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1.
Am J Physiol Renal Physiol ; 316(5): F1026-F1040, 2019 05 01.
Article in English | MEDLINE | ID: mdl-30810063

ABSTRACT

Conditional gene targeting using Cre recombinase has offered a powerful tool to modify gene function precisely in defined cells/tissues and at specific times. However, in mammalian cells, Cre recombinase can be genotoxic. The importance of including Cre-expressing control mice to avoid misinterpretation and to maximize the validity of the experimental results has been increasingly recognized. While studying the role of podocytes in the pathogenesis of glomerular basement membrane (GBM) thickening, we used Cre recombinase driven by the podocyte-specific podocin promoter (NPHS2-Cre) to generate a conditional knockout. By conventional structural and functional measures (histology by periodic acid-Schiff staining, albuminuria, and plasma creatinine), we did not detect significant differences between NPHS2-Cre transgenic and wild-type control mice. However, surprisingly, the group that expressed Cre transgene alone developed signs of podocyte toxicity, including marked GBM thickening, loss of normal foot process morphology, and reduced Wilms tumor 1 expression. GBM thickening was characterized by altered expression of core structural protein laminin isoform α5ß2γ1. RNA sequencing analysis of extracted glomeruli identified 230 genes that were significant and differentially expressed (applying a q < 0.05-fold change ≥ ±2 cutoff) in NPHS2-Cre mice compared with wild-type control mice. Many biological processes were reflected in the RNA sequencing data, including regulation of the extracellular matrix and pathways related to apoptosis and cell death. This study highlights the importance of including the appropriate controls for potential Cre-mediated toxicity in conditional gene-targeting experiments. Indeed, omitting the Cre transgene control can result in critical errors during interpretation of experimental data.


Subject(s)
Gene Targeting/adverse effects , Glomerular Basement Membrane/enzymology , Integrases/metabolism , Podocytes/enzymology , Animals , Gene Expression Regulation , Glomerular Basement Membrane/ultrastructure , Integrases/genetics , Intracellular Signaling Peptides and Proteins/genetics , Laminin/genetics , Laminin/metabolism , Membrane Proteins/genetics , Mice, Transgenic , Podocytes/ultrastructure , Promoter Regions, Genetic , Time Factors , WT1 Proteins/genetics , WT1 Proteins/metabolism
2.
Biol Cell ; 111(5): 109-120, 2019 May.
Article in English | MEDLINE | ID: mdl-30702162

ABSTRACT

Diabetic nephropathy is a major long-term complication of diabetes mellitus and one of the most common causes of end-stage renal disease. Thickening of the glomerular basement membrane, glomerular cell hypertrophy and podocyte loss are among the main pathological changes that occur during diabetic nephropathy, resulting in proteinuria. Injury to podocytes, which are a crucial component of the glomerular filtration barrier, seems to play a key role in the development of diabetic nephropathy. Recent studies have suggested that dysregulation of AMP-activated kinase protein, which is an essential cellular energy sensor, may play a fundamental role in this process. The purpose of this review is to highlight the molecular mechanisms associated with AMP-activated protein kinase (AMPK) in podocytes that are involved in the pathogenesis of diabetic nephropathy.


Subject(s)
AMP-Activated Protein Kinases/metabolism , Diabetic Nephropathies/enzymology , Diabetic Nephropathies/pathology , Podocytes/enzymology , Animals , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Humans , Hypertrophy , Podocytes/pathology , Signal Transduction
3.
Am J Physiol Renal Physiol ; 310(1): F27-40, 2016 Jan 01.
Article in English | MEDLINE | ID: mdl-26469975

ABSTRACT

Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing a mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of the tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in the obstructed kidney. Instead of acting directly on tPA in the kidney, EPO strongly increased the level of circulating microRNA (miR)-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target the tPA 3'-untranslated region and suppress tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1(+)CD44(+)CD11b(-)CD19(-) cells to secrete miR-144-containing EVs, which markedly suppressed tPA expression, as well as metalloproteinase 9 (MMP9) level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secrete miR-144, which, in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of the tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in CKD, is a potential therapeutic strategy for renal fibrosis.


Subject(s)
Bone Marrow Cells/drug effects , Erythropoietin/pharmacology , Extracellular Vesicles/drug effects , Glomerular Basement Membrane/drug effects , Kidney Diseases/prevention & control , Kidney Tubules/drug effects , MicroRNAs/metabolism , Tissue Plasminogen Activator/metabolism , Ureteral Obstruction/drug therapy , 3' Untranslated Regions , Animals , Binding Sites , Bone Marrow Cells/enzymology , Cell Line , Cytoprotection , Disease Models, Animal , Enzyme Activation , Enzyme Repression , Extracellular Vesicles/enzymology , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibrosis , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Kidney Diseases/enzymology , Kidney Diseases/genetics , Kidney Diseases/pathology , Kidney Tubules/enzymology , Kidney Tubules/pathology , Male , Matrix Metalloproteinase 9/metabolism , Mice , MicroRNAs/blood , MicroRNAs/genetics , Rats , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Tissue Plasminogen Activator/genetics , Ureteral Obstruction/enzymology , Ureteral Obstruction/genetics , Ureteral Obstruction/pathology
4.
PLoS One ; 9(6): e99083, 2014.
Article in English | MEDLINE | ID: mdl-24915008

ABSTRACT

It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes in vitro and in vivo. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.


Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/metabolism , Laminin/metabolism , Nephritis, Hereditary/enzymology , Nephritis, Hereditary/etiology , Animals , Biomechanical Phenomena/drug effects , Cells, Cultured , Enzyme Activation/drug effects , Focal Adhesion Protein-Tyrosine Kinases/antagonists & inhibitors , Gene Knockdown Techniques , Glomerular Basement Membrane/drug effects , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Glomerular Basement Membrane/ultrastructure , I-kappa B Proteins/metabolism , Interleukin-6/metabolism , Kinetics , Matrix Metalloproteinases/metabolism , Mice, Knockout , Morpholines/pharmacology , Morpholines/therapeutic use , NF-KappaB Inhibitor alpha , Nephritis, Hereditary/drug therapy , Nephritis, Hereditary/pathology , Podocytes/enzymology , Podocytes/pathology , Protein Kinase Inhibitors/pharmacology , RNA, Small Interfering/metabolism , Tetraspanin 24/metabolism
6.
J Ethnopharmacol ; 139(2): 454-61, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-22138658

ABSTRACT

AIM OF THE STUDY: To investigate the protective effects and the underlying mechanism of Eucommia lignans against hypertensive renal injury. MATERIAL AND METHODS: Ten-week-old Wistar Kyoto rats and age matched spontaneously hypertension rats were used in the study. The SHR were randomly divided into 4 groups (n=7 for each group) and received different treatment for 16 weeks, which including saline, Captopril, Epalrestat and Eucommia lignans, respectively. System blood pressures of the rats were monitored once every 4 weeks. N-Acetyl-ß-D-glucosaminidase (NAG) activity and the ratio of albumin and urinary creatinine were chosen as the indices of kidney function. Then the structure and renal collagen type III expression of glomerular basement membrane were observed by microscopy and the renal aldose reductase (AR) expression was measured by immunohistochemistry. In vitro, the proliferation of mesangial cells induced by AngII was assayed by MTT, and the mRNA expression of AR was measured by RT-real-time PCR. RESULTS: The renal function, evaluated by NAG enzyme activity and the ratio of albumin to urinary creatinine, was significantly ameliorated by Eucommia lignans treatment. Meanwhile, Eucommia lignans decreased both the protein (P<0.05) and the mRNA expressed lever of AR (P<0.05). Eucommia lignans also decreased the high expression of collagen type III in SHR (P<0.05) and inhibited the proliferation of renal mesangial cells induced by AngII (P<0.05). CONCLUSION: Eucommia lignans have protective effects against hypertensive renal injury, and the protective effects may be partly due to inhibition of aldose reductase.


Subject(s)
Aldehyde Reductase/antagonists & inhibitors , Antihypertensive Agents/pharmacology , Enzyme Inhibitors/pharmacology , Eucommiaceae , Hypertension/drug therapy , Kidney Diseases/prevention & control , Kidney/drug effects , Lignans/pharmacology , Plant Extracts/pharmacology , Acetylglucosaminidase/urine , Albuminuria/enzymology , Albuminuria/etiology , Albuminuria/prevention & control , Aldehyde Reductase/genetics , Aldehyde Reductase/metabolism , Animals , Antihypertensive Agents/isolation & purification , Biomarkers/urine , Blood Pressure/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Collagen Type III/metabolism , Creatinine/urine , Disease Models, Animal , Down-Regulation , Enzyme Inhibitors/isolation & purification , Eucommiaceae/chemistry , Glomerular Basement Membrane/drug effects , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Hypertension/complications , Hypertension/enzymology , Hypertension/physiopathology , Immunohistochemistry , Kidney/enzymology , Kidney/pathology , Kidney/physiopathology , Kidney Diseases/enzymology , Kidney Diseases/etiology , Kidney Diseases/physiopathology , Lignans/isolation & purification , Male , Plant Extracts/isolation & purification , Plants, Medicinal , RNA, Messenger/metabolism , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Time Factors
7.
J Endocrinol Invest ; 33(5): 292-6, 2010 May.
Article in English | MEDLINE | ID: mdl-19820293

ABSTRACT

OBJECTIVE: To observe the effects of simvastatin on urinary excretion of matrix metalloproteinase-9 (MMP- 9), renal expression of MMP-9, and investigate its possible renoprotective mechanisms in streptozotocin (STZ)-induced diabetic rats. METHOD: Twenty-four Wistar rats were divided into 3 groups: control healthy rats (group C, no.=8), untreated diabetic rats (group D, no.=8), and diabetic rats treated with simvastatin (20 mg/kg/d) (group S, no.=8). Peripheral blood glucose was tested weekly, glycosylated hemoglobin A1c (HbA1c), total cholesterol (TC), LDL cholesterol (LDL-C) levels, and urinary albumin (ALB) excretion rate as well as the urinary excretion rates of retinol-binding protein (RBP) and MMP-9 were tested at 8th week. The renal tissues of diabetic rats were obtained for evaluating kidney/ body weight ratio, observing renal pathological changes by electron microscope and examining the expression of renal MMP-9 mRNA by RT-PCR. RESULTS: There was no statistical difference on the change of peripheral blood TC and LDL-C between group C and group D. Peripheral blood glucose, HbA1c levels kidney/body weight ratio urinary excretion rates of ALB, RBP, and MMP-9 concurrently with the expression of renal MMP-9 mRNA were significantly higher in groups D and S compared with group C (p<0.01). Treatment with simvastatin significantly lowered peripheral blood TC, LDL-C, kidney/body weight ratio, urinary excretion rates of ALB, RBP, and MMP-9 as well as the expression of renal MMP-9 mRNA (p<0.01); however, there was no evident effect on the change of blood glucose and HbA1c levels between group D and group S. In addition, urinary excretion rate of MMP-9 showed positive correlations with the urinary ALB excretion and urinary RBP excretion. Pathological lesions of the glomeruli and epithelial cells foot processes (FP) was lightened by simvastatin. CONCLUSION: Simvastatin may has a potential therapeutic target in diabetic nephropathy, which may be partly attributed to down-regulating over-expression of MMP-9 in renal tissue.


Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/prevention & control , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Matrix Metalloproteinase 9/biosynthesis , Matrix Metalloproteinase Inhibitors , Simvastatin/pharmacology , Simvastatin/therapeutic use , Animals , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/pathology , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/pathology , Gene Expression Regulation, Enzymologic/drug effects , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Glycated Hemoglobin/metabolism , Kidney/pathology , Lipids/blood , Male , Matrix Metalloproteinase 9/genetics , Microscopy, Electron , Organ Size/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction
8.
PLoS One ; 4(12): e8474, 2009 Dec 29.
Article in English | MEDLINE | ID: mdl-20041189

ABSTRACT

BACKGROUND: Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease. METHODOLOGY/PRINCIPALS: In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis. FINDINGS: Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs. SIGNIFICANCE: These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure.


Subject(s)
Antibodies, Antinuclear/immunology , Autoimmunity/immunology , Kidney/enzymology , Kidney/pathology , Lupus Nephritis/immunology , Lupus Nephritis/pathology , Animals , Antibodies, Antinuclear/blood , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Disease Progression , Female , Gene Expression Regulation, Enzymologic , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/pathology , Glomerular Basement Membrane/ultrastructure , Glomerular Mesangium/enzymology , Glomerular Mesangium/pathology , Glomerular Mesangium/ultrastructure , Kidney/ultrastructure , Lupus Nephritis/enzymology , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Principal Component Analysis , Proteinuria/complications , Proteinuria/enzymology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Titrimetry
9.
Arthritis Res Ther ; 10(6): 229, 2008.
Article in English | MEDLINE | ID: mdl-19090960

ABSTRACT

Lupus nephritis is a major contributor to morbidity and mortality in systemic lupus erythematosus, but little is known about the pathogenic processes that underlie the progressive decay in renal function. A common finding in lupus nephritis is thickening of glomerular basement membranes associated with immune complex deposition. It has been speculated that alterations in the synthesis or degradation of membrane components might contribute to such changes, and thereby to initiation and progression of nephritis through facilitation of immune complex deposition. Matrix metalloproteinases (MMPs) are enzymes that are intimately involved in the turnover of major glomerular basement membrane constituents, including collagen IV and laminins. Alterations in the expression and activity of MMPs have been described in a number of renal diseases, suggesting their relevance to the pathogenesis of various glomerulopathies. The same is true for their natural inhibitors, the tissue inhibitor of metalloproteinase family. Recent data from our group have identified an increase in proteolytic activity within the glomerulus coinciding with the development of proteinuria in the mouse model of systemic lupus erythematosus. (NXB x NZW)F1 Here we review current understanding of MMP/tissue inhibitor of metalloproteinase function within the kidney, and discuss their possible involvement in the development and progression of lupus nephritis.


Subject(s)
Glomerular Basement Membrane/enzymology , Lupus Nephritis/enzymology , Lupus Nephritis/etiology , Matrix Metalloproteinases/physiology , Animals , Glomerular Basement Membrane/drug effects , Humans , Lupus Nephritis/drug therapy , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use
10.
J Biol Chem ; 283(28): 19432-9, 2008 Jul 11.
Article in English | MEDLINE | ID: mdl-18487197

ABSTRACT

The single 3-hydroxyproline residue in the collagen I polypeptides is essential for proper fibril formation and bone development as its deficiency leads to recessive osteogenesis imperfecta. The vertebrate prolyl 3-hydroxylase (P3H) family consists of three members, P3H1 being responsible for the hydroxylation of collagen I. We expressed human P3H2 as an active recombinant protein in insect cells. Most of the recombinant polypeptide was insoluble, but small amounts were also present in the soluble fraction. P3H1 forms a complex with the cartilage-associated protein (CRTAP) that is required for prolyl 3-hydroxylation of fibrillar collagens. However, coexpression with CRTAP did not enhance the solubility or activity of the recombinant P3H2. A novel assay for P3H activity was developed based on that used for collagen prolyl 4-hydroxylases (C-P4H) and lysyl hydroxylases (LH). A large amount of P3H activity was found in the P3H2 samples with (Gly-Pro-4Hyp)5 as a substrate. The Km and Ki values of P3H2 for 2-oxoglutarate and its certain analogues resembled those of the LHs rather than the C-P4Hs. Unlike P3H1, P3H2 was strongly expressed in tissues rich in basement membranes, such as the kidney. P3H2 hydroxylated more effectively two synthetic peptides corresponding to sequences that are hydroxylated in collagen IV than a peptide corresponding to the 3-hydroxylation site in collagen I. These findings suggest that P3H2 is responsible for the hydroxylation of collagen IV, which has the highest 3-hydroxyproline content of all collagens. It is thus possible that P3H2 mutations may lead to a disease with changes in basement membranes.


Subject(s)
Collagen Type IV/metabolism , Gene Expression Regulation, Enzymologic/physiology , Glomerular Basement Membrane/enzymology , Isoenzymes/biosynthesis , Membrane Glycoproteins/biosynthesis , Protein Processing, Post-Translational/physiology , Proteoglycans/biosynthesis , Animals , Collagen Type I/chemistry , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type IV/chemistry , Collagen Type IV/genetics , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Gene Expression , Glomerular Basement Membrane/chemistry , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Molecular Chaperones , Organ Specificity/physiology , Prolyl Hydroxylases , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Proteoglycans/chemistry , Proteoglycans/genetics , Proteoglycans/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Solubility
11.
Diabetologia ; 51(2): 372-82, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18058084

ABSTRACT

AIMS/HYPOTHESIS: Recent studies suggest that loss of heparan sulphate in the glomerular basement membrane (GBM) of the kidney with diabetic nephropathy is due to the increased production of heparanase, a heparan sulphate-degrading endoglycosidase. Our present study addresses whether heparan sulphate with different modifications is differentially reduced in the GBM and whether heparanase selectively cleaves heparan sulphate with different domain specificities. METHODS: The heparan sulphate content of renal biopsies (14 diabetic nephropathy, five normal) were analysed by immunofluorescence staining with four anti-heparan sulphate antibodies: JM403, a monoclonal antibody (mAb) recognising N-unsubstituted glucosamine residues; two phage display-derived single chain antibodies HS4C3 and EW3D10, defining sulphated heparan sulphate domains; and anti-K5 antibody, an mAb recognising unmodified heparan sulphate domains. RESULTS: We found that modified heparan sulphate domains (JM403, HS4C3 and EW3D10), but not unmodified domains (anti-K5) and agrin core protein were reduced in the GBM of kidneys from patients with diabetic nephropathy, compared with controls. Glomerular heparanase levels were increased in diabetic nephropathy kidneys and inversely correlated with the amounts of modified heparan sulphate domains. Increased heparanase production and loss of JM403 staining in the GBM correlated with the severity of proteinuria. Loss of modified heparan sulphate in the GBM as a result of degradation by heparanase was confirmed by heparan sulphate staining of heparanase-treated normal kidney biopsy specimens. CONCLUSIONS/INTERPRETATION: Our data suggest that loss of modified heparan sulphate in the GBM is mediated by an increased heparanase presence and may play a role in the pathogenesis of diabetes-induced proteinuria.


Subject(s)
Diabetic Nephropathies/enzymology , Glucuronidase/metabolism , Heparitin Sulfate/metabolism , Diabetic Nephropathies/metabolism , Fluorescent Antibody Technique , Glomerular Basement Membrane/enzymology , Glomerular Basement Membrane/metabolism , Humans , Immunohistochemistry
12.
Kidney Int ; 73(3): 278-87, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18046314

ABSTRACT

Heparan sulfate in the glomerular basement membrane has been considered crucial for charge-selective filtration. In many proteinuric diseases, increased glomerular expression of heparanase is associated with decreased heparan sulfate. Here, we used mice overexpressing heparanase and evaluated the expression of different heparan sulfate domains in the kidney and other tissues measured with anti-heparan sulfate antibodies. Glycosaminoglycan-associated anionic sites were visualized by the cationic dye cupromeronic blue. Transgenic mice showed a differential loss of heparan sulfate domains in several tissues. An unmodified and a sulfated heparan sulfate domain resisted heparanase action in vivo and in vitro. Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular basement membrane of transgenic mice, whereas glomerular ultrastructure and renal function remained normal. Heparanase-resistant heparan sulfate domains may represent remnant chains or chains not susceptible to cleavage. Importantly, the strong reduction of glycosaminoglycan-associated anionic sites in the glomerular basement membrane without development of a clear renal phenotype questions the primary role of heparan sulfate in charge-selective filtration. We cannot, however, exclude that overexpression of heparanase and heparan sulfate loss in the basement membrane in glomerular diseases contributes to proteinuria.


Subject(s)
Anions/metabolism , Glomerular Basement Membrane/metabolism , Glucuronidase/metabolism , Heparitin Sulfate/metabolism , Proteinuria/metabolism , Animals , Gene Expression , Glomerular Basement Membrane/enzymology , Glucuronidase/genetics , Glycosaminoglycans/metabolism , Humans , Kidney Function Tests , Kidney Glomerulus/ultrastructure , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Protein Structure, Tertiary
13.
J Am Soc Nephrol ; 17(9): 2513-20, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16899518

ABSTRACT

Heparan sulfate (HS) in the glomerular basement membrane (GBM) is important for regulation of the charge-dependent permeability. Heparanase has been implicated in HS degradation in several proteinuric diseases. This study analyzed the role of heparanase in HS degradation in Adriamycin nephropathy (AN), a model of chronic proteinuria-induced renal damage. Expression of heparanase, HS, and the core protein of agrin (to which HS is attached) was determined on kidney sections from rats with AN in different experiments. First, expression was examined in a model of unilateral AN in a time-course study at 6-wk intervals until week 30. Second, rats were treated with the hydroxyl radical scavenger dimethylthiourea (DMTU) during bilateral AN induction. Finally, 6 wk after AN induction, rats were treated with angiotensin II receptor type 1 antagonist (AT1A) or vehicle for 2 wk. Heparanase expression was increased in glomeruli of rats with AN, which correlated with HS reduction at all time points and in all experiments. Treatment with DMTU prevented the increased heparanase expression, the loss of GBM HS, and reduced albuminuria. Finally, treatment of established proteinuria with AT1A significantly reduced heparanase expression and restored glomerular HS. In conclusion, an association between heparanase expression and reduction of glomerular HS in AN was observed. The effects of DMTU suggest a role for reactive oxygen species in upregulation of heparanase. Antiproteinuric treatment by AT1A decreased heparanase expression and restored HS expression. These results suggest involvement of radicals and angiotensin II in the modulation of GBM permeability through HS and heparanase expression.


Subject(s)
Doxorubicin/toxicity , Glomerular Basement Membrane/enzymology , Glucuronidase/biosynthesis , Nephrosis/chemically induced , Proteinuria/chemically induced , Reactive Oxygen Species/pharmacology , Renin-Angiotensin System/physiology , Agrin/biosynthesis , Angiotensin Receptor Antagonists , Animals , Enzyme Induction , Gene Expression Regulation/drug effects , Heparitin Sulfate/metabolism , Imidazoles/pharmacology , Kidney Tubules/enzymology , Male , Rats , Rats, Wistar , Tetrazoles/pharmacology , Thiourea/analogs & derivatives , Thiourea/pharmacology
14.
Kidney Int ; 69(9): 1570-7, 2006 May.
Article in English | MEDLINE | ID: mdl-16541018

ABSTRACT

Diabetic glomerulopathy has been linked to shifts in balance between the synthetic and degradative pathways of the glomerular basement membrane (GBM), a key player in the permselectivity properties of the glomerular wall. The goal of this study was to trace the expression and localization of membrane type-1 metalloprotease (MT1-MMP) and its activating enzyme furin, key proteins involved in basement membrane turnover, in short- and long-term diabetic rat renal tissues. Quantitative immunogold was carried out for MT1-MMP and furin and their expression was evaluated in renal tissues of young and old, control and diabetic rats. To corroborate immunocytochemical findings, Western blots were performed on glomerular lysates. Electron microscopy revealed that the overall expression of MT1-MMP and furin is reduced in plasma membranes of all glomerular cell types of old normoglycemic animals, a phenomenon that is exacerbated in long-term diabetic animals. This observation supports the prevailing theory that diabetes fosters acceleration in the aging process. Interestingly, while biochemical results confirmed a decrease in MT1-MMP expression, an increase in furin was observed. Immunocytochemical studies resolved this discrepancy by tracing the increased furin expression in endoplasmic reticulum and Golgi membranes of podocytes, indicating that furin is retained in the secretory pathway in a diabetic environment. Disturbances at the molecular level of the otherwise tightly regulated MT1-MMP/furin interactions found at the cell surface must account for a lack in extracellular matrix remodeling, increased deposition of GBM material, and loss of glomerular filtration integrity.


Subject(s)
Diabetic Nephropathies/enzymology , Furin/metabolism , Glomerular Basement Membrane/enzymology , Kidney Glomerulus/enzymology , Matrix Metalloproteinases/metabolism , Animals , Endoplasmic Reticulum/enzymology , Furin/analysis , Glomerular Basement Membrane/ultrastructure , Golgi Apparatus/enzymology , Kidney Glomerulus/ultrastructure , Male , Matrix Metalloproteinases/analysis , Matrix Metalloproteinases, Membrane-Associated , Rats , Rats, Sprague-Dawley
15.
Saudi Med J ; 23(11): 1380-5, 2002 Nov.
Article in English | MEDLINE | ID: mdl-12506300

ABSTRACT

OBJECTIVES: The aim of the present study was to examine the effect of exposing rats to ischemia-reperfusion while breathing 100% oxygen or room air, to find the effect of glycine on renal sodium-potassium adenosine triphosphatase (Na+-K+ATPase) and endogenous antioxidant enzymes, superoxide dismutase and catalase, also to ascertain the effect of ischemia-reperfusion on renal nitric oxide and lipid peroxides. METHODS: This study was carried out at King Saud University, Riyadh, Kingdom of Saudi Arabia, over a period of 11 months, February to December 2001. All previous measurements were carried out on the renal homogenate after 60 minutes ischemia, then after reperfusion while animals breathed room air or 100% oxygen and also after glycine treatment. RESULTS: The activity of Na+-K+ATPase, catalase and superoxide dismutase concentration was decreased significantly in the ischemic rats compared to the control, a further decrease was found after 20 minutes of reflow while breathing room air. Breathing 100% oxygen resulted in a significant decrease in catalase and Na+-K+ATPase activity and concentration of superoxide dismutase, glycine caused insignificant change of these enzymes after ischemia-index of lipid peroxidation and nitric oxide they were significantly elevated following reperfusion while rats breathed room air and further elevation was noticed after breathing 100% oxygen. However, potassium and creatinine did not change in all study groups, showed significant decrease after ischemia and ischemia-reperfusion may be due to marked Na+ loss in urine and lack of Na+ reabsorption. The inhibition of superoxide dismutase and catalase can be explained by increased reactive oxygen species during reperfusion and hyperoxia, also due to nitric oxide production and lipid peroxidation as shown by high malondialdehyde. Lack of Na+K+ATPase can be contributed to loss of antioxidant enzymes, nitric oxide production, and high reactive oxygen species. CONCLUSION: Hyperoxia in ischemia-reperfusion induces severe damage to cellular defence mechanisms and enhances reactive oxygen species injury. Glycine, as antioxidant, is involved in kidney protection from massive injury induced by ischemia-reperfusion, protects renal antioxidant enzymes and Na+-K+ATPase, normalizes malondialdehyde, and nitric oxide levels. This data further supports the possible role of glycine therapy as an adjunct in the treatment of renal failure.


Subject(s)
Free Radical Scavengers/pharmacology , Glomerular Basement Membrane/enzymology , Glycine/pharmacology , Kidney/blood supply , Reperfusion Injury/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Lipid Peroxidation , Male , Rats , Rats, Wistar
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