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No. 11
Reports
It is difficult to assign a value for hydraulic or osmotic fluid permeability of the retinal pigment epithelium, since the precise hydrostatic pressure or osmotic pressure difference across the RPE is not
known. Elevation of intraocular pressure also
changes the suprachoroidal hydrostatic pressure,
such that the pressure drop across the RPE may not
change appreciably. Similarly, unstirred layer effects
in the extravascular choroid following systemic mannitol injection would cause an underestimation of the
true osmotic fluid permeability of the RPE.
It would appear that under normal circumstances,
osmotic and hydrostatic forces play a definite but
small role in fluid transport across the RPE. However, following damage to the RPE, such as with sodium iodate, the importance of these factors may be
enhanced.4 One might also speculate that a similar
situation could occur following retinal cryopexy
where the hydraulic permeability of the RPE would
be increased and the protein content of the extravascular choroid would be increased.
Key words: blood-retinal barrier, retinal pigment epithelium, permeability, carboxyfluorescein, mannitol, intraocular pressure, cynomolgus monkey
From the Department of Ophthalmology, University of Minnesota, Minneapolis, Minnesota. Dr. Tsuboi's current address is: Department of Ophthalmology, Osaka University Medical School,
Osaka 553, Japan. Supported by the James S. Adams Scholars
Award from Research to Prevent Blindness, Inc., and NIH grant
EY-03277 (JEP). Submitted for publication: August 10, 1987; ac-
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cepted June 13, 1988. Reprint requests: Jonathan E. Pederson,
MD, Box 493 UMHC, University of Minnesota, Minneapolis, MN
55455.
References
1. Pederson JE and MacLellan HM: Experimental retinal detachment: I. Effect of subretinal fluid composition on reabsorption rate and intraocular pressure. Arch Ophthalmol
100:1150, 1982.
2. Negi A and Marmor MF: Quantitative estimation of metabolic
transport of subretinal fluid. Invest Ophthalmol Vis Sci
27:1564, 1986.
3. Negi A and Marmor MF: Effects of subretinal and systemic
osmolality on the rate of subretinal fluid resorption. Invest
Ophthalmol Vis Sci 25:616, 1984.
4. Negi A, Kawano S, and Marmor MF: Effects of intraocular
pressure and other factors on subretinalfluidresorption. Invest
Ophthalmol Vis Sci 28:2099, 1987.
5. Tsuboi S and Pederson JE: Acetazolamide effect on the inward
permeability of the blood-retinal barrier to carboxyfluorescein.
Invest Ophthalmol Vis Sci 28:92, 1987.
6. Pederson JE, Cantrill HE, and Cameron JD: Experimental
retinal detachment: II. Role of the vitreous. Arch Ophthalmol
100:1155, 1982.
7. Brubaker RF and Coakes RL: Use of a xenon flash tube as the
excitation source in a new slitlamp fluorophotometer. Am J
Ophthalmol 86:474, 1978.
8. Tsuboi S and Pederson JE: Permeability of the blood-retinal
barrier to carboxyfluorescein in eyes with rhegmatogenous retinal detachment. Invest Ophthalmol Vis Sci 28:96, 1987.
9. Tsuboi S and Pederson JE: Permeability of the isolated dog
retinal pigment epithelium to carboxyfluorescein. Invest Ophthalmol Vis Sci 27:1767, 1986.
10. Tsuboi S, Pederson JE, and Toris CB: Functional recovery of
retinal pigment epithelial damage in experimental retinal detachment. Invest Ophthalmol Vis Sci 28:1788, 1987.
Investigative Ophthalmology & Visual Science, Vol. 29, N o . 11, N o v e m b e r 1988
Copyright © Association for Research in Vision and Ophthalmology
Oculor Renin-Angiorensin: Immunohistochemical Evidence
For the Presence of Prorenin in Eye Tissue
Stephen J. Sromek,* Ingolf H. L. Wallow,* Richard P. Doy.f and Edward N. Ehrlichf
Angiotensin II (A2) is a vasoconstrictor generated by the
renin-angiotensin system. A2 appears to act also as an angiogenic factor. Recent evidence suggests that renin is synthesized at many tissue sites and may generate A2 locally.
Local A2 may have important functions in the normal and
diseased eye. We examined eight human eyes by immunostaining with an antibody to prorenin, the biosynthetic precursor of renin. In all eyes, prorenin staining was extensive
in the pars plicata of the ciliary body suggesting that the
ciliary body synthesizes renin and this renin may be part of
an ocular A2 generating system. Invest Ophthalmol Vis Sci
29:1749-1752,1988
In the classic patnway angiotensin 11 (A2) is generated within the circulation by sequential cleavage of
liver-derived angiotensinogen. Renin cleaves this
substrate, forming angiotensin I (A 1). Converting enzyme subsequently converts Al into A2, a potent
vasoconstrictor and stimulant of the synthesis of the
mineralocorticoid aldosterone. A2 thereby affects
blood pressure and electrolyte homeostasis. The classic pathway has generally been thought of as a renal
feedback loop responding to afferent glomerular arteriolar pressure.1 However, recent observations sug-
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1988
Fig. 1. Photomicrograph of human renal cortex, stained with
anti-prorenin using the avidin-biotin-peroxidase technique. Juxtaglomerular tissue right of center shows prominent staining (XI50).
gest that this system may have function in addition to
the systemic or renal control of blood pressure and
electrolytes. As recently reviewed, A2 appears to have
Fig. 2. (A) Human ciliary body, pars plicata. Staining is present
in and along the basal portion of the nonpigmented ciliary epithelium (X230). (B) Adjacent section of same, treated with antibody
preabsorbed with peptide antigen. No staining is evident (X320).
Vol. 29
angiogenic activity stimulating vessel growth in a
rabbit cornea model of neovascularization.2 In addition, the synthesis of "local" extrarenal renin, which
may generate A2, has been described in various
organs.3'4 The precise function of local A2 is unknown.
Evidence has been presented that A2 may also be
generated in the eye. Both renin- and angiotensinconverting enzyme activity have been found in the
aqueous, ciliary body, retina and choroid of several
species, including man.5'6 In addition, the retinal vasculature was shown to contain A2 receptors, and to
constrict in response to externally administered A2.7
This report is the first description of immunostaining
of normal ocular tissue with an antibody to a component of the renin-angiotensin system. We began with
an antibody to prorenin, the biosynthetic precursor
ofrenin.
Materials and Methods. Monoclonal antibody specific to human prorenin was raised by immunization
with a peptide identical to the carboxyl terminal sequence of the human renin prosegment which has
been used to raise polyclonal antisera specific for
human prorenin.8 The antibody used in this study,
257B11A9, is an I g d which binds human amniotic
fluid prorenin with an apparent dissociation constant
of 0.35 nM and has no cross-reactivity with active
human renin, or other aspartate proteases (Day RP,
Gourley J, Duello T, unpublished observations).
Antibody was used at 10 ^g/ml. Formalin-fixed,
paraffin-embedded sections were stained using the
avidin-biotin-peroxidase technique (Vector Laboratories, Burlingame, CA) as previously described.9
Sections were stained with and without pretreatment
with 0.4% pepsin in 0.10 N HC1 for 5 min at 37°C.
Moderate improvement in contrast was observed
when sections were pretreated with protease, although no change in staining pattern or grading occurred. Negative controls consisted of replacement of
the primary antibody with nonimmune ascites fluid
(MOPC21,10 Mg/ml5 Bionetics Laboratory Products,
Kensington, MD) or with antibody that had been
preabsorbed with 100 Mg/nal of the peptide antigen
for 48 hr at 4°C (Day RP, Gourley J, Duello T, unpublished observations).
Tissue: Eight donor eyes from eight patients were
selected from the University of Wisconsin Eye Bank
according to the following criteria: less than 5 hr from
time of death to the time offixation,donation from a
Madison hospital to facilitate chart review, four patients under 40 years of age, and four patients over 40
years of age. Clinical information is summarized in
Results. Human kidney obtained at autopsy was
stained for a positive control in every experiment.
Results. The typical staining pattern obtained with
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No. 11
Reports
the monoclonal anti-prorenin antibody (aPR) in kidney is shown in Figure 1. Only areas consistent with
juxtaglomerular cells were stained, and the staining
was completely blocked by preabsorption of antibody
with peptide antigen.
In ocular tissue the striking finding was specific
staining of the ciliary body of all eyes examined. The
pars plicata of eight of eight eyes (Fig. 2A) and the
pars plana of six of eight eyes (Fig. 3 A) were positive.
The pattern was similar in all cases. Staining was limited to the basal portion of the nonpigmented ciliary
epithelium, beginning in the pars plicata and extending onto the pars plana with a decreasing intensity, so
that by approximately the posterior third staining was
not evident (Fig. 3B). No staining of the ciliary body
was detected in any of the negative control sections
(Fig. 2B).
Two other areas were frequently positive. A thin
layer of stain was evident on the inner surface of the
internal limiting membrane of the retina posterior to
the equator in five of eight eyes, and stain was present
on the outer and inner surface of the lens capsule in
eight of eight eyes. Although this stain was not seen
with control or preabsorbed serum, the possibility of
nonspecific "edge" staining cannot be completely
ruled out.9
The optic nerve and ciliary nerves stained in all
eyes. However, this stain was present with preabsorbed serum, and we have observed similar nonspecific staining of these structures with other monoclonal antibodies (unpublished data).
Clinical and staining information is briefly summarized in Table 1. Staining of ocular tissue was observed in a wide range of age groups, both sexes, and
in normotensive and hypertensive patients. No patient had been treated with an angiotensin converting
enzyme inhibitor.
Discussion. In this paper we demonstrate immunoreactivity in normal ocular tissue using an antibody
that reacts with prorenin (PR), an early component in
the angiotensin II (A2) generating system. PR staining was located primarily in the ciliary body, and
appeared concentrated in the basal portion of the
nonpigmented epithelium. Its presence in the eye is a
new finding and suggests that this proenzyme is synthesized and/or stored within the nonpigmented ciliary epithelium. PR may also be activated here since a
previous demonstration has shown renin enzyme activity within the ciliary body.5 We cannot rule out the
possibility that plasma components of the renin-angiotensin system are filtered by the ciliary body and
that our PR staining would then reflect "accumulating" plasma PR. However, if plasma PR were filtered
by the ciliary body, one would have to postulate selective concentration by the ciliary epithelium since
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Fig. 3. (A) Human ciliary body, pars plana. In this instance
staining with prorenin antibody (arrow) is accentuated through
pretreatment with protease (X230). (B) Human ciliary body, posterior pars plana. Staining for prorenin (arrow) decreases from left to
right (pars plana to ora) (X250).
plasma PR levels under most physiologic circumstances are low. Also, if our PR staining reflected
filtration from the plasma, one would expect a gradient of staining decreasing from the vascular core of
pars plicata and pars plana toward the vitreal surface.
Such a gradient was clearly absent. Regardless of its
source, whether locally produced and/or selectively
concentrated, the presence of PR within the nonpigmented ciliary epithelium deserves increased attention.
The physiologic and pathophysiologic significance
of the new finding remains to be specified by further
work. Others have raised the concept that locally
formed A2 may affect the retinal vasculature.7 Demonstration of renin in ciliary body and retina and of
angiotensin converting enzyme in ciliary body, retina
and aqueous is consistent with their proposal.56 Our
finding would fit with this concept if PR or renin
were secreted into the aqueous and transported to the
retina by posterior aqueous flow. Presence in the eye
of the enzymes necessary for A2 generation and presence of specific receptors in the retina for A2-me-
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Vol. 29
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / November 1988
Table 1. Summary of clinical data and immunostaining
ID
Age
(yrs)
Sex
Cause of death
HC-21
HC-10
HC-13
VU
18
21
M
M
M
HC-34
27
F
HC-51
45
M
GP-1
57
F
HC-49
58
M
HC-50
64
M
Asphyxiation
Massive head injury
Closed head injury,
elevated
intracranial
pressure
Closed head injury,
elevated
intracranial
pressure
Progressive renal
failure and
hepatobiliary
obstruction
secondary to
metastatic
adrenal cancer
Septic shock,
ischemic bowel
Small cell CA
metastatic to
brain
Pneumonia
1
Pars
plicata
Pars
plana
ILM*
+
+
+
+
+
+
+
Hypotension
treated with
dopamine
+
—
—
No history of
hypertension, but
B/P elevated
during week prior
to death
+
+
—
History of untreated
hypertension
—
+
+
+
+
+
+
—
+
+
Comment
—
Hypotension
treated with
dopamine
+
Internal limiting membrane.
diated vasoconstnction make it reasonable to suspect
that the eye contains a local renin angiotensin system
influencing retinal vasoconstnction and blood flow.
Key words: prorenin, renin, angiotensin, ciliary body, immunostaining
From the Department of "Ophthalmology and flnternal Medicine, University of Wisconsin Medical School, Madison, Wisconsin. Supported by a grant from the Diabetes Research and Education Foundation (SJS), NIH grant EY-01634 (IHLW), a grant from
the Wisconsin Affiliate, American Heart Association (RPD), and
by an unrestricted grant from Research to Prevent Blindness, Inc.,
New York, New York. Submitted for publication: November 30,
1987; accepted May 20,1988. Reprint requests: Stephen J. Sramek,
MD, PhD, Department of Ophthalmology, Clinical Sciences
Center, 600 Highland Avenue, Madison, WI 53792.
References
1. Oparil S and Haber E: The renin-angiotensin system. N Engl J
Med 291:389, 1974.
2. Fernandez LA, Twickler J, and Mead A: Neovascularization
produced by angiotensin II. J Lab Clin Med 105:141, 1985.
3. Re RN: Cellular biology of the renin-angiotensin systems.
Arch Int Med 144:2037, 1984.
4. Day R and Huber E: The biochemistry of renin. In Handbook
of Hypertension, Vol. 8: Pathophysiology of HypertensionRegulatory Mechanisms, Zanchatti A and Tarozi RC, editors.
Amsterdam, Elsevier Science Publishers B.V., 1986, pp.
315-364.
5. Igic R and Kojovic V: Angiotensin I converting enzyme (kininase II) in ocular tissues. Exp Eye Res 30:299, 1980.
6. Igic R, Robinson CJG, Milosevic A, Wilson C, and Erdos EG:
Angiotensin I converting enzyme activity in the choroid plexus
and in the retina. In Central Actions of Angiotensin and Related Hormones, Buckley JP and Ferrario CM, editors. New
York, Pergamon Press, 1977, pp. 23-27.
7. Ferrari-Dileo G, Davis EB, and Anderson DR: Angiotensin
binding sites in bovine and human retinal blood vessels. Invest
Ophthalmol Vis Sci 28:1747, 1987.
8. Kim SJ, Hirose S, Miyazaki H, Ueon N, Higashimori K, Morinaga S, Kimura T, Sakakibara S, and Murakami K: Identification of plasma inactive renin as prorenin with a site-directed
antibody. Biochem Biophys Res Commun 126:641, 1986.
9. Sramek SJ, Wallow IHL, Bindley C, and Sterken GW: Fibronectin distribution in the rat eye. Invest Ophthalmol Vis Sci
28:500, 1987.
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