Sun Woo Lim

Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney

Improving the ability of the kidney to tolerate ischemic injury has important implications. We investigated the effect of recombinant human erythropoietin (rHuEPO) treatment on subsequent ischemia/reperfusion (I/R) injury and evaluated the role of heat shock protein (HSP) 70 in rHuEPO‐induced renal protection. rHuEPO (3000 U/kg) was administered 24 h before I/R injury, and rats were killed at 24, 48, and 72 h after I/R injury. Pretreatment of rHuEPO resulted in the following: i) decreased serum creatinine level; ii) decreased tubular cell apoptosis and necrosis, measured by DNA fragmentation analysis and TUNEL staining and histomorphological criteria; iii) decreased tubular cell proliferation as determined by proliferating cell nuclear antigen expression; iv) increased bcl‐2 protein and decreased caspase 3 activity; and v) decreased JNK expression. rHuEPO treatment increased HSP70 expression in a dose‐dependent manner in normal rat kidneys, and inhibition of HSP70 expression by quercetin eliminated the renoprotective effect of rHuEPO in ischemic kidneys. Our study demonstrates that rHuEPO has a protective effect on subsequent I/R injury and that this effect is associated with induction of HSP70. Our study provides a new avenue for therapy to prevent renal damage after I/R injury.

Attenuation of Interstitial Inflammation and Fibrosis by Recombinant Human Erythropoietin in Chronic Cyclosporine Nephropathy

Background: Evidence suggests that recombinant human erythropoietin (rHuEPO) protects neurons and cardiomyocytes from acute insults. We investigated the protective effect of rHuEPO on cyclosporine (CsA)-induced renal injury. Methods: CsA (15 mg/kg/day) was given to rats for 1 or 4 weeks, and rHuEPO was concurrently administered at a dose of 100 units/kg (thrice weekly). Effects of rHuEPO on CsA-induced renal injury were evaluated with tubulointerstitial fibrosis (TIF) score, macrophage infiltration, expression of proinflammatory and profibrotic cytokines, and apoptotic cell death. Results: Administration of rHuEPO decreased TIF score and the number of macrophages, which increased significantly in CsA-treated rat kidneys. At the molecular level, rHuEPO treatment decreased proinflammatory mediators (osteopontin and C-reactive protein) and profibrotic mediators (transforming growth factor-β1 and transforming growth factor-β1-inducible gene-h3). Increased apoptotic cell death in CsA-treated rat kidneys was significantly decreased with rHuEPO cotreatment, and apoptosis-related genes were regulated in favor of cell survival (increased Bcl-2 and suppressed caspase-3). Conclusion: rHuEPO has a renoprotective effect against chronic CsA-induced renal injury.

Cyclosporine-induced Renal Injury Induces Toll-like Receptor and Maturation of Dendritic cells

Background. The toll-like receptor (TLR) is stimulated by not only pathogen-associated molecular patterns but also endogenous TLR ligands provided by injured cells. The influence of cyclosporine A (CsA)-induced renal injury on TLR expression and subsequent signaling pathway was evaluated. Methods. Induction of chronic CsA nephropathy was made by administering CsA (15 mg/kg/day) for 28 days in rats. The TLR2 and TLR4 mRNA and protein expression, TLR-signaling pathway (MYD88, NF-&kgr;B and AP-1), putative TLR ligand (heat shock protein 70 [HSP70]), and maturation of dendritic cells were evaluated in CsA-treated rat kidneys. Results. Long-term CsA treatment upregulated TLR2 and TLR4 mRNA and protein expression on renal tubular cells, and these were accompanied by increased MYD88, NF-&kgr;B and AP-1 expression. Putative TLR ligand (HSP70) was also significantly increased in CsA-treated rat kidney compared with vehicle-treated rat kidney. CsA-treatment increased expression of TNF-α mRNA, the number of dendritic cells, and expression of MHC class II antigen. Double-labeling of markers of dendritic cells and MHC class II antigen revealed that matured dendritic cells increased in CsA-treated rat kidney. Conclusions. CsA-induced renal injury stimulates components of innate immunity, and this finding suggests close association between CsA-induced renal injury and activation of innate immunity.

Rosiglitazone Protects Against Cyclosporine-Induced Pancreatic and Renal Injury in Rats

Rosiglitazone (RGTZ) has protective effect against various types of injury. This study was performed to evaluate the effect of RGTZ on pancreatic and renal injury caused by cyclosporine (CsA). CsA (15 mg/kg) and RGTZ (3 mg/kg) were administered alone and together to the rats for 28 days. The effect of RGTZ on CsA‐induced pancreatic injury was evaluated by intraperitoneal glucose tolerance test (IPGTT), plasma insulin concentrations and pancreatic β‐cell morphology. The effect of RGTZ on CsA‐induced renal injury was evaluated by assessing renal function and pathology; mediators of inflammation and fibrosis such as angiotensin II (AngII), osteopontin (OPN) and transforming growth factor‐beta1 (TGF‐β1) and apoptotic cell death. Four weeks of CsA treatment caused diabetes, renal dysfunction, typical pathologic lesions (arteriolopathy, interstitial fibrosis and inflammatory cells infiltration) and apoptotic cell death. RGTZ treatment decreased blood glucose concentration, increased plasma insulin concentration and preserved pancreatic β islet mass. RGTZ treatment improved renal function and histopathology. Pro‐inflammatory and pro‐fibrotic molecules such as AngII, OPN and TGF‐β1, and apoptotic cell death also decreased with RGTZ treatment. These data suggest that RGTZ has a protective effect against CsA‐induced pancreatic and renal injury.

Prolonged Endoplasmic Reticulum Stress Induces Apoptotic Cell Death in an Experimental Model of Chronic Cyclosporine Nephropathy

Background/Aims: Apoptosis contributes to cyclosporine (CsA)-induced renal cell death. This study tested the effects of CsA-induced endoplasmic reticulum (ER) stress on apoptotic cell death in an experimental model of chronic CsA nephropathy. Methods: CsA (15 mg/kg per day) was given to rats for 7 or 28 days. The ER stress response was evaluated with BiP expression, and the proapoptotic response was assessed with CHOP and caspase 12 expression. ER structure was evaluated by transmission electron microscopy, and apoptotic cell death was detected with TUNEL staining. Results: Short-term treatment of CsA for 7 days activated both the ER stress response (induction of BiP mRNA and protein) and the proapoptotic response (upregulation of caspase 12 and CHOP mRNAs and proteins). However, long-term treatment with CsA for 28 days decreased BiP and further increased CHOP. The imbalance between the two responses coincided with the timing of the appearance of apoptotic cell death and the disruption of the ER structure. Conclusion: Prolonged CsA-induced ER stress causes apoptotic cell death by depleting molecular chaperones and activating the proapoptotic pathway.

Downregulation of Renal Sodium Transporters and Tonicity-Responsive Enhancer Binding Protein by Long-Term Treatment with Cyclosporin A

Tonicity-responsive enhancer binding protein (TonEBP) is a transcriptional activator that is regulated by ambient tonicity. TonEBP protects the renal medulla from the deleterious effects of hyperosmolality and regulates the urinary concentration by stimulating aquaporin-2 and urea transporters. The therapeutic use of cyclosporin A (CsA) is limited by nephrotoxicity that is manifested by reduced GFR, fibrosis, and tubular defects, including reduced urinary concentration. It was reported recently that long-term CsA treatment was associated with decreased renal expression of TonEBP target genes, including aquaporin-2, urea transporter, and aldose reductase. This study tested the hypothesis that long-term CsA treatment reduces the salinity/tonicity of the renal medullary interstitium as a result of inhibition of active sodium transporters, leading to downregulation of TonEBP. CsA treatment for 7 d did not affect TonEBP or renal function. Whereas expression of sodium transporters was altered, the medullary tonicity seemed unchanged. Conversely, 28 d of CsA treatment led to downregulation of TonEBP and overt nephrotoxicity. The downregulation of TonEBP involved reduced expression, cytoplasmic shift, and reduced transcription of its target genes. This was associated with reduced expression of active sodium transporters-sodium/potassium/chloride transporter type 2 (NKCC2), sodium/chloride transporter, and Na(+),K(+)-ATPase-along with increased sodium excretion and reduced urinary concentration. Infusion of vasopressin restored the expression of NKCC2 in the outer medulla as well as the expression and the activity of TonEBP. It is concluded that the downregulation of TonEBP in the setting of long-term CsA administration is secondary to the reduced tonicity of the renal medullary interstitium.

Combined Effects of Losartan and Pravastatin on Interstitial Inflammation and Fibrosis in Chronic Cyclosporine-induced Nephropathy

Background. Statins and angiotensin II type I receptor blockers have synergistic effects on vascular smooth–muscle-cell proliferation and the progression of renal diseases. We evaluated whether combined treatment with losartan (LSRT) and pravastatin (PRVT) affords superior protection compared with their respective monotherapies in treating chronic cyclosporine (CsA)-induced nephropathy in rats. Methods. Rats maintained on a low salt diet were given vehicle, CsA (15 mg/kg), CsA and LSRT (10 mg/kg), CsA and PRVT (5 mg/kg), or a combination of CsA, LSRT, and PRVT for 28 days. Basic parameters (renal function, systolic blood pressure, serum high-sensitivity C-reactive protein [hs-CRP], and lipid profiles), histopathology (arteriolopathy, tubulointerstitial fibrosis, and inflammatory cell infiltration), and inflammatory and fibrotic factors (intrarenal CRP, angiotensin II, osteopontin, and transforming growth factor [TGF]-&bgr;1) were studied. Results. LSRT or PRVT treatment significantly attenuated the histopathologic changes induced by CsA, and combined treatment with LSRT and PRVT further decreased these parameters compared with giving each drug alone. Increased levels of angiotensin II, intrarenal CRP, osteopontin, and TGF-&bgr;1 in CsA-treated rat kidney were reduced by treatment with either LSRT or PRVT and were further decreased by the combination of the two drugs. There were no significant differences in systolic blood pressure or serum lipid parameters between groups. Conclusions. Combined treatment with LSRT and PRVT provided synergistic effects in attenuating inflammatory and fibrotic processes in a rat model of chronic CsA-induced nephropathy, and this effect was independent of their hypolipidemic and hypotensive actions.

Protective effect of peroxisome proliferator activated receptor gamma agonists on diabetic and non‐diabetic renal diseases

SUMMARY:  Peroxisome proliferator activated receptor gamma (PPARγ) agonist has not only antidiabetic effect but also a protective effect against various types of injury of the kidney. The protective effects of PPARγ agonists are observed in diabetic nephropathy and non‐diabetic renal diseases such as 5/6 ablation model of renal failure, experimental glomerulonephritis, ischemia‐reperfusion injury, hypertensive nephropathy and cyclosporin‐induced renal injury. The mechanism of renoprotection by PPARγ agonist is multifactorial. Anti‐fibrotic and anti‐inflammatory effects, suppression of renin‐angiotensin system, vascular protective effect and antiapoptotic effect were proposed.

Chronic cyclosporine nephropathy is characterized by excessive autophagosome formation and decreased autophagic clearance.

Background The study was performed to investigate the influence of cyclosporine A (CsA)–induced renal injury on autophagy in an experimental model of chronic CsA nephropathy. Methods Three dosages of CsA (7.5, 15, and 30 mg/kg/day) were administered to mice for 4 weeks. The formation of autophagosomes was measured with microtubule-associated protein 1 light chain 3 phospholipid-conjugated form (LC3-II) and beclin-1, and the ability of autophagic clearance was examined with sequestosome-1 (p62). Autophagic vacuoles were visualized and counted using electron microscopy. Double immunolabeling of LC3-II and active caspase-3 was performed to evaluate the association between autophagy and apoptosis. Oxidative stress was evaluated by measuring urinary 8-hydroxy-2′-deoxyguanosine excretion, demonstrating oxidative DNA damage. Antioxidative drugs, pravastatin and N-acetylcysteine, were used to evaluate the role of CsA-induced oxidative stress on autophagy. Results CsA treatment increased the expressions of LC3-II and beclin-1 in the kidney in a dose-dependent manner. The number of p62-positive cells was also significantly increased in a CsA dose–dependent manner. Electron microscopy revealed excessive autophagic vacuoles in the CsA group compared with the vehicle group. Expression of active caspase-3 was increased in a CsA dose–dependent manner and was colocalized with LC3-II in the injured area of CsA-treated kidneys. Concurrent pravastatin or N-acetylcysteine treatment reduced urinary excretion of 8-hydroxy-2′-deoxyguanosine and subsequently decreased LC3-II expression and the number of p62-positive cells compared with the CsA group. Conclusions Chronic CsA nephropathy is a state of excessive autophagic vacuoles and decreased autophagic clearance. Oxidative stress may play an importation role in the induction of autophagy.

Influence of angiotensin II on expression of toll-like receptor 2 and maturation of dendritic cells in chronic cyclosporine nephropathy.

Background. Angiotensin (Ang) II plays an important role in immune regulation. We evaluate the influence of the renin-angiotensin system (RAS) in the innate immune response caused by cyclosporine A (CsA)-induced renal injury. Methods. Two separate studies were performed in Sprague Dawley rats. First, losartan (LSRT, 10 mg/kg per day) was concurrently administered with CsA (15 mg/kg per day) for 28 days. Second, AngII (435 ng/kg/min) was infused with or without LSRT for 14 days. Results. AngII blockade with LSRT decreased toll-like receptor (TLR) 2 mRNA and protein expression, expression of tumor necrosis factor (TNF)-&agr; mRNA, and expression of major histocompatibility complex class II antigen, which was upregulated in CsA-induced renal injury. The increased number of matured dendritic cells (DCs) in CsA-induced renal injury was also decreased by concomitant treatment of LSRT. Direct infusion of AngII increased TNF-&agr; mRNA, TLR2 mRNA, and protein and the number of DCs, compared with the control rat kidney. In contrast, concomitant treatment of LSRT decreased all parameters. Conclusion. AngII plays a pivotal role in activating the innate immune response in CsA-induced renal injury.