MiR-107-3p Knockdown Alleviates Endothelial Injury in Sepsis via Kallikrein-Related Peptidase 5.

INTRODUCTION: Endothelial injury is a major characteristic of sepsis and contributes to sepsis-induced multiple-organ dysfunction. In this study, we investigated the role of miR-107-3p in sepsis-induced endothelial injury. METHODS: Human umbilical vein endothelial cells (HUVECs) were exposed to 20 µg/mL of lipopolysaccharide (LPS) for 6-48 h. The levels of miR-107-3p and kallikrein-related peptidase 5 (KLK5) were examined. HUVECs were treated with LPS for 12 h and subsequently transfected with miR-107-3p inhibitor, KLK5 siRNA, or cotransfected with KLK5 siRNA and miR-107-3p inhibitor/negative control inhibitor. Cell survival, apoptosis, invasion, cell permeability, inflammatory response, and the Toll-like receptor 4/nuclear factor κB signaling were evaluated. In addition, the relationship between miR-107-3p and KLK5 expression was predicted and verified. RESULTS: LPS significantly elevated miR-107-3p levels, which peaked at 12 h. Conversely, the KLK5 level was lower in the LPS group than in the control group and was lowest at 12 h. MiR-107-3p knockdown significantly attenuated reductions in cell survival and invasion, apoptosis promotion, hyperpermeability and inflammation induction, and activation of the NF-κB signaling caused by LPS. KLK5 knockdown had the opposite effect. Additionally, KLK5 was demonstrated as a target of miR-107-3p. MiR-107-3p knockdown partially reversed the effects of KLK5 depletion in LPS-activated HUVECs. CONCLUSIONS: Our findings indicate that miR-107-3p knockdown may protect against sepsis-induced endothelial cell injury by targeting KLK5. This study identified a novel therapeutic target for sepsis-induced endothelial injury.

Human Urinary Kallidinogenase Pretreatment Inhibits Myocardial Inflammation and Apoptosis after Coronary Microembolization by Activating PI3K/Akt/FoxO1 Axis.

BACKGROUND: As a fatal cardiovascular complication, coronary microembolization (CME) results in severe cardiac dysfunction and arrhythmia associated with myocardial inflammation and apoptosis. Human urinary kallidinogenase (HUK) can provide a protective function for cardiomyocytes by improving microcirculation. However, the therapeutic effects and underlying mechanisms of HUK in CME-induced myocardial injury remain unclear. AIMS: We evaluated the effect of HUK on cardiac protection in a rat model of CME and whether it could restrain myocardial inflammation and apoptosis, and alleviate CME-induced myocardial injury. METHODS: We established the CME model by injecting 42 µm inert plastic microspheres into the left ventricle of rats in advance, then the rats were randomly and equally divided into CME, CME + HUK (the dose of HUK at 0.016 PNA/kg/day), CME + HUK + LY (the dose of LY294002 at 10 mg/kg, 30 minutes before modeling), and Sham operation groups. Cardiac function, the serum levels of myocardial injury biomarkers, myocardial inflammation and apoptosis-related genes were measured; and the myocardial histopathological examination was performed at 12 h after the operation. RESULTS: The results revealed that HUK effectively reducing myocardial inflammation, apoptosis, and myocardial infarction area; and improving CME-induced cardiac injury by activating the PI3K/Akt/FoxO1 axis. In addition, these cardioprotective effects can be reduced by the PI3K specific inhibitor LY294002, suggesting that the aforementioned protective effects may be related to activation of the PI3K/Akt/FoxO1 axis. CONCLUSIONS: HUK seems to control inflammatory infiltration and cardiomyocyte apoptosis significantly to improve CME-induced cardiac injury via regulating the PI3K/Akt/FoxO1 axis.

Kallikrein-related peptidase-8 (KLK8) aggravated hypoxia-induced right ventricular hypertrophy by targeting P38 MAPK/P53 signaling pathway.

Right ventricular (RV) hypertrophy and further heart failure are major co-morbidities, resulting in the premature death of patients with hypoxic pulmonary hypertension (HPH). The regulatory effects of kallikrein-related peptidase (KLK) family members on cardiac function have been extensively studied. However, to the best of the authors' knowledge, the regulatory effects of KLK8 on RV hypertrophy caused by HPH have yet to be reported. The aim of the present study was to assess KLK8 expression in the RV tissue of HPH-modeled rats, and to further explore the effects and underlying mechanism of KLK8 in regulating the hypertrophy of hypoxia-induced H9c2 cardiomyocytes. In HPH model rats, increases in the right ventricle hypertrophy index, the right ventricular systolic pressure, cardiac output, as well as pulmonary artery wall thickness were observed. Western blot analysis revealed that KLK8 expression and MAPK/p53 signaling activity were enhanced in the RVs of rats in an RV HPH rat model. In hypoxia-induced H9c2 cardiomyocytes, KLK8 overexpression promoted cardiomyocyte hypertrophy, whereas KLK8 silencing showed the opposite results. KLK8 overexpression increased the expression levels of ventricular hypertrophy markers, including atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain 7, which were blocked upon addition of the p38 MAPK inhibitor, SB202190. Conversely, KLK8 silencing caused a decrease in the expression levels of the ventricular hypertrophy markers, which were further reduced via inhibition of the p38 MAPK/p53 signaling pathway. Taken together, the results of the present study have shown that KLK8 may subtly regulate RV hypertrophy, and therefore KLK8 may be a promising therapeutic target for treating HPH-induced RV hypertrophy.

The Effect of the Angiotensin-Converting Enzyme Inhibitor on Bone Health in Castrated Hypertensive Rats Is Mediated via the Kinin-Kallikrein System.

Background: In previous studies, angiotensin-converting enzyme inhibitor (ACEI) use was associated with increased bone loss, while an angiotensin II type I receptor blocker had no effect on bone loss in elder subjects, which suggested that the effect of ACEI on bone loss was not mediated through the classical renin-angiotensin system. In this study, we set to investigate whether the effect of ACEI on bone deterioration was mediated via the kinin-kallikrein system. Methods: Six-month-old male and female spontaneously hypertensive rats were used. The effect of captopril on blood pressure, serum Ang II, and bradykinin concentration was measured in intact rats. Ovariectomy and orchidectomy were performed to establish an osteoporosis model in female and male rats, respectively. Captopril and the bradykinin receptor blocker icatibant (HOE140) were administered after operation for 12 weeks. Serum Ang II and bradykinin concentration, bone turnover markers, bone mineral density (BMD), and bone microarchitecture were evaluated. Femur samples were subjected to a mechanical test. Results: Captopril decreased blood pressure and serum Ang II concentration and increased serum bradykinin concentration in intact rats (P < 0.05). After castration, captopril decreased serum Ang II concentration (P < 0.05); in female rats, icatibant increased serum Ang II concentration (P < 0.05). Captopril increased serum bradykinin concentration (P < 0.05); in male rats, icatibant decreased serum bradykinin concentration (P < 0.05). Captopril increased the rat urine deoxypyridinoline-creatinine ratio (DPD/Cr) and serum osteocalcin concentration (P < 0.05). Icatibant decreased urine DPD/Cr in male rats (P < 0.05) and increased osteocalcin concentration in female rats (P < 0.05). Captopril increased cancellous BMD in castrated hypertensive rats (P < 0.05), and icatibant further increased cancellous BMD (P < 0.05), which was due to the increased trabecular bone number. In mechanical testing, ACEI increased bone strength (P < 0.05), and icatibant further improved it (P < 0.05). Conclusion: ACEI decreased bone deterioration in both male and female hypertensive rats, and the bradykinin receptor blocker further decreased bone deterioration.

Uterine kallikrein and arterial bradykinin activities and uterine arterial proliferation in response to acute estradiol-17ß exposure in ovariectomized ewes.

Estradiol-17ß (E2) increases kallikrein in rodent and human reproductive tissues. Kallikrein specific activity is increased in the porcine uterus when conceptus E2 is secreted at maternal recognition of pregnancy. When kallikrein acts on kininogen to liberate bradykinin, angiogenic and vasoactive factors are released. The uterus of ovariectomized ewes administered E2 undergoes rapid vascular changes via different patterns of angiogenic and vasoactive factors. Our hypothesis was that E2 would increase the specific activity and protein secretion of tissue kallikrein in endometrial explants culture media (ECM) and ewes exposed to E2 would have uterine arteries that would be more sensitive to the vasodilatory effects of bradykinin. Ovariectomized ewes received 100 mg of E2 implants for 0, 12, 24, or 48 h. After treatment, uterine weights were determined, and caruncles were processed for ECM. Uterine weights and uterine weight per ewe body weight were significantly greater in the 12 and 24 h ewes compared with the 0 h ewes, with the 48 h ewes being similar to the 24 h ewes. There were no statistically significant differences in caruncular tissue kallikrein protein secretion among the treatment groups. There was a tendency (P = 0.09) for duration of E2 exposure to influence tissue kallikrein specific activity where kallikrein activity was greater (P ≤ 0.05) in the 12 and 48 h ewes compared with the 0 h ewes, with 24 h ewes being intermediate (unprotected F test). Uterine arteries from ewes with E2 for 24 and 48 h had more sensitivity to bradykinin, via the bradykinin receptor 2, than uterine arteries from ewes with 0 or 12 h E2 exposure. We fail to reject our hypothesis as E2 did elicit a positive response in tissue kallikrein specific activity and bradykinin response. Further investigations are needed to determine how kallikrein and bradykinin may be involved in vascular remodeling of the ovine uterus.

MiR-194-3p modulates the progression of colorectal cancer by targeting KLK10.

BACKGROUND: A rich history of studies have manifested the importance of miRNAs to cancer progression, while miR-194-3p has been seldom explored. OBJECTIVE: The purpose of this study is to unearth the way the KLK10/miR-194-3p axis modulates colorectal cancer (CRC). METHODS: Differentially expressed genes of CRC in TCGA database were analyzed. Western blot and qRT-PCR were employed to test protein and mRNA expressions of two researched genes. Their targeting was confirmed using dual-luciferase. Biological behaviors of cells were tested by a series of cellular functional assays. RESULT: Remarkably low miR-194-3p expression and high KLK10 expression were observed in cancer cells. Overexpressing miR-194-3p hindered the progression of CRC cells. Overexpression of miR-194-3p significantly weakened the promoting effect of upregulated KLK10 on cell migration, invasion and proliferation. Their targeting was verified by dual -luciferase assay. Therefore, miR-194-3p hindered cell behaviors of CRC through KLK10. CONCLUSION: This investigation casts new light on the treatment of CRC through the KLK10/miR-194-3p axis.

Human urinary kininogenase reduces the endothelial injury by inhibiting Pyk2/MCU pathway.

The injury of endothelial cells is one of the initiating factors in restenosis after endovascular treatment. Human urinary kallidinogenase (HUK) is a tissue kallikrein which is used for ischemia-reperfusion injury treatment. Studies have shown that HUK may be a potential therapeutic agent to prevent stenosis after vascular injury, however, the precise mechanisms have not been fully established. This study is to investigate whether HUK can protect endothelial cells after balloon injury or H2O2-induced endothelial cell damage through the proline-rich tyrosine kinase 2 (Pyk2)/mitochondrial calcium uniporter (MCU) pathway. Intimal hyperplasia, a decrease of pinocytotic vesicles and cell apoptosis were found in the common carotid artery balloon injury and H2O2-induced endothelial cell damage, Pyk2/MCU was also up-regulated in such pathological process. HUK could prevent these injuries partially via the bradykinin B2 receptor by inhibiting Pyk2/MCU pathway, which prevented the mitochondrial damage, maintained calcium balance, and eventually inhibited cell apoptosis. Furthermore, MCU expression was not markedly increased if Pyk2 was suppressed by shRNA technique in the H2O2 treatment group, and cell viability was significantly better than H2O2-treated only. In short, our results indicate that the Pyk2/MCU pathway is involved in endothelial injury induced by balloon injury or H2O2-induced endothelial cell damage. HUK plays an protective role by inhibiting the Pyk2/MCU pathway in the endothelial injury.

Human urinary kallidinogenase in acute ischemic stroke: A single-arm, multicenter, phase IV study (RESK study).

AIMS: Human urinary kallidinogenase (HUK) has shown favorable efficacies in acute ischemic stroke (AIS) treatment. We sought confirmation of the safety and efficacy of HUK for AIS in a large population. METHODS: RESK study enrolled patients with AIS of anterior circulation to receive HUK infusion. The primary endpoint was the incidence of treatment-emergent adverse events (AEs). Secondary endpoints assessed neurological and functional improvements and stroke recurrent rate. RESULTS: Of 1206 eligible patients, 1202 patients received at least one dose of HUK infusion and 983 (81.5%) completed the study. The incidence of treatment-emergent AEs and serious AEs were 55.99% and 2.41%, respectively. Pre-specified AEs of special interest occurred in 21.71% of patients, but the majority were mild and unrelated to therapy. Hypertension, age, treatment time, and drug combination were identified to be associated with drug-related blood pressure reduction. Neurological and functional evaluations revealed favorable outcomes from baseline to post-treatment assessment. The cumulative recurrence rate of stroke was 2.50% during the 90-day assessment. CONCLUSION: HUK had an acceptable safety and tolerability profile in AIS patients. Besides, HUK demonstrated the neurological and functional improvements in AIS, further confirming its clinical efficacy in a real-world large population.

PSMA-D4 Radioligand for Targeted Therapy of Prostate Cancer: Synthesis, Characteristics and Preliminary Assessment of Biological Properties.

A new PSMA ligand (PSMA-D4) containing the Glu-CO-Lys pharmacophore connected with a new linker system (L-Trp-4-Amc) and chelator DOTA was developed for radiolabeling with therapeutic radionuclides. Herein we describe the synthesis, radiolabeling, and preliminary biological evaluation of the novel PSMA-D4 ligand. Synthesized PSMA-D4 was characterized using TOF-ESI-MS, NMR, and HPLC methods. The novel compound was subject to molecular modeling with GCP-II to compare its binding mode to analogous reference compounds. The radiolabeling efficiency of PSMA-D4 with 177Lu, 90Y, 47Sc, and 225Ac was chromatographically tested. In vitro studies were carried out in PSMA-positive LNCaP tumor cells membranes. The ex vivo tissue distribution profile of the radioligands and Cerenkov luminescence imaging (CLI) was studied in LNCaP tumor-bearing mice. PSMA-D4 was synthesized in 24% yield and purity >97%. The radio complexes were obtained with high yields (>97%) and molar activity ranging from 0.11 to 17.2 GBq mcmol-1, depending on the radionuclide. In vitro assays confirmed high specific binding and affinity for all radiocomplexes. Biodistribution and imaging studies revealed high accumulation in LNCaP tumor xenografts and rapid clearance of radiocomplexes from blood and non-target tissues. These render PSMA-D4 a promising ligand for targeted therapy of prostate cancer (PCa) metastases.

Design, Synthesis, Computational, and Preclinical Evaluation of natTi/45Ti-Labeled Urea-Based Glutamate PSMA Ligand.

Despite promising anti-cancer properties in vitro, all titanium-based pharmaceuticals have failed in vivo. Likewise, no target-specific positron emission tomography (PET) tracer based on the radionuclide 45Ti has been developed, notwithstanding its excellent PET imaging properties. In this contribution, we present liquid-liquid extraction (LLE) in flow-based recovery and the purification of 45Ti, computer-aided design, and the synthesis of a salan-natTi/45Ti-chelidamic acid (CA)-prostate-specific membrane antigen (PSMA) ligand containing the Glu-urea-Lys pharmacophore. The compound showed compromised serum stability, however, no visible PET signal from the PC3+ tumor was seen, while the ex vivo biodistribution measured the tumor accumulation at 1.1% ID/g. The in vivo instability was rationalized in terms of competitive citrate binding followed by Fe(III) transchelation. The strategy to improve the in vivo stability by implementing a unimolecular ligand design is presented.

Effect of kallikrein on microcirculation of rats with pancreatic ischemia reperfusion injury (IRI).

The common pathway for pancreatitis onset is pancreatic ischemia reperfusion injury (IRI), which plays an especially significant role in the evolution process from acute edematous pancreatitis (AP) towards severe acute pancreatitis (SAP). This study explored the effect of Kallikrein (PK) on pancreatic ischemia reperfusion injury (IRI). Male Wistar rats were taken as study objects, and a SAP -IRI combined model was established through retrograde infusion of 5% sodium taurocholate in biliopancreatic duct combining 30 min splenic artery clipping; drug intervention was carried out by pumping PK into rat caudal vein. Pancreatic microcirculation blood flow, pancreatic micro vascular permeability, hemorheological change and levels of adherence factors CD18 and CD54 were determined respectively. PK can obviously improve pancreatic microcirculation blood flow volume and velocity of IRI rats and expand arteriole; expand diameter of pancreatic blood capillary so that perfusion state tends to be stable; decrease pancreatic micro vascular permeability, reduce rat whole blood viscosity, erythrocyte deformation index and rigidity index; SAP-IRI combination reduces expression levels of white cell adhesion factor CD18 and vascular endothelial cell adhesion cell CD54 in rats. In conclusion, PK is an effective method of improving SAP pancreatic IRI microcirculation.

Influences of urinary kallidinogenase on neuronal apoptosis in cerebral infarction rats through Nrf2/ARE oxidative stress pathway.

OBJECTIVE: To investigate the influences of urinary kallidinogenase on neuronal apoptosis in rats with cerebral infarction through the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) oxidative stress pathway. MATERIALS AND METHODS: A total of 30 male rats were divided into group A (model control group), group B (rat model of cerebral infarction) and group C (rat model of cerebral infarction + medical treatment with urinary kallidinogenase). The percentage of cerebral infarct volume and the apoptosis of brain cells in the three groups of rats were detected via 2,3,5-Triphenyltetrazolium chloride (TTC) staining, the pathological morphology of brain tissues in the three groups of rats was observed via hematoxylin and eosin (HE) staining, and the protein levels of Nrf2 and superoxide dismutase 1 (SOD1) in the brain tissues in the three groups of rats were measured using the Western blotting assay. RESULTS: The degree of neurological deficit in group B was remarkably higher than that in group A (p<0.05), and it was markedly decreased in group C compared to that in group B, displaying statistically significant differences (p<0.05). Compared to that in group A, the cell apoptosis was significantly aggravated in group B, while a remarkably alleviated cell apoptosis was observed in group C compared to that of group B, and the differences were statistically significant (p<0.05). The cerebral infarct volume accounted for 34.87% of the whole brain volume in group B, and a mild cerebral infarction was detected in group C, with a percentage of cerebral infarct volume of 21.14%. Group B showed a more evident increase in the cerebral infarct volume than in group C (p<0.05). Compared to those of group A, pyknotic nuclei and neuron staining of brain tissue cells were evidently increased, and the neuronal cell injury was aggravated in group B. Moreover, prominently decreased pyknotic nuclei and neuron staining (p<0.05) as well as mild neuronal cell injury (p<0.05) were detected in group C compared to those in group B. The levels of Nrf2 and SOD1 protein in the brain tissues in group B were remarkably lower than those of group C (p<0.05). CONCLUSIONS: Urinary kallidinogenase can inhibit the neuronal apoptosis in rats and protect the rats from cerebral infarction, whose mechanism is associated with the activation of the Nrf2/ARE oxidative stress pathway.

The kallikrein-like activity of Heloderma venom is inhibited by carbon monoxide.

Lizards in the genus Heloderma are the most ancient venomous reptiles, with a traceable lineage nearly 100 million years old. The proteome of the venom of three of the remaining species (Heloderma suspectum, H. exasperatum, H. horridum) are very conserved, with kallikrein-like activity present to cause critical hypotension to immobilize and outright kill prey. Kallikrein-like activity would be expected to activate the contact protein pathway of coagulation, which would be detectable with thrombelastography in human plasma. Thus, it was proposed to determine if kallikrein-like activity could be detected with thrombelastography, and if this activity could be inhibited by carbon monoxide (CO) via a putative heme-based mechanism. Procoagulant activity of each venom was assessed via thrombelastography with normal plasma, and kallikrein-like activity confirmed with FX-depleted plasma. Venom was then exposed to carbon monoxide releasing molecule-2 (CORM-2) or its inactive releasing molecule to assess CO inhibition. All three venoms demonstrated kallikrein-like activity with the same potency and inhibition of activity by CO. In conclusion, the present work documented that procoagulant, kallikrein-like activity containing venoms of the oldest species of venomous reptiles was inhibited by CO, potentially via heme modulation. This is also the first identification and characterization of a kallikrein-like enzyme utilizing coagulation factor-depleted plasma to assess venom that inflicts hypotension. Future investigations will continue to define the vulnerability of venom enzymatic activities to CO.

Effect of Kallikrein-related Peptidase KLK1 on Ameliorating Spermatogenesis Regeneration in Busulfan-induced Azoospermic Mice and Promoting Mouse Spermatogonial Stem Cell Proliferation In Vitro.

OBJECTIVES: To investigate the effect of kallikrein-related peptidase KLK1 on azoospermic mice induced by busulfan and mouse spermatogonial stem cell. METHODS: Mice were treated with a single intraperitoneal injection of busulfan, and 4 weeks later, they received a daily intraperitoneal injection of KLK1 at different doses for another 4 weeks. Eight weeks after the busulfan treatment, all mice were sacrificed and their testes were collected for histological evaluation, immunostaining and protein extraction. In vitro, immortalized mouse spermatogonial stem cells, namely C18-4 cells, were treated with KLK1 for proliferation assays. RESULTS: Histological evaluation of testes, epididymis and epididymal fluid showed that KLK1-treated mice had better spermatogenesis than the control group. Immunostaining showed that tissue samples from testes of KLK1-treated mice had more PLZF- and SCP3-positive cells per seminiferous tubule as well as more PNA-positive cells in the seminiferous tubules. Western blots revealed higher expression levels of PCNA in KLK1-treated mice than in control mice. C18-4 cells treated with KLK1 had a higher proliferation rate and higher expression levels of PCNA, Cyclin A and Cyclin E, and the level of phosphorylated ERK2 were increased after KLK1 treatment. CONCLUSION: Collectively, KLK1 can improve spermatogenesis in azoospermic mice, and KLK1 can promote the proliferation of mouse spermatogonial stem cells via activating ERK1/2 and cell cycle proteins Cyclin A and Cyclin E. This study could offer novel approach and provide new targets for the treatment of azoospermia.

Pancreatic kininogenase improves erectile function in streptozotocin-induced type 2 diabetic rats with erectile dysfunction.

Erectile dysfunction (ED) associated with type 2 diabetes is a severe problem that requires effective treatment. Pancreatic kininogenase (PK) has the potential to improve the erectile function of ED patients. This study aims to investigate the effect of PK on erectile function in streptozotocin-induced type 2 diabetic ED rats. To achieve this goal, we divided male Sprague-Dawley rats into five groups. One group was not treated, and the other four groups were treated with saline, sildenafil, PK or sildenafil, and PK, respectively, for 4 weeks after the induction of type 2 diabetic ED. Then, intracavernous pressure under cavernous nerve stimulation was measured, and penile tissue was collected for further study. Endothelial nitric oxide synthase levels, smooth muscle content, endothelium content, cyclic guanosine monophosphate (cGMP) levels in the corpus cavernosum, and neuronal nitric oxide synthase levels in the dorsal penile nerve were measured. Improved erectile function and endothelium and smooth muscle content in the corpus cavernosum were observed in diabetic ED rats. When treating diabetic ED rats with PK and sildenafil at the same time, a better therapeutic effect was achieved. These data demonstrate that intraperitoneal injection of PK can improve erectile function in a rat model of type 2 diabetic ED. With further research on specific mechanisms of erectile function improvement, PK may become a novel treatment for diabetic ED.

The Therapeutic Effect of Pancreatic Kininogenase on Treatment of Diabetic Peripheral Neuropathy in Patients with Type 2 Diabetes.

Background: To determine the therapeutic efficacy and cost-effective of pancreatic kininogenase (PKase) on treatment of diabetic peripheral neuropathy (DPN) compared with Prostaglandin E1 (PGE1) in patients with type 2 diabetes. Methods: 104 patients with DPN receiving standard glucose control therapy were randomly assigned into 3 groups: Group-A received PKase treatment, Group-B received PGE1 treatment, and Group-C received only standard glucose control therapy. Michigan neuropathy screening instrument (MNSI) score, neurophysiology examination, and nerve conduction velocity were measured. Results: Standard glucose control therapy significantly reduced hyperglycemia to a similar level in all groups. Questionnaire grading and neurophysiology examination both indicated that no significant difference was found at the end of treatment between Groups -A and -B. Except for the ulnar nerve sensory conduction velocity that was significantly improved in Group-B, the remaining nerve conduction velocity (regardless of sensory or motor nerve conduction velocities) was improved to a similar level in Groups -A and -B. Group-A had significantly reduced questionnaire grading and better improvement in motor nerve conduction velocity of the common peroneal nerve, ulnar nerve, and sensory nerve conduction velocity of the sural nerve as compared with Group-C. However, the medical cost of PKase was only 18.9% of that of PGE1 during one course of treatment. Conclusions: PKase has the similar therapeutic efficacy as PGE1 on treatment of DPN in patients with type 2 diabetes. However, the medical cost of PKase is one fifth of that of PGE1. Thus, PKase is a cost-effective drug for treatment of DPN.

Exogenous kallikrein protects against diabetic nephropathy.

The kallikrein-kinin system has been shown to be involved in the development of diabetic nephropathy, but specific mechanisms are not fully understood. Here, we determined the renal-protective role of exogenous pancreatic kallikrein in diabetic mice and studied potential mechanisms in db/db type 2 diabetic and streptozotocin-induced type 1 diabetic mice. After the onset of diabetes, mice were treated with either pancreatic kallikrein (db/db+kallikrein, streptozotocin+kallikrein) or saline (db/db+saline, streptozotocin+saline) for 16 weeks, while another group of streptozotocin-induced diabetic mice received the same treatment after onset of albuminuria (streptozotocin'+kallikrein, streptozotocin'+saline). Db/m littermates or wild type mice were used as non-diabetic controls. Pancreatic kallikrein had no effects on body weight, blood glucose and blood pressure, but significantly reduced albuminuria among all three groups. Pathological analysis showed that exogenous kallikrein decreased the thickness of the glomerular basement membrane, protected against the effacement of foot process, the loss of endothelial fenestrae, and prevented the loss of podocytes in diabetic mice. Renal fibrosis, inflammation and oxidative stress were reduced in kallikrein-treated mice compared to diabetic controls. The expression of kininogen1, tissue kallikrein, kinin B1 and B2 receptors were all increased in the kallikrein-treated compared to saline-treated mice. Thus, exogenous pancreatic kallikrein both prevented and ameliorated diabetic nephropathy, which may be mediated by activating the kallikrein-kinin system.

The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents.

AIMS: The isolated human umbilical vein is a robust contractile bioassay for ligands of the bradykinin (BK) B2 receptor (B2R), also extendable to B1 receptor (B1R) pharmacology. We hypothesized that, as a freshly isolated vessel, it also contains traces of plasma proteins that may confer responses to exogenous proteases via the formation of kinins. MAIN METHODS: Rings of human umbilical veins were mounted in organ baths containing Krebs buffer maintained at 37°C and purified proteases were introduced in the bathing fluid along with additional drugs/proteins that permit mechanistic analysis of effects. KEY FINDINGS: The previously described contractile response to human recombinant tissue kallikrein (KLK-1, 1-10nM) is not influenced by metabolic inhibitors, suggesting its dependence on a preexisting reservoir of low molecular weight-kininogen (LK). Active plasma kallikrein (apK, ≤5nM) was inactive in fresh tissues, unless high molecular weight-kininogen (HK, 39-197nM) replenishment was applied. The effects of KLK-1 and HK+apK are abolished by pretreating tissues with icatibant, but not with tranexamic acid. C1-esterase inhibitor inhibited only HK+apK. Purified plasmin and neutrophil proteinase-3 produced small contractions in the presence of HK only, and tissue plasminogen activator, none. B1R stimulation was pharmacologically evidenced in response to KLK-1 if LK was supplied. SIGNIFICANCE: The pharmacology of KLK-1 and HK+apK in the human isolated umbilical vein is essentially based on the activity of locally generated kinins and this assay models the inhibitory action of some therapeutic agents active in angioedema states. Proteases that indirectly generate kinins have little activity in the system.

Pancreatic Kininogenase Ameliorates Renal Fibrosis in Streptozotocin Induced-Diabetic Nephropathy Rat.

BACKGROUND/AIMS: We aimed to evaluate whether pancreatic kininogenase (PKase) can relieve renal fibrosis and investigate its mechanisms in diabetic nephropathy (DN) rats Methods: We established streptozotocin (STZ) induced-DN rats. After treatment with PKase for 4 weeks, urinary weight, urinary protein content and blood glucose concentration were detected, and then renal histopathological changes were examined using Hematoxylin and Eosin (H&E) and Masson's thrchrome staining. In addition, the expressions of miR-433, transforming growth factor-ß1 (TGF-ß1) and antizyme inhibitor 1 (Azin1) were detected by qRT-PCR and/or western blotting. RESULTS: PKase reduced urinary weight, urinary protein contents and blood glucose concentrations. PKase treated DN rats exhibited less renal fibrosis than untreated DN rats (P < 0.05). Furthermore, the expression levels of TGF-ß and miR-433 were reduced (P < 0.05), while Azin1 expression was increased in renal tissues of PKase treated DN rats compared with untreated DN rats (P < 0.05). CONCLUSIONS: PKase might not only inhibit the development of DN by reducing urinary weight, urinary protein content and blood glucose concentration in DN rats, but also relieve renal fibrosis in DN rats through inhibiting the expression of TGF-ß1, and miR-433 and Azin1 might involve in this process.

Human urine kininogenase attenuates balloon-induced intimal hyperplasia in rabbit carotid artery through transforming growth factor ß1/Smad2/3 signaling pathway.

OBJECTIVE: Effective treatments against restenosis after percutaneous transluminal angioplasty and stenting are largely lacking. Human tissue kallikrein gene transfer has been shown to be able to attenuate neointima formation induced by balloon catheter. As a tissue kallikrein in vivo, human urinary kininogenase (HUK) is widely used to prevent ischemia-reperfusion injury. However, the effects of HUK on neointima formation have not been explored. We therefore investigated whether HUK could alleviate balloon catheter-induced intimal hyperplasia in rabbits fed with high-fat diets. METHODS: The effects of HUK on neointima and atherosclerosis formation were analyzed by hematoxylin-eosin staining and immunohistochemical staining in balloon-injured carotid arteries of rabbits. Local inflammatory response was evaluated by detecting the gene expression of tumor necrosis factor α and interleukin 1ß with real-time quantitative polymerase chain reaction plus the invasion of macrophages with immunohistochemical staining. Western blotting was employed to investigate the effects of HUK on activities of endothelial nitric oxide synthase (eNOS), transforming growth factor ß1 (TGF-ß1), and Smad signaling pathway. The long-term effect of HUK on intimal hyperplasia of the injured carotid artery was assessed by angiography. RESULTS: Quantitative image analysis showed that intravenous administration of HUK for 14 days significantly decreased the intimal areas and intima area/media area ratios (day 14, 54% decrease in intimal area and 58% decrease in intima area/media area ratios; day 28, 63% and 85%). Significant decreases were also noted in macrophage foam cell-positive area after 7-day or 14-day administration of HUK (day 7, 69% decrease in intimal area and 78% decrease in media area; day 14, 79% and 60%; day 28, 68% and 44%). Actin staining for smooth muscle cells in neointima at 2 months showed similar results (vascular smooth muscle cell-positive area of neointima, 28.21% ± 5.58% vs 43.78% ± 8.36%; P < .05). Real-time quantitative polymerase chain reaction or Western blot analysis showed that HUK reduced expression of tumor necrosis factor α, interleukin 1ß, TGF-ß1, and p-Smad2/3 but increased the expression of p-eNOS. Angiography analysis showed that 14-day administration of HUK significantly decreased the degree of stenosis (26.8% ± 7.1% vs 47.9% ± 5.7%; P < .01) at 2 months after balloon injury. CONCLUSIONS: Our results indicate that HUK is able to attenuate atherosclerosis formation and to inhibit intimal hyperplasia by downregulating TGF-ß1 expression and Smad2/3 phosphorylation, upregulating eNOS activity. HUK may be a potential therapeutic agent to prevent stenosis after vascular injury.