(Received for publication, August 1, 1994; and in revised form, September 27, 1994)
From the
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.
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 -antitrypsin and
kallikrein-binding protein(10, 11) .
Kallikrein-binding protein (KBP) (
)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
-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.
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.
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.
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.
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U17869[GenBank].