure 6, C and D).

Figure 6.

Kidney content of Ang peptides after chronic AngII infusion is altered in APA-KO mice. Quantification of tissue content of (A) AngI and (B) AngII performed by LC-MS/MS in whole-kidney homogenates from wild-type (wt) or APA-KO mice treated 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) UACR and (D) SBP) is shown.

Effect of Chronic AngII Infusion on Kidney Morphology in APA-KO Mice

Glomerular and vascular lesions are common morphologic features described in this model.^30,34,35 Sections from wild-type and APA-KO mice treated with vehicle showed no evidence of glomerular damage after 4 weeks. Only a few wild-type mice treated with AngII showed discrete evidence of segmental sclerosis and microaneurysms. In contrast, AngII-treated APA-KO mice exhibited significantly more pronounced segmental and global sclerosis and glomerular collapse (Figure 7). Affected glomeruli were almost exclusively found at the juxtamedullary region. Because of the known effects of AngII in cell hypertrophy^36,37 and on juxtamedullary glomeruli volume,³⁸ we assessed for glomerular size but did not find a significant difference in glomerular volume among treatment groups.

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 μm.

Sections from wild-type and APA-KO mice treated with vehicle control showed no evidence of tubulointerstitial damage. Mild patchy tubular atrophy, microcystic tubular dilation, and interstitial fibrosis were found in AngII-treated wild-type mice, whereas APA-KO mice infused with AngII acquired significantly worse tubulointerstitial injury, including scattered large protein lakes (Figure 8). These lesions were found either surrounding an affected glomerulus within the juxtamedullary region or at the subcapsular region depicting a radial pattern, perhaps after a vascular bed distribution.

Figure 8.

Histologic examination shows exacerbated tubulointerstitial 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 (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 μm in yellow; 2 mm in white.

Effect of Chronic AngII Infusion on Podocyte Protein Expression in APA-KO Mice

Because AngII has been reported to induce podocyte injury in association with a decrease in nephrin expression,^39,40 we examined the effect of chronic AngII infusion on nephrin. We found a reduction in nephrin expression in AngII-treated APA-KO mouse kidneys by Western blotting in whole-kidney homogenates as well as immunofluorescence in tissue sections (Figure 9). Zona occludens 1 (ZO-1) was used as a colocalizing 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 cytoskeletal rearrangement in response to AngII.⁴¹

Figure 9.

Podocyte protein expression changes observed in APA-KO mice after chronic AngII infusion. Representative images depicting the effect of chronic infusion of AngII on (A) nephrin and β-actin as examined by Western blotting in whole-kidney homogenates and (B) nephrin and ZO-1 expression as examined by immunofluorescence in 5 µm kidney sections. Graph shown next to the Western blot reflects quantification of band densities from five individual mouse kidney homogenates from the wild-type (wt) control (Ctrl) group and six of each of the other three treatment groups. *P<0.01.

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).

Figure 10.

RAS peptidase expression does not change in APA-KO mice after chronic AngII infusion. Representative images depicting the effect of chronic infusion of AngII on expression of NEP, ACE2, and APN as examined by immunofluorescence. Glomerular parietal epithelial and tubular epithelial cell distribution was noted for all three enzymes, with faint podocyte localization. No difference in degree of staining was observed across treatment groups. Ctrl, control; wt, wild type. Scale bars, 200 μm.

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 population of glomeruli is recognizable at advanced stages of disease. Strong APA staining was observed in the surviving intact glomeruli as well as the segmental areas where the glomerular capillary loops remain intact, whereas virtually no APA staining was found in globally sclerotic glomeruli. Using enzyme histochemistry, a similar adaptive pattern of APA activity was reported in advanced human CKD.⁴² The authors labeled the glomeruli displaying high APA activity as “highly resistant” because of their normal morphology, despite the severe damage of the surrounding tissue, and implicated APA in mechanisms of cellular adaptation to progressive nephron loss.^42,43 Our findings are in line with those observations and provide a protein abundance correlate to their functional studies. In addition, the pattern of APA expression did not seem to correspond to nonspecific podocyte hypertrophy, because the expression of GLEPP-1, a podocyte marker, was not enhanced in the surviving glomeruli at advanced stages. In agreement with our findings, others reported gradual fall in GLEPP-1 expression in a rat model of podocyte injury.^44,45 Thus, augmentation of podocyte APA abundance seems to be an adaptive response to progressive glomerulosclerosis.

To further investigate the role of APA in mechanisms of adaptation to glomerular injury, we subjected APA-KO mice to a glomerular injury model. Compared with wild-type controls, APA-KO mice exhibited persistent albuminuria and glomerular hyalinosis in response to NTS, suggesting that APA participates in glomerular repair mechanisms. Because APA is an angiotensinase and AngII can promote podocyte loss and injury,^44,46 it is plausible that the lack of recovery to the NTS injection in APA-KO mice corresponds to an exacerbated AngII-mediated mechanism.

Our studies using a chronic AngII infusion model show that mice fully deficient in APA exhibit increased susceptibility to AngII-mediated kidney injury as manifested by changes in albuminuria, kidney morphology, and nephrin expression. These findings were observed in association with increased intrarenal abundance of AngII in APA-KO mice infused with AngII. Thus, our data suggest that the increased susceptibility to AngII-mediated kidney injury may correspond to reduced metabolism of AngII. Intrarenal AngII content in vehicle-treated APA-KO mice was not increased, suggesting that, under basal conditions and in the absence of an amplified RAS state, other mechanisms of AngII degradation might be sufficient to counterbalance APA deficiency. However, this equilibrium may be lost during a perturbation that leads to amplification in AngII generation or enhanced AngII delivery to the kidney.

Despite pronounced hypertension, only minimal renal parenchymal damage was observed in AngII-treated wild-type mice, likely due to resistance inherent to their background strain.⁴⁷ In contrast, although acquiring a similar degree of 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 susceptible 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 hybrid 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 injury.⁵³ Although systemic hypertension is a critical element driving the pathogenesis of AngII-dependent glomerulosclerosis, local nonpressor effects of AngII in the kidney are thought to play an independent role.^54–56 Thus, the greater renal parenchymal injury in AngII-treated APA-KO mice likely resulted from local intrarenal effects of AngII amplified by APA deficiency.

A vast array of evidence indicates that AngII can directly exert deleterious effects in podocyte biology, such as derangements of the slit diaphragm composition^40,57,58 or the cytoskeleton,^38,59,60 activation of profibrotic pathways,⁶¹ apoptosis,^62–64 and podocyte detachment.⁴⁴ In our studies, AngII infusion induced attenuation of nephrin expression in APA-KO mice but not in wild-type mice. These findings suggest that the noxious effects of AngII on podocyte slit diaphragm integrity of BALB/c mice were enhanced in a state of global APA deficiency. Because APA deficiency led to greater kidney AngII content, direct effects of AngII on podocytes were likely amplified. In support of this contention, AngII has been shown to reduce nephrin expression⁴⁰ and mRNA⁵⁷ in mouse podocytes in vitro as well as nephrin mRNA abundance after acute subcutaneous AngII injection in rats.⁵⁷ The observed decrease in nephrin expression should not be viewed as a sole mediator of the albuminuric glomerular phenotype. Rather, it is interpreted as a surrogate indicator for distressed glomeruli caused by AngII. The lack of nephrin attenuation in wild-type AngII-treated mice likely corresponded to the relative renal resistance of BALB/c mice to the AngII infusion model.⁴⁷

Interestingly, vehicle-treated APA-KO mice were found to have increased kidney AngI abundance. Because APA is the primary glomerular peptidase responsible for the conversion of AngI to Ang2–10,⁶⁵ the greater content of AngI in APA-KO mice likely corresponds to diminished AngI metabolism. In AngII-treated APA-KO mice, the intrarenal AngI content was not increased. This may pertain to the known negative feedback mechanism by which AngII inhibits renin release via stimulation of Angtype 1 receptors. Thus, AngII infusion may suppress renin release, thereby diminishing AngI generation from angiotensinogen. Altogether, our findings of intrarenal AngI content are in accordance with the known RAS feedback loops. Interestingly, we did not detect any compensatory increase in kidney expression of ACE2, NEP, or APN in our studies. ACE2 has been implicated as a renoprotective molecule in models of diabetic kidney disease.^66–68 Nonetheless, peptidomic and network modeling approaches have revealed dominance of APA over ACE2 as Ang peptidase.^12,69

In conclusion, deficiency of APA leads to increased susceptibility to glomerular injury as shown by the NTS injection model and the AngII-mediated renal parenchymal injury model. In lieu of the relative abundance and enzymatic activity of APA in the kidney and the adaptive changes in glomerular APA expression during glomerulosclerosis, our findings suggest a key role of APA in maintenance of intrarenal RAS equilibrium.

Concise Methods

Animal Care

Our protocols were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee and in accordance with the procedures and practices of the National Insititutes of Health Guide for the Care and Use of Laboratory Animals. All rodents were housed at a certified facility at 22°C and in a 12:12-hour light/dark cycle, allowed free access to tap water, fed a standard diet, and provided environmental enrichment. All surgeries were performed under isoflurane anesthesia (5% induction and 2% maintenance) at 37°C and perioperative buprenorphine.

Rodents

BALB/c APA-KO (ENPEP−/−) mice were provided by Renata Pasqualini (University of New Mexico, Albaquerque, NM). Generation of APA-KO mice was previously reported in detail.⁷⁰ Wild-type BALB/c control mice were purchased from Envigo (Indianapolis, IN). Male FHH rats were an in-house colony established from animals obtained from Charles River Laboratories (Wilmington, MA). Male Wistar rats were purchased from Envigo (Indianapolis, IN).

Human Tissue

Our study protocol was approved by the Medical University of South Carolina Institutional Review Board. Archived formalin-fixed, paraffin-embedded kidney specimens were processed for examination by immunohistochemistry. Five-μm sections were obtained from biopsy cores from three healthy donors and five patients with diagnosis of idiopathic FSGS.

Glomerular Injury Model

Mice were retro-orbitally injected with 50 μl NTS (PTX-001S; Probetex Inc., San Antonio, TX) at 12 weeks of age as previously reported.^29,71 Pilot studies were carried out to select the dosage. Urine samples were collected at preinjection and 24 and 96 hours postinjection. Kidney tissues were collected at euthanasia.

AngII Infusion Model

An established model for AngII-mediated hypertensive kidney injury was used with some modifications.³⁰ Eight- to 12-week-old male mice underwent osmotic minipump implantation (Alzet, Palo Alto, CA) for delivery of AngII (400 mg/kg per minute) for 4 weeks. SBP was measured with a tail cuff sphygmomanometer at baseline and weekly throughout the experiment.

Albuminuria

Mice were placed in metabolic cages for 24-hour urine collection weekly. UACR was used as a marker of glomerular injury. Urine albumin and creatinine were measured by ELISA (Exocell, Philadelphia, PA) as per the manufacturer’s protocol. Samples were diluted (1:10–1:100) before assay. For the NTS model, 2.5-μl urine aliquots were 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% acetic acid for 30 minutes as previously reported.^72,73

Immunohistochemistry for Kidney Morphology and RAS Peptidase Expression

Parenchymal renal injury was assessed by immunohistochemistry. Kidneys harvested at euthanasia were fixed in 4% paraformaldehyde and paraffin embedded. Morphology was examined in sections stained with hematoxylin-eosin, Jones silver, periodic acid–Schiff, and Masson trichrome. Expression RAS peptidases and GLEPP-1 were examined using an antigen retrieval technique. Briefly, 4- to 5-μm sections were deparaffinized in xylene, washed in ethanol gradient, and microwaved for 20 minutes with Cymatin Citrate (pH 6.0) for antigen retrieval (DAKO, Carpinteria, CA) followed by washes with 3% hydrogen peroxide, PBS, and 2.5% horse serum (ImmPRESS Reagent Peroxidase Anti-Goat Ig Kit; Vector Laboratories, Burlingame, CA). Primary antibodies were goat anti-APA (anti–BP-1; Abcam Laboratories, Cambridge, MA), 1:167 (mouse specimens) and 1:150 (rat and human specimens); mouse anti-NEP (anti-CD10; Vector Laboratories), 1:50; goat anti-ACE2 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:50; rabbit anti-APN (anti-CD13; Origene, Rockville, MD), 1:250; and mouse anti-GLEPP-1 (gift from Roger Wiggins, University of Michigan, Ann Arbor, MI), 1:50. The anti–GLEPP-1 antibody was not reactive in mouse tissue. Tissues were mounted with Cytoseal 60 (ThermoFisher Scientific, Carlsbad, CA).

Scoring of specimens were adjudicated blindly by a trained pathologist. Glomerular damage was rated by percentage of affected glomeruli and determined by the presence of segmental or global sclerosis or hyalinosis, glomerular collapse, microaneurysms, pericapsular fibrosis, mesangial hypercellularity, or parietal epithelial cell hyperplasia. Glomerulomegaly was assessed by measurement of glomerular volume using the software AxioVision 4.6 (Dublin, CA) and standard morphometric equations.⁷⁴ An average of 15 glomeruli per tissue section per kidney was obtained. Tubulointerstitial 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

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 4′,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–β-actin (Sigma-Aldrich, St. Louis, MO), 1:5000.

APA Activity by Fluorometry

Mouse kidney homogenates (n=3 per group) were prepared with triethanolamine sucrose buffer (10 mM triethanolamine and 250 mM sucrose, pH 7.6) and diluted in assay buffer (25 mM Tris, 50 mM CaCl₂, and 0.2 M NaCl, pH 8.0) to a 2 μg/μl concentration. Lysates were pretreated with or without 4-amino-4-phosphonobutyric acid (glutamate phosphonate, 10 μM final; APA inhibitor^77,78; gift from Robert Speth, Nova Southeastern University, Fort Lauderdale FL). The fluorogenic substrate H-Glu-AMC (Bachem, Torrance, CA) was added to 100 μg protein per well at a final concentration of 100 μM. Excitation and emission were measured for 30 minutes at 380 and 460 nm, respectively. Recombinant APA (0.02 μg; R&D Systems, Minneapolis, MN) was used as a positive control.

APA Activity by LC-MS/MS

Conversion of AngII to AngIII was examined in suspensions of glomeruli isolated from wild-type and APA-KO mouse kidneys as previously reported with some modifications.⁶⁵ Isolation of glomeruli was undertaken following a magnetic beads–based method.⁷⁹ Two thousand glomeruli per 1 ml were incubated in Krebs buffer with 1 μM AngII for 60 minutes. Buffer aliquots were serially obtained and analyzed by LC-MS/MS to search for generation of AngIII. Each aliquot was centrifuged at 20,000×g for 10 minutes. Supernatant (25 μl) was mixed 1:1 with stable isotope–labeled AngIII in 0.4% formic acid to a final concentration of 22.5 fmol/μl. Samples were loaded into the autosampler, and 4 μl supernatant was injected onto an ACQUITY UPLC M-Class HSS T3 Column (1.8 μm; 75 μm×150 mm; Waters Corp., Milford, MA) at 10 μl/min with 0.2% formic acid in mass spectrometry–grade water. Peptides were separated at 10 μl/min using an M-Class UPLC (Waters Corp.) from 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 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 standard. Estimates of concentration were made against an external standard curve (0.5–250 fmol on column) in Targetlynx (Waters Corp.). Lower limit of quantification was determined as 10× SD of five blank injections.

Ang Peptide Content by LC-MS/MS

Tissue abundance of Ang peptides was determined using a Triple-ToF 5600 Mass Spectrometer (SCIEX, Framingham, MA) using previously published methods with some modifications.⁸⁰ Frozen whole kidney was pulverized in liquid nitrogen, and 40–190 mg were transferred to a glass dounce for homogenization in 60% methanol in water containing 1 mM phenathroline. Stable isotope–labeled standards for AngII, AngIII, AngIV, and Ang2–10 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. Comparisons between groups were performed using two-way ANOVA with post hoc analysis, repeated measures ANOVA, paired t tests, or Fischer exact tests when appropriate. Correlations were assessed by Spearman test. A P value <0.05 was deemed significant.

Disclosures

J.C.Q.V. has served on advisory boards for Mallinckrodt Pharmaceuticals, Alexion Pharmaceuticals, and Relypsa Inc.