Article pubs.acs.org/molecularpharmaceutics
Mechanistic Studies of a Novel Mycophenolic Acid−Glucosamine Conjugate That Attenuates Renal Ischemia/Reperfusion Injury in Rat Xiaohong Wang, Menghua Xiong, Yingchun Zeng, Xun Sun, Tao Gong, and Zhirong Zhang* Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Southern Renmin Road, No. 17, Section 3, Chengdu 610041, P. R. China ABSTRACT: Renal ischemia/reperfusion (I/R) injury causes high mortality and morbidity during renal procedures, yet current drugs should be used at high doses or for long periods due to lack of tissue specificity. In previous work we described a novel mycophenolic acid−glucosamine conjugate (MGC) that targets the proximal tubule epithelium, where it efficiently reduces renal I/R injury in rats and promotes recovery from reperfusion. Here we perform mechanistic studies of MGC in rats that suggest that the conjugate works by repressing the activation of renal inosine-5′-monophosphate dehydrogenase 2 (IMPDH2), thereby inhibiting the proliferation and accumulation of lympholeukocytes in the proximal tubules. In addition, MGC appears to inhibit inflammation through various pathways, including inhibition of free oxygen radical production, upregulation of bone morphogenetic protein-7, and downregulation of complement protein 3, TLR 4, intracellular adhesion molecules in the endothelium, proinflammatory cytokines (e.g., TNF-α, IL-6, IL-1, TGF-β), and chemotactic cytokines [e.g., monocyte chemoattractant protein-1 (MCP-1) and IL-8]. These findings suggest that MGC specifically targets the proximal tubules and acts through numerous mechanisms to substantially mitigate I/R injury in rats; this conjugate may provide a more effective alternative to current combination therapy. KEYWORDS: ischemia/reperfusion, mycophenolic acid−glucosamine conjugate, inflammation, renal targeting
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rats. Given these promising findings, we wanted to explore the mechanisms by which MGC attenuates I/R injury. Here we describe results suggesting that MGC significantly reduces renal I/R injury by inhibiting proinflammatory cytokines and other factors that promote immunocyte proliferation and infiltration, as well as by reducing the production of reactive oxygen species. These results suggest that MGC works via several mechanisms simultaneously to reduce I/R injury. Together with our previous work showing that the compound specifically targets the proximal tubule, our present findings suggest that MGC may prove superior to current combination therapies.
INTRODUCTION Renal ischemia/reperfusion (I/R) injury, a frequent cause of acute renal failure in renal transplant patients and a contributor to renal damage during revascularization procedures and after hypoperfusion, is associated with high mortality and morbidity in the clinic.1 Treatment is quite expensive, requires hospitalization,2,3 and is only partially effective.4 Both the immunosuppressant mycophenolic acid (MPA) and its prodrug mycophenolate mofetil may relieve renal I/R injury, but treatment often requires extremely high dosages and prolonged administration because these drugs do not target the kidneys. Combination therapy with several drugs may prove more efficient at mitigating the effects of I/R injury,5−8 but this may also increase the risk of side effects and make rational treatment planning difficult. Hence finding more effective drugs that target the kidney may significantly improve treatments for I/R injury. I/R injury is mediated by inflammation as well as alterations in kidney morphology, hemodynamics, tubule cell metabolism, and microvasculature.9,10 Since cells of the proximal tubules are key sites of injury,11 they seem a suitable target for designing new drugs or drug delivery systems. In previous work, our group identified a novel mycophenolic acid−glucosamine conjugate (MGC) that efficiently and substantially reduced renal I/R injury in rats. Through its interaction with megalin receptors, MGC targeted the proximal convoluted tubules12 in HK-2 cells, while it accumulated selectively in the kidney in © XXXX American Chemical Society
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MATERIALS AND METHODS Materials. Male Sprague−Dawley rats were provided by the West China Experimental Animal Center of Sichuan University (China). Levels of malondialdehyde (MDA) were assayed using a commercial kit (catalog no. KGT004, KeyGEN BioTECH, Nanjing, China). Levels of nitric oxide synthase (NOS) were assayed using a commercial kit (catalog no. KGT020-1, KeyGEN BioTECH). Levels of superoxide dismutate (SOD) Received: April 17, 2014 Revised: August 11, 2014 Accepted: August 19, 2014
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dx.doi.org/10.1021/mp500282g | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
KGT020-1, KeyGEN BioTECH) according to the manufacturer’s instructions. In brief, isolated rat kidneys were cleaned with cold PBS, homogenized, and diluted 1:10 in cold PBS. The diluted homogenate was centrifuged at 1000g for 10 min at 4 °C, and aliquots (50 μL) of supernatant were assayed for NOS. These levels were normalized to the protein concentration in each sample. Superoxide Dismutase (SOD) Assay. Levels of superoxide dismutate (SOD), which serve as an index of superoxide levels, were assayed using a commercial kit (catalog no. KGT001100, KeyGEN BioTECH) according to the manufacturer’s instructions. In brief, isolated rat kidneys were cleaned with cold PBS, homogenized, and diluted 1:10 in cold PBS. The diluted homogenate was centrifuged at 12000g for 5 min at 4 °C, and aliquots (100 μL) of supernatant were assayed for SOD. These levels were normalized to the protein concentration in each sample. Catalase Assay. Levels of catalase, which serve as an index of hydrogen peroxide, were assayed using a commercial kit (catalog no. KGT017, KeyGEN BioTECH) according to the manufacturer’s instructions. In brief, isolated rat kidneys were cleaned with cold PBS, homogenized, and diluted 1:10 in cold PBS. The diluted homogenate was centrifuged at 600g for 10 min at 4 °C, and aliquots (50 μL) of supernatant were assayed for catalase levels. These levels were normalized to the protein concentration in each sample. ELISA Determinations. A commercial ELISA kit (R&D System, America) was used to assay levels of free oxygen radicals, TNF-α, IL-1, IL-2, IL-6, IL-8, leukotriene, PAF, ICAM-1, IMPDH2, KIM-1, and complement protein C3 in kidney homogenate from I/R and sham-operated rats. Aliquots (10 μL) of experimental samples were diluted into a final volume of 50 μL in sample diluent. To these wells was added 50 μL of horseradish peroxide (HPR)-conjugated reagent. Blank control wells were prepared in parallel in the same way, except that 50 μL of PBS was added instead of HRP conjugate. Plates were gently mixed by shaking and then incubated for 60 min at 37 °C. Plates were washed with wash solution, 50 μL of chromogen solution A and 50 μL of chromogen solution B were added to each well, and the plates were incubated for another 15 min at 37 °C. Stop solution was added to each well, which made the solution in the wells change immediately from blue to yellow. Within 15 min of adding stop stolution, optical density (OD) of all wells was measured at 450 nm, relative to an OD of 0 for the blank well. Immunohistochemistry. Fixed and embedded kidney sections were deparaffinized and incubated in antigen retrieval buffer (citrate, pH 6) for 40 min at 95 °C. Endogenous peroxidase activity was blocked by incubating sections with 3% H2O2 for 15 min at room temperature in the dark. Sections were then washed with PBS three times (3 min per wash) and then incubated for 45 min at 37 °C with antibodies against one of the following antigens: KIM-1 (1:100; R&D, AF3689), IMPHD2 (1:200; Epitomics, 5814-1), MCP-1 (1:200; Abcam, ab25124), TNF-α (1:100; Abcam, ab6671), IL-1 (1:50; Abcam, ab109555), IL-2 (1:100; Santa Cruz, sc-7896), IL-6 (1:100; Abcam, ab6672), IL-8 (1:50; Abcam, ab7747), TGF-β (1:100; Abcam, ab92486), ICAM-1 (1:100; Abcam, ab2213), leukotriene (1:100; GeneTex, GTX13346), TLR4 (1:100; Abcam, ab22048), C3 (1:50; Novus, NBP1-05140), or BMP-7 (1:200; Abcam, ab56023). Sections were washed with PBS three times, then incubated for 45 min at 37 °C with 50−100 μL of Agent A (HRP-conjugated
were assayed using a commercial kit (catalog no. KGT001100, KeyGEN BioTECH). Levels of catalase were assayed using a commercial kit (catalog no. KGT017, KeyGEN BioTECH). Commercial ELISA kits (R&D System, America) were used to assay levels of free oxygen radicals, TNF-α, IL-1, IL-2, IL-6, IL-8, leukotriene, PAF, ICAM-1, IMPDH2, KIM-1, and complement protein C3. Immunohistochemistry antigens: KIM-1 (R&D, AF3689), IMPHD2 (Epitomics, 5814-1), MCP-1 (Abcam, ab25124), TNF-α (Abcam, ab6671), IL-1 (Abcam, ab109555), IL-2 (Santa Cruz, sc-7896), IL-6 (Abcam, ab6672), IL-8 (Abcam, ab7747), TGF-β (Abcam, ab92486), ICAM-1 (Abcam, ab2213), leukotriene (GeneTex, GTX13346), TLR4 (Abcam, ab22048), C3 (Novus, NBP105140), or BMP-7 (Abcam, ab56023). Kidney protein concentration was estimated using the BCA protein assay kit (Pierce, USA). Stained slides were viewed on an upright metallurgical microscope (Nikon Eclipse 80i, Nikon Instech Co. Ltd., Kawasaki, Kanagawa, Japan) fitted with a Nikon camera to take photomicrographs. Rat Model of Renal I/R Injury. Male Sprague−Dawley rats (240 ± 20 g) were maintained in a germ-free environment and allowed free access to food and water. All animal experiments were approved by the Animal Ethical Experimentation Committee of Sichuan University and were performed in accordance with the National Act on the Use of Experimental Animals of the People’s Republic of China. Rats were divided into four groups of 5 animals each: the Sal-I/R group, which was administered normal saline via caudal vein injection 3 days before renal ischemia was induced as described below; the MPA-I/R group, which received mycophenolic acid (46.8 μmol/kg/day) via caudal vein injection 3 days before renal ischemia was induced; the MGC-I/R group, which received MGC (46.8 μmol/kg/day) via caudal vein injection 3 days before renal ischemia; and the sham-operated group, which underwent mock renal ischemia induction described below. All rats were anesthetized with chloral hydrate (280 mg·kg−1) delivered intraperitoneally, and they remained under anesthesia during the 35 min midline laparotomy. After laparotomy, rats in the three I/R groups were subjected to bilateral renal ischemia, during which the renal blood vessels were kept occluded for 35 min using bulldog clamps. Then the renal clamps were removed, the kidneys were observed for 5 min to confirm their reflow, and finally the incision was sutured. Sham-operated rats were treated in the same way as the I/R animals, except that the renal blood vessels were not occluded. After the abdomens were sutured shut, animals were allowed to recover for 24 h. Then their kidneys were removed, fixed in 4% buffered formalin, and embedded in paraffin. Photomicrographs were taken with a microscope camera at 400× magnification. Lipid Peroxidation Assay. Levels of malondialdehyde (MDA), which serve as an index of lipid peroxidation, were assayed using a commercial kit (catalog no. KGT004, KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. In brief, isolated rat kidneys were cleaned with cold phosphate-buffered saline (PBS), homogenized, and diluted 1:10 in cold PBS. The diluted homogenate was centrifuged at 12000g for 5 min at 4 °C, and aliquots (200 μL) of supernatant were assayed for MDA. These levels were normalized to the protein concentration in each sample. Nitric Oxide Synthase (NOS) Assay. Levels of nitric oxide synthase (NOS), which serve as an index of nitric oxide (NO) production, were assayed using a commercial kit (catalog no. B
dx.doi.org/10.1021/mp500282g | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Molecular Pharmaceutics
Article
Figure 2. Renal expression of IMPDH2 measured in rats subjected to induced ischemia and reperfusion (I/R) following pretreatment with saline (Sal-I/R), mycophenolic acid (MPA-I/R), or mycophenolic acid− glucosamine conjugate (MGC-I/R). IMPDH2 levels were assayed by ELISA at 24 h after I/R. Sham-operated rats were treated in the same way as Sal-I/R animals except that I/R was not induced. (upper panel) ELISA determinations of IMPDH2. Data are mean ± SD of 5 animals per condition. *P < 0.05, vs MPA-I/R rats; ##P < 0.01, vs Sal-I/R rats. (lower panel) Representative photomicrographs of IMPDH2 immunostaining in sham-operated (A), Sal-I/R (B), MPA-I/R (C), and MGC-I/R (D) rats. IMPDH2 expression increased after I/R, and this increase was partially prevented by pretreatment with MPA or MGC, with MGC blocking the I/R-induced increase to a greater extent. Photomicrographs were taken with a microscope camera at 400× magnification.
Figure 1. Renal expression of KIM-1 measured in rats subjected to induced ischemia and reperfusion (I/R) following pretreatment with saline (Sal-I/R), mycophenolic acid (MPA-I/R), or mycophenolic acid−glucosamine conjugate (MGC-I/R). KIM-1 levels were assayed by ELISA at 24 h after I/R. Sham-operated rats were treated in the same way as Sal-I/R animals except that I/R was not induced. (upper panel) ELISA determinations of KIM-1. Data are mean ± SD of 5 animals per condition. #P < 0.05, vs Sal-I/R. (lower panel) Representative photomicrographs of KIM-1 immunostaining in shamoperated (A), Sal-I/R (B), MPA-I/R (C), and MGC-I/R (D) rats. KIM-1 expression, indicating injured tubular cells, increased after I/R, and MGC prevented this increase to a greater extent than MPA did. MGC also prevented I/R-induced dilation of tubules. Photomicrographs were taken with a microscope camera at 400× magnification.
blood vessels were occluded for 35 min and the animal was allowed to recover for 24 h. Prior to ischemia and reperfusion, animals were pretreated with saline as a negative control (Sal-I/R), MPA as a positive control (MPA-I/R), or our experimental conjugate MGC (MGC-I/R). A sham-operated group of animals were treated like the Sal-I/R group except that the renal blood vessels were not occluded, so I/R was not induced. Levels of kidney injury molecule 1 (KIM-1), proliferating and dedifferentiated tubular epithelial cells, are useful because ischemia triggers loss of tubular epithelial polarity and development of interstitial fibrosis. The levels were significantly higher in the Sal+I/R group than in the sham group at 24 h after the procedure, based on ELISA (Figure 1). Pretreatment with MPA led to levels that were 19% lower than those in the Sal-I/R group, a difference that did not achieve statistical significance. However, pretreatment with MGC reduced KIM-1 expression by 47% relative to the level in the Sal-I/R group. Immunohistochemistry of kidney sections showed that I/R induced KIM-1 production in injured tubular cells, and that
ChemMate Envision reagent) from the anti-rabbit/rat general immunohistochemical test kit (Envision Detection Kit, catalog no. GK500705). The sections were washed again three times with PBS (3 min per wash). A color reaction was developed by adding 50−100 μL of 3,3-diaminobenzidine (Sigma), and sections were counterstained with hematoxylin. The levels of tubular expression of MCP-1, TLR4, BMP-7, and TGF-β were determined by computerized morphometry of approximately 30 interstitial rectangular fields per section at 400× magnification. Data Analysis. Quantitative data was expressed as mean ± SD and analyzed by one-way ANOVA. Later, individual comparisons were performed using the Tukey method (post hoc test), with a p value
