Corresponding author J. Chao: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA. Email: firstname.lastname@example.org
Tissue kallikrein, a serine proteinase, produces the potent vasodilator kinin peptide from kininogen substrate. The levels of tissue kallikrein are reduced in humans and animal models with hypertension, cardiovascular and renal disease. Using transgenic and somatic gene transfer approaches, we investigated the role of the tissue kallikrein–kinin system in cardiovascular, renal and central nervous systems. A single injection of the human tissue kallikrein gene in plasmid DNA or an adenoviral vector resulted in a prolonged reduction of blood pressure and attenuation of hypertrophy and fibrosis in the heart and kidney of several hypertensive animal models. Furthermore, enhanced kallikrein–kinin levels after gene transfer exerted beneficial effects, with protection against cardiac remodelling, renal injuries, restenosis, cerebral infarction and neurological deficits in normotensive animal models without haemodynamic effects, indicating direct actions of kallikrein independent of its ability to lower blood pressure. The effects of kallikrein were mediated by the kinin B2 receptor, as the specific B2 receptor antagonist icatibant abolished the actions of kallikrein. Moreover, kallikrein–kinin exhibited pleiotropic effects by inhibiting apoptosis, inflammation, hypertrophy and fibrosis, and promoting angiogenesis and neurogenesis in the heart, kidney, brain and blood vessel. Exogenous administration of kallikrein also led to increased nitric oxide (NO)/cGMP and cAMP levels, and reduced NAD(P)H oxidase activities, superoxide formation and pro-inflammatory cytokine levels. These results indicate a novel role of kallikrein–kinin through the kinin B2 receptor as an antioxidant and anti-inflammatory agent in protection against stroke, cardiovascular and renal disease, and may uncover new drug targets for the prevention and treatment of heart failure, vascular injury, end-stage renal disease and stroke in humans.
Tissue kallikrein belongs to a subgroup of serine proteinases and processes low molecular weight kininogen substrates to release vasoactive kinin peptides (Bhoola et al. 1992). The well-recognized function of tissue kallikrein is mediated by lysyl-bradykinin (Lys-BK or kallidin) and bradykinin (BK). Kinins are then degraded by enzymes such as kininases I and II and neutral endopeptidase to produce a number of kinin metabolites. Intact kinins bind to bradykinin B2 receptors, whereas kinin metabolites, such as Des-Arg9-BK or Des-Arg10-Lys-BK, bind to bradykinin B1 receptors. The B2 receptor is constitutively expressed, while the B1 receptor is expressed at low levels in various organs and is induced by trauma or inflammation (Marceau, 1995). The binding of kinins to their respective receptors activates signalling pathways such as NO–cGMP and prostacyclin–cAMP, which trigger a broad spectrum of biological effects including vasodilation, smooth muscle contraction and relaxation, inflammation and pain (Regoli et al. 1990). The tissue kallikrein–kinin system (KKS) can be blocked by icatibant (Hoe 140, a specific B2 receptor antagonist) or Des-Arg9-[Leu8]-BK (a specific B1 receptor antagonist) (Regoli et al. 1990; Bhoola et al. 1992). Furthermore, tissue kallikrein activity can be inhibited by kallistatin, a specific tissue kallikrein inhibitor that forms a covalently linked complex with tissue kallikrein (Chao et al. 1986; Zhou et al. 1992). Kallistatin has also been found to have direct actions independent of its binding to tissue kallikrein, such as reduction of blood pressure, stimulation of neointima formation and inhibition of angiogenesis and tumour growth (Chao et al. 1997; Miao et al. 2000, 2002). The KKS and renin–angiotensin system (RAS) are linked by angiotensin-converting enzyme (ACE), a dipeptidase, which is the same enzyme as kininase II. The beneficial effects of ACE inhibition in hypertension, cardiovascular and renal disease can be partially attributed to kinin accumulation, as icatibant can partially abolish these effects (Martorana et al. 1990; Linz & Scholkens, 1992). Thus, the effect of the KKS can counter-regulate the detrimental actions of the RAS.
Tissue kallikrein in hypertension
Abnormality of renal kallikrein levels has long been documented in the pathogenesis of hypertension (Elliot & Nuzum, 1934; Margolius, 1989). Epidemiological studies have identified an inverse relationship between urinary or renal kallikrein levels and blood pressure in patients with essential hypertension (Zinner et al. 1976, 1978). Furthermore, a study using family pedigrees indicated that a dominant gene expressed as renal or urinary kallikrein may be associated with a reduced risk of hypertension (Berry et al. 1989). Moreover, human tissue kallikrein gene promoter is highly polymorphic and the alleles of the kallikrein gene promoter are associated with hypertension and hypertension-associated end-stage renal disease (Song et al. 1997; Yu et al. 2002). Additionally, our recent study shows that the kallikrein promoter polymorphism regulates gene expression and modifies blood pressure in response to dietary salt intake (Song et al. 2005). These findings suggest that expression of the kallikrein gene may serve as a marker for linkage analysis in populations with salt-sensitive hypertension and renal disease. Reduced urinary kallikrein excretion has also been described in a number of genetically hypertensive rat models (Favaro et al. 1975; Powers et al. 1984; Gilboa et al. 1989; Bouhnik et al. 1992; Madeddu et al. 1997). Kinin appears to play a role in blood pressure regulation in spontaneously hypertensive rats (SHRs) fed a salt-deficient diet, as the administration of a BK receptor antagonist caused an increase in blood pressure (Gavras & Gavras, 1988). Furthermore, kininogen-deficient (Brown Norway Katholiek) rats, which cannot generate kinin, are susceptible to the development of salt-induced hypertension (Majima et al. 1994). Restriction fragment length polymorphisms (RFLPs) have been mapped in the rat tissue kallikrein gene in SHRs, and a kallikrein RFLP has been shown to co-segregate with high blood pressure in the F2 offspring of SHR and normotensive Brown Norway rat crosses (Woodley-Miller et al. 1989; Pravenec et al. 1991), suggesting a close linkage between the kallikrein gene locus and the hypertensive phenotype.
Genetic manipulation of the KKS in animal models and blood pressure regulation
Using a transgenic strategy, we have shown that mice and rats overexpressing human tissue kallikrein were permanently hypotensive throughout their lifetime compared to their control littermates (Wang et al. 1994; Song et al. 1996; Silva et al. 2000). Administration of aprotinin (a tissue kallikrein inhibitor) or icatibant to the transgenic mice raised their blood pressures to normal levels (Wang et al. 1994; Song et al. 1996). These results indicate that the expression of functional human tissue kallikrein can permanently alter the blood pressure setting in the transgenic animals and that the effect is mediated by the kinin B2 receptor. This conclusion is further reinforced by a study showing that transgenic mice overexpressing human kinin B2 receptor are hypotensive when compared to their control littermates (Wang et al. 1997). Moreover, ablation of the B2 receptor gene in mice results in elevated systolic blood pressure in response to salt loading (Alfie et al. 1996). In contrast, the kinin B1 receptor-deficient mice are healthy, fertile and normotensive (Pesquero et al. 2000). Collectively, these findings provide the first direct evidence demonstrating the role of tissue kallikrein through the kinin B2 receptor in blood pressure regulation in vivo and lend support to the hypothesis that elevated levels of tissue kallikrein–kinin may offer a protective effect in preventing the development of hypertension and cardiovascular and renal complications.
Kallikrein protein administration and gene delivery reduces blood pressure in hypertensive patients and animal models
One strategy to elucidate the potential role of kallikrein–kinin in hypertension is to enhance kallikrein levels via kallikrein protein or gene delivery. Previous studies showed that oral administration of purified pig pancreatic kallikrein resulted in a temporary lowering of blood pressure of hypertensive patients (Overlack et al. 1980, 1981). This hypotensive effect required repeated administrations of the enzyme, and the antihypertensive effect disappeared shortly after the treatment was discontinued. Somatic gene delivery results in continuous expression of the gene of interest for an extended period of time, and is therefore a potential therapeutic tool in treating cardiovascular and renal disease. To investigate the impact of supplying tissue kallikrein by gene delivery, we injected human tissue kallikrein plasmid cDNA constructs into SHRs and showed that a single injection of the kallikrein gene caused a prolonged reduction of systolic blood pressure for up to 8 weeks (Wang et al. 1995; Xiong et al. 1995; Chao et al. 1996). The extent of blood pressure reduction was dependent on the dose of DNA injected, the gender of the animals, the promoter used in the gene construct and the route of injection. Moreover, kallikrein gene transfer in fructose-induced hypertensive rats normalized not only systolic blood pressure but also serum insulin levels, suggesting a protective role of the KKS against hypertension and associated insulin resistance in type 2 diabetes (Zhao et al. 2003).
To achieve high efficiency expression of tissue kallikrein, we delivered an adenoviral vector containing the human tissue kallikrein cDNA into animal models. Systemic delivery of the human tissue kallikrein gene produced a rapid and profound reduction of blood pressure for 3 weeks in hypertensive animal models including the SHR and Dahl salt-sensitive (DSS), deoxycorticosterone acetate (DOCA)-salt, 2 kidney 1 clipped (2K1C) and 5/6 nephrectomy rats (Jin et al. 1997; Yayama et al. 1998; Chao et al. 1998a; Dobrzynski et al. 1999; Wolf et al. 2000). Kallikrein gene transfer was also effective in causing reduction of blood pressure using several non-systemic delivery routes, including intramuscular, intraportal vein, and intracerebroventricular injections (Xiong et al. 1995; Chao et al. 1996; Wang et al. 1998). Expression of recombinant human tissue kallikrein was measured with highest levels found at 3–5 days after gene delivery and rapidly reduced soon thereafter. In contrast, the adeno-associated viral (AAV) vector is attractive for long-term gene expression. A single intravenous injection of the recombinant AAV vector encoding the human tissue kallikrein gene into SHRs resulted in persistent expression of recombinant human kallikrein and a significant reduction of the systolic blood pressure for more than 5 months (Wang et al. 2004). AAV-mediated delivery of the kallikrein gene also rendered protection against cardiac remodelling and renal injury in SHRs.
Kallikrein–kinin and cardiac protection
Studies using ACE inhibitors showed a protective effect of endogenous kinins in the development of cardiac hypertrophy and neointimal vascular injury (Linz & Scholkens, 1992; Farhy et al. 1993). Bradykinin administration attenuated infarct size in an isolated perfused heart model of ischaemia–reperfusion injury (Bell & Yellon, 2003). Furthermore, genetic manipulation of KKS components clearly demonstrated a role of this system in cardiac function. Tissue kallikrein knockout mice are normotensive, but develop cardiovascular abnormalities early in adulthood (Meneton et al. 2001). In addition, ablation of the kinin B2 receptor gene in mice caused dilated cardiomyopathy followed by cardiac failure (Emanueli et al. 1999). In contrast, overexpression of human tissue kallikrein in transgenic rats resulted in reduction of isoprenaline (isoproterenol)-induced cardiac hypertrophy and fibrosis, and these effects were abolished by icatibant (Silva et al. 2000). Moreover, systemic delivery of the kallikrein gene attenuated hypertension, cardiac hypertrophy and fibrosis in pressure- and volume-overload hypertensive rat models such as SHR, 2K1C, DSS and DOCA-salt rats, as well as in fructose-induced hypertensive rats (Jin et al. 1997; Yayama et al.1998; Chao et al. 1998a,b; Dobrzynski et al.1999; Zhao et al. 2003; Bledsoe et al. 2003). Kallikrein gene transfer also attenuated cardiac hypertrophy and fibrosis in normotensive rats after myocardial infarction without affecting haemodynamic parameters (Agata et al. 2002). Furthermore, kallikrein gene transfer improved cardiac function, and reduced myocardial infarct size, incidence of ventricular fibrillation and apoptosis after acute ischaemia–reperfusion via activation of Akt-GSK-3 and Akt-Bad-14-3-3 signalling pathways (Yoshida et al. 2000; Yin et al. 2005). Icatibant abolished these beneficial effects. Taken together, these results indicate that kallikrein, through the B2 receptor, plays an important role in cardioprotection.
Protection of vascular injury by kallikrein gene delivery
Kinin inhibits the proliferation of cultured primary vascular smooth muscle cells, and transfection of the kallikrein gene into isolated aortic segments results in a time-dependent secretion of recombinant human tissue kallikrein, coinciding with significant increases in NO and cGMP levels (Murakami et al. 1999a). In addition, local delivery of the kallikrein gene into rat left common carotid artery after balloon angioplasty was shown to result in a significant reduction in intima/media ratio at the injured vessel (Murakami et al. 1999a,b). The inhibitory effect of kallikrein on neointima formation was blocked by l-NAME, a NO synthase inhibitor, and by icatibant, indicating a kinin–NO-dependent event. Moreover, systemic delivery of the kallikrein gene into a mouse model of arterial remodelling induced by a permanent alteration in shear stress conditions resulted in a reduction of neointima formation (Emanueli et al. 2000). The protective action of kallikrein gene transfer was significantly reduced in kinin B2 knockout mice, but amplified in transgenic mice overexpressing human kinin B2 receptor, compared to wild-type mice (Emanueli et al. 2000). Furthermore, in streptozotocin-induced diabetic mice, local delivery of the kallikrein gene halted the progression of microvascular rarefaction in hindlimb skeletal muscle by inhibiting apoptosis and promoting vascular regeneration (Emanueli et al. 2002). Collectively, these results indicate a protective role of the kallikrein–kinin system in vascular injury.
Kallikrein–kinin and renal protection
Reduced renal kallikrein excretion has been documented in patients with mild renal disease and more markedly reduced in patients with severe renal failure (Price, 1982; Naicker et al. 1999). Because urinary kallikrein originates in the kidney, reduced urinary kallikrein levels would suggest impaired renal function. Intravenous infusion of a subdepressor dose of rat urinary kallikrein via osmotic minipump reduced renal damage without affecting the blood pressure of DSS rats fed a high-salt diet, and the effect was abolished by icatibant (Uehara et al. 1994; Hirawa et al. 1999). We further showed that adenovirus-mediated kallikrein gene delivery enhanced renal function and reduced renal damage in DSS, 2K1C and 5/6 nephrectomy hypertensive rats (Chao et al. 1998a; Yayama et al. 1998; Wolf et al. 2000). Moreover, kallikrein not only attenuated but also reversed salt-induced renal fibrosis and glomerular hypertrophy, as well as reduced inflammatory cell accumulation in the interstitium and vasculature of DSS rats (Zhang et al. 2004; authors' unpublished results). Furthermore, kallikrein–kinin restored NO production and significantly inhibited salt-induced NADH oxidase activity, superoxide formation and leucocyte adhesion molecule expression. These protective effects of kallikrein were abolished by co-administration with icatibant. This unique ability of kallikrein–kinin to repair renal tubular damage may have general implications, as we also observed a similar renal protection in normotensive rats with gentamycin-induced nephrotoxicity (Murakami et al. 1998). These results indicate a novel role of kallikrein–kinin in protection against salt- and drug-induced renal injury by inhibiting oxidative stress and inflammation.
Kallikrein–kinin in protection against stroke
There are two types of strokes, pressure-induced haemorrhagic stroke and ischaemia–reperfusion-induced stroke. Our previous study showed that kallikrein–kinin plays a protective role in haemorrhagic stroke as delivery at an early age of the kallikrein gene into DSS rats fed a high-salt diet significantly reduced stroke-induced mortality, blood pressure elevation and aortic hypertrophy (Zhang et al. 1999). Focal brain ischaemia is the most common event leading to ischaemic stroke in humans. To determine the potential protective role of kallikrein–kinin in ischaemic stroke, we first employed a focal cerebral ischaemic rat model with middle cerebral artery occlusion (MCAO). We showed that delivery of the kallikrein gene via local injection through the intracerebroventricular route immediately after MCAO significantly reduced ischaemia-induced neurological deficits, cerebral infarct volume and apoptosis, while promoting the survival and migration of glial cells into the ischaemic core (Xia et al. 2004).
Stroke-induced neurological deficits and mortality are often associated with timing of treatment after the onset of stroke. The therapy approved for the treatment of acute ischaemic stroke is intravenous recombinant tissue-type plasminogen activator initiated within 3 h of symptom onset (Fisher, 2002). We employed a more practical approach for the treatment of ischaemic stroke by systemic delivery of the human tissue kallikrein gene. Our studies showed that delayed delivery of the kallikrein gene via intravenous injection after the onset of ischaemic stroke protected against cerebral ischaemic injury. The beneficial effects of kallikrein–kinin include reducing neurological dysfunction, infarct size, neuronal/glial cell apoptosis and inflammatory cell infiltration, and promoting angiogenesis and neurogenesis in the ischaemic brain. The neuroprotective effects of kallikrein were accompanied by reduced oxidative stress and activation of Akt-mediated signalling pathways. Similarly, a continuous supply of tissue kallikrein via protein infusion through minipump reduced ischaemia–reperfusion-induced neurological dysfunction, cerebral infarct size and inflammatory cell accumulation in the ischaemic brain after MCAO, and icatibant abolished these beneficial effects. These combined results indicate a novel function of kallikrein–kinin through the kinin B2 receptor in protection against ischaemic stroke-induced brain injuries.
Role of kinin B2 receptor in anti-inflammation
Kinin is well known to be a pro-inflammatory agent (Couture et al. 2001). Our previous studies showed that kallikrein gene transfer reduced renal inflammatory cell infiltration identified by haematoxylin and eosin staining in DSS rats and rats with chronic renal failure (Chao et al. 1998b; Wolf et al. 2000). Moreover, we recently demonstrated that kallikrein gene delivery or protein infusion inhibits macrophage/monocyte accumulation along with reduced pro-inflammatory cytokines in the heart, kidney and brain of animals induced by ischaemic–reperfusion, high-salt loading or drug treatment (authors' unpublished results). The effect of kallikrein on anti-inflammation appears to be dependent on a specific kinin receptor. In a rat model of streptozotocin-induced diabetes, Des-Arg9-BK (an agonist of B1 receptor) was shown to increase mononuclear cell and neutrophil migration. In addition, pretreatment with icatibant potentiated the effect of the B1 receptor agonist on leucocyte migration, suggesting that the B2 receptor may exert a protective effect in inflammatory cell migration (Couture et al. 2001). Similarly, we observed (J. Chao & L. Chao, unpublished observations) that the inhibitory effect of kallikrein on inflammatory cell accumulation was blocked by icatibant, indicating an anti-inflammatory role of kinin through the B2 receptor. In contrast, there is a reduced accumulation of polymorphonuclear leucocytes in inflamed tissues of B1 knockout mice, suggesting a pro-inflammatory effect of kinin mediated by the B1 receptor (Pesquero et al. 2000). Furthermore, a recent study showed that B2 receptor activation may cause tissue inflammatory injury in wild-type mice, whereas B2 receptor activation may prevent exacerbated tissue injury in B1 knockout mice (Souza et al. 2004). These results suggest that kinin B2 receptor activation can have dual effects in pro-inflammatory and anti-inflammatory reactions. Our studies indicate a new role of kallikrein–kinin through the kinin B2 receptor in protection against the inflammatory response in the cardiovascular, renal and central nervous systems.
Roles of kallikrein–kinin in hypertension, cardiovascular and renal injuries and stroke