Inhibitor of protein kinase N3 suppresses excessive bone resorption in ovariectomized mice.

INTRODUCTION: The long-term inhibition of bone resorption suppresses new bone formation because these processes are coupled during physiological bone remodeling. The development of anti-bone-resorbing agents that do not suppress bone formation is urgently needed. We previously demonstrated that Wnt5a-Ror2 signaling in mature osteoclasts promoted bone-resorbing activity through protein kinase N3 (Pkn3). The p38 MAPK inhibitor SB202190 reportedly inhibited Pkn3 with a low Ki value (0.004 µM). We herein examined the effects of SB202190 on osteoclast differentiation and function in vitro and in vivo. MATERIALS AND METHODS: Bone marrow cells were cultured in the presence of M-csf and GST-Rankl to differentiate into multinucleated osteoclasts. Osteoclasts were treated with increasing concentrations of SB202190. For in vivo study, 10-week-old female mice were subjected to ovariectomy (OVX). OVX mice were intraperitoneally administered with a Pkn3 inhibitor at 2 mg/kg or vehicle for 4 weeks, and bone mass was analyzed by micro-CT. RESULTS: SB202190 suppressed the auto-phosphorylation of Pkn3 in osteoclast cultures. SB202190 significantly inhibited the formation of resorption pits in osteoclast cultures by suppressing actin ring formation. SB202190 reduced c-Src activity in osteoclast cultures without affecting the interaction between Pkn3 and c-Src. A treatment with SB202190 attenuated OVX-induced bone loss without affecting the number of osteoclasts or bone formation by osteoblasts. CONCLUSIONS: Our results showed that Pkn3 has potential as a therapeutic target for bone loss due to increased bone resorption. SB202190 is promising as a lead compound for the development of novel anti-bone-resorbing agents.

Protein Kinase N Family Negatively Regulates Constitutive Androstane Receptor-Mediated Transcriptional Induction of Cytochrome P450 2b10 in the Livers of Mice.

In receptor-type transcription factors-mediated cytochrome P450 (P450) induction, few studies have attempted to clarify the roles of protein kinase N (PKN) in the transcriptional regulation of P450s. This study aimed to examine the involvement of PKN in the transcriptional regulation of P450s by receptor-type transcription factors, including the aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor. The mRNA and protein levels and metabolic activity of P450s in the livers of wild-type (WT) and double-mutant (D) mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations [PKN1 T778A/T778A; PKN3 -/-] were determined after treatment with activators for receptor-type transcription factors. mRNA and protein levels and metabolic activity of CYP2B10 were significantly higher in D mice treated with the CAR activator phenobarbital (PB) but not with 1,4-bis((3,5-dichloropyridin-2-yl)oxy)benzene compared with WT mice. We examined the CAR-dependent pathway regulated by PKN after PB treatment because the extent of CYP2B10 induction in WT and D mice was notably different in response to treatment with different CAR activators. The mRNA levels of Cyp2b10 in primary hepatocytes from WT and D mice treated with PB alone or in combination with Src kinase inhibitor 1 (SKI-1) or U0126 (a mitogen-activated protein kinase inhibitor) were evaluated. Treatment of hepatocytes from D mice with the combination of PB with U0126 but not SKI-1 significantly increased the mRNA levels of Cyp2b10 compared with those from the corresponding WT mice. These findings suggest that PKN may have inhibitory effects on the Src-receptor for activated C kinase 1 (RACK1) pathway in the CAR-mediated induction of Cyp2b10 in mice livers. SIGNIFICANCE STATEMENT: This is the first report of involvement of PKN in the transcriptional regulation of P450s. The elucidation of mechanisms responsible for induction of P450s could help optimize the pharmacotherapy and improve drug development. We examined whether the mRNA and protein levels and activities of P450s were altered in double-mutant mice harboring both PKN1 kinase-negative knock-in and PKN3 knockout mutations. PKN1/3 negatively regulates CAR-mediated induction of Cyp2b10 through phosphorylation of a signaling molecule in the Src-RACK1 pathway.

PKN1 controls the aggregation, spheroid formation, and viability of mouse embryonic fibroblasts in suspension culture.

The role of protein kinase N1 (PKN1) in cell aggregation and spheroid formation was investigated using mouse embryonic fibroblasts (MEFs) deficient in kinase activity caused by a point mutation (T778A) in the activation loop. Wild type (WT) MEFs formed cell aggregates within a few hours in suspension cultures placed in poly-2-hydroxyethylmethacrylate (poly-HEMA) coated flat-bottom dishes. By contrast, PKN1[T778A] (PKN1 T778A/T778A homozygous knock-in) MEFs showed significantly delayed aggregate formation and higher susceptibility to cell death. Video analysis of suspension cultures revealed decreased cell motility and lesser frequency of cell-cell contact in PKN1[T778A] MEFs compared to that in WT MEFs. Aggregate formation of PKN1[T778A] MEFs was compensated by shaking the cell suspension. When cultured in U-shaped ultra-low attachment well plates, initially larger-sized and loosely packed aggregates of WT MEFs underwent compaction resulting in a single round spheroid. On the other hand, image-based quantitative analysis of PKN1[T778A] MEFs revealed irregular compaction with decreased roundness, solidity, and sphericity within 24 h. Flow cytometry of PKN1[T778A] MEFs revealed decreased surface-expression of N-cadherin and integrins α5 and αV. These results suggest that kinase activity of PKN1 controls cell aggregation and spheroid compaction in MEF suspension culture, possibly by regulating the cell migration and cell-surface expression of N-cadherin and integrins.

Inverse J-shaped relation between coronary arterial calcium density and mortality in advanced chronic kidney disease.

BACKGROUND: The coronary artery calcium (CAC) score from cardiac computed tomography (CT) is a composite of CAC volume and CAC density. In the general population, CAC volume is positively and CAC density inversely associated with cardiovascular disease (CVD) events, implying that decreased CAC density reflects atherosclerotic plaque instability. We analysed associations of CAC indices with mortality risk in patients with end-stage renal disease [chronic kidney disease Stage 5 (CKD5)]. METHODS: In 296 CKD5 patients undergoing cardiac CT (median age 55 years, 67% male, 19% diabetes, 133 dialysed), the Framingham risk score (FRS), presence of CVD and protein-energy wasting (PEW; subjective global assessment) and high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) were determined at baseline. During follow-up for a median of 35 months, 51 patients died and 75 patients underwent renal transplantation. All-cause mortality risk was analysed with competing-risk regression models. Vascular calcification was analysed in biopsies of the arteria epigastrica inferior in 111 patients. RESULTS: Patients in the middle tertile of CAC density had the highest CAC score, CAC volume, age, CVD, PEW, FRS, hsCRP and IL-6. In competing risk analysis, the middle {subhazard ratio [sHR] 10.7 [95% confidence interval (CI) 2.0-57.3]} and high [sHR 8.9 (95% CI 1.5-51.8)] tertiles of CAC density associated with increased mortality, independent of CAC volume. The high tertile of CAC volume, independent of CAC density, associated with increased mortality [sHR 8.9 (95% CI 1.5-51.8)]. Arterial media calcification was prominent and associated with CAC volume and CAC density. CONCLUSIONS: In CKD5, mortality increased linearly with higher CAC score and CAC volume whereas for CAC density an inverse J-shaped pattern was observed, with the crude mortality rate being highest for the middle tertile of CAC density. CAC volume and CAC density were associated with the extent of arterial media calcification.

Skin autofluorescence, arterial stiffness and Framingham risk score as predictors of clinical outcome in chronic kidney disease patients: a cohort study.

BACKGROUND: The risk of cardiovascular disease (CVD) is predicted by Framingham's CVD risk scores (FRS) but the high CVD-related mortality in patients with chronic kidney disease (CKD) is only partially explained by traditional CVD risk markers. Therefore, there is a need to explore whether other CVD risk markers may improve risk prediction. Although arterial stiffness measured by augmentation index (AIx) and tissue content of advanced glycation end-products (AGEs) measured by skin autofluorescence (SAF) are two biomarkers that associate with CVD and mortality in CKD, it is not known how they compare with FRS. We evaluated associations between SAF, AIx and FRS, and their associations with CVD and mortality in CKD patients. METHODS: SAF (AGE Reader) and AIx (SphygmoCor; adjusted for 75 heart beats per minute) were measured in 261 clinically stable and extensively phenotyped patients with CKD Stage 5 (median age 56 years, 66% male, 20% diabetes; 130 non-dialysed, 93 patients on peritoneal dialysis and 38 patients on haemodialysis). Multivariate receiver operator characteristics (ROC) curve analysis and multivariate Cox models followed by C-statistics were used to evaluate CVD-related and all-cause mortality risk associated with SAF, AIx and FRS during follow-up for median 25 months with 46 deaths. RESULTS: In multivariate regression analysis, SAF associated with FRS, haemoglobin, fat body mass index and CVD, and inversely with per cent handgrip strength (HGS). AIx associated with FRS, and inversely with per cent HGS. Associations of SAF and AIx with high-sensitivity C-reactive protein (hsCRP), serum albumin, statin therapy and renal replacement therapy were not statistically significant. In ROC analysis, area under the curve (AUC) for CVD mortality ranged from AUC = 0.72 (AIx and FRS, respectively) to AUC = 0.78 (FRS + AIx), and for all-cause mortality from AUC = 0.70 (AIx) to AUC = 0.79 (FRS + AIx). In multivariate Cox analysis, after adjusting for 1-standard deviation (1-SD) of FRS, 1-SD increase of SAF associated with all-cause mortality and 1-SD increase of AIx associated with CVD mortality and all-cause mortality. After further adjustments for hsCRP, albumin and presence of CVD, AIx (but not SAF) remained independently associated with CVD mortality, hazard ratio (HR) 2.14 [95% confidence interval (95% CI) 1.18-3.89] and all-cause mortality, HR 1.74 (95% CI 1.16-2.60). CONCLUSIONS: In patients with CKD Stage 5, SAF and aortic stiffness associated with mortality, independently of FRS. After adjusting for additional confounders including inflammation, aortic stiffness remained as an independent predictor of outcome. Since the contribution of SAF and aortic stiffness compared with FRS in ROC curve analysis was relatively modest, this underlines the importance of traditional CVD risk factors in CKD.

PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts.

PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2-/- embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2-/- embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2-/- phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.

Serum albumin, inflammation, and nutrition in end-stage renal disease: C-reactive protein is needed for optimal assessment.

Low serum albumin (S-Alb) is a frequent feature of end-stage renal disease (ESRD) that independently predicts mortality. Serum albumin has mainly been considered a biomarker of visceral protein and immunocompetence status, fundamental to nutritional assessment. However, low S-albumin level is associated with persistent systemic inflammation and many bodies of evidence show that S-Alb has a limited role as a marker of nutritional status. We reported that a low S-Alb concentration was an independent risk factor for poor outcome in ESRD only in the presence of systemic inflammation. Moreover, the relationships between inflammatory biomarkers and outcome are confounded also by alterations in body composition (such as obese sarcopenia) and oxidative stress. Taken together, S-Alb alone should not be used as a proxy of the nutritional status in a dialysis patient. Its association with dietary intake is poor and low S-Alb values are most often non-nutritional in origin. When analyzing S-Alb to predict mortality risk in ESRD, it should always be combined with measurement of hsCRP.

Lung Dysfunction and Mortality in Patients with Chronic Kidney Disease.

BACKGROUND/AIMS: Lung dysfunction associates with increased mortality but the impact of chronic kidney disease (CKD) is less clear. We evaluated lung function and its association with mortality among individuals with normal to severely reduced glomerular filtration rate (GFR). METHODS: 404 individuals representing GFR category G1 (n=31; GFR >90 mL/min/1.73 m2), G2 (n=46), G3 (n=33), G4 (n=49) and G5 (n=245; GFR< 15 mL/min/1.73 m2) underwent spirometry yielding lung function indices forced vital capacity (FVC), forced expiratory volume in the first second (FEV1) and peak expiratory flow (PEF). Associations of lung function indices expressed as percentages of predicted values (%FEV1, %FVC and %PEF) with 5-year mortality were analyzed by competing-risk regression models. RESULTS: The prevalence of obstructive (6% in G1 and 11% in G5) and especially restrictive (9% in G1 to 36% in G5) lung dysfunction increased with declining GFR and with higher comorbidity burden. In patients (n=22) with protein-energy wasting, inflammation and cardiovascular disease, the prevalence of restrictive lung function was 64%. The highest tertiles of % FEV1 and %FVC associated with lower sub-hazard ratios (sHR) for all-cause mortality, 0.49 (95% CI, 0.27-0.88)) and 0.56 (95% CI, 0.32-0.98), and that of %FEV1 also with lower cardiovascular mortality risk (sHR 0.16; 95%CI 0.04-0.69) after adjusting for multiple confounders. Restrictive lung dysfunction (FEV1/FVC ≥ 0.70, and %FVC < 80) associated with increased mortality risk (sHR 1.80, 95%CI, 1.04-3.13) while the association with obstructive lung impairment was not statistically significant. CONCLUSION: Lung dysfunction and in particular restrictive lung dysfunction associates with degree of renal function impairment and presence of comorbidities, and is an independent predictor of increased mortality in CKD patients.

S6 kinase phosphorylated at T229 is involved in tau and actin pathologies in Alzheimer's disease.

The 70-kDa ribosomal protein S6 kinase (S6K), a serine/threonine kinase that modulates the phosphorylation of the 40S ribosomal protein S6, regulates cell cycle progression and is known as a tau kinase in Alzheimer's disease (AD). In AD brains, neurofibrillary tangles (NFTs) have been shown to be positively stained with antibodies against S6K proteins phosphorylated at T389 (pT389-S6K) or T421/S424 (pT421/S424-S6K) by the mammalian target of rapamycin and mitogen-activated protein kinase pathways, respectively. However, there is little information available about S6K proteins directly phosphorylated at T229 (pT229-S6K) by the PI3K-PDK1 pathway. In the present study, we investigated the distribution of pT229-S6K in post mortem human brain tissues from elderly (control) and patients with AD using immnunoblotting and immunohistochemistry. pT229-S6K immunoreactivity was localized to small granular structures in neurons and endothelial cells in control and AD brains. In AD brains, intense pT229-S6K immunoreactivity was detected in 16.3% of AT8-positive NFTs, neuropil threads, and dystrophic neurites in the hippocampus and other vulnerable brain areas. In addition, Hirano bodies were also positive for pT229-S6K but were negative for pT389-S6K or pT421/S424-S6K. The present results indicate that S6K phosphorylation via the PI3K-PD1 pathway is involved in tau pathology in NFTs and abnormal neurites as well as actin pathology in Hirano bodies.

Turning off of GluN2B subunits and turning on of CICR in hippocampal LTD induction after developmental GluN2 subunit switch.

NMDA receptors (NMDARs) are essential for the induction of synaptic plasticity that mediates activity-dependent refinement of neural circuits during development. GluN2B subunits of NMDARs are abundant at synapses in the immature hippocampus and begin to be replaced by GluN2A subunits with the help of casein kinase 2 activity in the second postnatal week, the critical period for the GluN2 subunit switch (Sanz-Clemente et al. (2000) Neuron 67:984-996). However, the physiological role of GluN2B subunits in the hippocampus during this critical period has not been elucidated. Here, we report that GluN2B subunits mediate the induction of long-term depression (LTD) in the CA1 region of the hippocampus only until this period. Ifenprodil and Ro25-6981, selective inhibitors of NMDARs containing GluN2B subunits, blocked LTD in postnatal Day 11-14 (P11-14) rat hippocampal slices but not in P18-22 hippocampus. Just a few days after P14, synaptic NMDAR currents became narrower than those at P11-14, and calcium influx through NMDARs must be reduced. We found that calcium-induced calcium release (CICR) through ryanodine receptors starts to support the induction of NMDAR-dependent LTD at P18-22. Intracellular application of thapsigargin and ryanodine, inhibitors of Ca2+ -ATP pumps on internal stores and ryanodine receptors, respectively, did not at all affect LTD in the hippocampus at P11-14 but completely blocked LTD in the P18-22 hippocampus. Therefore, calcium influx through NMDAR with GluN2B subunits is sufficient to induce LTD at P11-14, after which CICR compensates for the decrease in calcium influx during LTD induction.

Protein kinase N1 is a novel substrate of NFATc1-mediated cyclin D1-CDK6 activity and modulates vascular smooth muscle cell division and migration leading to inward blood vessel wall remodeling.

Toward understanding the mechanisms of vascular wall remodeling, here we have studied the role of NFATc1 in MCP-1-induced human aortic smooth muscle cell (HASMC) growth and migration and injury-induced rat aortic wall remodeling. We have identified PKN1 as a novel downstream target of NFATc1-cyclin D1/CDK6 activity in mediating vascular wall remodeling following injury. MCP-1, a potent chemoattractant protein, besides enhancing HASMC motility, also induced its growth, and these effects require NFATc1-dependent cyclin D1 expression and CDK4/6 activity. In addition, MCP-1 induced PKN1 activation in a sustained and NFATc1-cyclin D1/CDK6-dependent manner. Furthermore, PKN1 activation is required for MCP-1-induced HASMC growth and migration. Balloon injury induced PKN1 activation in NFAT-dependent manner and pharmacological or dominant negative mutant-mediated blockade of PKN1 function or siRNA-mediated down-regulation of its levels substantially suppressed balloon injury-induced smooth muscle cell migration and proliferation resulting in reduced neointima formation. These novel findings suggest that PKN1 plays a critical role in vascular wall remodeling, and therefore, it could be a promising new target for the next generation of drugs for vascular diseases, particularly restenosis following angioplasty, stent implantation, or vein grafting.

Removal of calciprotein particles from the blood using an adsorption column improves prognosis of hemodialysis miniature pigs.

Hyperphosphatemia is a major risk for poor prognosis in patients with end-stage renal disease. However, the molecular mechanism behind this link remains elusive. We and others have demonstrated that serum phosphorus levels correlate positively with circulating levels of calciprotein particles (CPPs). CPPs are colloidal mineral-protein complexes containing insoluble calcium-phosphate precipitates and have been reported to induce calcification in cultured vascular smooth muscle cells and inflammatory responses in cultured macrophages. Hence, we hypothesize that CPPs may be responsible for disorders associated with hyperphosphatemia. Using hyperphosphatemic miniature pigs receiving hemodialysis, here we show that removal of CPPs from the blood with a newly developed CPP adsorption column improves survival and alleviates complications including coronary artery calcification, vascular endothelial dysfunction, metastatic pulmonary calcification, left ventricular hypertrophy, and chronic inflammation. The present study identifies CPPs as an effective therapeutic target and justifies clinical trials to determine whether the CPP adsorption column may be useful as a medical device for improving clinical outcomes of hemodialysis patients.

Repeated Epstein-Barr virus-associated hemophagocytic syndrome in a lupus nephritis patient.

A 55-year-old Japanese female was diagnosed with systemic lupus erythematosus (SLE) and developed nephrotic syndrome. She was diagnosed with lupus nephritis by a percutaneous renal biopsy. She was treated with intravenous steroid pulse therapy twice, but it proved to be ineffective. She achieved a complete remission after intravenous cyclophosphamide pulse (CPAIV) therapy. Thereafter, her lupus nephritis was well controlled and demonstrated only a low activity. However, she suffered Epstein- Barr virus (EBV)-associated hemophagocytic syndrome (HPS) twice, and in each case she was treated with anticancer drugs and achieved a complete remission. This was a rare case of lupus nephritis who showed repeated EBV-associated HPS.

Development of Wernicke's encephalopathy during initiation of hemodialysis in an elderly non-alcoholic patient.

A 79-year-old man with chronic renal failure developed general fatigue and loss of appetite. He was diagnosed with endstage renal disease and was started on hemodialysis (HD). The symptoms improved immediately, but the mental status deteriorated gradually, reaching Glasgow Coma Scale (GCS) 5. Computed tomography showed no significant intracranial lesion, but magnetic resonance images showed symmetric high-intensity changes in the periaqueductal area, suggestive of Wernicke's encephalopathy (WE). He was immediately treated with intravenous infusion of thiamine. Five days later, the mental status level improved up to GCS 14, and the above MRI findings disappeared. To our knowledge, this is the first report describing the clinical outcome of a non-alcoholic patient who developed WE during initiation of HD. WE should be suspected in patients who are on chronic HD as well as those on initiation of HD with unexplained neurological abnormalities.

The effects for inflammatory responses by CPP with different colloidal properties in hemodialysis patients.

Calciprotein particles (CPPs) are colloids composed of solid-phase calcium-phosphate and serum protein fetuin-A. CPPs form a polydispersed system with different particle size and density. CPPs with specific physical properties can induce calcification and innate immune responses in cultured cells. In hemodialysis patients, blood CPP levels were reported to correlate with vascular calcification and inflammation. However, little is known about relation between these disorders and physical properties of CPPs. Here, we show that the association between physical properties of plasma CPPs and serum levels of inflammatory cytokines/chemokines in 78 hemodialysis out-patients by cross-sectional study. Patients with cardiovascular disease (CVD) had significantly higher high density CPP (H-CPP) levels than patients without CVD but not low density CPP (L-CPP). Seven cytokines/chemokines (EGF, eotaxin, IL-8, IP-10, MCP-1, MIP-1, MIP-1ß and TNFα) were detectable in the serum samples from > 95% of the patients. In multivariate regression analysis, H-CPP was positively associated with eotaxin after adjusting for age, gender, smoking, serum phosphate and FGF23. L-CPP was negatively associated with IL-8 after adjusting for age, gender, serum albumin, phosphate and FGF23. High H-CPP levels were associated with pro-inflammatory response, whereas L-CPPs were associated with anti-inflammatory response. CPPs with different physical properties may impact differently on pathophysiology in HD patients.

Hypotonic swelling-induced activation of PKN1 mediates cell survival in cardiac myocytes.

Hypotonic cell swelling in the myocardium is induced by pathological conditions, including ischemia-reperfusion, and affects the activities of ion transporters/channels and gene expression. However, the signaling mechanism activated by hypotonic stress (HS) is not fully understood in cardiac myocytes. A specialized protein kinase cascade, consisting of Pkc1 and MAPKs, is activated by HS in yeast. Here, we demonstrate that protein kinase N1 (PKN1), a serine/threonine protein kinase and a homolog of Pkc1, is activated by HS (67% osmolarity) within 5 min and reaches peak activity at 60 min in cardiac myocytes. Activation of PKN1 by HS was accompanied by Thr(774) phosphorylation and concomitant activation of PDK1, a potential upstream regulator of PKN1. HS also activated RhoA, thereby increasing interactions between PKN1 and RhoA. PP1 (10(-5) M), a selective Src family tyrosine kinase inhibitor, significantly suppressed HS-induced activation of RhoA and PKN1. Constitutively active PKN1 significantly increased the transcriptional activity of Elk1-GAL4, an effect that was inhibited by dominant negative MEK. Overexpression of PKN1 significantly increased ERK phosphorylation, whereas downregulation of PKN1 inhibited HS-induced ERK phosphorylation. Downregulation of PKN1 and inhibition of ERK by U-0126 both significantly inhibited the survival of cardiac myocytes in the presence of HS. These results suggest that a signaling cascade, consisting of Src, RhoA, PKN1, and ERK, is activated by HS, thereby promoting cardiac myocyte survival.

Left ventricular mass index is an independent determinant of diastolic dysfunction in patients on chronic hemodialysis: a tissue Doppler imaging study.

BACKGROUND: Diastolic heart failure is the most common clinical form of heart failure. Tissue Doppler imaging (TDI) is often used to quantitate left ventricular (LV) diastolic function. The purpose of this study was to identify the determinant(s) of diastolic dysfunction in patients with end-stage renal disease on hemodialysis (HD), using the TDI method. METHODS: The study subjects were 53 patients with end-stage renal disease and preserved LV systolic function on maintenance HD. LV function was assessed by conventional echocardiography. The ratio of early trans-mitral flow velocity to early mitral annular velocity (E/e') was measured by TDI. Patients were stratified into two groups based on E/e' value (≤15 and >15 groups). Arterial stiffness was evaluated by pulse wave velocity and cardio-ankle vascular index. RESULTS: Patients of the E/e' >15 group were older (p = 0.025). There were no significant differences in blood pressure, ejection fraction, E/A, deceleration time, and pulse wave velocity between the E/e' >15 and E/e' ≤15 groups. However, there were significant differences in LV mass index (LVMI; p < 0.001) and cardio-ankle vascular index (p = 0.048) between the two groups. Multiple regression analysis identified that LVMI was an independent determinant of E/e' (p = 0.003). CONCLUSIONS: Our findings suggest that LVMI is an independent determinant of LV diastolic dysfunction in patients on HD.

PKN2 is involved in aggregation and spheroid formation of fibroblasts in suspension culture by regulating cell motility and N-cadherin expression.

The role of Protein Kinase N2 (PKN2, also known as PRK2/PKNγ) in cell aggregate/spheroid formation in suspension culture was investigated using immortalized fibroblasts established from PKN2 flox/flox mouse embryos. PKN2 flox/flox cells formed cell aggregates in flat bottom low attachment well plates, such as 2% agar and poly-2-hydroxyethymethacrylate coated plates, however, Cre;PKN2 flox/flox cells in which PKN2 was depleted by the introduction of Cre-recombinase rarely formed aggregates. Time-lapse analysis revealed that the velocity of Cre;PKN2 flox/flox cell motility was significantly lower than that of PKN2 flox/flox in a low attachment flat-bottom plate, which likely resulted in a lower cell-cell contact frequency among Cre;PKN2 flox/flox cells. Conversely, Cre;PKN2 flox/flox cells could form initial cell aggregates in U-bottom low attachment well plates, however, the succeeding compaction process was delayed in Cre;PKN2 flox/flox cells with decreased roundness, although PKN2 flox/flox cells underwent compaction in a round shape spheroid within 24 h. Immunoblot analysis revealed that the preparation of the cell suspension from adherent conditions using trypsin/EDTA treatment significantly decreased the expression of N-cadherin in both PKN2 flox/flox and Cre;PKN2 flox/flox cells. The N-cadherin expression level recovered time-dependently; however, the recovery of N-cadherin was significantly delayed in Cre;PKN2 flox/flox cells compared to PKN2 flox/flox cells. Reverse transcription quantitative PCR revealed that N-cadherin mRNA in Cre;PKN2 flox/flox cells was significantly lower than that of PKN2 flox/flox cells. These results suggest that PKN2 controls the velocity of cell motility and the transcription of N-cadherin in fibroblasts, leading to cell aggregation and compaction for spheroid formation in suspension culture.

Development of an intracellularly acting inhibitory peptide selective for PKN.

PKNs form a subfamily of the AGC serine/threonine protein kinases, and have a catalytic domain homologous with that of PKC (protein kinase C) in the C-terminal region and three characteristic ACC (antiparallel coiled-coil) domain repeats in the N-terminal region. The preferred peptide phosphorylation motif for PKNs determined by a combinatorial peptide library method was highly similar to that of PKCs within a 10-amino-acid stretch. Previously reported PKN inhibitory compounds also inhibit PKCs to a similar extent, and no PKN selective inhibitors have been commercially available. We have identified a 15-amino-acid peptide inhibitor of PKNs based on amino acids 485-499 of the C-terminal region of the C2-like domain of PKN1. This peptide, designated as PRL, selectively inhibits the kinase activity of all isoforms of PKN (Ki=0.7 muM) towards a peptide substrate, as well as autophosphorylation activity of PKN in vitro, in contrast with PKC. Reversible conjugation by a disulfide bond of a carrier peptide bearing a penetration accelerating sequence to PRL, facilitated the cellular uptake of this peptide and significantly inhibited phosphorylation of tau by PKN1 at the PKN1-specific phosphorylation site in vivo. This peptide may serve as a valuable tool for investigating PKN activation and PKN-mediated responses.

Inter-leg systolic blood pressure difference predicts cardiovascular events and mortality in incident hemodialysis patients.

BACKGROUND: High inter-arm blood pressure difference, a marker of vascular disease, may be difficult to assess in hemodialysis patients with arm arteriovenous fistulae. We investigated if high inter-leg systolic blood pressure difference associates with cardiovascular events and increased mortality in hemodialysis patients. METHODS: Among 118 incident Japanese dialysis patients, bilateral leg blood pressure, arm blood pressure, brachial-ankle pulse wave velocity, and ankle-brachial index were measured, and the relative risk associated with inter-leg systolic blood pressure difference and other indices of vascular status was analyzed. RESULTS: During follow-up (median, 46 months), 18 deaths and 75 cardiovascular events occurred in 38 patients. Kaplan-Meier curves showed that higher inter-leg systolic blood pressure difference was associated with overall (log-rank 9.35, p = 0.002) and cardiovascular (log-rank 5.81, p = 0.02) mortality. The period from the start of dialysis therapy to the first cardiovascular event was shorter as inter-leg systolic blood pressure difference increased (log-rank 23.7, p < 0.0001). In Cox hazard models, inter-leg systolic blood pressure difference greater than median independently predicted deaths (relative risk, 3.8; 95% confidence interval, 1.3-13.9) and cardiovascular events (relative risk, 3.9; 95% confidence interval, 1.9-9.21) after adjustments for age, sex, nutritional status, and diabetes, whereas other indices were not related to the risks. For well-nourished, moderately malnourished, and severely malnourished patients, the cumulative number of cardiovascular events in the high-inter-leg systolic blood pressure difference patients were 4.96, 31.44, and 55.18 events per 100 patient-years. CONCLUSIONS: Higher inter-leg systolic blood pressure difference associated with increased risk of mortality and cardiovascular events suggesting that wider application of inter-leg systolic blood pressure difference measurements may be warranted in hemodialysis patients.