ABSTRACT
BACKGROUND: Serum amyloid A (SAA), a potent inflammatory mediator, and Janus kinase 2 (JAK2), an intracellular signaling kinase, are increased by diabetes. The aims were to elucidate: 1) a JAK2-mediated pathway for increased SAA in the kidneys of diabetic mice; 2) a JAK2-SAA pathway for inflammation in podocytes. METHODS: Akita diabetic mice (129S6) with podocyte JAK2 overexpression and angiotensin II infusion (4 weeks) were given a JAK1,2 inhibitor (LY03103801, 3 mg/kg/day orally for the last two weeks). Kidneys were immunostained for SAA isoform 3 (SAA3). SAA3 knockout and control mouse podocytes were exposed to advanced glycation end products (AGE) or exogenous SAA with JAK2 inhibition (Tyrphostin AG 490, 50µM). JAK2 activity (phosphorylation, Western blot, 1 hour) and mRNA for SAA3 and associated inflammatory genes (Cxcl5, Ccl2, and Ccl5) were measured by RT-PCR (20 hours). RESULTS: SAA3 protein was present throughout the diabetic kidney, and podocyte JAK2 overexpression increased tubulointerstitial SAA3 compared to wild type diabetic controls, 43% versus 14% (p = 0.007); JAK1,2 inhibition attenuated the increase in SAA3 to 15% (p = 0.003). Urine albumin-to-creatinine ratio (r = 0.49, p = 0.03), mesangial index (r = 0.64, p = 0.001), and glomerulosclerosis score (r = 0.51, p = 0.02) were associated with SAA3 immunostaining scores across mouse groups. Exposing podocytes to AGE or exogenous SAA increased JAK2 activity within one hour and mRNA for associated inflammatory genes after 20 hours. JAK2 inhibition reduced SAA3 mRNA expression in podocytes exposed to AGE or SAA. SAA3 knockout podocytes had >85% lower AGE-induced inflammatory genes. CONCLUSION: JAK1,2 inhibition reduced SAA and histological features of DKD in podocyte JAK2-overexpressing mice. In podocytes exposed to a diabetes-like condition, JAK2 inhibition reduced expression of SAA, while SAA knockout blocked expression of associated pro-inflammatory mediators. SAA may promote JAK2-dependent inflammation in the diabetic kidney.
Subject(s)
Diabetic Nephropathies/metabolism , Janus Kinase 2/metabolism , Serum Amyloid A Protein/metabolism , Animals , Cells, Cultured , Cholesterol/blood , Gene Knockout Techniques , Glycated Hemoglobin/metabolism , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Kidney/metabolism , Mice , Podocytes/metabolism , Protein Kinase Inhibitors/pharmacology , Serum Amyloid A Protein/genetics , Signal Transduction , Triglycerides/bloodABSTRACT
IN BRIEF Current therapeutic approaches are only moderately efficacious at preventing the progression of diabetic kidney disease (DKD). As the number of people with DKD continues to rise worldwide, there is an urgent need for novel therapies. A better understanding of the root causes and molecular mechanisms of DKD pathogenesis has enabled the identification of numerous new therapeutic targets, including advanced glycation end products, reactive oxygen species, protein kinase C, and serum amyloid A. Although experimental studies have illustrated the potential of such approaches, challenges in clinical translation remain a barrier in therapeutic development. Advances in preclinical safety and efficacy evaluations and improved delivery systems may aid in clinical translation of novel DKD therapies.
ABSTRACT
Inflammatory pathways are central mechanisms in diabetic kidney disease (DKD). Serum amyloid A (SAA) is increased by chronic inflammation, but SAA has not been previously evaluated as a potential DKD mediator. The aims of this study were to determine whether SAA is increased in human DKD and corresponding mouse models and to assess effects of SAA on podocyte inflammatory responses. SAA was increased in the plasma of people with DKD characterized by overt proteinuria and inversely correlated with estimated glomerular filtration rate (creatinine-based CKD-EPI). SAA was also elevated in plasma of diabetic mouse models including type 1 diabetes (streptozotocin/C57BL/6) and type 2 diabetes (BTBR-ob/ob). SAA mRNA (Nephromine) was increased in human DKD compared with non-diabetic and/or glomerular disease controls (glomerular fold change 1.5, P=0.017; tubulointerstitium fold change 1.4, P=0.021). The kidneys of both diabetic mouse models also demonstrated increased SAA mRNA (quantitative real-time PCR) expression compared with non-diabetic controls (type 1 diabetes fold change 2.9; type 2 diabetes fold change 42.5, P=0.009; interaction by model P=0.57). Humans with DKD and the diabetic mouse models exhibited extensive SAA protein deposition in the glomeruli and tubulointerstitium in similar patterns by immunohistochemistry. SAA localized within podocytes of diabetic mice. Podocytes exposed to advanced glycation end products, metabolic mediators of inflammation in diabetes, increased expression of SAA mRNA (fold change 15.3, P=0.004) and protein (fold change 38.4, P=0.014). Podocytes exposed to exogenous SAA increased NF-κB activity, and pathway array analysis revealed upregulation of mRNA for NF-κB-dependent targets comprising numerous inflammatory mediators, including SAA itself (fold change 17.0, P=0.006). Inhibition of NF-κB reduced these pro-inflammatory responses. In conclusion, SAA is increased in the blood and produced in the kidneys of people with DKD and corresponding diabetic mouse models. Podocytes are likely to be key responder cells to SAA-induced inflammation in the diabetic kidney. SAA is a compelling candidate for DKD therapeutic and biomarker discovery.
Subject(s)
Diabetic Nephropathies/blood , Inflammation/blood , Podocytes/metabolism , Serum Amyloid A Protein/metabolism , Animals , Antioxidants/pharmacology , Cells, Cultured , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 2/blood , Gene Expression/drug effects , Humans , Immunohistochemistry , Kidney/drug effects , Kidney/metabolism , Kidney/pathology , Kidney Diseases/blood , Mice, Inbred C57BL , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Podocytes/drug effects , Pyrrolidines/pharmacology , Recombinant Proteins/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/pharmacology , Thiocarbamates/pharmacologyABSTRACT
BACKGROUND: Overfeeding amino acids (AAs) increases cellular exposure to advanced glycation end-products (AGEs), a mechanism for protein intake to worsen diabetic kidney disease (DKD). This study assessed receptor for AGE (RAGE)-mediated apoptosis and inflammation in glomerular cells exposed to metabolic stressors characteristic of high-protein diets and/or diabetes in vitro with proof-of-concept appraisal in vivo. METHODS: Mouse podocytes and mesangial cells were cultured under control and metabolic stressor conditions: (i) no addition; (ii) increased AAs (4-6-fold>control); (iii) high glucose (HG, 30.5 mM); (iv) AA/HG combination; (v) AGE-bovine serum albumin (AGE-BSA, 300 µg/mL); (vi) BSA (300 µg/mL). RAGE was inhibited by blocking antibody. Diabetic (streptozotocin) and nondiabetic mice (C57BL/6J) consumed diets with protein calories of 20 or 40% (high) for 20 weeks. People with DKD and controls provided 24-h urine samples. RESULTS: In podocytes and mesangial cells, apoptosis (caspase 3/7 activity and TUNEL) increased in all metabolic stressor conditions. Both inflammatory mediator expression (real-time reverse transcriptase-polymerase chain reaction: serum amyloid A, caspase-4, inducible nitric oxide synthase, and monocyte chemotactic protein-1) and RAGE (immunostaining) also increased. RAGE inhibition prevented apoptosis and inflammation in podocytes. Among mice fed high protein, podocyte number (WT-1 immunostaining) decreased in the diabetic group, and only these diabetic mice developed albuminuria. Protein intake (urea nitrogen) correlated with AGE excretion (carboxymethyllysine) in people with DKD and controls. CONCLUSIONS: High-protein diet and/or diabetes-like conditions increased glomerular cell death and inflammation, responses mediated by RAGEs in podocytes. The concept that high-protein diets exacerbate early indicators of DKD is supported by data from mice and people.
Subject(s)
Apoptosis , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/pathology , Diabetic Nephropathies/etiology , Diet , Dietary Proteins/pharmacology , Inflammation/etiology , Animals , Blotting, Western , Case-Control Studies , Cells, Cultured , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/metabolism , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Glycation End Products, Advanced/genetics , Glycation End Products, Advanced/metabolism , Humans , Inflammation/metabolism , Inflammation/pathology , Inflammation Mediators/metabolism , Male , Mesangial Cells/metabolism , Mesangial Cells/pathology , Mice , Mice, Inbred C57BL , Podocytes/metabolism , Podocytes/pathology , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Receptor for Advanced Glycation End Products , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND/AIMS: High levels of glucose and/or amino acids increase advanced glycation end products (AGE) and activate protein kinase C (PKC), a key signal for injury in mesangial cells. The aim was to determine whether oxidative stress mediates bidirectional interactions between AGE and PKC ('cross-activation') in this model. METHODS: Rat mesangial cells were examined after 48 h of exposure to: high glucose (30.5 mM), increased amino acids designed to resemble a protein meal, the combination of both conditions, and control. Cells were treated with antioxidants (vitamin E, alpha-lipoic acid, N-acetylcysteine, apocynin, doses based on suppression of reactive oxygen species), PKC inhibitors (calphostin C orPLY379196, 100 nM), or AGE inhibitors (aminoguanidine or pyridoxamine 0.5 mM). RESULTS: Carboxymethyllysine, an AGE marker, increased twofold in mesangial cells exposed to the experimental conditions. Antioxidants and PKC inhibition prevented carboxymethyllysine increases. Likewise, antioxidants and AGE inhibition prevented PKC activation. Inhibition of carboxymethyllysine increases and PKC activation by apocynin indicates a primary role for NADPH oxidase in producing oxidative stress. Induction of transforming growth factor-beta(1) and fibronectin was inhibited by antioxidants and inhibitors of PKC and AGE. CONCLUSIONS: Oxidative stress mediated cross-activation between PKC and AGE in this mesangial cell model of diabetes and high protein diet.PKC may amplify cellular injury by promoting AGE accumulation.
Subject(s)
Diabetes Mellitus/metabolism , Dietary Proteins/administration & dosage , Glycation End Products, Advanced/metabolism , Mesangial Cells/enzymology , Oxidative Stress , Protein Kinase C/metabolism , Animals , Antioxidants/pharmacology , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/pharmacology , Female , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: In diabetes, high intake of dietary protein exacerbates responses associated with kidney damage. Increased levels of amino acids could injure cells by providing free amino groups for glycation reactions leading to advanced glycation end products (AGEs). METHODS: Rat mesangial cells were cultured with increased amino acids designed to resemble protein feeding, high glucose (30.5 mmol/L), and, the combination, amino acids/high glucose. AGEs, reactive oxygen species (ROS), protein kinase C (PKC) activity and production, and mitogen-activated protein (MAP) kinase-extracellular signal regulated kinase (ERK) 1,2 activity were measured. Inhibitors were used to determine roles of these processes in fibrosis and/or AGE formation. RESULTS: AGE immunostaining increased when cells were cultured in amino acids and was comparable to that observed with high glucose. In amino acids/high glucose, AGE immunostaining appeared even greater. Amino acids, high glucose, and amino acids/high glucose induced ROS production. Aminoguanidine and vitamin E prevented AGE accumulation and induction of protein and mRNA for fibrosis markers [transforming growth factor-beta1 (TGF-beta1), fibronectin, and collagen IV]. PKC and ERK 1,2 activity increased with amino acids, high glucose, and amino acids/high glucose. PKC-beta inhibition prevented ERK 1,2 activation and fibrosis induction. ERK 1,2 inhibition also blocked the fibrosis response. CONCLUSION: A profibrotic injury response occurred in mesangial cells exposed to amino acids, with or without high glucose, by formation of AGE, oxidative stress, and activation of the PKC-beta and MAP kinase-ERK 1,2 signal pathway. These observations provide new insight into cellular mechanisms of kidney damage produced by excess dietary protein, particularly in diabetes.
Subject(s)
Amino Acids/toxicity , Glomerular Mesangium/drug effects , Glycation End Products, Advanced/biosynthesis , Oxidative Stress , Protein Kinase C/physiology , Animals , Antioxidants/pharmacology , Cells, Cultured , Extracellular Signal-Regulated MAP Kinases/physiology , Female , Fibrosis , Glomerular Mesangium/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
High-protein diets exacerbate glomerular hyperfiltration and the progression of diabetic nephropathy. The purpose of this study was to determine whether amino acids also produce nonhemodynamic injury in the glomerulus. When rat mesangial cells were cultured with an amino acid mixture designed to replicate the composition in plasma after protein feeding, production of mRNA (Northern blot analysis) and/or protein (ELISA or Western blot analysis) for transforming growth factor-beta1 (TGF-beta1), fibronectin, thrombospondin-1 (TSP-1), and collagen IV were enhanced in a manner comparable to a culture with high glucose (30.5 mM). The bioactive portion of total TGF-beta (NRK assay) increased in response to amino acids. The TSP-1 antagonist LSKL peptide reduced bioactive TGF-beta and fibronectin, indicating the dependence of TGF-beta1 activation on TSP-1. DNA synthesis ([3H]thymidine incorporation), an index of cellular proliferation, increased in response to amino acids and was further enhanced by culture with increased levels of both amino acids and glucose. TGF-beta1 and matrix proteins increased when mesangial cells were cultured with excess l-arginine (2.08 mM) alone. Although l-arginine is the precursor of nitric oxide (NO), such responses to amino acids do not appear to be mediated through increased NO production. NO metabolites decreased in the media, and these responses to mixed amino acids or l-arginine were not prevented by NO synthase inhibition. In conclusion, amino acids induce indicators of response to injury in mesangial cells, even when hemodynamic stress is absent. In conditions associated with increased circulating amino acids, such as diabetes and/or a high-protein diet, direct cellular effects could contribute to glomerular injury.
Subject(s)
Amino Acids/pharmacology , Glomerular Mesangium/drug effects , Glomerular Mesangium/pathology , Animals , Arginine/pharmacology , Cell Division/drug effects , Cell Survival/drug effects , Extracellular Matrix Proteins/metabolism , Female , Glomerular Mesangium/metabolism , Glucose/pharmacology , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Rats , Rats, Sprague-Dawley , Thrombospondin 1/pharmacology , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1ABSTRACT
BACKGROUND: High protein diets and diabetes increase renal renin angiotensin system (RAS) activity, which is associated with glomerular injury. Aminopeptidase A (APA) is a cell surface metalloprotease that degrades angiotensin II (AII) in the mesangium. Mesangial cells (MC) also possess receptors for AII; the type 1 (AT1 receptor) promotes proliferation and fibrosis, while the type 2 (AT2 receptor) opposes these effects. We evaluated whether amino acids and glucose alter expression of APA, AT1 receptor and AT2 receptor in a manner that further augments RAS activity. METHODS: Confluent rat MC were grown in serum-free media for 48 hours prior to exposing to experimental conditions: control (C), high amino acids (HA, mixed amino acid solution added to raise concentrations 5- to 6-fold over C), high glucose (HG 30, mM glucose). Semi-quantitative RT-PCR was used to assess mRNA for APA, AT1 receptor, AT2 receptor, and beta-actin. Values are expressed relative to beta actin. RESULTS: Both HA and HG reduced APA mRNA (HG 1.13 plus minus 0.19, HA 1.12 plus minus 0.16 versus C 1.27 plus minus 0.16 P < 0.05, N = 8). HA increased AT1 receptor mRNA (HA 2.11 plus minus 0.43 versus C 1.14 plus minus 0.28 P < 0.05, N = 8). HG increased AT2 receptor mRNA (HG 1.31 plus minus 0.43 versus C 0.82 plus minus 0.33 P < 0.05, N = 6). CONCLUSIONS: A reduction of APA, in response to high levels of amino acids or glucose, could contribute to increased AII as a result of decreased degradation in MC. The effect of amino acids to increase AT1 receptor expression may further enhance adverse hemodynamic and pro-fibrotic actions of AII. Conversely, glucose increased AT2 receptor expression, which could modulate responses mediated by the AT1 receptor.
Subject(s)
Amino Acids/pharmacology , Glucose/pharmacology , Glutamyl Aminopeptidase/biosynthesis , Mesangial Cells/drug effects , Mesangial Cells/physiology , Receptor, Angiotensin, Type 1/biosynthesis , Receptor, Angiotensin, Type 2/biosynthesis , Animals , Cells, Cultured , Rats , Rats, Sprague-DawleyABSTRACT
Increased dietary protein and circulating amino acids raise glomerular filtration rate (GFR) and pressure. In diabetes, this glomerular hyperfiltration response is augmented. The purpose of this study was to determine whether glucagon mediates the augmented GFR response to amino acids in diabetes and whether the responses to amino acids and glucagon depend on prostaglandins. Patients with type 1 diabetes mellitus (n = 12) and normal control subjects (n = 12) were studied in a series of six experiments, each on different occasions. Baseline GFR was not significantly increased, but filtration fraction was higher in diabetes. In response to amino acid infusion, GFR increased more and filtration fraction was greater among those with diabetes. Their augmented GFR response to amino acids was not inhibited by octreotide or indomethacin. Participants with diabetes also had enhanced GFR and renal plasma flow responses to glucagon infusion, both of which were inhibited by indomethacin. Glomerular hyperfiltration responses induced by amino acids or glucagon occur by divergent pathways in diabetes; only the response to glucagon is prostaglandin dependent.