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From the Department of Vascular Biology, The Scripps Research Institute, La Jolla, California
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although the liver appears to be the principal site of PC synthesis,12 a few recent studies suggest that it also may be produced extrahepatically.13,14 For example, several components of the PC anticoagulant system have been shown to be synthesized in the male reproductive tissues. In this regard, PC and protein S (a co-factor of APC15 ) have been detected in Leydig cells of the human testis,16,17 whereas PC inhibitor was detected throughout the male reproductive system18 and in tubular cells of the human kidney.19 These observations thus raise the possibility that the PC pathway may contribute to the maintenance of tubular fluidity in the kidney and that it may be involved in various reproductive processes.
In this report, quantitative reverse transcription-polymerase chain reaction (RT-PCR)20,21 is used to more completely identify extrahepatic sites of PC synthesis in the mouse and to determine whether expression of this potent anticoagulant molecule is altered in murine models of thrombotic disease. Although PC gene expression was the highest in the liver, high concentrations also were apparent in the kidneys and testes, and low but significant levels were demonstrated in the epididymis, brain, and lung. The kidneys of mice with autoimmune lupus nephritis (MRL lpr/lpr), with diabetic nephropathy (db/db), and with sepsis after endotoxin (lipopolysaccharide (LPS)) treatment expressed considerably lower amounts of PC mRNA than the normal controls. The decreased renal expression of PC may increase the local procoagulant potential of the kidney and thus contribute to the fibrin-mediated progression of renal damage associated with chronic kidney diseases and sepsis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female MRL lpr/lpr mice22 and their normal counterparts (MRL +/+) were obtained from Scripps Clinic Rodent Breeding Colony (La Jolla, CA), whereas male obese/diabetic mice (C57BL/KsJ db/db)23,24 and their lean counterparts (C57BL/KsJ +/?) were obtained from The Jackson Laboratories (Bar Harbor, ME). Adult mice (2 to 6 months old) were sacrificed, and various tissues were surgically removed and immersed in chilled 4% paraformaldehyde. The tissues were fixed in 4% paraformaldehyde by incubation at 4°C overnight, embedded in paraffin blocks, and sectioned at a thickness of 2 to 5 µm using a microtome. The sections were then mounted onto polylysine slides and stored at room temperature pending analysis. Portions of the freshly removed tissues also were minced and then immediately frozen in liquid nitrogen for preparation of total RNA. Total RNA was prepared by the acid guanidinium thiocyanate-phenol-chloroform method25 and then quantitated by measuring absorption at 260 nm. The integrity of the 18S and 28S ribosomal RNA was monitored by inspection under UV light after electrophoresing 10 µg of total RNA through a 1.2% agarose/formaldehyde gel.
In separate experiments, LPS (50 µg/mouse; E. coli serotype O111:B4; Sigma Chemical Co., St. Louis, MO) was diluted in 200 µl of saline (Baxter, Deerfield, IL) and injected intraperitoneally into adult male CB6 mice (BALB/c/ByJ x C57BL6/J; Scripps Clinic Rodent Breeding Colony). Control mice were injected with an equivalent volume of saline alone. At 2, 4, 8, and 24 hours after LPS, the mice were sacrificed, and kidney and liver tissues were removed as described above.
Quantitative RT-PCR
We have developed quantitative RT-PCR assays to determine the concentration of specific mRNAs in murine tissues,21,26 and these procedures were adapted to the analysis of PC mRNA. Briefly, a synthetic DNA template to be used as a standard for the quantitation of PC mRNA was first constructed. This template consisted of the sequence of an upstream primer for mouse PC (5'- GGCAGACGACCACATGCGCTGCAAGTCCAC-3') followed by the sequence of a downstream primer on the complementary strand (5'-CCAGGAAGTGTGGATGAGCACCCCTCCGCA-3').27 The complementary RNA (cRNA) standard was then in vitro transcribed from this template using the Riboprobe Gemini II (Promega, Madison, WI), and a fixed amount was mixed with 1 µg of total tissue RNA and then reverse transcribed using a GeneAmp RNA PCR kit (Perkin-Elmer/Cetus, Norwalk, CT). Serial twofold dilutions of the RT mixture were amplified using PC (described above) or ß-actin-specific primers (sense, 5'-TGGAATCCTGTGGCATCCATGAAAC-3'; antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3')28 in the presence of 32P-end-labeled sense primer (5 x 105 cpm). After PCR amplification for 28 cycles (ie, denaturation at 95°C for 1 minute, primer annealing at 60°C for 1 minute, and extension at 72°C for 1 minute), 20-µl aliquots of the PCR products were electrophoresed on a 2.5% agarose gel. The appropriate bands corresponding to the standard cRNA product (189 bp for PC; 293 bp for ß-actin) and the target mRNA product (241 bp for PC; 349 bp for ß-actin) were excised from the gel, and the incorporated radioactivity was determined using a scintillation counter. The number of molecules of PC mRNA was then determined by extrapolation using the cRNA standard curve as previously described.20 Variations in sample loading were assessed by measuring ß-actin mRNA.
Riboprobe Preparation
A BamHI/KpnI fragment of the mouse PC cDNA was obtained by RT-PCR from 1 µg of total mouse liver RNA using the following specific primers: 5'-GGATCCAAGAGATGCGGCCAGGCAGC and 3'-GGTACCCCGAAAGAAGACCACC. The resulting fragment containing nucleotides 150 to 1249 of mouse PC cDNA27 was subcloned into the vector pGEM-3Z (Promega). This vector was linearized and used as a template for in vitro transcription of radiolabeled antisense or sense riboprobes using SP6 or T7 RNA polymerase (Promega), respectively, in the presence of 35S-labeled UTP (>1200 Ci/mmol; Amersham Corp., Arlington Heights, IL). Templates were removed by digestion with RQ1 DNase (Promega) for 15 minutes at 37°C, and the riboprobes were purified by phenol extraction and ethanol precipitation. Mouse urokinase-type plasminogen activator (u-PA) mRNA was used as a control in some experiments. The riboprobes for mouse u-PA were prepared as described previously.21
In Situ Hybridization
In situ hybridizations were performed as described previously.21,29 After hybridization, the slides were dehydrated by immersion in a graded alcohol series containing 0.3 mol/L NH4Ac and then dried and either placed directly on XAR-5 film (Eastman Kodak, Rochester, NY) for regional in situ autoradiography or coated with emulsion for high-resolution analysis. For regional autoradiography, the films were developed and photographed after incubation in the dark at room temperature for 2 weeks. For high-resolution analysis, the slides were coated with NTB2 emulsion (Kodak; 1:2 in water) and exposed in the dark at 4°C for 4 to 12 weeks. The slides were developed for 2 minutes in D19 developer (Kodak), fixed, washed in water, and counterstained with hematoxylin and eosin. No specific hybridization signal could be detected in parallel sections using 35S-labeled sense probes for nonspecific hybridization in each experiment (data not shown).
Immunohistochemistry
Immunohistochemical staining was performed using the HISTOSTAIN-SP Kit (Zymed Laboratories, South San Francisco, CA) as described previously.21 Briefly, the paraffin-embedded tissues were deparaffinized, treated with 2% hydrogen peroxide to quench endogenous peroxidase activity, and dehydrated. The sections were then permeabilized by sequential treatment with 0.2% and 0.5% Triton X-100 in Tris-buffered saline. To unmask tissue antigens, sections were incubated at 37°C with prewarmed 0.23% (w/v) pepsin (2830 U/mg; Worthington Biochemical Corp., Freehold, NJ) in 0.01 N HCl for 8 minutes. After incubation with 10% normal goat serum for 30 minutes, the slides were incubated with primary rabbit antibodies (25 µg/ml of anti-human PC immunoglobulin G; Sigma Chemical Co.) containing 0.1% bovine serum albumin for 16 to 18 hours at 4°C, followed by incubation for 1 hour at 25°C. According to the manufacturer, this polyclonal antibody shows no reaction with PC-depleted human plasma using immunoblotting methods. In control experiments, tissues were incubated with preimmune (normal) rabbit immunoglobulin G instead of primary antibody. The slides were then washed and treated sequentially with biotinylated goat anti-rabbit immunoglobulin G (Zymed Laboratories), streptavidin-peroxidase conjugate (Zymed Laboratories) and aminoethylcarbazole chromagen containing 0.03% hydrogen peroxide (Zymed Laboratories). After rinsing in distilled water for 3 minutes, the slides were counterstained with Gill modified hematoxylin for 20 seconds, rinsed well with tap water, and mounted in GVA mounting solution (Zymed Laboratories).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Experiments were performed to determine the concentration of PC
mRNA in various mouse tissues. Total RNA was extracted from murine
tissues, and the concentration of PC mRNA was determined by
quantitative RT-PCR (see Materials and Methods). In these experiments,
1 µg of total tissue RNA and a fixed amount of the cRNA standard (ie,
1 x 107 molecules for the liver; 1 x
106 molecules for the kidney and testis; and 1 x
105 molecules for the epididymis, brain, and lung) were
combined and then quantitated by RT-PCR. The results are shown in
Figure 1
. As expected, the liver
contained the highest concentration of PC mRNA (1.3 ± 0.24
x 107 molecules of PC mRNA/µg total tissue RNA).
However, the kidney and the testis also contained relatively high
amounts, representing 35% and 22%, respectively, of the amount
detected in the liver. Low but significant levels of PC mRNA also were
detected in the epididymis (1.7% of the liver), brain (1.1% of the
liver), and lung (0.8% of the liver). PC mRNA was not detected in the
heart, thymus, spleen, gut, pancreas, adrenal, skeletal muscle, adipose
tissue, uterus, and aorta (data not shown).
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To identify the cellular sites of PC synthesis in the above
tissues, tissue sections were hybridized to 35S-labeled
probes and analyzed by regional in situ autoradiography and
by high-resolution in situ hybridization (Figure 2)
. The tissue sections also were stained
immunohistochemically using rabbit anti-human PC antibody as described
in Materials and Methods (Figures 2 and 3)
. As expected, hepatocytes in the liver
expressed abundant PC mRNA and antigen (data not shown). In the kidney,
PC mRNA was expressed exclusively in the cortex (Figure 2A)
, in
contrast to the expression of u-PA mRNA, which was localized primarily
to the medulla (Figure 2B
21). High-resolution in
situ hybridization analysis revealed that the PC mRNA in the renal
cortex was localized specifically to epithelial cells of the proximal
and distal convoluted tubules (Figure 2
, C and D). Immunohistochemical
staining demonstrated similar localization of PC antigen (red stain) in
the kidney (Figure 2
, E and F). No expression of PC mRNA or antigen was
observed in glomerular endothelial cells or mesangial cells or in
epithelial cells of the straight portions of tubules in the renal
medulla. In the testis, PC mRNA was detected in spermatogenic cells
present in both seminiferous tubules (Figure 2G)
and in efferent ducts
(data not shown). Epithelial cells in the epididymis also expressed
abundant PC mRNA (Figure 2H)
and antigen (not shown). However, no
signal was detected in interstitial cells or in Leydig cells in the
testis (not shown).
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, red-brown stain), in pyramidal neurons in the
cerebral cortex (Figure 3B)
, in neurons in the pyramidal layer of the
hippocampal cortex (Figure 3
, C and D), and in Purkinje cells lining
the outer surface of the granular layer in the cerebellar cortex
(Figure 3
, E and F). No specific staining for PC antigen was observed
in tissues that did not contain detectable levels of PC mRNA when
analyzed by RT-PCR (ie, in the heart, thymus, spleen, adrenal gland,
and aorta; data not shown). Although PC synthesis was previously
demonstrated in human umbilical vein endothelial cells,30
no PC mRNA or antigen was observed in endothelial cells in any of the
tissues examined (data not shown). Decreased Renal Expression of PC mRNA in Mice with Kidney Disease
Figure 1
demonstrates that the kidney is a major site of PC
synthesis. The kidney is also one of the primary sites of thrombosis in
a variety of disorders. We therefore used quantitative RT-PCR (Figure 4)
and in situ hybridization
(Figure 5)
to determine whether PC gene
expression is altered in the kidneys of mice with renal disease (ie,
female MRL lpr/lpr mice, a model of lupus nephritis; db/db mice, a
model of type II diabetes; and LPS-treated mice, a model of sepsis). No
significant differences in PC mRNA levels were observed in kidneys from
lupus-prone or diabetes-prone mice 2 months of age (Figure 4
, A and C).
These observations are consistent with the fact that histopathological
changes in the kidneys of female MRL lpr/lpr mice and db/db mice
usually do not become apparent until 4 to 6 months of
age.22,31-33
However, marked decreases in PC mRNA (50 to
60% reduction) were observed in kidneys of these mice at 5 to 6 months
of age (Figure 4
, A and C). These age-related decreases in PC
expression seem to be specific for the diseased kidneys, given that no
changes in PC mRNA levels were observed in similarly aged control
kidneys or in livers from the control versus the
disease-prone animals (Figure 4
, B and D). In situ
hybridization analysis revealed that the reduction of PC mRNA in the
lupus-prone mice occurred specifically in the tubular epithelial cells
of kidneys having the characteristic histopathological changes
associated with lupus nephritis (compare Figure 5
, A and B). Similarly,
the kidneys of 6-month-old db/db mice (ie, with pathological changes
characteristic of diabetic nephropathy including mesangial matrix
accumulation in glomeruli and hyaline material in the interstitium;
Figure 5D
) also showed decreased expression of PC mRNA in tubular cells
(Figure 5D)
compared with their normal counterparts (Figure 5C)
.
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, E and F). This was a transient
effect, with PC mRNA levels returning to control levels by 24 hours
(Figure 4
, E and F). High-resolution in situ hybridization
demonstrated that LPS caused a marked and specific decrease in PC mRNA
in tubular epithelial cells of the kidney (compare Figure 5
, E and F)
and in hepatocytes of the liver (compare Figure 5
, G and H). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The level of extrahepatic PC mRNA expression was the highest in the
kidney and testis, representing 35% and 22%, respectively, of that in
the liver (Figure 1)
. Histological approaches revealed abundant amounts
of PC mRNA and antigen in the epithelial cells of the convoluted
tubules in the renal cortex (Figure 2)
. Interestingly, in the human
kidney, renal tubular cells also appear to synthesize PC
inhibitor,19
and they are the major source of u-PA, another
target of the PC inhibitor.35
However, the cells
responsible for the synthesis of u-PA are located in the straight
portion of tubules in the renal medulla36
(Figure 2)
. The
different patterns of expression of PC and u-PA in the kidney (Figure 2)
imply that different portions of the renal tubules may synthesize
different anticoagulant and/or fibrinolytic proteins. It should be
noted that protein S is also synthesized in the kidney, at least in the
rat.14,37,38
The relatively high concentration of PC in the
kidney and the demonstration that the kidney also produces the PC
inhibitor, u-PA and protein S, suggest that the PC pathway may function
as a local anticoagulant in the renal circulation.
In the testis, PC mRNA and antigen were detected in spermatogenic cells
and in epithelial cells of the epididymis in the mouse (Figure 2)
in
agreement with the human studies. However, we did not detect it in
Leydig cells in the testis, as observed in the human
case.16
The different PC mRNA expression pattern in human
and murine testis may reflect species variations. In any case, these
results raise the possibility that the PC present in reproductive
tissues may contribute to the fluidity of the seminal plasma.
Low concentrations of PC mRNA also were demonstrated in the lung
(Figure 1)
, and PC antigen was specifically detected in bronchial
epithelial cells (Figure 3)
. However, no specific signal for PC mRNA
was detected in these cells. The function of PC in the lung remains to
be determined. Low levels of PC mRNA also were detected in the murine
brain (Figure 1)
. Immunohistochemical staining analysis revealed that
PC antigen was expressed specifically in pyramidal neurons in the
cerebral cortex and in the hippocampal area and in Purkinje cells in
the cerebellum (Figure 3)
. Again, we were unable to demonstrate a
specific signal for PC mRNA in these cells. The failure to demonstrate
PC mRNA in specific cells of the lung and brain suggests that its
concentration in these cells is below the detection level of in
situ hybridization. Although the function of PC in the brain is
unknown, a number of other PC pathway genes appear to be expressed in
the central nervous system in vivo. These include
prothrombin,39
protein S,37
and thrombomodulin,
an important co-factor for the activation of PC.40
Interestingly, the distribution of PC antigen in the murine cerebrum is
very similar to that observed for protein S in rabbits.37
These observations suggest that the PC pathway may act as a local
anticoagulant in the cerebral and cerebellar circulation. It should be
noted that tissue plasminogen activator is also synthesized by a
variety of neurons in the brain41
and appears to be induced
in Purkinje neurons after cerebellar motor learning.42
Thus, the plasminogen activator-plasmin system may also contribute to
neuronal plasticity. In this context, it has been reported that APC
inactivates plasminogen activator inhibitor-1,5,6
the
primary inhibitor of plasminogen activators. Taken together, these
observations raise the possibility that the PC system contributes to
the physiology of the central nervous system.
Experiments were performed in an attempt to relate changes in the
extrahepatic expression of PC to tissue function and disease. The
kidney was selected for these studies not only because of its
relatively high level of expression of PC, but also because of the
availability of numerous murine models of renal disease. Thus, we
investigated the expression of PC in three different murine models of
renal disease (ie, in autoimmune lupus nephritis, in diabetic
nephropathy, and in acute renal injury in sepsis). Significant
decreases in PC mRNA levels were observed in kidneys from lupus-prone
(MRL lpr/lpr) and diabetes-prone (db/db) mice compared with control
kidneys (Figures 4 and 5)
. Again, these decreases in PC expression
seemed to be specific for the diseased kidneys, because no differences
in PC mRNA levels were observed in similarly aged control kidneys or in
livers of control, lupus-prone, or diabetes-prone mice (Figure 4)
.
Fibrin deposits are often detected in the renal microvasculature of lupus nephritic lesions,43 and a fibrin cap and occluded capillaries are often formed in the glomeruli of diabetic nephropathy. These alterations may lead to tubular dysfunction in these renal pathologies.44 Fibrin itself may promote the progression of renal damage by occluding microvessels, by inducing glomerular cell proliferation, and by a direct cytotoxic effect on mesangial cells.45 In this regard, the observed reduction in renal PC expression may result in an increase in the local procoagulant potential of the kidney, leading to a predisposition to renal microthrombosis and in the development of renal injuries under these pathological conditions. Interestingly, PC activity has been reported to decrease in patients with chronic renal insufficiency46 and uremia,47 possibly because of the presence of an inhibitor. Although these observations are consistent with the possibility that the observed reduction in PC expression could contribute to the development of several types of human renal disease, other reports show increased PC antigen in nephrotic syndrome.48,49 Thus, elucidation of the exact role of the PC system in human renal disease will require detailed analyses of PC activity, antigen, and mRNA.
Marked decreases in PC mRNA expression also were demonstrated in
kidneys from endotoxin-treated mice (Figures 4 and 5)
. However, in this
instance, the decrease was not restricted to the kidneys, because PC
gene expression in the liver was reduced to a similar extent.
Interestingly, plasma PC levels were observed to be consistently
decreased in patients with sepsis.50,51
Although it was
suggested that these decreases resulted from increased PC consumption,
our observations support the alternative hypothesis that endotoxin may
act by decreasing the rate of PC synthesis in the liver and kidney, the
two organs that contribute most to the composition of the plasma.
Endotoxin has been reported to alter the expression of a variety of
hemostatic genes. For example, endotoxin administration frequently
results in an increase in tissue factor procoagulant activity and in
antifibrinolytic potential (ie, plasminogen activator inhibitor-1
activity) in vitro52,53
and in
vivo.54,55
Moreover, PC activation and/or
thrombomodulin expression decreases when endothelial cells in culture
are exposed to endotoxin52
or tumor necrosis
factor-
.56,57
We previously showed that microvascular
fibrin deposition was selectively induced in the kidney by endotoxin
injection into normal mice, primarily because of the dramatic induction
of plasminogen activator inhibitor-1 and tissue factor and the
reduction of u-PA in the kidney.21
The decrease in renal PC
expression after endotoxin injection may also contribute to the
increase in local procoagulant potential, eventually leading to fibrin
deposition in the renal microvasculature. The molecular mechanism by
which PC expression is decreased in these renal pathologies is unknown.
However, in preliminary studies, we observed that injection of tumor
necrosis factor- into normal mice also decreased PC mRNA levels in
the liver and kidney (data not shown). Tumor necrosis factor- is
known to be one of the primary inflammatory mediators in sepsis, and
its expression appears to be elevated in lupus
nephritis58,59
and in obesity/non-insulin-dependent
diabetes mellitus.60
Taken together, these results suggest
that tumor necrosis factor- may be one of the cytokines involved in
the decreased expression of PC under these pathological conditions.
In summary, our data not only demonstrate PC gene expression in a variety of murine tissues, but also show that the expression of this potent anticoagulant molecule is altered in thrombotic diseases of the kidney. Our results thus suggest that PC may contribute to the evolution of these pathologies. The nature of factors that control the expression and activity of PC in the kidney and other tissues, and the exact function of the PC pathway in physiological and pathological processes of the kidney, testis, epididymis, brain, and lung, remain to be determined.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health Grant HL-47819 (to DJL).
Accepted for publication May 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-thrombin. J Clin Invest 1979, 64:761-769
and lipopolysaccharide. J Clin Invest 1996, 97:37-46[Medline]
. J Biol Chem 1989, 264:10396-10401: comparison of transcriptional and posttranscriptional mechanisms. Blood 1991, 77:542-550: direct role in obesity-linked insulin resistance. Science 1993, 259:87-91This article has been cited by other articles:
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  M. J Schultz and M. Levi Pulmonary coagulopathy: a potential therapeutic target in different forms of lung injury Thorax, July 1, 2007; 62(7): 563 - 564. [Full Text] [PDF]
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 F. Odet, A. Verot, and B. Le Magueresse-Battistoni The Mouse Testis Is the Source of Various Serine Proteases and Serine Proteinase Inhibitors (SERPINs): Serine Proteases and SERPINs Identified in Leydig Cells Are under Gonadotropin Regulation Endocrinology, September 1, 2006; 147(9): 4374 - 4383. [Abstract] [Full Text] [PDF]
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F. Odet, R. Guyot, P. Leduque, and B. Le Magueresse-Battistoni Evidence for Similar Expression of Protein C Inhibitor and the Urokinase-Type Plasminogen Activator System during Mouse Testis Development Endocrinology, March 1, 2004; 145(3): 1481 - 1489. [Abstract] [Full Text] [PDF]
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 T. Sato, T. Miwa, H. Akatsu, N. Matsukawa, K. Obata, N. Okada, W. Campbell, and H. Okada Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not J. Immunol., July 15, 2000; 165(2): 1053 - 1058. [Abstract] [Full Text] [PDF]
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 M. B. Gingrich, C. E. Junge, P. Lyuboslavsky, and S. F. Traynelis Potentiation of NMDA Receptor Function by the Serine Protease Thrombin J. Neurosci., June 15, 2000; 20(12): 4582 - 4595. [Abstract] [Full Text] [PDF]
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