Kidney International, Vol. 42 (1992), pp. 1457—1461

RAPID COMMUNICATION

Vascular permeability factor mRNA and protein expression in human kidney LAWRENCE F. BROWN, BRYGIDA BERSE, KATHI TOGNAZZI, ELEANOR J. MANSEAU, LIVINGSTON VAN DE WATER, DONALD R. SENGER, HAROLD F. DVORAK, and SEYMOUR ROSEN Departments of Pathology, Beth Israel Hospital and Harvard Medical School, and the Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts, USA

Vascular permeability factor mRNA and protein expression in human

kidney. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent microvascular permeabil-

ity-enhancing mediator as well as a selective mitogen for vascular endothelium. In this study, in situ hybridization and immunohistochem-

derived growth factor [9, 10] and more substantial homology to another newly described protein, placenta growth factor [13].

We have recently demonstrated significant levels of VPF mRNA in extracts of both guinea pig and human kidney by

istry co-localized VPF mRNA and protein to glomerular visceral Northern blotting [14]. In guinea pig kidneys, VPF mRNA was epithelial cells in human kidneys. Northern analysis confirmed the localized to the glomeruli, and most likely to glomerular epithepresence of VPF mRNA of expected size. The finding of VPF in renal glomerular epithelium identifies a potent mediator of permeability and endothelial proliferation whose role in renal physiology and pathology requires investigation.

Vascular permeability factor (VPF) is a dimeric glycoprotein

hum, by in situ hybridization. Others have also localized VPF/VEGF to glomeruli in mice [15] and rats [16]. In this study,

we have performed in situ hybridization with antisense VPF RNA probes and immunohistochemistry with affinity purified anti-VPF antibodies in order to determine more precisely its cellular localization in the human kidney.

of Mr 34,000 to 42,000, consisting of two disulfide-linked peptide chains having identical N-termini [1, 2]. As its name

Methods

Preparation of single-stranded RNA probes for in situ hybridization activity, such that, on a molar basis, it is some 50,000 times more potent than histamine [1]. VPF was first identified in The single-stranded RNA probe for localizing VPF mRNA by tumor cell conditioned media [3] and has been purified to in situ hybridization has been described previously [14]. Briefly, homogeneity from guinea pig and human sources [1, 4]. VPF we employed reverse transcription-PCR to amplify a 204-bp acts directly on cultured vascular endothelium to induce a rapid VPF eDNA fragment from human fibrosarcoma cell line increase in free cytosolic calcium, apparently by activating a HT1O8O. This 204-bp region, which is common to all known phosphoinositide-specific phospholipase C [5]. It also stimu- VPF splicing variants, was subcloned into the polylinker region lates the release of von Willebrand factor from endothelial cells of plasmid pGEM3Zf(+) (Promega, Madison, Wisconsin, [5] and induces the expression of endothelial cell tissue factor USA). The identity of the cloned insert was confirmed by DNA sequencing. To prepare RNA probes, the pGEM construct activity [6]. Concurrent with these studies, a protein selectively mito- containing the human VPF insert was linearized with Hindlil genie for vascular endothelial cells was purified from cell and transcribed in vitro from the T7 polymerase promoter to culture systems in several laboratories and named vascular yield the antisense probe; alternatively, the same construct was endothelial growth factor (VEGF) [7—i 1]. Protein and cDNA linearized with EcoRI and transcribed from the SP6 promoter to sequencing, as well as other lines of evidence, indicate that yield the corresponding sense (control) probe. Transcription VPF and VEGF are products of the same gene [9, 10]. The reactions were carried out using a Riboprobe Gemini II kit VPF/VEGF gene transcript may be alternatively spliced [12], (Promega) in the presence of (a-35S)UTP (Amersham Corp., but whether alternatively spliced forms have different biologic Arlington Heights, Illinois, USA). activities has not been determined. There is limited homology In situ hybridization between the VPF/VEGF gene and the gene encoding plateletimplies, VPF possesses potent vascular permeability-enhancing

Received for publication August 31, 1992 and in revised form September 16, 1992 Accepted for publication September 17, 1992

© 1992 by the International Society of Nephrology

Grossly and microscopically normal-appearing portions of five nephrectomy specimens for tumor were used in this study. Segments of kidney that included both cortex and medulla were

fixed for four hours in 4% paraformaldehyde in phosphate buffered saline, pH 7.6 (PBS) at 4°C and were then transferred to a solution of 30% sucrose in PBS overnight at 4°C. Tissue 1457

Brown et al: VPF in human kidney

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was then frozen in OCT compound (Miles Diagnostics, Elkhart, Indiana, USA) and stored at —70°C. Four pm frozen sections

were subjected to in situ hybridization (ISH) as previously described [17]. Other kidney specimens were frozen fresh in OCT compound and frozen sections were subsequently fixed for 15 minutes in 4% paraformaldehyde solution prior to in situ hybridization.

Inimunohistochemistry A synthetic peptide corresponding to amino acid residues I through 26 of human VPF was synthesized by Multiple Peptide Systems, San Diego, California, USA. The peptide sequence, in single letter code was: APMAEGGGQNHHEVVKFMDVYQRSYC with an amide replacing a carboxyl group at the C-terminus. The peptide was coupled to keyhole limpet hemocyanin

and rabbits were immunized as previously described for a similar peptide based on guinea pig sequence [1]. Anti-peptide antibody was affinity purified on columns prepared by conjuga-

tion of 5 mg peptide to 1 g of CnBr-activated Sepharose

28S— 18S—

(Pharmacia). Typically, 20 ml of antiserum was passed over the

column after which the column was washed with phosphate buffered saline. Bound antibody was eluted with 15 ml of 0.1 M acid glycine buffer, pH 2.5. Eluted antibody was immediately diluted in 2 ml of 1 M Tris buffer, pH 8.0, and dialyzed against phosphate buffered saline. Affinity-purified antibody concentrations were typically 0.5 mg/ml. The anti-peptide antibody bound VPF in ELISA assays and on immunoblots.

Immunohistochemistry was performed on 4 m unfixed frozen sections or frozen sections of tissue fixed in paraformalde-

hyde using the affinity purified anti-VPF antibody with an

3-actin

avidin-biotin peroxidase conjugate protocol (Vector Lab, Burlingame, California, USA). Normal rabbit IgG diluted to an equivalent concentration was substituted for the primary antiFig. 1. Northern blot of RNA isolated from a normal adult human body as a control. kidney and from human fibrosarcoma HTIO8O cells probed with 32Plabeled VPF cDNA (top panel) and 13-actin cDNA (bottom panel). The

Northern hybridization positions of 28S and 18S rRNA species on the original gel are marked. For Northern analysis, the human VPF cDNA insert from the

pGEM construct described above was labeled with (a-32P) dCTP (New England Nuclear, Boston, Massachusetts, USA) to known VPF variants. RNA from human fibrosarcoma cell line a specific activity of ——2 x l0 cpm/tg DNA using the random HT1O8O, known to be a rich source of VPF, served as a positive hexamer labeling method [18]. Total RNA was prepared from control. human kidney and from cultured HT1O8O cells using a guanidiIn situ hybridization was performed on five human kidneys in nium isothiocyanate lysis-CsCl gradient method [19]. RNA order to localize VPF mRNA at the cellular level (Fig. 2). In all samples (20 g per lane) were size-fractionated on 1.2% agarose cases, antisense probe strongly labeled a population of glomerular containing 6% formaldehyde and blotted on Nytran membranes cells. When these were situated peripherally in the glomerular (Schleicher and Schuell, Keene, New Hampshire, USA). Hy- tuft, they could be confidently identified as visceral epithelial bridization was performed as previously described 114]. Final cells. More centrally situated cells, morphologically identical to

washes were carried out at high stringency (0.1 x SSC, 0.1% labeled peripheral cells, were also labeled and these were SDS at 65°C). Blots were exposed to Kodak XAR-2 film with an probably also epithelial cells. Glomerular endothelial cells were intensifying screen at —70°C for two days. To control for total not significantly labeled with the VPF probe, but rare tubular mRNA content and lack of degradation, the blots were stripped epithelial cells were labeled lightly. No specific cellular labeling and subsequently hybridized with human /3-actin probe. was seen with sense control probes, and nonspecific background was low. Results

Northern blots of RNA preparations from three different human kidneys, including both cortex and medulla, were probed with VPF cDNA, demonstrating significant levels of VPF mRNA of expected size, -—-4.5 kb (Fig, 1). The cDNA

Immunohistochemical staining with affinity purified antibod-

ies to the human VPF N-terminus demonstrated definitively that glomerular visceral epithelial cells contained VPF (Fig. 3). Staining was cytoplasmic and highlighted the epithelial cells' extensive elongate processes which surrounded glomerular probe used in these experiments was designed to recognize all capillary endothelial cells. All visceral glomerular epithelial

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Fig. 2. In situ hybridization of glomeruli from normal adult human kidneys. A,B. VPF antisense riboprobe labeling of a glomerulus as visualized

by brightfield (A) and darkfield (B) microscopy (x300). Note intense labeling of individual cells at the periphery of the glomerulus and within the glomerulus. Immediately surrounding tubular epithelium shows little consistent labeling. C,D. VPF sense riboprobe application to an adjacent section of kidney visualized by brightfield (C) or darkfield (D) microscopy (x300). Note low background and lack of specific cellular labeling.

cells stained with apparently equal intensity. In contrast, endo-

labeling was far less than that seen in glomeruli. No convincing

thelial cells, parietal glomerular epithelial cells and tubular tubular staining for VPF was seen by immunohistochemistry. epithelium did not stain. No specific cellular staining was seen These data indicate that renal glomerular epithelial cells with control rabbit IgG. express VPF mRNA. Notably, the pattern of immunohistochemical staining for VPF in kidneys differs somewhat from Discussion that described in tumors [20]. Tumor cells stained relatively The data presented here indicate that both VPF mRNA and weakly with antipeptide antibodies to VPF, possibly because VPF protein are present in readily detected amounts in normal VPF was constitutively secreted and not stored; however, adult human kidneys. Northern analysis performed on whole endothelial cells adjacent to tumors which did not express VPF kidney RNA revealed a VPF mRNA transcript of expected size mRNA nonetheless stained intensely for VPF, suggesting that (—4,5 kb). In situ hybridization and immunohistochemistry tumor-secreted VPF accumulated on nearby target endothelocalized both VPF mRNA and VPF protein to glomerular hum. In contrast to tumors, no such VPF accumulation was visceral epithelium. Rare tubular epithelial cells also hybridized observed in normal renal glomerular capillaries, however, the weakly with antisense riboprobes to VPF mRNA, but the possibility that a small amount of VPF was present on these

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Fig. 3. Immunohistochemistry of normal human kidney with an antipeptide antibody to the N-terminus of human VPF. Note intense staining of glomerular visceral epithelial cells and their elongate cytoplasmic processes in A (x400) and, at higher power (x600) in B.

capillaries could not be excluded given the intense glomerular epithelial staining and the limited resolution of light microscopy. The roles that VPF plays in normal renal physiology and in renal disease have not been investigated, but it is likely that, as in other organs and tissues, VPF acts on endothelial cells and mediates functions such as vascular permeability and endothe-

hal cell division. This view is reinforced by the intimate anatomic relationships that exist between the glomerular visceral epithelial cells that synthesize VPF and the glomerular

contributes to the synthesis and maintenance of the glomerular

basement membrane. The epithelial slit pore, a specialized membrane between the foot processes, is so situated that it too

could have a role in limiting molecular filtration. Injury to visceral epithelium is a crucial component in the development of proteinuria and glomerulosclerosis in experimental chronic hyperfiltration models [23, 24]. It is interesting to speculate that glomerular epithelial injury leads to increased VPF synthesis and/or secretion and thereby to abnormal glomerular permeability. VPF may thus be found to be an important mediator of renal

vascular endothelium; secreted VPF needs only cross the glomerular dysfunction, and VPF itself, or inhibitors of its intervening glomerular basement membrane to reach glomeru- activities, may provide novel treatments for at least some lar endothehjal cells. Labeled VPF has been shown to bind to

endothelial cells in the renal cortex (most intensely in the

glomerular disorders.

VPF/VEGF is also known as a selective endothelial cell

glomeruli) and medulla [21], suggesting that endothelial cells in mitogen. VPF/VEGF could have a role in maintaining glomerular endothelial cell number and function. In certain pathologthe kidney express receptors for VPF. VPF is a potent microvascular permeability-enhancing agent ical states, VPF/VEGF might serve to stimulate glomerular in skin, peritoneal wall, subcutaneous tissue, skeletal muscle endothelial cell division.

and likely in other organs that have not as yet been tested;

In addition to VPF, normal kidneys express a number of

therefore, it is tempting to speculate that VPF may have a role other growth factors. Transforming growth factor /31 and /32 in regulating normal renal glomerular permeability. Filtration of mRNA have been detected in normal rat glomeruli but cellular plasma is the major function of the glomerulus and is critical to localization was not defined [25]. In addition, epidermal growth renal function. Most investigators feel that the glomerular factor mRNA has been localized to the thick ascending limb of basement membrane and endothelium are primarily responsible Henle and the early distal convoluted tubular epithelium by in for the selective permeability exhibited by the glomerulus, situ hybridization in the mouse [261. Insulin-like growth factor-i regulating molecular passage on the basis of size and charge synthesis has been localized to collecting ducts by immunohis[22]. The role of the glomerular epithelial cell in permeability tochemistry and RNA analysis of isolated segments of the rat regulation has been less clear. The visceral epithelial cell nephron [27]. Basic fibroblast growth factor mRNA has been

Brown et a!: VPF in human kidney

identified in cultured rat glomerular epithelial cells by reverse transcriptase PCR [281. The actions and possible interactions of these growth factors with VPF need to be explored in order to gain a better understanding of the regulation of renal function. In summary, data presented here establish the presence of VPF/VEGF in the visceral glomerular epithelium of human kidneys. The precise roles of VPF in normal renal physiology and in renal disease remain to be determined, but given what is

known about the biology of VPF/VEGF, VPF could play important roles in both the regulation of glomerular permeability and in glomerular endothelial cell growth.

1461

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Acknowledgments This work was supported by U.S. Public Health Service NIH grants CA50453 (to HFD), CA 43967 (to DRS), and by grants from the BIH Pathology Foundation, Inc.

Reprint requests to Lawrence F. Brown, M.D., Department of

Pathology, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts 02215, USA.

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