Expression of Aminopeptidase A, an Angiotensinase, in Glomerular Mesangial Cells
Abstract Glomerular mesangial cells are known to express angiotensin II type 1 receptors and contract in response to circulating and/or locally produced angiotensin II. In addition, stimulation of mesangial cell matrix protein synthesis by elevated levels of angiotensin II is known to contribute to the development of glomerulosclerosis. Previously, we reported that mesangial cells were positively immunostained with antiserum directed against aminopeptidase A, the principal angiotensinase in the metabolism of angiotensin II. Here we demonstrate directly that aminopeptidase A is expressed in mesangial cells cultured from rat kidney. First, cultured mesangial cells had measurable aminopeptidase A enzymatic activity. Second, immunoblots for aminopeptidase A were positive for isolated glomeruli and mesangial cells, although two bands were seen for mesangial cells (≈138 and 144 kD), and only the larger band was seen for isolated glomeruli and kidney. Third, Northern blot hybridizations of total RNA from mesangial cells or kidney were positive and labeled similarly sized bands. Fourth, reverse transcription–polymerase chain reaction amplification of mesangial cell total RNA yielded a partial cDNA of the expected size that was confirmed by sequencing to be identical to rat kidney aminopeptidase A. These results indicate that aminopeptidase A is expressed within mesangial cells. These results further suggest that metabolism of angiotensin II by aminopeptidase A could play a protective role in minimizing the adverse effects of angiotensin II stimulation of mesangial cells.
The renin-angiotensin system is known to play a critical role in the maintenance of blood pressure and blood volume.1 Elevated activity of the renin-angiotensin system has been shown to be involved in several forms of hypertension.2 3 In the kidney, low levels of the bioactive octapeptide Ang II decrease renal plasma flow and glomerular filtration and increase proximal tubule reabsorption of sodium, actions that together reduce urinary excretion of sodium and water.4 One of the principal target cells in the kidney for circulating or locally produced Ang II is the glomerular mesangial cell. Mesangial cells encircle glomerular capillaries and have been shown to contract in response to Ang II,5 possibly through an AT1 receptor–mediated mechanism.6 7 Contraction of mesangial cells decreases the glomerular ultrafiltration coefficient (Kf), possibly by decreasing the surface area of glomerular capillaries, thereby decreasing the glomerular filtration rate.8 Ang II has also been shown to stimulate mesangial cell synthesis of matrix proteins both in vitro and in vivo9 10 11 and also through an AT1 receptor–mediated mechanism.9 11 The resultant thickening of the mesangium in vivo has been linked to development of glomerulosclerosis in a variety of diseases, including renal hypertension and diabetes.12 13
Although a great deal is known regarding the synthesis of Ang II, relatively less is known about its degradation. The first step in the metabolism of Ang II in the circulation is selective hydrolysis to Ang III,14 a less potent vasoconstrictor. The principal enzyme that hydrolyzes the N-terminal aspartyl residue of Ang II is APA (EC 22.214.171.124). Ang III is further hydrolyzed by a series of aminopeptidases, carboxypeptidases, and endopeptidases.14 High levels of APA are found within the kidney, where it has been localized histochemically to proximal tubules and glomeruli.15 We have previously shown, by immunohistochemistry and in situ hybridization, that APA is localized to proximal tubule brush border and glomerular mesangial cells.16 Here, using a variety of approaches, we sought to confirm the presence of APA within cultured mesangial cells.
Isolation of Glomeruli
Rat kidney glomeruli were isolated according to the method of Fujiwara et al,17 with some modifications as previously described.16 Briefly, male Sprague-Dawley rats (200 to 250 g) were killed by decapitation, and the kidneys were removed and placed in ice-cold DPBS, pH 7.4. The cortex was dissected, minced with a tissue chopper into 100-μm pieces, and then suspended in cold DPBS. The suspension was passed successively with DPBS washes through 200- and 150-μm sieves, and the material was collected on a 50-μm sieve. This material was confirmed by light microscopy to be enriched with glomeruli.
Mesangial Cell Culture
Mesangial cells (passages 5 through 8) were kindly provided by Dr Joel Neugarten (Albert Einstein College of Medicine, New York, NY). Two independently isolated mesangial cell preparations were used for these experiments. Cells were cultured under standard conditions18 for no more than five additional passages.
APA Enzyme Assay
APA enzymatic activity was assayed with α-glutamyl-2-naphthylamide (Bachem Bioscience) as substrate. Specific activity was expressed as units per milligram protein, where 1 U equals the hydrolysis of 1 nmol substrate per hour. Values were expressed as mean±SEM. Cultured mesangial cells were scraped from cell culture plates and mildly homogenized with a Teflon pestle in a Brinkman test tube in 50 mmol/L Tris-HCl buffer, pH 7.5.
Antiserum against kidney APA has been characterized previously.16 Immunoblots with APA antiserum were conducted using kidney cortex membranes with some modification of the method described by Harlowe and Lane.19 Briefly, rat kidney membranes were prepared in buffer containing 60 mmol/L Tris-HCl buffer (pH 7.2), 2% SDS, 100 mmol/L dithiothreitol, and 0.01% Coomassie brilliant blue. Samples were boiled for 5 minutes and then centrifuged at 10 000g for 10 minutes. The resulting supernatants were separated on a 10% SDS-polyacrylamide gel (40 μg per sample) and then transferred to a nylon membrane (Immobilon membrane, Millipore) in the presence of a transfer buffer containing 25 mmol/L Tris base, 192 mmol/L glycine, and 15% methanol at 70 V for 1 hour. The membrane was treated with a blocking buffer containing 5% nonfat dry milk and 0.02% sodium azide with agitation at 37°C for 1 hour. The blocked membrane was washed with PBS 2×5 minutes and then incubated overnight at 4°C with antiserum against APA (1:1000 or 1:3000 dilution). The membrane was washed with DPBS 4×5 minutes and then incubated with peroxidase-labeled goat secondary antibody against rabbit IgG at 37°C with agitation for 3 hours. The membrane was washed with DPBS 4×5 minutes and incubated with 10 mL 50 mmol/L Tris-HCl (pH 7.6) containing 6 mg diaminobenzidine and 10 μL 30% hydrogen peroxide for 5 minutes. The membrane was then washed with DPBS and dried. Immunoblots of samples of outer medulla and glomeruli were studied by the same method. A more sensitive immunoblot method using ECL (Amersham) was used for mesangial cell protein. This method allows for greater dilution of the primary antiserum, thus reducing background and cross-reactivity.
Reverse Transcription–Polymerase Chain Reaction
Total RNA from cultured mesangial cells was obtained according to the method of Chomczynski and Sacchi20 and digested with DNAse. cDNA was synthesized from mesangial cell RNA with an antisense oligonucleotide primer based on the rat APA cDNA sequence corresponding to nucleotides 1797 through 1815 (5′-CATCTCCGCTAAGATTAGC-3′) and murine reverse transcriptase at 37°C for 30 minutes. For PCR amplification of the APA cDNA, the antisense oligonucleotide primer was used together with a sense primer corresponding to nucleotides 1443 through 1461 (5′-CAAGACTGGATAACACCAG-3′). For PCR amplification of the AT1 receptor, oligonucleotide primers based on the AT1A cDNA sequence and used previously to generate both AT1A and AT1B partial cDNAs were used.21 The antisense oligonucleotide was from the sixth transmembrane domain (nucleotides 757 through 777, 5′-GAATATTTGGTGGGGGACCCA-3′), and the sense primer was from the second transmembrane domain (nucleotides 250 through 270, 5′-TGGGCAGTCTATACCGCTATG-3′). For PCR, 30 cycles were conducted in the presence of Taq DNA polymerase (Promega Corp) in the following sequence: first cycle, 5 minutes at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C; subsequent cycles, 1 minute at 94°C, 2 minutes at 55°C, and 3 minutes at 72°C. The PCR products were then separated on a 1% agarose gel, and the bands were examined under UV illumination. PCR controls consisted of amplification with RNA without the RT step.
The PCR-amplified product using the APA primers was subcloned into a plasmid (TA cloning vector, Invitrogen) and used for transformation of INV αF–competent Escherichia coli. Positive transformants were selected by growing bacteria on LB plates in the presence of kanamycin. Plasmids were isolated from minicultures of bacterial colonies and then sequenced directly by the dideoxynucleotide chain termination method with 35S-dATP, sequence-specific oligonucleotide primers, and Sequenase (version 2.0 T7 DNA polymerase, US Biochemical Corp).
Northern blots were conducted by size fractionation of 10 μg mesangial cell or rat kidney total RNA on an agarose-formaldehyde gel and transferal to nitrocellulose membranes (NitroPlus, Micron Separations Inc). The blots were then hybridized with a partial cDNA of rat kidney APA as previously described.16
Isolated glomeruli were shown previously to have approximately threefold higher levels of APA activity than the kidney cortex.16 Consistent with this difference, immunoblots of isolated glomeruli and kidney cortex yielded a positively stained band at ≈140 kD, with higher levels seen in isolated glomeruli than from kidney cortex (Fig 1⇓).
Since previous immunohistochemical evidence suggested that glomerular mesangial cells were positively stained by antiserum against rat kidney APA,16 APA activity was measured in cultured mesangial cells. Mesangial cells had APA specific activity of 0.73±0.23 μmol·h−1·mg protein−1 (n=3). This activity was comparable to that seen in LLC-PK1 cells, a renal epithelial cell line with proximal tubule cell properties, which had APA specific activity of 1.21±0.33 μmol·h−1·mg protein−1 (n=3). These activities are less than that from kidney tissues but may be related to reduced expression in cultured cells.
Immunoblots of mesangial cell protein with antiserum against APA by the sensitive ECL method yielded labeling of two similarly sized bands at ≈138 and 144 kD, whereas kidney protein yielded a single major band at ≈144 kD (Fig 2⇓). The much stronger immunostaining with kidney membranes is consistent with the much higher APA enzymatic activity seen in kidney membranes compared with mesangial cells. The nature of the doublet pattern of staining in immunoblots with mesangial cell protein was not clear. The pattern was not due to cross-reactivity of the antiserum to either aminopeptidase M or dipeptidyl peptidase IV (as seen in Fig 1⇑), since cross-reactivity was not encountered with kidney membranes under the conditions used for the ECL method (data not shown).
Northern blot analysis with kidney or mesangial cell total RNA yielded a positively labeled band ≈4.4 kb in size (Fig 3⇓), similar to what has been reported previously.16 In addition, RT-PCR amplification of mesangial cell RNA with oligonucleotide primers based on the rat kidney APA cDNA sequence yielded a PCR product of the predicted size (Fig 4⇓). Amplification of the same RNA with AT1A receptor primers also yielded a product of appropriate size (Fig 4⇓). Incubation of each PCR product with appropriate restriction enzymes resulted in complete digestion of the PCR product and generation of restriction fragments of the predicted size (Fig 4⇓). The complete digestion of the PCR products indicates that the reaction material in each case was most likely to be a single molecular species and not a mixture of two or more. However, to confirm the molecular identity of the RT-PCR product generated with APA oligonucleotide primers, the PCR products were subcloned into a plasmid vector and sequenced from both directions. Sequencing confirmed that the material was a partial cDNA of rat kidney APA (Fig 5⇓). The partial cDNAs were 78% and 88% identical to the corresponding sequences from human and mouse APA, respectively.22 23
We previously reported that antiserum against rat kidney APA positively stained cells in rat kidney glomeruli that were morphologically consistent with staining of mesangial cells.16 However, because of the use of minimal fixation, we were unable to identify the cells unequivocally as being mesangial cells. Since the conditions to selectively grow mesangial cells in culture are well established and permit study of mesangial cells without potential confounding effects by contaminating epithelial or endothelial cells, we sought to determine whether rat mesangial cells expressed APA. In addition to the usual markers,18 the identity of the cultured cells as being mesangial was confirmed by the ability of RNA from the cultured cells to yield an AT1 receptor PCR product. The PCR product was completely digested with a restriction enzyme specific for the AT1A sequence,21 indicating that the AT1A gene is the major AT1 receptor gene expressed in mesangial cells. This is also consistent with a recent report in which an AT1A receptor knockout animal had diminished AT1 receptor binding in glomeruli.24
Cultured mesangial cells had APA specific enzyme activity that was less than that seen in kidney homogenates but comparable to levels in a renal epithelial cell line with proximal tubule-like properties. Proximal tubules are known to express high levels of APA in the luminal brush border.15 The phenomenon of a lower level of expression of a specific protein in cultured cells compared with native tissues is frequently seen with cultured cells. This process is likely to involve a de-adaptation by the cells due to an absence of the proper induction conditions in vitro.25 Northern blots of mesangial cell RNA yielded a band of similar size to that seen with kidney RNA. In addition, RT-PCR of mesangial cell mRNA yielded a partial cDNA that was identical in sequence to APA cloned from a rat kidney cDNA library (M.T. et al, unpublished observations). The cDNAs for human and mouse APA have been cloned.22 23 The partial cDNA from mesangial cells was very similar to human (78%) and mouse (88%) APA at the nucleotide level. The proteins encoded by the partial cDNA from rat would be 70% and 82% identical to the equivalent regions of the human and mouse proteins, respectively.
Somewhat surprisingly, immunoblots of mesangial cell protein with APA antiserum yielded labeling of two bands very similar in size, the larger of which was identical to the labeled band from whole kidney. The nature of these two bands is not known. As noted above, it is unlikely that the second band is a result of cross-reactivity of the antiserum to aminopeptidase M or dipeptidyl peptidase IV, since cross-reactivity was not seen in kidney membrane protein under the same conditions. Likewise, since Northern blots of mesangial cell and kidney RNA yielded a single band identical in size, it is unlikely that there are multiple genes. However, since the genomic organization of the APA gene has not been reported, the possibility of multiple genes cannot be excluded. Another possible explanation is that there is a single gene that is alternatively spliced in mesangial cells to yield two proteins. The fact that sequencing of the partial cDNA RT-PCR product identified only one form does not exclude this possibility, since the primers may have amplified RNA from a region that is shared by the two transcripts. Cloning of the APA gene may help resolve these possibilities.
The demonstration that APA is expressed on mesangial cells is consistent with findings that Ang III is the major degradation product of mesangial cells exposed to Ang II.9 In general, Ang III has lower affinity than Ang II at mesangial cell AT1 receptors in binding experiments using APA-resistant peptide radioligands.7 26 Thus, it would appear that hydrolysis of Ang II to Ang III at the mesangial cell surface would serve to diminish the effects of Ang II. In this regard, it is possible that APA plays a protective role to limit the contractile and growth-promoting activities of Ang II on mesangial cells. The regulation of APA levels within mesangial cells may therefore have physiological significance with regard to the progression of Ang II–mediated glomerulosclerosis.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|Ang III||=||(des-Asp1)–Ang II|
|AT1||=||angiotensin II type 1 receptor|
|DPBS||=||Dulbecco’s phosphate-buffered saline|
|RT-PCR||=||reverse transcription–polymerase chain reaction|
|SDS-PAGE||=||sodium dodecyl sulfate–polyacrylamide gel electrophoresis|
This work was supported by grants from the National Institutes of Health (HL-42585 and HL-50685). We wish to thank Dr Joel Neugarten (Department of Medicine, Albert Einstein College of Medicine, Bronx, NY) for contributing the mesangial cell cultures used for these experiments.
Reprint requests to Dr Dennis P. Healy, Department of Pharmacology, Box 1215, Mount Sinai School of Medicine, One Gustave L. Levy Pl, New York, NY 10029.
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