Soon Bae Kim

AMP-activated protein kinase inhibits TGF-β-, angiotensin II-, aldosterone-, high glucose-, and albumin-induced epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a novel mechanism that promotes renal fibrosis. Transforming growth factor-β (TGF-β), angiotensin II, aldosterone, high glucose, and urinary albumin are well-known causes of EMT and renal fibrosis. We examined whether and how activation of AMP-activated protein kinase (AMPK) suppressed EMT induced by the above agents in tubular epithelial cells. All experiments were performed using HK-2 cells. Protein expression was measured by Western blot analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. Exposure of tubular cells to TGF-β (10 ng/ml), angiotensin II (1 μM), aldosterone (100 nM), high glucose (30 mM), and albumin (5 mg/ml) for 5 days induced EMT, as shown by upregulation of α-smooth muscle actin and downregulation of E-cadherin. ROS and NADPH oxidase 4 (Nox4) expression were increased, and antioxidants such as tiron and N-acetylcysteine inhibited EMT induction. Metformin (the best known clinical activator of AMPK) suppressed EMT induction through inhibition of ROS via induction of heme oxygenase-1 and endogenous antioxidant thioredoxin. An AMPK inhibitor (compound C) and AMPK small interfering RNA blocked the effect of metformin, and another AMPK activator [5-aminoimidazole-4-carboxamide-1β riboside (AICAR)] exerted the same effects as metformin. In conclusion, AMPK activation might be beneficial in attenuating the tubulointerstitial fibrosis induced by TGF-β, angiotensin II, aldosterone, high glucose, and urinary albumin.

Exogenous Nitric Oxide Inhibits VCAM-1 Expression in Human Peritoneal Mesothelial Cells

Leukocyte adhesion to mesothelium is an important step during peritonitis, which is mediated by adhesion molecules including vascular cell adhesion molecule-1 (VCAM-1). We investigated the effect of exogenous nitric oxide (NO) on VCAM-1 expression in cultured human peritoneal mesothelial cells and its signal transduction pathway. Mesothelial cells were exposed to tumor necrosis factor-α (TNF-α) in the presence or absence of NO donors, 3-morpholino-sydnonimine (SIN-1) and nitroprusside (NP). VCAM-1 mRNA and protein expression were measured by Northern blot analysis and flow cytometry. Nuclear factor-ĸB (NF-ĸB) binding activity was determined by electrophoretic mobility shift assay. Both SIN-1 and NP inhibited the TNF-α induced VCAM-1 mRNA expression in a dose dependent manner (0.25–2 mM). SIN-1 also suppressed the cell surface expression of VCAM-1 molecule. Furthermore, SIN-1 and NP inhibited the VCAM-1 mRNA expression induced by interleukin-1β or lipopolysaccharide as well. NF-ĸB inhibitor, PDTC dose dependently inhibited the TNF-α induced VCAM-1 mRNA expression. SIN-1 inhibited the TNF-α- induced NF-ĸB binding activity. Analogue of cGMP (8-bromo-cGMP) had no significant effect on TNF-α-induced VCAM-1 mRNA expression and guanylate cyclase inhibitor (ODQ) also had no significant influence on the inhibitory effect of SIN-1. These results suggest that exogenous NO inhibits VCAM-1 expression via suppression of NF-ĸB through a cGMP-independent pathway.

Effects of Hypoxia on the Extracellular Matrix Production of Cultured Rat Mesangial Cells

Glomerular changes in patients with cyanotic congenital heart and chronic lung diseases and in persons living at a high altitude might be related to hypoxemia. This study was carried out to examine the effects of hypoxia on the extracellular matrix production by cultured rat mesangial cells (CRMC). Subconfluent CRMC monolayers were grown with 10% oxygen (hypoxia) and 20% oxygen (control) for 1, 3, and 5 days. The production of type IV collagen (CIV), fibronectin (FN), and laminin (LN) by CRMC was evaluated by flow cytometry and immunofluorescent microscopy. Total RNA was extracted and Northern blotting and hybridization were performed with cDNAs for CIV, FN, and LN. The surface expression of CIV on FC was higher in hypoxia than under control conditions at day 5 (158% of control). The surface expression of FN was also higher in hypoxia at day 3 (303%) and at day 5 (332%). The surface expression of LN was lower at day 1 (71%). Immunofluorescent microscopy showed similar changes with flow cytometry. The mRNA level for CIV and FN was maximal at day 5 with 206 and 305% of control, respectively. Hypoxia had little effect on LN mRNA expression. These results show that hypoxia stimulates the synthesis of extracellular matrix of cultured rat mesangial cells. Hypoxia may contribute to the development of glomerular changes in cyanotic congenital heart diseases, chronic lung diseases, and in persons living at a high altitude.

Annexin-I inhibits PMA-induced c-fos SRE activation by suppressing cytosolic phospholipase A2 signal

Annexin‐I (ANX‐I) is a 37‐kDa protein with a calcium‐dependent phospholipid‐binding property. Previously we have observed the inhibition of cytosolic phospholipase A2 (cPLA2) by ANX‐I in the studies using purified recombinant ANX‐I, and proposed a specific interaction model for the mechanism of cPLA2 inhibition by ANX‐I [Kim et al. (1994) FEBS Lett. 343, 251–255]. Here we have studied the role of ANX‐I in the cPLA2 signaling pathway by transient transfection assay. The stimulation of Rat2 fibroblast cells with phorbol 12‐myristate 13‐acetate (PMA) induced the c‐fos serum response element (SRE). The SRE stimulation by PMA was dramatically reduced by (1) pretreatment with a cPLA2‐specific inhibitor, arachidonyltrifluoromethyl ketone, or (2) co‐transfection with antisense cPLA2 oligonucleotide, indicating that the SRE activation was through cPLA2 activation. Co‐transfection with an ANX‐I expression vector also reduced the SRE stimulation by PMA, suggesting the inhibition of cPLA2 by ANX‐I. The active domain of ANX‐I was mapped using various deletion mutants. ANX‐I(1–113) and ANX‐I(34–346) were fully active, whereas ANX‐I(114–346) abolished the activity. Therefore the activity was in the amino acid 34 to 113 region, which corresponds to the conserved domain I of ANX‐I.

Treatment of hepatitis B virus associated glomerulonephritis with recombinant human alpha interferon

To evaluate the therapeutic effect of recombinant human alpha-interferon (alpha-IFN) on hepatitis B virus associated glomerulonephritis (HBV-GN) and the relationship between the seroconversion of viral antigens and the change of proteinuria, the hepatitis B viral markers and urinary protein were monitored during alpha-IFN treatment in 8 male adult patients who (1) were positive in serum HBsAg and HBeAg, (2) had chronic hepatitis, (3) had persistent proteinuria > 1 g/day, and (4) showed glomerulonephritis on kidney biopsy. alpha-IFN was given at a dose of 3 million units, subcutaneously, three times a week for 6 months. Kidney biopsy specimens showed membranoproliferative glomerulonephritis (MPGN) in 4 patients, mesangial proliferative glomerulonephritis (MesPGN) in 2, and membranous glomerulonephritis (MGN) in 2 patients. Seven of the 8 patients received a 6-month course of alpha-IFN therapy; 1 patient with MGN quitted therapy 2 months after the initial dose because of side effects. In 5 of the 7 patients who received a 6-month therapy, serum HBeAg disappeared, and anti-HBe appeared during the therapy. In 2 of these 5 patients, HBeAg reappeared, in 1 during alpha-IFN therapy and in 1 9 months after the last dose of alpha-IFN. The hepatitis B viral markers of the patient who received a 2-month therapy did not change. HBs antigenemia persisted in all patients. In all 4 patients with MPGN, serum HBeAg was transiently or persistently converted to negative, but the proteinuria persisted. Both patients with MesPGN showed remission of proteinuria; however, only 1 patient had seroconversion of HBeAg. In 2 patients with MGN, proteinuria persisted. In conclusion, alpha-IFN at the doses given was not effective in MPGN type of HBV-GN. Improvement of proteinuria was achieved in MesPGN patients without disappearance of HBs antigenemia which is the finding against the possible role of HBsAg in the pathogenesis of this type of HBV-GN.

Proinflammatory Cytokine-Induced NF-κB Activation in Human Mesangial Cells Is Mediated through Intracellular Calcium but Not ROS: Effects of Silymarin

Background: It is not fully understood whether intracellular calcium and/or reactive oxygen species (ROS) are involved in nuclear factor-ĸB (NF-ĸB) activation by proinflammatory cytokines. Silymarin exhibits anti-inflammatory and antioxidant effects but the effect of silymarin in human mesangial cells is largely unknown. Method: NF-ĸB binding activity was measured by electrophoretic mobility shift assay. Intracellular calcium was monitored by confocal microscopy using Fluo-3 and intracellular ROS production was determined by flow cytometry. Monocyte chemoattractant protein-1 (MCP-1) expression was measured by Northern blot analysis and ELISA. Results: NF-ĸB was activated within 30 min by tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β). Intracellular ROS was not produced until 30 min and also antioxidants such as N-acetylcysteine and tiron had no effect on the TNF-α- or IL-1β-induced NF-ĸB activation. Intracellular calcium was increased by TNF-α and IL-1β. Furthermore, a calcium chelator, BAPTA-AM, attenuated the NF-ĸB activation. Silymarin dose-dependently inhibited the TNF-α- or IL-1β-induced NF-ĸB activation and MCP-1 expression. Silymarin also inhibited TNF-α-induced intracellular calcium. Conclusions: Induction of NF-ĸB within 30 min by TNF-α- and IL-1β was mediated through intracellular calcium but not ROS. Silymarin inhibited TNF-α-induced calcium-dependent NF-ĸB activation irrespective of its antioxidant effect.

Usefulness of Segmental Bioimpedance Ratio to Determine Dry Body Weight in New Hemodialysis Patients: A Pilot Study

Background: The ratio of bioimpedance in the right leg (rl-RBI) may be helpful in adjusting dry body weight (DBW) in new hemodialysis (HD) patients. Methods: rl-RBI was calculated as follows: rl-RBI = impedance at 50 kHz/impedance at 500 kHz, as measured by bioimpedance spectroscopy (BIS). Theoretically, rl-RBI is inversely related to extracellular water. A reference range of rl-RBI was obtained from 137 chronic but stable HD patients already achieving DBW. In 34 new HD patients (females:males = 16:18; age 49 ± 12 years), DBW(s) were stepwise adjusted under the guidance of rl-RBI by modifying the amount of ultrafiltration. Results: The target range of rl-RBI was defined as 1.106–1.150. rl-RBI before the first HD was 1.115 ± 0.027. At the study endpoint, when the target range of rl-RBI was achieved, pretibial pitting edema and pulmonary edema were resolved without any episode of muscle cramping or intradialytic hypotension. Along with an increase in rl-RBI, pre-HD blood pressure tended to decrease at systole (p = 0.072) and diastole (p = 0.005). The cardiothoracic ratio also decreased significantly (p = 0.004). Conclusion: The measurement of rl-RBI by BIS is worthy of further evaluation as an objective and applicable index for determining DBW in new HD patients.

C-Reactive Protein Induces NF-κB Activation through Intracellular Calcium and ROS in Human Mesangial Cells

Background: C-reactive protein (CRP) is known to have a direct proinflammatory effect in endothelial cells. However, little is known about the effect of CRP in intrinsic renal cells. We investigated the effects of CRP on the nuclear factor-ĸB (NF-ĸB) activation and monocyte chemoattractant protein-1 (MCP-1) gene expression in human mesangial cells and also examined whether intracellular calcium and reactive oxygen species (ROS) were involved in the CRP- induced NF-ĸB activation. Methods: NF-ĸB binding activity and MCP-1 mRNA expression were measured by electrophoretic mobility shift assay and Northern blot analysis, respectively.Intracellular calcium was monitored by confocal microscopy using calcium sensitive dye, Fluo-3 and intracellular ROS production was determined, using 2′,7′-dichlorofluorescin diacetate. Results: CRP increased NF-ĸB binding activity in a dose-dependent manner (12.5–100 µg/ml), which was induced within 1 h after incubation and peaked around 3 h. CRP also increased the MCP-1 mRNA expression via activation of NF-ĸB. Both intracellular calcium and ROS was induced by CRP. Calcium chelator, BAPTA-AM and anti-oxidants such as N-acetylcysteine and tiron suppressed CRP-induced NF-ĸB activation. Conclusion: CRP exerted a proinflammatory effect in human mesangial cells by inducing MCP-1 gene expression via NF-ĸB activation, which was mediated, at least in part, through intracellular calcium and ROS.

Experiences with acute kidney injury complicating non-fulminant hepatitis A.

Aim:u2003 To describe the clinical features and to identify factors related to development of acute kidney injury in acute hepatitis A patients.

Desmopressin improves platelet function in uremic patients taking antiplatelet agents who require emergent invasive procedures.

Uremia is associated with platelet dysfunction and can cause a bleeding tendency resulting in a major bleeding event after an invasive procedure or surgery that may be aggravated by antiplatelet agents. We prospectively investigated the potential of desmopressin to improve platelet dysfunction and to lower bleeding risk after emergent invasive procedures in uremic patients taking antiplatelet drugs. Twenty-three patients were enrolled with a mean age of 60.2u2009±u200911.7xa0years. Baseline blood urea nitrogen and creatinine were 70.5u2009±u200929.4 and 10.02u2009±u20094.52xa0mg/dL, respectively. Twenty-one patients took aspirin. All patients were infused with desmopressin before their invasive procedures, which were a central catheter insertion for emergent hemodialysis in 13 patients, percutaneous nephrostomy in 7 patients, and angiography through arm or leg vessels in 3 patients. After desmopressin infusion, both the hematocrit and platelet count were slightly decreased without changes in prothrombin time or activated partial thrombin time. Collagen/epinephrine-closure time was significantly shortened from 252.7u2009±u200940.7 to 144.6u2009±u200951.0xa0s (pu2009<u20090.001). There were minimal bleeding in 20 patients and mild bleeding in 3 patients. None experienced severe bleeding event or required additional intervention for bleeding control. There were no adverse events including the decrease of serum sodium concentration. In conclusion, a single infusion of desmopressin before invasive procedures in uremic patients on antiplatelet drugs appeared to be well tolerated and improved platelet dysfunction measured by collagen/epinephrine-closure time.