Kallikrein 1 Research Articles

Analysis of IGHA1 and other salivary proteins post half marathon in female participants.

High-intensity exercise (HIE), such as that in marathons and triathlons, suppresses transient local and systemic immunity. Serum and salivary immunoglobulin heavy constant alpha 1 (IGHA1) are major markers of immunosuppression by HIE. Although much is known about the systemic immunosuppressive response, little is known about its local response in the oral cavity, lungs, bronchial tubes, and skin. The oral cavity allows bacteria or viruses to enter the body. Saliva covers the epidermis of the oral cavity and plays an important role in the local stress response by preventing infection. In this study, we examined the properties of saliva secreted during the local stress response for half-marathon (HM) induced IGHA1 protein expression using quantitative proteomics. The Exercise Group (ExG) (19 healthy female university students) participated in a HM race. The Non-Exercise Group (NExG) (16 healthy female university students) did not participate in the ExG. The ExG saliva samples were collected 1 h pre and 2 h and 4 h post-HM. The NExG saliva samples were collected at the same time intervals. The saliva volume, protein concentration, and relative IGHA1 expression were analyzed. In addition, 1 h pre and 2 h post- HM saliva samples were analyzed by iTRAQ. The identified factors in iTRAQ were analyzed for the ExG and the NExG using western blotting. We identified kallikrein 1 (KLK1), immunoglobulin kappa chain (IgK), and cystatin S (CST4) as suppression factors, as well as IGHA1, which has been reported to be an immunological stress marker. IGHA1 (p = 0.003), KLK1 (p = 0.011), IGK (p = 0.002), and CST4 (p = 0.003) were suppressed 2 h post-HM compared with their levels pre HM, and IGHA1 (p < 0.001), KLK1 (p = 0.004), and CST4 (p = 0.006) were suppressed 4 h post-HM. There was also a positive correlation between IGHA1, IGK, and CST4 levels at 2 and 4 h post-HM. In addition, KLK1 and IGK levels at 2 h post-HM were positively correlated. Our study demonstrated that the salivary proteome is regulated, and antimicrobial proteins are suppressed post-HM. These results suggest that oral immunity was transiently suppressed post-HM. The positive correlation of each protein at 2 and 4 h post-HM suggests that the suppressed state was similarly regulated up to 4 h after a HM. The proteins identified in this study may have applications as stress markers for recreational runners and individuals who perform moderate to HIE on a regular basis.

Mechanisms of thrombosis pathogenesis and prevention vary in patients with ovarian and endometrial cancers.

e17554 Background: Thrombosis in patients with gynecologic cancers worsens the outcome of antitumor treatment and is one of the leading causes of their death. The kallikrein-kinin system (KKS) is involved in both the regulation of thrombus formation and the development of cancer. The purpose of this study was to analyze characteristics of the KKS components in the blood in patients with endometrial cancer (EM) and ovarian cancer (OC) with and without secondary thrombosis (VTEC). Methods: The study included 39 patients, mean age 58.0±4.2 years: main groups – patients with OC T1c-3cN0M0 (n = 10) or EC T1a-2N0M0 (n = 9) with VTEC; comparison groups - patients with OC (n = 10) or EC (n = 10) without VTEC. EC was represented by adenocarcinomas (G1-G3), OC by serous carcinomas (90%) and clear cell adenocarcinomas (10%). The control group included healthy women of the corresponding age (n = 10). Blood levels of kallikrein 1 (K1), kallikrein 14 (K14), and kininogen (KG) were measured by ELISA after the surgery. Results: EC patients with VTEC showed 1.5 times (p &lt; 0.05) higher blood levels of K1, compared to donors, while other indices were unchanged. EC patients without VTEC had 4 times lower KG levels, compared to donors and EC+VTEC. In OC, regardless of the presence or absence of VTEC, levels of K1 in the blood increased, as well as in women with EC+VTEC, by 1.5 times (p &lt; 0.05) compared with donors, which was combined with a twofold increase in KG in OC+VTEC and a decrease in KG by 1.4 times in OC patients without VTEC. Blood levels of K14 increased only in OC patients without VTEC by an average of 1.9 times (p &lt; 0.05) compared with donors and OC+VTEC. Conclusions: The revealed changes in some KKS components in the blood demonstrate the similarity (K1 increase) and differences (KG increase only in OC) in the pathogenesis of thrombosis associated with gynecologic cancers. The mechanisms of protection against VTEC in the early postoperative period also showed common (KG decrease) and specific features associated with the characteristics of cancer (K14increase only in OC). The development of therapy aimed at correcting the identified disorders will allow an antitumor treatment program for this category of patients with maximum efficiency improving their quality of life and survival.

Analysis of the association of ANO3/MUC15, COL4A4, RRBP1, and KLK1 polymorphisms with COPD susceptibility in the Kashi population.

ObjectiveChronic obstructive pulmonary disease (COPD) is a complex, multifactorial, polygenic disease. The rate of occurrence of COPD in the Kashi population (Uyghur) is significantly higher than that observed nationwide. The identification of COPD-related genes in the Chinese Uyghur population could provide useful insights that could help us understand this phenomenon. Our previous whole-exome sequencing study of three Uyghur families with COPD demonstrated that 72 mutations in 55 genes might be associated with COPD; these included rs15783G > A in the anoctamin 3 (ANO3) gene/mucin 15 (MUC15) gene, rs1800517G > A in the collagen type IV alpha 4 chain (COL4A4) gene, rs11960G > A in the ribosome binding protein 1 (RRBP1) gene, and rs5516C > G in the kallikrein 1 (KLK1) gene. This case–control study aimed to further validate the association of the four mutations with COPD in the Chinese Uyghur population.MethodsSanger sequencing was used for the genotyping of four polymorphisms (ANO3/MUC15 rs15783, COL4A4 rs1800517, RRBP1 rs11960, and KLK1 rs5516) in 541 unrelated Uyghur COPD patients and 534 Uyghur healthy controls. We then conducted stratified analyses based on the smoking status and airflow limitation severity, to explore the correlation between selected gene polymorphisms and COPD.ResultsANO3/MUC15 rs15783 and KLK1 rs5516 polymorphisms could significantly reduce COPD risk (p < 0.05), but COL4A4 rs1800517 and RRBP1 rs11960 polymorphisms were not correlated with COPD in the entire population. In a stratified analysis of smoking status, non-smokers with the ANO3/MUC15 rs15783G/G genotype (OR = 0.63, p = 0.032) or COL4A4 rs1800517 allele G (OR = 0.80, p = 0.023) had a reduced risk of COPD. Smokers with the RRBP1 rs11960A/G genotype had a lower risk of COPD (OR = 0.41, p = 0.025). The KLK1 rs5516G > C polymorphism was associated with a decreased risk of COPD (OR < 1, p < 0.05), irrespective of the smoking status of individuals. No significant association with COPD severity was observed in individuals with these four polymorphisms (p > 0.05).ConclusionWe identified four previously unreported mutations (ANO3/MUC15 rs15783, COL4A4 rs1800517, RRBP1 rs11960, and KLK1 rs5516) that might decrease the COPD risk in individuals with different smoking statuses in the Chinese Uyghur population. Our findings provide new light for the genetic risk factors associated with the occurrence of COPD.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12890-022-01975-3.

Heparin Blocks the Inhibition of Tissue Kallikrein 1 by Kallistatin through Electrostatic Repulsion.

Kallistatin, also known as SERPINA4, has been implicated in the regulation of blood pressure and angiogenesis, due to its specific inhibition of tissue kallikrein 1 (KLK1) and/or by its heparin binding ability. The binding of heparin on kallistatin has been shown to block the inhibition of KLK1 by kallistatin but the detailed molecular mechanism underlying this blockade is unclear. Here we solved the crystal structures of human kallistatin and its complex with heparin at 1.9 and 1.8 Å resolution, respectively. The structures show that kallistatin has a conserved serpin fold and undergoes typical stressed-to-relaxed conformational changes upon reactive loop cleavage. Structural analysis and mutagenesis studies show that the heparin binding site of kallistatin is located on a surface with positive electrostatic potential near a unique protruded 310 helix between helix H and strand 2 of β-sheet C. Heparin binding on this site would prevent KLK1 from docking onto kallistatin due to the electrostatic repulsion between heparin and the negatively charged surface of KLK1, thus blocking the inhibition of KLK1 by kallistatin. Replacement of the acidic exosite 1 residues of KLK1 with basic amino acids as in thrombin resulted in accelerated inhibition. Taken together, these data indicate that heparin controls the specificity of kallistatin, such that kinin generation by KLK1 within the microcirculation will be locally protected by the binding of kallistatin to the heparin-like glycosaminoglycans of the endothelium.

Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals

Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Novel risk genes and mechanisms implicated by exome sequencing of 2572 individuals with pulmonary arterial hypertension

BackgroundGroup 1 pulmonary arterial hypertension (PAH) is a rare disease with high mortality despite recent therapeutic advances. Pathogenic remodeling of pulmonary arterioles leads to increased pulmonary pressures, right ventricular hypertrophy, and heart failure. Mutations in bone morphogenetic protein receptor type 2 and other risk genes predispose to disease, but the vast majority of non-familial cases remain genetically undefined.MethodsTo identify new risk genes, we performed exome sequencing in a large cohort from the National Biological Sample and Data Repository for PAH (PAH Biobank, n = 2572). We then carried out rare deleterious variant identification followed by case-control gene-based association analyses. To control for population structure, only unrelated European cases (n = 1832) and controls (n = 12,771) were used in association tests. Empirical p values were determined by permutation analyses, and the threshold for significance defined by Bonferroni’s correction for multiple testing.ResultsTissue kallikrein 1 (KLK1) and gamma glutamyl carboxylase (GGCX) were identified as new candidate risk genes for idiopathic PAH (IPAH) with genome-wide significance. We note that variant carriers had later mean age of onset and relatively moderate disease phenotypes compared to bone morphogenetic receptor type 2 variant carriers. We also confirmed the genome-wide association of recently reported growth differentiation factor (GDF2) with IPAH and further implicate T-box 4 (TBX4) with child-onset PAH.ConclusionsWe report robust association of novel genes KLK1 and GGCX with IPAH, accounting for ~ 0.4% and 0.9% of PAH Biobank cases, respectively. Both genes play important roles in vascular hemodynamics and inflammation but have not been implicated in PAH previously. These data suggest new genes, pathogenic mechanisms, and therapeutic targets for this lethal vasculopathy.

Tissue kallikrein regulates alveolar macrophage apoptosis early in influenza virus infection.

Host cell proteases are involved in influenza pathogenesis. We examined the role of tissue kallikrein 1 (KLK1) by comparing wild-type (WT) and KLK1-deficient mice infected with influenza H3N2 virus. The levels of KLK1 in lung tissue and in bronchoalveolar lavage (BAL) fluid increased substantially during infection. KLK1 did not promote virus infectivity despite its trypsin-like activity, but it did decrease the initial virus load. We examined two cell types involved in the early control of pathogen infections, alveolar macrophages (AMs) and natural killer (NK) cells to learn more about the antiviral action of KLK1. Inactivating the Klk1 gene or treating WT mice with an anti-KLK1 monoclonal antibody to remove KLK1 activity accelerated the initial virus-induced apoptotic depletion of AMs. Intranasal instillation of deficient mice with recombinant KLK1 (rKLK1) reversed the phenotype. The levels of granulocyte-macrophage colony-stimulating factor in infected BAL fluid were significantly lower in KLK1-deficient mice than in WT mice. Treating lung epithelial cells with rKLK1 increased secretion of this factor known to enhance AM resistance to pathogen-induced apoptosis. The recruitment of NK cells to the air spaces peaked 3 days after infection in WT mice but not in KLK1-deficient mice, as did increases in several NK-attracting chemokines (CCL2, CCL3, CCL5, and CXCL10) in BAL. Chronic obstructive pulmonary disease (COPD) patients are highly susceptible to viral infection, and we observed that the KLK1 mRNA levels decreased with increasing COPD severity. Our findings indicate that KLK1 intervenes early in the antiviral defense modulating the severity of influenza infection. Decreased KLK1 expression in COPD patients could contribute to the worsening of influenza.

Novel SNPs of WNK1 and AKR1C3 are associated with preeclampsia

Preeclampsia is a hypertensive disorder of pregnancy and is one of the most common causes of poor perinatal outcomes. Preeclampsia increases the risk of hypertension in the future. Variants of WNK1 (lysine deficient protein kinase 1), ADRB2 (β2 adrenergic receptor), NEDD4L (ubiquitin-protein ligase NEDD4-like), KLK1 (kallikrein 1) contribute to hypertension, and AKR1C3 (aldo-keto reductase family1 member C3), is associated with preeclampsia. The association of single nucleotide polymorphisms (SNPs) in these five candidate preeclampsia susceptibility genes and the related traits in Chinese individuals were investigated. In this study, 13 SNPs of the five genes were genotyped in 276 preeclampsia patients and 229 age- and area-matched normal pregnancies in women of Chinese Northern Han origin. The 95% confidence interval (CI) and odds ratio (OR) were estimated by binary logistic regression. No obvious linkage disequilibrium or haplotypes were observed among these SNPs. Those with GG genotype and allele G of AKR1C3 (rs10508293) had a decreased risk of preeclampsia (adjusted OR = 3.011, 95% CI = 1.758–5.159, and adjusted OR = 1.745, 95% CI = 1.349–2.257, respectively). The AA genotype and allele A of WNK1 (rs1468326) were significantly associated with an increased risk in preeclampsia (adjusted OR = 2.307, 95% CI = 1.206–3.443, and adjusted OR = 1.663, 95% CI = 1.283–2.157, respectively). The findings indicate that the GG genotype of AKR1C3 rs10508293 is associated with decreased risk for preeclampsia and the AA genotype of WNK1 rs1468326 are related with an increased risk for preeclampsia.

Identification of potential key genes and high-frequency mutant genes in prostate cancer by using RNA-Seq data.

The aim of the present study was to identify potential key genes and single nucleotide variations (SNVs) in prostate cancer. RNA sequencing (RNA-seq) data, GSE22260, were downloaded from the Gene Expression Omnibus database, including 4 prostate cancer samples and 4 normal tissues samples. RNA-Seq reads were processed using Tophat and differentially-expressed genes (DEGs) were identified using the Cufflinks package. Gene Ontology enrichment analysis of DEGs was performed. Subsequently, Seqpos was used to identify the potential upstream regulatory elements of DEGs. SNV was analyzed using Genome Analysis Toolkit. In addition, the frequency and risk-level of mutant genes were calculated using VarioWatch. A total of 150 upregulated and 211 downregulated DEGs were selected and 25 upregulated and 17 downregulated potential upstream regulatory elements were identified, respectively. The SNV annotations of somatic mutations revealed that 65% were base transition and 35% were base transversion. At frequencies ≥2, a total of 17 mutation sites were identified. The mutation site with the highest frequency was located in the folate hydrolase 1B (FOLH1B) gene. Furthermore, 20 high-risk mutant genes with high frequency were identified using VarioWatch, including ribosomal protein S4 Y-linked 2 (RPS4Y2), polycystin 1 transient receptor potential channel interacting (PKD1) and FOLH1B. In addition, kallikrein 1 (KLK1) and PKD1 are known tumor suppressor genes. The potential regulatory elements and high-frequency mutant genes (RPS4Y2, KLK1, PKD1 and FOLH1B) may have key functions in prostate cancer. The results of the present study may provide novel information for the understanding of prostate cancer development.

Genome-wide transcriptional profiling identifies potential signatures in discriminating active tuberculosis from latent infection.

To better understand the host immune response involved in the progression from latent tuberculosis infection (LTBI) to active tuberculosis (TB) and identify the potential signatures for discriminating TB from LTBI, we performed a genome-wide transcriptional profile of Mycobacterium tuberculosis (M.TB)–specific antigens-stimulated peripheral blood mononuclear cells (PBMCs) from patients with TB, LTBI individuals and healthy controls (HCs). A total of 209 and 234 differentially expressed genes were detected in TB vs. LTBI and TB vs. HCs, respectively. Nineteen differentially expressed genes with top fold change between TB and the other 2 groups were validated using quantitative real-time PCR (qPCR), and showed 94.7% consistent expression pattern with microarray test. Six genes were selected for further validation in an independent sample set of 230 samples. Expression of the resistin (RETN) and kallikrein 1 (KLK1) genes showed the greatest difference between the TB and LTBI or HC groups (P < 0.0001). Receiver operating characteristic curve (ROC) analysis showed that the areas under the curve (AUC) for RETN and KLK1 were 0.844 (0.783–0.904) and 0.833 (0.769–0.897), respectively, when discriminating TB from LTBI. The combination of these two genes achieved the best discriminative capacity [AUC = 0.916 (0.872–0.961)], with a sensitivity of 71.2% (58.7%–81.7%) and a specificity of 93.6% (85.7%–97.9%). Our results provide a new potentially diagnostic signature for discriminating TB and LTBI and have important implications for better understanding the pathogenesis involved in the transition from latent infection to TB activation.

Potential diagnostic biomarkers for chronic kidney disease of unknown etiology (CKDu) in Sri Lanka: a pilot study

BackgroundIn Sri Lanka, there exists chronic kidney disease of both known (CKD) and unknown etiologies (CKDu). Identification of novel biomarkers that are customized to the specific causative factors would lead to early diagnosis and clearer prognosis of the diseases. This study aimed to find genetic biomarkers in blood to distinguish and identify CKDu from CKD as well as healthy populations from CKDu endemic and non-endemic areas of Sri Lanka.MethodsThe expression patterns of a selected panel of 12 potential genetic biomarkers were analyzed in blood using RT-qPCR. Fold changes of gene expressions in early and late stages of CKD and CKDu patients, and an apparently healthy population of a CKDu endemic area, Girandurukotte (GH) were calculated relative to apparently healthy volunteers from a CKDu non-endemic area, Kandy (KH) of Sri Lanka, using the comparative CT method.ResultsSignificant differences were observed between KH and early stage CKDu for both the insulin-like growth factor binding protein 1 (IGFBP1; p = 0.012) and kidney injury molecule-1 (KIM1; p = 0.003) genes, and KH and late stage CKD and CKDu for the glutathione-S-transferase mu 1 (GSTM1; p < 0.05) gene. IGFBP1 and KIM1 genes showed significant difference between the early and late stage CKDu (p < 0.01). The glutamate cysteine ligase catalytic subunit (GCLC) gene had significantly different expression between KH and all the other study groups (p < 0.01). The GH group was significantly different from the KH group for the oxidative stress related genes, G6PD, GCLC and GSTM1 (p < 0.01), and also the KIM1 gene (p = 0.003). IGFBP1, insulin-like growth factor binding protein 3 (IGFBP3), fibronectin 1 (FN1) and KIM1 showed significant correlations with serum creatinine, and IGFBP1, KIM1 and kallikrein 1 (KLK1) with eGFR (p < 0.05).ConclusionA panel consisting of IGFBP1, KIM1, GCLC and GSTM1 genes could be used in combination for early screening of CKDu, whereas these genes in addition with FN1, IGFBP3 and KLK1 could be used to monitor progression of CKDu. The regulation of these genes has to be studied on larger populations to validate their efficiency for further clinical use.

Discovery of a new isomannide-based peptidomimetic synthetized by Ugi multicomponent reaction as human tissue kallikrein 1 inhibitor

Human kallikrein 1 (KLK1) is the most extensively studied member of this family and plays a major role in inflammation processes. From Ugi multicomponent reactions, isomannide-based peptidomimetic 10 and 13 where synthesized and showed low micromolar values of IC50 for KLK1 The most active compound (10) presented competitive mechanism, with three structural modifications important to interact with active site residues which corroborates its KLK1 inhibition. Finally, the most active compound also showed good ADMET profile, which indicates compound 10 as a potential hit in the search for new KLK1 inhibitors with low side effects.

Risk factors for the development of essential hypertension in a Mongolian population of China: a case-control study.

Lifestyle, habits, diet, and genetics are all important factors associated with the prevalence of hypertension. Many association studies have been performed in the Chinese Han population, whereas data explaining the high prevalence of hypertension in the Mongolian population remain scarce. In the present study, we aimed to determine the factors associated with the development of essential hypertension in Mongolians. A total of 194 hypertensive cases and 201 controls from Dongwu County were enrolled in the study. Demographics, anthropometric and blood biochemical parameters, food intake, lifestyle, habits, education, occupation, and family history were recorded for each subject. Genotype and allele frequencies of six single nucleotide polymorphisms (SNPs) of the kallikrein 1 (KLK1) gene were also examined. Mean body mass index, waistline, hipline, blood sugar, blood urea nitrogen, creatinine, uric acid, total cholesterol, triglyceride, and low-density lipoprotein levels were all significantly higher in the hypertensive group (P<0.01). Hypertensives consumed less milk, vegetables, and fruits, and had higher cigarette, alcohol, and salt intake (P<0.05). There were also less regular physical exercisers and manual workers among the hypertensive group (P<0.05). The mean inheritance rank of the hypertensive group was higher than that of controls (P<0.05). There were no differences in the distribution of genotype and allele frequencies of the six SNPs between the hypertensive and control groups (P>0.05). These results suggest that dietary history and habits have the most important influence on the development of essential hypertension in the Mongolian population.

KLK1 A1789G gene polymorphism and the risk of coronary artery stenosis in the Chinese population

Previous reports have shown that polymorphism of the human tissue kallikrein 1 (KLK1) A1789G gene is associated with susceptibility to hypertension. The current study aimed to confirm the association between the polymorphism in KLK1 and coronary artery stenosis (CAS). A total of 458 patients with CAS and 482 controls were used in a case-control study carried out between January 2008 and January 2011 at the Qilu Hospital (Jinan, China). Analyses of the KLK1 A1789G genotype were performed, and the logistic regression model was used to assess the odds ratio related to CAS. The results showed that the frequencies of the AA, AG, and GG genotypes were 11.4, 50.2, and 38.4%, respectively, in patients with CAS, and 21.2, 47.7, and 31.1%, respectively, in controls. Compared with the AA genotype, the GG and AG/GG genotypes were associated with a significantly increased risk of CAS. Furthermore, the AG and GG genotypes combined with smoking showed a remarkable increase in the risk for CAS. In conclusion, polymorphism of the KLK1 A1789G gene is associated with CAS, and smoking combined with the KLK1 GG genotype was significantly associated with an increased risk of CAS. This information is extremely important to prevention strategies for CAS.

Coupling of epithelial Na+ and Cl− channels by direct and indirect activation by serine proteases

The mammalian collecting duct (CD) is continuously exposed to urinary proteases. The CD expresses an epithelial Na(+) channel (ENaC) that is activated after cleavage by serine proteases. ENaC also exists at the plasma membrane in the uncleaved form, rendering activation by extracellular proteases an important mechanism for regulating Na(+) transport. Many exogenous and a small number of endogenous extracellular serine proteases have been shown to activate the channel. Recently, kallikrein 1 (KLK1) was shown to increase γENaC cleavage in the native CD indicating a possible direct role of this endogenous protease in Na(+) homeostasis. To explore this process, we examined the coordinated effect of this protease on Na(+) and Cl(-) transport in a polarized renal epithelial cell line (Madin-Darby canine kidney). We also examined the role of native urinary proteases in this process. Short-circuit current (I(sc)) was used to measure transport of these ions. The I(sc) exhibited an ENaC-dependent Na(+) component that was amiloride blockable and a cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl(-) component that was blocked by inhibitor 172. Apical application of trypsin, an exogenous S1 serine protease, activated I(ENaC) but was without effects on I(CFTR). Subtilisin an exogenous S8 protease that mimics endogenous furin-type proteases activated both currents. A similar activation was also observed with KLK1 and native rat urinary proteases. Activation with urinary proteases occurred within minutes and at protease concentrations similar to those in the CD indicating physiological significance of this process. ENaC activation was irreversible and mediated by enhanced cleavage of γENaC. The activation of CFTR was indirect and likely dependent on activation of an endogenous apical membrane protease receptor. Collectively, these data demonstrate coordinated stimulation of separate Na(+) and Cl(-) transport pathways in renal epithelia by extracellular luminal proteases. They also indicate that baseline urinary proteolytic activity is sufficient to modify Na(+) and Cl(-) transport in these epithelia.

A single nucleotide polymorphism in exon 3 of the kallikrein 1 gene is associated with large but not small abdominal aortic aneurysm

Objective Abdominal aortic aneurysm (AAA) is a late onset degenerative condition with an inherited component thought to be due to multiple risk alleles. A locus on chromosomes 19q13 has been previously associated with AAA. The gene encoding kallikrein 1 ( KLK1) is located on chromosome 19q13 and the single nucleotide polymorphism (SNP) rs5516 has been previously shown to lead to structural changes in the KLK1 transcription regulatory region. The aim of this study was to investigate whether rs5516 was associated with AAA and aortic diameter. Methods We performed a case–control study on two independent subject groups from Western Australia ( n = 1304) and Queensland ( n = 325) of which 609 and 225 had an AAA, respectively. In addition, we analysed RNA extracted from abdominal aortic biopsies from 12 patients undergoing AAA surgery and 6 organ donors. Results After adjusting for other risk factors the G allele of the rs5516 polymorphism was associated with large but not small AAA using a dominant model in the Western Australian men and a recessive model in Queensland subjects. In subjects with large AAA the G allele was associated with aortic diameter. The short splice variant of KLK1 was upregulated within AAA compared to control biopsies. Conclusion This study suggests that a genetic polymorphism in KLK1 may contribute to the risk of developing later stage AAA.

Polymorphisms in genes &amp;lt;i&amp;gt;CTSB, CTSD, CAPN2, KLK1&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;TGFB1&amp;lt;/i&amp;gt; not associated with susceptibility to atypical or classical ovine scrapie

Abstract. In the present study polymorphisms in the genes cathepsin B (CTSB), cathepsin D (CTSD), calpain, large polypeptide L2 (CAPN2), kallikrein 1 (KLK1) and transforming growth factor β1 (TGFB1) were investigated for association with scrapie susceptibility in sheep. Therefore single nucleotide polymorphisms in the respective genes were identified and examined for a potential impact on the gene function with different computer programs. Samples of 72 atypical and 104 classical scrapie cases as well as of 443 clinically healthy flock mates were genotyped by PCR-based screening methods. Neither allele frequencies nor genotype frequencies showed significant differences between scrapie positive sheep and control animals in any of the investigated genes.

Effects of protein coding polymorphisms in the kallikrein 1 gene on baseline blood pressure and antihypertensive response to irbesartan in Chinese hypertensive patients

The aim of this study was to determine the association between coding variants in the human tissue kallikrein 1 (KLK1) gene and baseline blood pressure (BP) and antihypertensive response to irbesartan treatment in Chinese hypertensive patients. A total of 1061 hypertensives were recruited and received daily oral dosage of 150 mg irbesartan for 4 weeks. Predose BPs, BPs and blood irbesartan concentrations at postdose on the 28th day were all measured. Common functional single-nucleotide polymorphisms (SNPs) in the KLK1 gene were genotyped. On the basis of the HapMap data of Han Chinese in the Beijing population, two non-synonymous polymorphisms with minor allele frequency>0.1, SNP rs5517 (Glu162Lys) and rs5516 (Gln121Glu), were selected. Those with GG genotype in the rs5516 locus had higher average baseline systolic BP (SBP) than CC subjects (β±s.e.: 5.0±2.3, P=0.033); and no associations of rs5517 with baseline BP (diastolic BP (DBP) and SBP) and BP responses, or rs5516 with baseline DBP and BP response were observed. In a haplotype-based association test for the KLK1 gene, the Haplo-special score analyses identified that haplotype AG was marginally associated with SBP response (specific score: 1.75 for P=0.08), but not with DBP response. We did not find any associations between haplotypes (GC and AC) and BP responses. The Haplo-GLM analyses showed that, compared with haplotype GC subjects, the subjects with haplotype AG had a marginally greater SBP response (adjusted β±s.e.: 1.81±0.97, P=0.06), but DBP response did not differ. This study suggests that rs5516 in the KLK1 gene may be involved in the development of essential hypertension and in the regulation of SBP-lowering response to irbesartan in Chinese hypertensives.

Bradykinin and high glucose promote renal tubular inflammation

The role of the kallikrein-kinin system in diabetic nephropathy remains controversial. High-glucose (HG) super-induced interleukin (IL)-6, CCL-2, transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and B(2)K receptor (B(2)KR) mRNA in cultured proximal tubular epithelial cells (PTEC), whereas bradykinin (BK) upregulated IL-6, CCL-2 and TGF-beta mRNA. HG activated mitogen-activated protein kinase (MAPK) p42/p44 and protein kinase C (PKC) signals, whereas BK only activated MAPK. Tubular expression of these mediators and tissue kallikrein 1 (KLK1) was confirmed in human diabetic kidney biopsies. Inhibition of MAPK p42/p44 by PD98059 partially reduced HG and BK induction of IL-6, CCL-2 and TGF-beta, whereas inhibition of PKC by staurosporine partially reduced HG- but not BK-induced overexpression of these cytokines and that of VEGF. Staurosporine and PD98059 synergistically reduced the effect of HG on IL-6, CCL-2 and TGF-beta expression. The B(2)KR blocker, icatibant, downregulated BK- and HG-induced MAPK p42/p44 but not HG-induced PKC activation and partially reduced both HG- and BK-induced IL-6, CCL-2 and TGF-beta secretion. HG stimulated expression of KLK1 and low-molecular-weight kininogen (LMWK) and its downstream effects were attenuated by aprotinin (tissue kallikrein inhibitor). The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, rosiglitazone, attenuated HG-induced PKC but not HG- or BK- induced MAPK p42/44 activation and reduced HG-stimulated VEGF, along with IL-6, CCL-2 and TGF-beta secretion. Rosiglitazone plus icatibant further reduced these effects of HG. In conclusion, HG stimulates tubular proinflammatory, profibrotic and angiogenic signals, which is partly mediated through BK via MAPK signalling and partly through PKC independent of BK. The potential therapeutic role of complementary B(2)KR blockade and PPAR-gamma activation deserves clinical investigation.

Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.