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Recent experimental and clinical studies suggested that apart from playing an essential role in blood pressure homeostasis, aldosterone is involved in the pathophysiology of cardiovascular and renal diseases by inducing structural changes in the heart, kidney, and vessel wall. The interindividual variation of aldosterone response to antihypertensive treatment is considerable, and is at least partially explained by genetic variation. In this study, we investigated aldosterone response to two antihypertensive drugs-a thiazide diuretic and an angiotensin receptor blocker (ARB). Genetic variations in 50 candidate genes were tested for association with aldosterone response in four independent samples: African American (AA) responders to a diuretic (n = 289), AA responders to an ARB (n = 252), European American (EA) responders to a diuretic (n = 295) and EA responders to an ARB (n = 300). Linear regression was used to test the association with inclusion of age, sex, and body mass index as covariates. The results indicated the existence of one or more variants in the kininogen gene (KNG) that influence interindividual variation in aldosterone response. The significant association was replicated in three of four studied groups. The single nucleotide polymorphism rs4686799 was associated in AA and EA responders to the diuretic (P = 0.04 and P = 0.07, respectively), and rs5030062 and rs698078 were significantly associated in EA responders to the diuretic (P = 0.05 and P = 0.01) and EA responders to the ARB (P = 0.04 and P = 0.02). Although the clinical implication of KNG gene variation to antihypertensive drug response is yet to be determined, this novel candidate locus provides important new insights into drug response physiology.

We isolated PCR, RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE-PCR)-, and RT-PCR-generated clones from mouse kininogen family transcripts. DNA sequencing indicated that the clones were from two distinct genes. One set (K1) is from the previously reported mouse kininogen gene. The second set (K2) has an open reading frame, is 93% identical to K1 in the overlapping nucleotide sequence, and, unlike T-kininogens in the rat, encodes a bradykinin motif identical to K1. We discovered that K2 exists with two different 5' ends. We used RT-PCR to determine the distribution and relative abundance of K1 and K2 mRNA in mouse tissues. K2 is transcribed and K1 and K2 are generally both expressed in the same tissues; however, they differ in their regulation of the alternative splicing event that yields either low-molecular-weight kininogen (LMWK) or high-molecular-weight kininogen (HMWK). For example, in the liver K1 is expressed as both HMWK and LMWK, whereas K2 is only expressed as LMWK. Conversely, in the kidney K2 is strongly expressed as both HMWK and LMWK, whereas K1 is not expressed as HMWK and expressed only very weakly as LMWK.

Brown Norway (BN) and BN Katholiek (BN/Ka) rat strains are both susceptible to develop lesions in the internal elastic lamina (IEL) of the aorta. BN/Ka rats are characterized by a single point mutation in the kininogen gene leading to deficiency in high- and low-molecular-weight kininogen. Recently, a suggestive quantitative trait locus for lesions in the IEL of the abdominal aorta was identified in an F2 intercross between Dahl salt-sensitive (SS) and BN rats, implicating kininogen as a positional candidate gene. Therefore, BN and BN/Ka rat strains represent ideal model organisms with which to study the contribution of kininogen to the genetic predisposition to IEL lesion formation and to characterize the early events underlying vascular remodeling. Here we present data demonstrating that genetic kininogen deficiency promotes the formation of aneurysms in the abdominal aorta but not the development of atherosclerosis upon 12-wk treatment with an atherogenic diet. Aneurysm formation was associated with an enhanced elastolysis, increased expression of MMP-2 and MMP-3, downregulation of TIMP-4, and with FasL- and caspase-3-mediated apoptosis. Kininogen-deficient animals also featured changes in plasma cytokines compatible with apoptotic vascular damage, i.e., upregulation of IFN-gamma and downregulation of GM-CSF and IL-1beta. Finally, in response to atherogenic diet, kininogen-deficient animals developed an increase in HDL/total cholesterol index, pronounced fatty liver and heart degeneration, and lipid depositions in aortic media without atherosclerotic plaque formation. These findings suggest that genetic kininogen deficiency renders vascular tissue prone to aneurysmatic but not to atherosclerotic lesions.

Using Brown Norway Katholiek (BNK) rats, which are deficient in kininogen (kinin precursor) due to a mutation in the kininogen gene, we examined the role of endogenous kinins in 1) normal cardiac function; 2) myocardial infarction (MI) caused by coronary artery ligation; 3) cardiac remodeling in the development of heart failure (HF) after MI; and 4) the cardioprotective effect of angiotensin-converting enzyme inhibitors (ACEI) on HF after MI. Two months after MI, rats were randomly treated with vehicle or the ACEI ramipril for 2 mo. Brown Norway rats (BN), which have normal kininogen, were used as controls. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), end-diastolic pressure (EDP), and ejection fraction (EF) as well as myocardial infarct size (IS), interstitial collagen fraction (ICF), cardiomyocyte cross-sectional area (MCA), and oxygen diffusion distance (ODD) were measured. We found that 1) cardiac hemodynamics, function, and histology were the same in sham-ligated BN and BNK rats; 2) IS was similar in BN and BNK; 3) in rats with HF treated with vehicle, the decrease in LVEF and the increase in LVEDV, LVESV, LVEDP, ICF, MCA, and ODD did not differ between BNK and BN; and 4) ACEI increased EF, decreased LVEDV and LVESV, and improved cardiac remodeling in BN-HF rats, and these effects were partially blocked by the bradykinin B(2) receptor antagonist icatibant (HOE-140). In BNK-HF rats, ACEI failed to produce these beneficial cardiac effects. We concluded that in rats, lack of kinins does not influence regulation of normal cardiac function, myocardial infarct size, or development of HF; however, kinins appear to play an important role in the cardioprotective effect of ACEI, since 1) this effect was significantly diminished in kininogen-deficient rats and 2) it was blocked by a B(2) kinin receptor antagonist in BN rats.

High potassium intake is known to attenuate hypertension, glomerular lesion, ischemic damage, and stroke-associated death. Our recent studies showed that expression of recombinant kallikrein by somatic gene delivery reduced high blood pressure, cardiac hypertrophy, and renal injury in hypertensive animal models. The aim of this study is to explore the potential role of the tissue kallikrein-kinin system in blood pressure reduction and renal protection in spontaneously hypertensive rats (SHR) on a high-potassium diet. Young SHR were given drinking water with or without 1% potassium chloride for 6 wk. Systolic blood pressure was significantly reduced beginning at 1 wk, and the effect lasted for 6 wk in the potassium-supplemented group compared with that in the control group. Potassium supplement induced 70 and 40% increases in urinary kallikrein levels and renal bradykinin B2 receptor density, respectively (P < 0.05), but did not change serum kininogen levels. Similarly, Northern blot analysis showed that renal kallikrein mRNA levels increased 2.7-fold, whereas hepatic kininogen mRNA levels remained unchanged in rats with high potassium intake. No difference was observed in beta-actin mRNA levels in the kidney or liver of either group. Competitive RT-PCR showed a 1.7-fold increase in renal bradykinin B2 receptor mRNA levels in rats with high potassium intake. Potassium supplement significantly increased water intake, urine excretion, urinary kinin, cAMP, and cGMP levels. This study suggests that upregulation of the tissue kallikrein-kinin system may be attributed, in part, to blood pressure-lowering and diuretic effects of high potassium intake.

The tissue kallikrein-kinin system has been implicated in regulating blood pressure and electrolyte homeostasis. To understand the function of this system, we identified the expression and cellular localization of its components including tissue kallikrein, kallistatin, kininogen, and bradykinin B1 and B2 receptors in human adrenal gland. Reverse transcription-polymerase chain reaction followed by Southern blot analysis showed that these five components of this system were all expressed in human adrenal gland. In situ hybridization histochemistry with respective digoxigenin-labeled antisense riboprobes revealed localization of kallikrein transcript throughout the adrenal cortex and medulla except the zona glomerulosa, whereas kallistatin mRNA was only localized in the zona fasciculata. Low-molecular-weight kininogen and B2 receptor mRNAs were colocalized in the zona glomerulosa and zona fasciculata and also in the zona reticularis and chromaffin cells but to a lesser degree. The B1 receptor mRNA was stained in the zona fasciculata and medulla. These results show the expression and differential colocalization of the components of the tissue kallikrein-kinin system and reveal the potential action sites of this system in the adrenal gland.

This study was designed to determine whether the kallikrein-kinin system exerts a protective action in hypertension induced by chronic inhibition of nitric oxide synthase. N omega-nitro-L-arginine methyl ester (L-NAME, 40 mg/100 ml water) was given orally to Sprague-Dawley rats, while controls received regular tap water. Hepatic kininogen mRNA levels in the L-NAME-treated group were 2.9- and 2.5-fold higher at 3 and 4 wk, respectively, compared with control rats, whereas kallikrein-binding protein (KBP) mRNA levels were 82% and 45% of the values found in control rats at 3 and 4 wk, respectively. There was no significant change in hepatic alpha 1-antitrypsin mRNA levels under the same conditions. At 3 and 4 wk post L-NAME treatment, renal kallikrein mRNA levels were 2.5- and 3.4-fold higher than in controls, whereas renal beta-actin mRNA levels were similar between groups. Changes in the transcript levels of renal kallikrein, kininogen, and KBP were consistent with their protein levels. Immunoreactive total kininogen and low-Mr kininogen levels in sera and tissue kallikrein levels in kidney were significantly higher in the L-NAME-treated group, whereas KBP levels in the circulation were lower compared with controls. Systolic blood pressure was increased by 58 +/- 4 mmHg after 4 wk of L-NAME treatment. This effect was enhanced in rats given L-NAME in combination with HOE-140, a bradykinin B2-receptor antagonist, at the dose of 100 micrograms/day ip (79 +/- 5 vs. 58 +/- 4 mmHg, P < 0.05). This difference was confirmed by direct measurement of mean blood pressure (MBP). An intra-arterial bolus injection of 200 ng bradykinin significantly decreased MBP of L-NAME-treated rats, and this effect was blunted in the group treated with the bradykinin antagonist (-29 +/- 3 vs. -9 +/- 2 mmHg, P < 0.01). These results suggest that enhanced kallikrein and kininogen synthesis may have a protective role against the cardiovascular effects induced by chronic inhibition of nitric oxide synthesis.

Kininogen is the precursor of the kinin peptide, which binds to kinin receptors and mediates a broad spectrum of physiological effects. To understand the function of kinin in the kidney, we have identified the cellular localization of the human low-molecular-weight (LMW) kininogen and bradykinin B2 receptor mRNAs in the human kidney by in situ hybridization histochemistry. Kininogen mRNA was found in the juxtaglomerular cells, mesangial areas, epithelium of parietal and visceral (podocytes) layers of Bowman's capsule, proximal and distal tubules, thin and thick segments of Henle's loop, collecting ducts, and the endothelial cells of the blood vessels. B2 receptor mRNA was colocalized with kininogen mRNA in the kidney except the podocytes. The most intense signals were observed in the distal tubules and collecting ducts for both kininogen and B2 receptor mRNAs. No signals were observed in the interstitial cells and macula densa. Control sections did not stain with either the kininogen or B2 receptor sense riboprobe. A Northern blot showed that the expression of LMW kininogen is in the liver and the kidney. Reverse transcription-polymerase chain reaction Southern blot showed expression of B2 receptor mRNA in the endothelial cells, renal proximal tubular cells, and kidney. Our results show the sites of action of kinin in the human kidney and provide further insight into the physiological role of the kallikrein-kinin system on renal function.