Seonghun Kim

The monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is involved in peritoneal dialysis-related epithelial–mesenchymal transition of peritoneal mesothelial cells

Epithelial–mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) has a role in the process of peritoneal fibrosis (PF), a serious complication in peritoneal dialysis (PD) patients. Even though monocyte chemoattractant protein-1 (MCP-1) was demonstrated to directly increase extracellular matrix (ECM) synthesis, the role of the MCP-1/CCR2 system in PD-related EMT and ECM synthesis in cultured human PMCs (HPMCs) and in an animal model of PD has never been elucidated. In vitro, HPMCs were exposed to 5.6 mM glucose (NG), NG+MCP-1 (10 ng/ml) (NG+MCP-1), or 100 mM glucose (HG) with or without CCR2 inhibitor (RS102895) (CCR2i) or a dominant-negative mutant MCP-1-expressing lentivirus (LV-mMCP-1). In vivo, PD catheters were inserted into 60 Sprague-Dawley rats, and saline (Control, C) (N=30) or 4.25% PD solution (PD) (N=30) was infused for 4 weeks. Twenty rats from each group were treated with empty LV or LV-mMCP-1 intraperitoneally. Snail, E-cadherin, α-smooth muscle actin (α-SMA), and fibronectin protein expression in HPMCs and the peritoneum was evaluated by western blot analysis. Compared with NG cells, Snail, α-SMA, and fibronectin expression was significantly increased, while E-cadherin expression was significantly decreased in HPMCs exposed to HG and NG+MCP-1, and these changes were significantly abrogated by CCR2i (P<0.05). In addition, MCP-1-induced EMT was significantly attenuated by anti-TGF-β1 antibody. In PD rats, Snail and fibronectin expression was significantly increased in the peritoneum, whereas the ratios of E-cadherin/α-SMA protein expression were significantly decreased (P<0.05). The thickness of the peritoneum and the intensity of Masson’s trichrome staining in the peritoneum were also significantly higher in PD rats than in C rats (P<0.05). These changes in PD rats were significantly abrogated by LV-mMCP-1. These findings suggest that the MCP-1/CCR2 system is directly involved in PD-related EMT and ECM synthesis and that this is mediated, at least in part, via TGF-β1.

Graft Selection in Anterior Cruciate Ligament Reconstruction for Smoking Patients

Background: There has been no previous study regarding graft selection in anterior cruciate ligament (ACL) reconstruction for smoking patients. Purpose: To compare the clinical outcomes of ACL reconstruction between smokers and nonsmokers and to find an optimal graft in ACL reconstruction with regard to clinical outcomes for smoking patients. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 487 patients who underwent unilateral ACL reconstruction were retrospectively reviewed. Included patients were divided into 2 groups according to their history of smoking. Group 1 was composed of patients who had never smoked (n = 322), and group 2 consisted of patients who had reported smoking before ACL reconstruction and during rehabilitation (n = 165). Additionally, each group was divided into 4 subgroups according to the selected graft type (bone–patellar tendon–bone autograft, hamstring [semitendinosus-gracilis] tendon autograft, quadriceps tendon–bone autograft, or Achilles tendon–bone allograft). Patients were assessed for knee instability with the Lachman and pivot-shift tests as well as anterior translation measured by the KT-2000 arthrometer. Functional outcomes were evaluated with the Lysholm knee score, International Knee Documentation Committee (IKDC) subjective score, and IKDC objective grade. Results: The minimum follow-up period was 24 months. At the final follow-up evaluation, there were significant mean between-group differences regarding the side-to-side difference in anterior translation (group 1, 2.15 ± 1.11 mm; group 2, 2.88 ± 1.38 mm; P < .001), Lysholm knee score (group 1, 90.25 ± 6.18; group 2, 84.79 ± 6.67; P < .001), IKDC subjective score (group 1, 89.16 ± 5.01; group 2, 83.60 ± 7.48; P < .001), and IKDC objective grade (group 1, grade A = 151, B = 130, C = 36, D = 5 patients; group 2, grade A = 48, B = 71, C = 37, D = 9 patients; P < .001). With regard to differences in outcomes between the selected grafts within each group, the Achilles tendon–bone allograft showed the worst outcomes, with statistically significant mean differences for smoking patients in the side-to-side difference in anterior translation (3.59 ± 1.26 mm), Lysholm knee score (81.05 ± 2.82), and IKDC subjective score (79.73 ± 4.29). Conclusion: Unsatisfactory outcomes with regard to stability and functional scores were shown in the smoking group compared with the nonsmoking group. In smokers, the patients receiving an Achilles tendon–bone allograft had poorer outcomes than those with autografts. The bone–patellar tendon–bone autograft is recommendable for ACL reconstruction in a smoking patient.

Clinical Outcomes for Reconstruction of the Posterolateral Corner and Posterior Cruciate Ligament in Injuries With Mild Grade 2 or Less Posterior Translation Comparison With Isolated Posterolateral Corner Reconstruction

Background: There have been no previous studies showing clinical outcomes according to treatment options of posterior cruciate ligament (PCL) injury with mild grade 2 or less posterior translation (<7 mm) combined with posterolateral rotatory instability. Purpose: To compare the clinical outcomes of posterolateral corner (PLC) reconstruction with or without simultaneous PCL reconstruction in PCL injuries with mild posterior translation. Study Design: Cohort study; Level of evidence, 3. Methods: A total of 46 patients with a PCL injury with mild posterior translation combined with posterolateral rotatory instability were retrospectively reviewed. Twenty-two patients had undergone isolated PLC reconstruction (group A), and 24 patients had undergone simultaneous reconstruction of the PCL and PLC (group B). Each patient was assessed for knee instability with the dial test at 30° and 90° as well as with varus and posterior stress radiography and were evaluated with the Lysholm knee score and International Knee Documentation Committee (IKDC) subjective and objective grading. Results: In all cases, the minimum follow-up period was 24 months. At the final follow-up evaluation, no significant side-to-side difference was found on varus stress radiography (group A, 1.55 ± 0.78 mm vs group B, 1.35 ± 1.00 mm; P = .458) or the dial test (at 30°: group A, 4.00° ± 1.83° vs group B, 4.04° ± 1.30°; P = .929; at 90°: group A, 3.64° ± 1.18° vs group B, 3.67° ± 1.37°; P = .937). However, group B showed a significant improvement compared with group A on posterior stress radiography (group A, 0.16 ± 0.44 mm vs group B, –1.44 ± 0.74 mm; P < .001), Lysholm knee score (group A, 18.36 ± 8.73 vs group B, 23.42 ± 7.44; P = .040), IKDC subjective score (group A, 25.51 ± 7.11 vs group B, 33.08 ± 5.89; P < .001), and IKDC objective score (group A preoperatively: grade C = 19 patients, grade D = 3; group B preoperatively: grade C = 20, grade D = 4; group A postoperatively: grade B = 11, grade C = 11; group B postoperatively: grade A = 12, grade B = 9, grade C = 3) (P < .001). Conclusion: Simultaneous reconstruction of the PCL and PLC is recommended when addressing PCL injuries with mild grade 2 or less posterior translation combined with posterolateral rotary instability.

Effect of Cigarette Smoking on the Clinical Outcomes of ACL Reconstruction

BACKGROUND We compared the clinical outcomes of nonsmokers, current smokers, and former smokers following reconstruction of the anterior cruciate ligament (ACL) and investigated the association between the amount of smoking and outcomes following ACL reconstruction. METHODS We retrospectively reviewed the records of 251 patients who underwent unilateral ACL reconstruction with use of bone-patellar tendon-bone autograft between January 2002 and August 2009. Patients were divided into three groups according to smoking history: Group 1, nonsmokers; Group 2, current smokers; and Group 3, former smokers. Preoperative values and twenty-four-month postoperative findings were compared among the groups. The stability of the ACL was evaluated with use of the Lachman test and the pivot-shift test, and anterior translation was tested with a KT2000 arthrometer. Functional outcomes were assessed on the basis of the Lysholm score and the International Knee Documentation Committee (IKDC) subjective score and objective grade. RESULTS The three groups differed significantly in terms of postoperative knee translation, Lysholm score, and IKDC subjective score. The mean side-to-side difference in anterior translation (and standard deviation) was 2.08 ± 1.08 mm in Group 1 (nonsmokers), 2.65 ± 1.31 mm in Group 2 (smokers), and 2.15 ± 1.05 mm in Group 3 (former smokers) (p = 0.003). The mean Lysholm score was 90.5 ± 6.5 in Group 1, 86.0 ± 7.1 in Group 2, and 89.8 ± 6.3 in Group 3 (p < 0.001). The mean IKDC subjective score was 89.3 ± 5.1 in Group 1, 84.9 ± 7.5 in Group 2, and 88.5 ± 4.2 in Group 3 (p < 0.001). However, the difference in the IKDC subjective score among the three groups did not demonstrate a minimal clinically important difference. A dose-dependent association was noted between pack-years of exposure and postoperative anterior translation (estimate, 0.039; p = 0.015) and IKDC objective grade (odds ratio, 1.083; p = 0.002). A comparison of the three subgroups of smokers showed a significant difference in anterior translation (a mean side-to-side difference in anterior translation of 2.31 ± 1.17 mm for the light smokers, 2.60 ± 1.14 mm for the moderate smokers, and 3.29 ± 1.55 mm for the heavy smokers; p = 0.038). The three subgroups also differed significantly in terms of the proportion of cases by IKDC objective grade; among the light smokers, thirteen (42%) were grade A, fifteen (48%) were grade B, two (7%) were grade C, and one (3%) was grade D; among the moderate smokers, seven (35%) were grade A, eight (40%) were grade B, four (20%) were grade C, and one (5%) was grade D; and among the heavy smokers, one (6%) was grade A, eight (44%) were grade B, eight (44%) were grade C, and one (6%) was grade D (p = 0.013). CONCLUSIONS Cigarette smoking appeared to have a negative effect on subjective and objective outcomes of ACL reconstruction, and heavy smokers showed greater knee instability. Patients who had stopped smoking at least one month prior to ACL reconstruction had no significant difference in outcomes compared with patients who had never smoked. LEVEL OF EVIDENCE Prognostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Does Anterior Laxity of the Uninjured Knee Influence Clinical Outcomes of ACL Reconstruction

BACKGROUND The purpose of this study was to evaluate the association between the postoperative outcomes of anterior cruciate ligament (ACL) reconstruction and the anterior laxity of the uninjured knee. METHODS We retrospectively reviewed 163 patients who had undergone unilateral ACL reconstruction from January 2002 to August 2009. Patients were divided into three groups according to the anterior laxity of the contralateral, normal knee in 30° of knee flexion as measured with a KT2000 arthrometer exerting a force of 134 N: <5 mm for Group 1, 5 to 7.5 mm for Group 2, and >7.5 mm for Group 3. Anterior laxity of the uninjured knee was assessed preoperatively, and anterior laxity of the reconstructed knee was assessed at twenty-four months postoperatively. Anterior stability of the knee was also assessed with use of the Lachman and pivot-shift tests. Functional outcomes were assessed with the Lysholm score and the International Knee Documentation Committee (IKDC) score. RESULTS The three groups differed significantly with respect to the postoperative side-to-side difference in anterior laxity (p = 0.015), Lysholm score (p < 0.001), and IKDC subjective score (p < 0.001). The mean side-to-side difference in anterior laxity of the reconstructed knee was 2.1 ± 1.3 mm in Group 1, 2.2 ± 1.3 mm in Group 2, and 2.9 ± 1.4 mm in Group 3. The postoperative Lysholm score was 91.8 ± 4.5 in Group 1, 90.3 ± 5.5 in Group 2, and 85.4 ± 6.6 in Group 3. The postoperative IKDC subjective score was 89.3 ± 6.4 in Group 1, 87.9 ± 6.0 in Group 2, and 82.6 ± 8.2 in Group 3. Post hoc testing showed that Group 3 had significantly greater anterior laxity (p ≤ 0.039) and lower functional scores (p ≤ 0.001) compared with Groups 1 and 2. CONCLUSIONS Greater anterior laxity of the uninjured knee was associated with poorer stability and functional outcomes after ACL reconstruction. Excessive anterior laxity of the uninjured knee thus appears to represent a risk factor for inferior outcomes.

Podocyte hypertrophy precedes apoptosis under experimental diabetic conditions

Podocyte hypertrophy and apoptosis are two hallmarks of diabetic glomeruli, but the sequence in which these processes occur remains a matter of debate. Here we investigated the effects of inhibiting hypertrophy on apoptosis, and vice versa, in both podocytes and glomeruli, under diabetic conditions. Hypertrophy and apoptosis were inhibited using an epidermal growth factor receptor inhibitor (PKI 166) and a pan-caspase inhibitor (zAsp-DCB), respectively. We observed significant increases in the protein expression of p27, p21, phospho-eukaryotic elongation factor 4E-binding protein 1, and phospho-p70 S6 ribosomal protein kinase, in both cultured podocytes exposed to high-glucose (HG) medium, and streptozotocin-induced diabetes mellitus (DM) rat glomeruli. These increases were significantly inhibited by PKI 166, but not by zAsp-DCB. In addition, the amount of protein per cell, the relative cell size, and the glomerular volume were all significantly increased under diabetic conditions, and these changes were also blocked by treatment with PKI 166, but not zAsp-DCB. Increased protein expression of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase, together with increased Bax/Bcl-2 ratios, were also observed in HG-stimulated podocytes and DM glomeruli. Treatment with either zAsp-DCB or PKI 166 resulted in a significant attenuation of these effects. Both PKI 166 and zAsp-DCB also inhibited the increase in number of apoptotic cells, as assessed by Hoechst 33342 staining and TUNEL assay. Under diabetic conditions, inhibition of podocyte hypertrophy results in attenuated apoptosis, whereas blocking apoptosis has no effect on podocyte hypertrophy, suggesting that podocyte hypertrophy precedes apoptosis.

(The) effect of statin on epithelial-mesenchymal transition in peritoneal mesothelial cells

Background Statins have recently been highlighted for their pleiotropic actions distinct from cholesterol-lowering effects. Despite this interest, it is currently unknown whether statin therapy inhibits peritoneal dialysis (PD)-related epithelial-mesenchymal transition (EMT). Methods In vitro, human peritoneal mesothelial cells (HPMCs) were exposed to 5.6 mM glucose (NG) or 100 mM glucose (HG) with or without simvastatin (1 µM). In vivo, PD catheters were inserted into 32 Sprague-Dawley rats, and saline (C, n = 16) or 4.25% peritoneal dialysis fluid (PDF) (PD, n = 16) was infused for 4 weeks. Eight rats from each group were treated with 5 mg/kg/day of simvastatin intraperitoneally. Changes in the protein expression of EMT markers such as E-cadherin, α-SMA, Snail, and fibronectin in HPMCs and the peritoneum were evaluated by Western blot analysis and immunofluorescence or immunohistochemical staining. We also explored whether activation of the mevalonate pathway and its downstream small GTPases were involved in dialysis-related peritoneal EMT and could be inhibited by statin treatment. Results Compared to NG cells, E-cadherin expression was significantly decreased, while α-SMA, Snail, and fibronectin expression were significantly increased in HPMCs exposed to HG, and these changes were abrogated by simvastatin (p<0.05). In addition, the cobblestone-like appearance of normal HPMCs was converted into a fibroblast-like morphology after HG treatment, which was reversed by simvastatin. These EMT-like changes were also observed in HPMCs treated with geranyl-geranyl pyrophosphate (5 µM). HG significantly increased the protein expression of RhoA and Rac1 in the membrane fractions, and these increases were ameliorated by simvastatin (p<0.05). In PD rats, E-cadherin in the peritoneum was significantly decreased, whereas α-SMA, Snail, and fibronectin expression were significantly increased (p<0.05) compared to C rats. The thickness of the mesothelial layer in the peritoneum were also significantly greater in PD rats than in C rats (p<0.05). These changes of the peritoneum in PD rats were significantly attenuated by simvastatin. Conclusion This study demonstrated that PD-related EMT was mediated via the mevalonate pathway, and statin treatment inhibited the EMT changes in HG-treated HPMCs and PDF-stimulated PD rats. These findings suggest that statins may be a promising therapeutic strategy for preservation of peritoneal membrane integrity in long-term PD patients.

Periostin-Binding DNA Aptamer Treatment Ameliorates Peritoneal Dialysis-Induced Peritoneal Fibrosis

Peritoneal fibrosis is a major complication in peritoneal dialysis (PD) patients, which leads to dialysis discontinuation. Periostin, increased by transforming growth factor β1 (TGF-β1) stimulation, induces the expression of extracellular matrix (ECM) genes. Aberrant periostin expression has been demonstrated to be associated with PD-related peritoneal fibrosis. Therefore, the effect of periostin inhibition by an aptamer-based inhibitor on peritoneal fibrosis was evaluated. In vitro, TGF-β1 treatment upregulated periostin, fibronectin, α-smooth muscle actin (α-SMA), and Snail expression and reduced E-cadherin expression in human peritoneal mesothelial cells (HPMCs). Periostin small interfering RNA (siRNA) treatment ameliorated the TGF-β1-induced periostin, fibronectin, α-SMA, and Snail expression and restored E-cadherin expression in HPMCs. Similarly, the periostin-binding DNA aptamer (PA) also attenuated fibronectin, α-SMA, and Snail upregulation and E-cadherin downregulation in TGF-β1-stimulated HPMCs. In mice treated with PD solution for 4 weeks, the expression of periostin, fibronectin, α-SMA, and Snail was significantly increased in the peritoneum, whereas E-cadherin expression was significantly decreased. The thickness of the submesothelial layer and the intensity of Masson’s trichrome staining in the PD group were significantly increased compared to the untreated group. These changes were significantly abrogated by the intraperitoneal administration of PA. These findings suggest that PA can be a potential therapeutic strategy for peritoneal fibrosis in PD patients.

Periostin-binding DNA aptamer treatment attenuates renal fibrosis under diabetic conditions

Diabetic nephropathy, the major cause of chronic kidney disease, is associated with progressive renal fibrosis. Recently, accumulation of periostin, an extracellular matrix protein, was shown to augment renal fibrosis. Aptamers have higher binding affinities without developing the common side effects of antibodies. Thus, we evaluated the effect of periostin inhibition by an aptamer-based inhibitor on renal fibrosis under diabetic conditions. In vitro, transforming growth factor-β1 (TGF-β1) treatment significantly upregulated periostin, fibronectin, and type I collagen mRNA and protein expressions in inner medullary collecting duct (IMCD) cells. These increases were attenuated significantly in periostin-binding DNA aptamer (PA)-treated IMCD cells exposed to TGF-β1. In vivo, PA treatment attenuated the increased blood urea nitrogen levels in the diabetic mice significantly. Fibronectin and type I collagen mRNA and protein expressions increased significantly in the kidneys of diabetic mice: PA administration abrogated these increases significantly. Immunohistochemistry and Sirius Red staining also revealed that fibronectin expression was significantly higher and tubulointersititial fibrosis was significantly worse in diabetic mice kidneys compared with control mice. These changes were ameliorated by PA treatment. These findings suggested that inhibition of periostin using a DNA aptamer could be a potential therapeutic strategy against renal fibrosis in diabetic nephropathy.

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.