muramidase and Fibrosis

Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tubular cells and reduced unwanted systemic effects. In our approach kinase inhibitors are linked to the renal carrier lysozyme using a platinum-based linker that binds drugs via a coordinative linkage. Many kinase inhibitors contain aromatic nitrogen atoms able to bind to this linker without the need of prior derivatization. The resulting drug-lysozyme conjugates are rapidly filtered in the glomerulus into the tubular lumen and subsequently reabsorbed via the endocytic pathway for clearance of low-molecular weight proteins. An important property of the formed conjugates is their in vivo stability and the sustained drug release profile within target cells. This review summarizes the state-of-the-art of drug targeting to the kidney. Furthermore, we will highlight recent results obtained with kinase inhibitor-lysozyme conjugates targeted to different kinases, i.e. the transforming growth factor (TGF)-beta-receptor kinase, p38 MAPkinase and Rho-associated kinase. Both in vitro and in vivo results demonstrated their efficient tubular uptake and beneficial therapeutic effects, superior to treatment with free kinase inhibitors. These proof-of-concept studies clearly indicate the feasibility of drug targeting for improving the renal specificity of kinase inhibitors.

Topics: Animals; Drug Delivery Systems; Fibrosis; Gene Targeting; Humans; Kidney; Kidney Diseases; Kidney Tubules; Muramidase; Protein Kinase Inhibitors

In this paper, the effects of three isoflavones including daidzein, genistein, and puerarin on fibrillation of hen egg-white lysozyme were investigated by various analytical methods. The results demonstrated that all isoflavones could effectively inhibit the fibrillogenesis of hen egg-white lysozyme and destabilized the preformed fibrils of hen egg-white lysozyme in a dose-dependent manner. To further understand the inhibition mechanism, molecular modeling was carried out. The docking results demonstrated that the isoflavones could bind to two key fibrogenic sites in hen egg-white lysozyme through van der Waals force, electrostatic forces, and hydrogen bonding, as well as σ-π stacking. By these means, isoflavones could not only obviously enhance the hydrophobicity of the binding sites, but also greatly stabilize the native state of HEWL, which was able to postpone the fibrosis process of hen egg-white lysozyme.

Topics: Animals; Binding Sites; Chickens; Fibrosis; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Isoflavones; Models, Molecular; Molecular Docking Simulation; Muramidase; Protein Binding; Static Electricity

Diabetic nephropathy (DN) is one of the most common and serious complications of diabetes, and is the most important cause of death for diabetic patients. Baicalin (BAI) has anti-oxidative, anti-inflammatory and anti-apoptotic activities, which play a role in attenuating insulin resistance and protecting the kidney. Moreover, cell-specific targeting of renal tubular cells is an approach to enhance drug accumulation in the kidney.. Forty-five Sprague-Dawley rats were divided into four groups. A diabetes model was created using streptozotocin (STZ) intraperitoneally injection. The four groups included: Control group (n = 10), DN (n = 15), BAI treatment (BAI; n = 10) and BAI-LZM treatment (BAI-LZM; n = 10) groups. In the current study, the renoprotection and anti-fibrotic effects of BAI-lysozyme (LZM) conjugate were further investigated in rats with DN induced by STZ compared with BAI treatment alone.. The results suggest that BAI-LZM better ameliorates renal impairment, metabolic disorder and renal fibrosis than BAI alone in rats with DN, and the potential regulatory mechanism likely involves inhibiting inflammation via the nuclear factor-κB signaling pathway, inhibiting extracellular matrix accumulation via the transforming growth factor-β/Smad3 pathway and regulating cell proliferation via the insulin-like growth factor (IGF)-1/IGF-1 receptor/p38 Mitogen-activated protein kinase (MAPK) pathway. BAI and the kidney-targeted BAI-LZM can utilize the body's cytoprotective pathways to reactivate autophagy (as indicated by the autophagy markers mechanistic target of rapamycin and sirtuin 1 to ameliorate DN outcomes.. Our data support the traditional use of S. baicalensis as an important anti-DN traditional chinese medicine (TCM), and BAI, above all BAI-LZM, is a promising source for the identification of molecules with anti-DN effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Blood Glucose; Cholesterol; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Drug Carriers; Drug Delivery Systems; Fibrosis; Flavonoids; Insulin; Kidney; Malondialdehyde; Muramidase; Rats; Triglycerides

Renal fibrosis is a serious clinical problem resulting in the greatest need for renal replacement therapy. No adequate preventive or curative therapy is available that could be clinically used to target renal fibrosis specifically. The search for new efficacious treatment strategies is therefore warranted. Although in vitro models using homogeneous cell populations have contributed to the understanding of the pathogenetic mechanisms involved in renal fibrosis, these models poorly mimic the complex in vivo milieu. Therefore, we here evaluated a precision-cut kidney slice (PCKS) model as a new, multicellular ex vivo model to study the development of fibrosis and its prevention using anti-fibrotic compounds. Precision-cut slices (200-300 μm thickness) were prepared from healthy C57BL/6 mouse kidneys using a Krumdieck tissue slicer. To induce changes mimicking the fibrotic process, slices were incubated with TGFβ1 (5 ng/ml) for 48 h in the presence or absence of the anti-fibrotic cytokine IFNγ (1 µg/ml) or an IFNγ conjugate targeted to PDGFRβ (PPB-PEG-IFNγ). Following culture, tissue viability (ATP-content) and expression of α-SMA, fibronectin, collagen I and collagen III were determined using real-time PCR and immunohistochemistry. Slices remained viable up to 72 h of incubation, and no significant effects of TGFβ1 and IFNγ on viability were observed. TGFβ1 markedly increased α-SMA, fibronectin and collagen I mRNA and protein expression levels. IFNγ and PPB-PEG-IFNγ significantly reduced TGFβ1-induced fibronectin, collagen I and collagen III mRNA expression, which was confirmed by immunohistochemistry. The PKCS model is a novel tool to test the pathophysiology of fibrosis and to screen the efficacy of anti-fibrotic drugs ex vivo in a multicellular and pro-fibrotic milieu. A major advantage of the slice model is that it can be used not only for animal but also for (fibrotic) human kidney tissue.

Topics: Animals; Biomarkers; Collagen Type II; Drug Delivery Systems; Extracellular Matrix; Fibronectins; Fibrosis; Fluorescein-5-isothiocyanate; Histocompatibility Antigens Class II; Humans; Interferon-gamma; Kidney; Mice, Inbred C57BL; Models, Biological; Muramidase; Polyethylene Glycols; Receptor, Platelet-Derived Growth Factor beta; Time Factors; Tissue Survival; Transforming Growth Factor beta1; Treatment Outcome

Activated proximal tubular cells play an important role in renal fibrosis. We investigated whether sunitinib and a kidney-targeted conjugate of sunitinib were capable of attenuating fibrogenic events in tubulointerstitial fibrosis.. A kidney-targeted conjugate was prepared by linkage of a sunitinib analog (named 17864) via a platinum-based linker to the kidney-specific carrier lysozyme. Pharmacological activity of 17864-lysozyme was evaluated in human kidney proximal tubular cells (HK-2); the capability of the kidney-directed conjugate to accumulate in the kidneys was studied in mice. Potential antifibrotic effects of a single-dose treatment were evaluated in the unilateral ureteral obstruction (UUO) model in mice.. The 17864-lysozyme conjugate and its metabolites strongly inhibited tyrosine kinase activity. Upon intravenous injection, 17864-lysozyme rapidly accumulated in the kidneys and provided sustained renal drug levels for up to 3 days after a single dose. Renal drug level area under the curve was increased 28-fold versus an equimolar dose of sunitinib malate. Daily treatment of UUO mice with a high dose of sunitinib malate (50 mg/kg) resulted in antifibrotic responses, but also induced drug-related toxicity. A single dose of 17864-lysozyme (equivalent to 1.8 mg/kg sunitinib) was safe but showed no antifibrotic effects.. Multikinase inhibitors like sunitinib can be of benefit in the treatment of fibrotic diseases, provided that their safety can be improved by strategies as presented in this paper, and sustained renal levels can be achieved.

Topics: Animals; Area Under Curve; Cell Line; Cell Survival; Drug Carriers; Drug Delivery Systems; Fibrosis; Humans; Immunohistochemistry; Indoles; Kidney; Kidney Diseases; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Muramidase; Organoplatinum Compounds; Protein-Tyrosine Kinases; Pyrroles; Receptor, Platelet-Derived Growth Factor beta; Sunitinib

Cell-specific targeting to renal tubular cells is an interesting approach to enhance the accumulation of drugs in the kidney. Low molecular weight proteins are rapidly filtered and extensively accumulate in proximal tubular cells. We therefore have used lysozyme (LZM, 14 kDa) as a tubular cell-specific carrier for the delivery of kinase inhibitors. Two different kinase inhibitors (LY364947 and erlotinib, directed to either the TGF-beta receptor kinase or the EGF receptor) were individually conjugated to LZM via a novel platinum-based linker (Universal Linkage System; ULS). The cellular handling and pharmacological efficacy of the conjugates were evaluated in cultured proximal tubular cells (HK-2 cells). Both conjugates were efficiently internalized via endocytosis. TGF-beta or EGF activated HK-2 cells showed a strong activation of the studied kinases and the conjugates inhibited these events, as was demonstrated by Western blotting of phosphorylated downstream mediators and quantitative gene expression analysis. In conclusion, we have developed tubular cell-specific kinase inhibitor-LZM conjugates via a novel linker strategy, which both showed to be effective in vitro. Future in vivo studies should show their potential for the treatment of renal diseases.

Topics: Cells, Cultured; Chemistry, Pharmaceutical; Drug Carriers; Endocytosis; Epidermal Growth Factor; ErbB Receptors; Erlotinib Hydrochloride; Fibrosis; Gene Expression Regulation; Humans; Intercellular Signaling Peptides and Proteins; Kidney Tubules, Proximal; Kinetics; Muramidase; Phosphorylation; Protein Kinase Inhibitors; Pyrazoles; Pyrroles; Quinazolines; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta

Activation of tubular epithelial cells by transforming growth factor-beta (TGF-beta) plays an important role in the pathogenesis of renal tubulointerstitial fibrosis. We developed a renally accumulating conjugate of a TGF-beta type-I receptor kinase inhibitor (TKI) and evaluated its efficacy in vitro and in vivo.. TKI was conjugated to the protein Lysozyme (LZM) via a platinum-based linker. TKI-LZM was evaluated in human tubular cells (HK-2) for its anti-fibrotic activity. Plasma, kidney and urine drug levels after a single intravenous dose of TKI-LZM in rats were determined by HPLC or immunodetection. Anti-fibrotic effects of TKI-LZM were examined in the unilateral ureteral obstruction (UUO) model.. TKI-LZM conjugate was successfully synthesized at an 1:1 drug/carrier ratio, and inhibited TGF-beta1-induced procollagen-1alpha1 gene expression in HK-2 cells. In vivo, TKI-LZM accumulated rapidly in tubular cells and provided a local depot for 3 days. Interestingly, a single dose of TKI-LZM inhibited the activation of tubular cells and fibroblasts in UUO rats and reduced renal inflammation. In contrast, free TKI at an equimolar (low) dosage exhibited little effects.. Inhibition of TGF-beta signaling by local drug delivery is a promising antifibrotic strategy, and demonstrated the important role of tubular activation in renal fibrosis.

Topics: Animals; Cell Line; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Drug Carriers; Epithelial Cells; Fibroblasts; Fibrosis; Humans; Injections, Intravenous; Kidney Diseases; Kidney Tubules, Proximal; Male; Muramidase; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrazoles; Rats; Rats, Wistar; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Transforming Growth Factor beta1; Ureteral Obstruction

During renal injury, activation of p38 mitogen-activated protein kinase (MAPK) in proximal tubular cells plays an important role in the inflammatory events that eventually lead to renal fibrosis. We hypothesized that local inhibition of p38 within these cells may be an interesting approach for the treatment of renal fibrosis. To effectuate this, we developed a renal-specific conjugate of the p38 inhibitor SB202190 [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] and the carrier lysozyme. First, we demonstrated that SB202190 inhibited the expression of albumin-induced proinflammatory (monocyte chemoattractant protein-1) and transforming growth factor (TGF)-beta1-induced profibrotic (procollagen-Ialpha1) genes over 50% in renal tubular cells (normal rat kidney-52E). Next, we conjugated SB202190 via a carbamate linkage to lysozyme. However, this conjugate rapidly released the drug upon incubation in serum. Therefore, we applied a new platinum(II)-based linker approach, the so-called universal linkage system (ULS), which forms a coordinative bond with SB202190. The SB202190-ULS-lysozyme remained stable in serum but released the drug in kidney homogenates. SB202190-ULS-lysozyme accumulated efficiently in renal tubular cells and provided a local drug reservoir during a period of 3 days after a single intravenous injection. Treatment with SB202190-ULS-lysozyme inhibited TGF-beta1-induced gene expression for procollagen-Ialpha1 by 64% in HK-2 cells. Lastly, we evaluated the efficacy of a single dose of the conjugate in the unilateral renal ischemia-reperfusion rat model. A reduction of intrarenal p38 phosphorylation and alpha-smooth muscle actin protein expression was observed 4 days after the ischemia-reperfusion injury. In conclusion, we have developed a novel strategy for local delivery of the p38 MAPK inhibitor SB202190, which may be of use in the treatment of renal fibrosis.

Topics: Animals; Biological Transport; Cisplatin; Fibrosis; Imidazoles; Kidney; Kidney Tubules, Proximal; Male; Muramidase; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Wistar; Reperfusion Injury

Some peptide growth factors produced by macrophages play a role in fibrosis following tissue injury, through the induction of myofibroblasts. In the present study, the appearance of macrophages and myofibroblast development in hepatic and renal fibrosis was determined by immunohistochemical analysis of tissue from 15 dogs. The hepatic and renal fibrosis was classified as grade I, II or III, depending on the extent (percentage) of fibrotic areas per unit area measured by morphometry with Azan-stained sections. The presence of alpha-smooth muscle actin-immunolabelled myofibroblasts was directly related to advancing grade of both hepatic and renal fibrosis. Lysozyme-immunolabelled macrophages also increased in number with increasing grade of hepatic and renal fibrosis. These findings indicate that myofibroblasts and lysozyme-positive macrophages may contribute to progressive fibrosis in canine liver and kidney disease. Interestingly, the number of macrophages recognized by AM-3K, a newly generated monoclonal antibody capable of labelling exuded macrophages and resident tissue macrophages in dogs, fell significantly in grades II and III of renal fibrosis. By contrast, in hepatic fibrosis there were no marked differences in the number of AM-3K-positive macrophages between grades. These findings suggest that there are functional differences between lysozyme- and AM-3K-positive macrophages.

Topics: Actins; Animals; Antibodies, Monoclonal; Cell Count; Dogs; Female; Fibroblasts; Fibrosis; Immunohistochemistry; Liver Cirrhosis; Macrophages; Male; Muramidase; Muscle, Smooth; Nephritis, Interstitial

Canine chronic hepatitis (CCH) is a progressive inflammatory disease of unknown etiology. To characterize the inflammatory infiltrate, 16 dogs with CCH were selected and classified into three groups based on the stage of fibrosis, as evaluated with Masson's trichrome stain. The inflammatory infiltrate in each liver section was immunohistochemically characterized and evaluated using CD3, lysozyme, lamba and kappa light chain, and alpha-smooth muscle actin antibodies. Numerous breeds were affected, and middle-aged females predominated in this select group. Necroinflammatory activity progressively increased and then waned as the hepatitis progressed to cirrhosis. CD3+ lymphocytes were the most numerous lymphoid cells in dogs with CCH. Degenerate hepatocytes were occasionally surrounded by CD3+ lymphocytes. Necrosis was positively correlated with the number of CD3+ lymphocytes. The lamba and kappa light chain-positive cell infiltrate was variable but generally mild. A positive correlation between the lambda and kappa light chain-positive cells and the portal alpha-smooth muscle actin was found. The number of alpha-smooth muscle actin-positive cells (myofibroblasts) in portal triads and fibrous septa was positively correlated with the stage of fibrosis. In contrast, no correlation was found between the number of lysozyme-positive cells (Kupffer cells) and the stage of fibrosis. These results further support the idea of an immune-mediated process in CCH and suggest that periductular myofibroblasts play an important role in canine liver fibrogenesis.

Topics: Actins; Animals; CD3 Complex; Dog Diseases; Dogs; Female; Fibroblasts; Fibrosis; Hepatitis, Animal; Hepatitis, Chronic; Immunoglobulin Light Chains; Immunohistochemistry; Kupffer Cells; Liver; Male; Muramidase; Neutrophils; Retrospective Studies; Statistics, Nonparametric

The objective of this study was to investigate the cellular tissue response to temporomandibular joint (TMJ) Proplast-Teflon disc material by morphologic and immunohistochemical means.. Twelve patients who had been subjected to TMJ discectomy combined with insertion of a Proplast-Teflon interpositional implant (PTIPI) were recalled for removal of the alloplastic disc. The time elapsed between the Proplast-Teflon disc implantation and its removal varied between 13 and 71 months (mean, 54.6 +/- 5.8 [SEM]) The implants and periimplant tissues were examined by light microscopy and immunohistochemically using a panel of monoclonal antibodies reactive with different subclasses of leukocytes. The sections were immunostained using the alkaline phosphatase-antialkaline phosphatase (APAAP) technique.. Fibrosis and a massive foreign body giant cell reaction were seen inside the heavily disrupted alloplastic implants and in the periimplant tissues. CD68-positive monocyte-derived cells dominated the reactive infiltrate in the implants and surrounding tissue. The CD68-positive cells also were partly positive for lysozyme. The lymphocytic infiltration contained no B cells.. This study of the PTIPI-induced tissue reaction gave no indication of a toxic or an immunologic pathogenesis. Mechanical stress seems important in the fragmentation of the implant and induction of the foreign body reaction. It is not yet known if this fragmentation is the major contributing factor.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Bite Force; Female; Fibrosis; Foreign-Body Reaction; Giant Cells; Humans; Immunoenzyme Techniques; Joint Prosthesis; Male; Middle Aged; Monocytes; Muramidase; Proplast; Reoperation; Stress, Mechanical; Temporomandibular Joint Disorders

In vivo cytotoxicity including cellular metabolic activity, lysozyme content and total protein content in rat bronchoalveolar lavage, capacity of interleukin-1 released from rat pulmonary cells and fibrogenic effects evaluated from rat lung dry weight, collagen content of the whole lung and pathological grading induced by mineral dust were assayed. The results showed that: (1) The relationship among in vivo cytotoxicity, interleukin-1 release, fibrogenic effects on the lung induced by mineral dusts correlated well with the free SiO2 content in mineral dusts in most (but not all) cases; (2) The biological harmful effects of mixed dusts were not simply the additive effect of single dust. In the group of WO3-SiO2 mixture, the fibrogenicity was mainly due to SiO2, tungsten trioxide (WO3) showed neither fibrogenic effect, nor significant potentiality to enhance SiO2 fibrogenicity, while in the group of SnO2-SiO2, SnO2 was suppressive to the effect of SiO2, although the contents of SiO2 in the two mixed dusts were similar.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Division; Cells, Cultured; Collagen; Fibroblasts; Fibrosis; Interleukin-1; Lung; Macrophage Activation; Macrophages, Alveolar; Male; Muramidase; Proteins; Rats; Rats, Wistar; Silicon Dioxide; Tin Compounds; Tungsten Compounds

A rare case of malignant T-cell lymphoma with manifold histologic appearances was described. The lymphoma occurred in the stomach of a 50-year-old Japanese male. Grossly, the lymphoma exhibited a deeply ulcerated mass. Histologically, in addition to diffuse infiltrate of large lymphoid cells with deeply indented nuclei, there were many epithelioid cell granulomas, remarkable tissue eosinophilia and stromal fibrosis, mimicking inflammatory disease. Immunohistochemical studies and a gene analysis demonstrated the T-cell phenotype.

Topics: Biomarkers, Tumor; Blotting, Southern; Cell Nucleus; DNA, Neoplasm; Eosinophilia; Fibrosis; Gene Rearrangement, beta-Chain T-Cell Antigen Receptor; Humans; Immunohistochemistry; Lymphoma, T-Cell; Male; Middle Aged; Muramidase; Phenotype; S100 Proteins; Stomach; Stomach Neoplasms