©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Intramuscular Delivery of Rat Kallikrein-binding Protein Gene Reverses Hypotension in Transgenic Mice Expressing Human Tissue Kallikrein (*)

(Received for publication, August 1, 1994; and in revised form, September 27, 1994)

Jian-xing Ma Zhirong Yang Julie Chao (§) Lee Chao

From the Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The tissue kallikrein-kinin system has been postulated to play a role in blood pressure regulation. The activity of tissue kallikrein is controlled by a number of factors in vivo. Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor which binds to and inhibits tissue kallikrein's activity in vitro. We have recently developed several hypotensive transgenic mouse lines which express human tissue kallikrein. In order to investigate the role of RKBP in blood pressure regulation, we delivered the RKBP to these transgenic mice by intramuscular injection. Expression of the RKBP was detected in skeletal muscle by reverse transcription-polymerase chain reaction and Southern blot analysis at 10, 20, 30, and 40 days post-injection. Immunoreactive RKBP levels in the muscle and serum of these mice were quantified by a RKBP-specific enzyme-linked immunosorbent assay and Western blot analysis. The levels of RKBP mRNA and immunoreactive protein were detectable at 10 days post-injection and increased significantly at 20 and 30 days. During this period, RKBP delivery significantly increased systemic blood pressure in the kallikrein transgenic mice to a level comparable to that of normotensive control mice. The RKBP and vector DNA delivery had no effect on the blood pressure of normotensive control mice. No serum antibodies to RKBP or its DNA were detected in the mice 40 days post injection. These results suggest that the increase of systemic blood pressure by RKBP delivery in these hypotensive transgenic mice may be mediated by inhibiting tissue kallikrein activity.


INTRODUCTION

Tissue kallikrein is a serine proteinase which processes kininogen and releases vasoactive kinin peptides(1) . Extensive studies have shown that the tissue kallikrein-kinin system is involved in blood pressure regulation(2, 3) . It was discovered in 1934 that urinary excretion of tissue kallikrein levels is reduced in essential hypertensive patients(4) . Epidemiologic studies showed that urinary kallikrein levels are inversely correlated with blood pressure (5, 6) , and high urinary kallikrein activity is correlated with a protective effect against hypertension(3) . Reduced urinary kallikrein or kinin excretion has also been described in a number of genetically hypertensive rats(7, 8) . Kallikrein gene polymorphism has been shown to co-segregate with the hypertensive phenotype in animal models(9) . These findings suggest that low renal kallikrein levels may contribute to the pathogenesis of hypertension. Reduced kallikrein activity could be attributed to a deficiency in protein synthesis, accelerated degradation, or increased kallikrein inhibitor activity.

Tissue kallikrein levels are regulated at both transcriptional and post-translational levels. At the post-translational level, the activity of tissue kallikrein may be controlled by protein factors such as alpha(1)-antitrypsin and kallikrein-binding protein(10, 11) . Kallikrein-binding protein (KBP) (^1)is a novel serine proteinase inhibitor (serpin) identified in serum and it forms a 1:1 stoichiometric complex with tissue kallikrein(11, 12) . The rate of complex formation between kallikrein and KBP is much faster than that between kallikrein and alpha(1)-antitrypsin(13) . KBP inhibited the amidolytic activity of tissue kallikrein in vitro(14) and the gene encoding rat kallikrein-binding protein (RKBP) has been cloned and characterized(15) . The recombinant RKBP expressed in Escherichia coli binds to tissue kallikrein and forms a high molecular weight SDS-stable complex, indicating covalent linkage between kallikrein and RKBP(12, 16) . The function of KBP to modulate tissue kallikrein in vivo has not been established.

Recent studies showed that an restriction fragment length polymorphism at or near the RKBP locus co-segregates with increased diastolic blood pressure of spontaneously hypertensive rats (SHR) after salt loading(17) . These findings suggest that RKBP may participate in blood pressure regulation. Two strains of transgenic mice bearing an overexpressed human tissue kallikrein gene have been recently established(18) . These transgenic mice have high levels of human tissue kallikrein in sera and various tissues, which results in sustained hypotension(18) . To study the potential function of RKBP in blood pressure regulation, we delivered the RKBP into skeletal muscle of kallikrein transgenic mice. In this study, we show that intramuscular injection of the RKBP reverses hypotension in these transgenic mice. These results suggest that the increase in systemic blood pressure by RKBP delivery in kallikrein transgenic mice might be mediated by inhibiting tissue kallikrein activity.


EXPERIMENTAL PROCEDURES

DNA Injection

pUC19 vector DNA and the plasmid DNA containing a MRE-RKBP construct were isolated by the alkaline lysis method. Supercoiled plasmid DNA was purified by CsCl-ethidium bromide gradient centrifugation (19) and diluted to 1 mg/ml in 0.9% NaCl. Plasmid DNA (250 µg/mouse) was injected at multiple sites into the quadriceps of mice using a 27 gauge needle. The same amount of pUC19 vector DNA was injected into control mice.

Tissue Homogenate Preparation and Protein Determination

Tissue homogenates were prepared from mouse muscle injected with DNA as described previously(18) . The protein concentration of the tissue extract was determined by the Lowry et al.(20) method and RKBP levels were measured by ELISA (16) . Sera of these mice were collected and subjected to RKBP-ELISA and Western blot analysis.

RT-PCR Southern Blot Analysis

Total RNA was extracted from fresh mouse muscle by guanidine-CsCl gradient centrifugation(19) . One µg of total RNA was reverse transcribed in a 10-µl reaction mixture containing 10 pmol of the 3` primer (GATCCTACTCAGATCAGCTTGC), 1 µl of 2.5 mM dNTP, 2 µl of 5 times reverse transcription buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl(2)) and 10 units of avian myeloblastosis virus reverse transcriptase (Life Technologies, Inc.). The RT reaction mixture was incubated at 37 °C for 1 h to allow synthesis of the first strand of cDNA. The cDNA was amplified using 50 pmol of the 5` primer (AGCATCTCAGCTGCCTTGGC) and 3` primer (as specified above), 5 µl of 10 times PCR buffer, 5 µl of 2.5 mM dNTP, and 2.5 units of Taq DNA polymerase and heated at 94 °C, 1 min; 55 °C, 2 min; 72 °C, 3 min for 30 cycles in a thermal cycler. One-fifth of the RT-PCR products was used for Southern blot analysis. A nested RKBP oligonucleotide primer (CAAGCTGGATTCCACCTGCTGC) was used as a probe for hybridization. The filter was washed twice in 2 times SSC at 60 °C and exposed to Kodak X-Omat film at -80 °C.

ELISA

Purified anti-RKBP IgG (2 mg/ml) was dialyzed against 0.1 M sodium bicarbonate buffer, pH 9.5, at 4 °C for 24 h and added to 10 µl of freshly prepared 0.1 M biotinyl-N-hydroxysuccinimide ester which was dissolved in dimethyl formamide(21) . The reaction was carried out at room temperature for 1 h and the mixture was dialyzed against phosphate-buffered saline (PBS). An equal volume of double-distilled glycerol was added and the biotin-labeled anti-RKBP IgG was stored at -70 °C. Microtiter plates (96-well) were coated with non-labeled anti-KBP IgG (2 µg/ml, 100 µl/well) overnight at 4 °C. The plates were then blocked with 200 µl of phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) containing 1% bovine serum albumin at 37 °C for 1 h. The plates were washed three times with PBS containing 0.1% Tween-20 (PBST). Purified rat KBP standard (0.04 ng to 2.5 ng), mouse serum and muscle samples in a total volume of 100 µl of PBS containing 0.05% Tween-20 and 0.5% gelatin (dilution buffer) were added to individual wells. The plates were incubated at 37 °C for 90 min. After incubation, the plates were washed three times with PBST. One hundred microliters of 1 µg/ml biotin-labeled anti-RKBP IgG in dilution buffer were added to each well. The reaction was carried out at 37 °C for 1 h. Excess labeled IgG was washed off with PBST after incubation. One-hundred microliters of 1 µg/ml peroxidase-avidin diluted in the dilution buffer were added in each well and the plates were incubated at 37 °C for 1 h. Following incubation, the plates were washed five times in PBST and once with PBS. The color reaction was performed by adding 100 µl/well of freshly prepared substrate solution (0.03% 2, 2`-azino-bis(3-ethyl benzthiazoline-6-sulfonic acid), and 0.03% H(2)O(2) in 0.1 M citrate buffer, pH 4.3). Plates were read at 414 nm with an ELISA reader.

Assays for Serum Antibodies in Mice

Immunological responses were monitored by measuring antibody production against RKBP protein or RKBP in the sera of transgenic mice after gene delivery. Serum antibodies binding to RKBP protein or its DNA were measured by ELISA as described previously(22) . Microtiter plates were coated overnight with either purified RKBP at 5 µg/ml in PBS at 4 °C or with the RKBP construct at 5 µg/ml in SSC at 37 °C. Mouse serum, which was serially diluted into PBS containing 0.05% Tween 20, was added to the plates. After incubation for 45 min at room temperature, plates were washed and peroxidase-conjugated goat anti-rat IgG was added. Substrate solution was added and plates were read on a Titertek plate reader.

Western Blot Analysis

RKBP levels in the muscle and serum of transgenic mice were analyzed semi-quantitatively by immunoblot using an antigen overlay method as described previously (16) . Mouse sera and muscle extracts were resolved on SDS-polyacrylamide gel electrophoresis and electrotransferred onto an Immobilon-P membrane. The membrane was blocked with BLOTTO solution (5% (w/v) nonfat dry milk in 10 mM sodium phosphate, pH 7.4, 0.14 M NaCl, 1 mMp-phenylmethylsulfonyl fluoride, 1 mg/liter thimerosal, 200 mg/liter NaN(3), and 0.01% antifoam A) for 1 h and then incubated with rabbit anti-RKBP antiserum (1:250 in BLOTTO solution) for 3 h with gentle shaking. The membrane was washed three times with BLOTTO, followed by incubation with I-RKBP for 1.5 h. The membrane was then washed three times with BLOTTO and once with phosphate-buffered saline (10 mM sodium phosphate, pH 7.4, 0.14 M NaCl), air-dried, and exposed to Kodak X-Omat film. All procedures were carried out at room temperature.

Blood Pressure Measurement

Systolic blood pressure was measured with a Programmed Electro-Sphygmomanometer PE-300 (Narco Bio-Systems, Division of International Biomedical, Inc., Houston, TX) using the tail-cuff method as described previously(18) . Calibration of the blood pressure device was carried out as described by the manufacturer. Unanesthetized mice were introduced into a small plastic holder mounted on a thermostatically controlled warm plate which was maintained at 37-38 °C during measurement. An average of five readings was taken for each animal. Blood pressure measured by the tail-cuff method was correlated with the cannulation method. There was no apparent change in blood pressure with age during the experimental period.

Statistical Analysis

Group data are expressed as mean ± S.E. Comparison of the data between two groups was made with the Student's t test. Differences were considered significant at a value of p < 0.05.


RESULTS

Construction of MRE-RKBP Vector

A full-length RKBP was cloned and sequenced from a rat genomic library. It is 9 kilobases in length and contains two functional promoters, one in the 5` flanking region and the other in the first intron(15) . The RKBP including 700 base pairs of the 5`-flanking region and 300 base pairs of the 3`-flanking region was released from a genomic phage clone and inserted into a pUC19 vector. In order to delete the native promoters of the RKBP and place the RKBP structure gene under the control of a metal responsive promoter-enhancer element (MRE) of the metallothionein gene(23) , we manipulated the gene as follows. A PCR was performed using two primers from the second exon of the RKBP. The 5` primer is at the beginning of the second exon and contains an imported BglII site at its 5` end. The 3` primer is 400 base pairs downstream from the 5` primer. The 400-base pair fragment between the two primers contains an EcoRI site. The PCR product was digested with BglII and EcoRI and ligated downstream of MRE in pUC19 vector at the same sites. This vector, containing an MRE and 5` part of the second exon of the RKBP, was linearized with EcoRI and ligated with the rest of the RKBP. In this MRE-RKBP construct, the 5`-flanking region, the first exon and the first intron including both promoters were deleted and the MRE is immediately upstream of the RKBP structural gene beginning with the second exon. The scheme for preparation of the MRE-RKBP construct is illustrated in Fig. 1.


Figure 1: Scheme for construction of the MRE-RKBP construct. To delete the native promoters of the RKBP, a PCR was performed to synthesize the 5` part of the second exon which contains an EcoRI site. The 5` PCR primer is at the beginning of the second exon and contains an imported BglII site to facilitate cloning. The PCR product was digested with BglII and EcoRI and fused with a metallothionein gene promoter (MRE). The remaining part of the RKBP, beginning with the EcoRI site in the second exon, was released by EcoRI and ligated with the 5` part of the second exon in the vector. In the final construct of MRE-RKBP, the RKBP, beginning with the second exon, is under the control of MRE. The positions of exons, introns, ATG codon and polyadenylation signal, AATAAA of the RKBP are indicated.



Detection of RKBP mRNA in Mouse Skeletal Muscle

RT-PCR Southern blot analysis shows that the RKBP is transcribed in the mouse muscle injected with MRE-RKBP plasmid DNA and not in control mice injected with vector DNA (Fig. 2). RKBP mRNA levels were detectable at 10 days post-injection and increased significantly at 20 and 30 days. At 40 days post injection, RKBP mRNA levels started to decline. The control mice injected with pUC19 vector did not hybridize with the nested RKBP oligonucleotide, indicating that the Southern blot was specific (Fig. 2). These results suggest that the expression of the RKBP is time-dependent.


Figure 2: RKBP mRNA levels in mouse muscle. Five µg of RNA from mouse skeletal muscle and a pair of RKBP cDNA-specific primers from two exons of the RKBP were used in the RT-PCR. The PCR products were probed with a nested primer in Southern blot analysis.



Detection of RKBP in Mouse Muscle and Serum

RKBP levels in mouse muscle extract and serum measured by a specific RKBP-ELISA are shown in Table 1. RKBP in the mouse muscle was 147.4 ng/mg at 10 days post-injection. The levels reached 475.9 and 825.8 ng/mg of protein at 20 and 30 days, respectively (Table 1). Similarly, secretion of RKBP into mouse serum reached high levels at 20 and 30 days post injection of the MRE-RKBP (Table 1). Linear displacement curves for immunoreactive RKBP in mouse muscle and serum were parallel with the standard curve of purified RKBP, indicating their immunological identity (Fig. 3). Control mice receiving pUC19 vector DNA contained no detectable RKBP in either muscle or in the circulation.




Figure 3: Enzyme-linked immunosorbent assay of RKBP in mouse muscle and serum. RKBP standard curve ranging from 0.4 to 25.0 ng/ml is shown by closed circles, and serially diluted mouse serum and muscle extracts are shown as solid triangles and solid squares, respectively.



Immunoreactive RKBP detected by an ELISA was further confirmed by Western blot analysis. The anti-RKBP antibody recognized a single protein of 60 kDa in the muscle or serum of mice receiving RKBP injections (Fig. 4). This immunoreactive protein has a molecular mass of 60 kDa, which is identical to the purified RKBP(12) . In the transgenic mice receiving pUC19 vector DNA, no immunoreactive RKBP was detected in either muscle or serum, indicating antibody specificity. The semiquantitative immunoblot showed that the highest level of RKBP expression occurs between 20 and 30 days post injection. These results are consistent with those of ELISA and RT-PCR Southern blot analysis and indicate that changes in the expression level of RKBP is time-dependent.


Figure 4: Western blot analysis of immunoreactive RKBP in mouse muscle and serum. One µl of serum and 80 µg of muscle protein from each mouse were resolved on SDS-polyacrylamide gel electrophoresis and electrotransferred onto an Immobilon-P filter which was blotted by an antigen-overlay method.



Intramuscular RKBP Injection Does Not Cause Immunoresponse

RKBP is an exogenous protein which is produced in mouse muscle and released into the circulation after delivery of the RKBP. In order to examine whether RKBP causes an immunoresponse in the recipient mouse, we examined the potential production of antibodies to RKBP and RKBP protein in mouse sera by solid phase ELISAs. The results show no detectable antibodies to either RKBP protein or its DNA in mouse sera collected at 10, 20, 30, and 40 days after RKBP delivery. These results indicate that the production of exogenous RKBP in transgenic mice did not result in immunogenicity of the recipients during the experimental period. No apparent change in body weight was observed, indicating that the intramuscular injection of the RKBP is nontoxic to the mice.

RKBP Reversed Hypotension in the Kallikrein Transgenic Mice

Results obtained from RKBP expression in mouse muscle and secretion of its encoded gene product into mouse serum led to the study of its biological activity. High expression levels of tissue kallikrein under the promoter control of MRE (18) in transgenic mice caused a significant reduction in blood pressure. These transgenic mice were chosen for the MRE-RKBP DNA injection to determine whether RKBP expression could reverse the hypotension caused by the tissue kallikrein transgene. There was a clear increase in blood pressure in the mice injected with the RKBP beginning on day 10. The increase in blood pressure reached a plateau at 20-30 days after the injection from 89.7 ± 2.0 to 106.8 ± 2.7 (n = 10, p < 0.01) and 107.6 ± 3.0 mm Hg (n = 10, p < 0.01), respectively (Fig. 5, Panel A). At these points, the blood pressure reached a level comparable to that of normotensive mice. The difference in blood pressure values between the two groups were significant (p < 0.01) throughout the post injection period. Blood pressure started to decline at 40 days post-injection. The results showed that the elevated blood pressure levels were maintained for more than 30 days after a single injection and the effect of the transferred RKBP was transient (Fig. 5, Panel A).


Figure 5: Blood pressure changes in the mice after RKBP delivery. Mean values (mm Hg) of blood pressure of the mice in a group of ten were plotted as a function of time after DNA injection. Panel A shows blood pressure of kallikrein transgenic mice and Panel B shows blood pressure of B6 normotensive mice after vector DNA (Control) or MRE-RKBP delivery. Blood pressures are shown as mean ± S.E. (n = 10).



To determine whether the effect of RKBP on blood pressure in the kallikrein transgenic mice was mediated by overexpressed human tissue kallikrein, we also delivered the RKBP expression vector into the normotensive control B6 mice. The B6 strain of mice was chosen for this study since they have the same genetic background as the kallikrein transgenic mice. At all points, no significant difference in the mean blood pressure was observed between the control and MRE-RKBP injected group (p > 0.1) (Fig. 5, Panel B). The results indicate that RKBP transfer caused an increase in the blood pressure of kallikrein transgenic mice without altering the blood pressure of normotensive mice.


DISCUSSION

In the present study, we showed that intramuscular injection of the rat kallikrein-binding protein gene construct into skeletal muscle reverses hypotension in kallikrein transgenic mice. Expression of the RKBPin these mice was identified at both mRNA and protein levels. The encoded product of the transferred RKBP was detected in the circulation and skeletal muscle. The effect of RKBP on blood pressure lasted for more than 30 days after one single injection. The effect of somatic RKBP delivery on blood pressure is time-dependent and is consistent with expression levels of RKBP which was quantified by ELISA, Western blot, and RT-PCR Southern blot analysis.

These results indicate that the tissue kallikrein-kinin system exerts an effect on blood pressure regulation in vivo. Tissue kallikrein processes kininogen to release bioactive kinin which has vasodilation activity(1, 24) . In kallikrein transgenic mice, the tissue kallikrein gene was expressed in a number of tissues(18) . The serum levels of human tissue kallikrein in kallikrein-transgenic mice were 11-115-fold higher than in normal human serum. High levels of tissue kallikrein may be beyond the control of endogenous kallikrein-binding protein in the transgenic mice, which may result in hypotension. When RKBP was supplied by intramuscular DNA injection, the hypotension resulting from the excess tissue kallikrein was reversed. In normal mice, the kallikrein level is low in the circulation and the level of endogenous kallikrein-binding protein exceeds that of tissue kallikrein. Additional kallikrein-binding protein provided by gene delivery did not exert further effect on blood pressure. As a result, RKBPtransfer increased blood pressure only in the kallikrein-transgenic mice but not in normal mice. Since rat KBP forms a covalent complex with human tissue kallikrein in vitro (data not shown), the increase in blood pressure caused by KBP delivery in transgenic mice expressing human kallikrein is likely mediated through the inhibition of kallikrein's activity in vivo.

Several gene transfer techniques have been developed in recent years. Because of its unique properties, skeletal muscle was chosen as one of the target tissues for gene transfer(25, 26, 27) . Direct intramuscular DNA injection has proved to be a simple and efficient way to deliver a gene construct in vivo. This method of gene delivery is not likely to cause chromosomal breakage or carcinogenesis since no viral vectors are involved to provide opportunity for chromosomal insertion. Also, the expression of a delivered gene can last for a long time, without causing any undesirable immunological response(28) . This method has been utilized to deliver and express foreign genes in skeletal muscle of mouse, rat and fish. The gene expression can be detected as early as 3 days post-injection and can be maintained for several months(27, 29, 30) . However, it was not known whether this gene transfer method can deliver adequate gene product into the circulation to exert its function(26, 31) . Our results demonstrated that the injected RKBP was expressed in muscle and its gene product was secreted into the circulation. The gene product produced was able to exert its biological effect by significantly increasing the blood pressure in animals. There was no significant change in body weight and activity of mice, indicating that the DNA injection was nontoxic to the recipient mice. This technique could find wide use in studying gene expression and function in vivo. Furthermore, this gene delivery method could be useful in gene therapy for certain inherited and acquired diseases that require delivering gene products into the circulation.


FOOTNOTES

(^1)
The abbreviations used are: KBP, kallikrein-binding protein; RKBP, rat KBP; RKBP, rat kallikrein-binding protein gene; SHR, spontaneously hypertensive rat; MRE, metal responsive promoter-enchancer element; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcription; PCR, polymerase chain reaction.

*
This work was supported by National Institutes of Health grants HL 44083 and HL 29397. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17869[GenBank].

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical University of South Carolina, 171 Ashley Ave., Charleston, SC 29425. Tel: 803-792-4321; Fax: 803-792-4322.


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