Serum amyloid A and Janus kinase 2 in a mouse model of diabetic kidney disease.

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.

Association of Serum Amyloid A with Kidney Outcomes and All-Cause Mortality in American Indians with Type 2 Diabetes.

BACKGROUND: Serum amyloid A (SAA) induces inflammation and apoptosis in kidney cells and is found to be causing the pathologic changes that are associated with diabetic kidney disease (DKD). Higher serum SAA concentrations were previously associated with increased risk of end-stage renal disease (ESRD) and death in persons with type 2 diabetes and advanced DKD. We explored the prognostic value of SAA in American Indians with type 2 diabetes without DKD or with early DKD. METHODS: SAA concentration was measured in serum samples obtained at the start of follow-up. Multivariate proportional hazards models were employed to examine the magnitude of the risk of ESRD or death across tertiles of SAA concentration after adjustment for traditional risk factors. The C statistic was used to assess the additional predictive value of SAA relative to traditional risk factors. RESULTS: Of 256 participants (mean ± SD glomerular filtration rate [iothalamate] = 148 ± 45 mL/min, and median [interquartile range] urine albumin/creatinine = 39 [14-221] mg/g), 76 developed ESRD and 125 died during a median follow-up period of 15.2 and 15.7 years, respectively. After multivariable proportional hazards regression, participants in the 2 highest SAA tertiles together exhibited a 53% lower risk of ESRD (hazard ratio [HR] 0.47, 95% CI 0.29-0.78), and a 30% lower risk of death (HR 0.70, 95% CI 0.48-1.02), compared with participants in the lowest SAA tertile, although the lower risk of death was not statistically significant. Addition of SAA to the ESRD model increased the C statistic from 0.814 to 0.815 (p = 0.005). CONCLUSIONS: Higher circulating SAA concentration is associated with a reduced risk of ESRD in American Indians with type 2 diabetes.

Serum amyloid a and risk of death and end-stage renal disease in diabetic kidney disease.

AIMS: To determine if serum levels of serum amyloid A (SAA) predict death and end-stage renal disease in a cohort of people with diabetic kidney disease. METHODS: In a longitudinal cohort study of 135 participants with type 2 diabetes and diabetic kidney disease, serum samples were assayed for SAA. Censored time-to-event analyses in Cox-proportional hazard models were utilized to assess SAA as a predictor of the primary outcome of death and end-stage renal disease. RESULTS: Participants were 73% Mexican-American (99/135) and 55% men (75/135), with a mean±SD age of 57±7.5years. At baseline, participants had hemoglobin A1c of 8.6±2.3%, systolic blood pressure of 153±27mm Hg, body mass index of 31±9kg/m2, median urine-albumin-to-creatinine ratio of 1861mg/g (interquartile range 720-3912mg/g), and estimated glomerular filtration rate of 55.7±22.3ml/min/1.73m2. Over a median duration of follow-up of 3.5years, 44% (60/135) of participants experienced a primary outcome event. The hazards ratio for the primary outcome was 3.03 (95% CI 1.43-6.40, p=0.003) in the highest (>1.0 µg/ml) compared to the lowest (<0.55 µg/ml) SAA tertile in a model adjusted for urine-albumin-to-creatinine ratio, estimated glomerular filtration rate, age, sex, and race/ethnicity. Addition of SAA as a covariate improved the model C-statistic (Δ c=0.017). CONCLUSIONS: In a longitudinal cohort study of participants with type 2 diabetes and DKD, higher levels of serum SAA predicted higher risk of death and ESRD. SAA is a promising targetable biomarker for DKD.

Novel Therapies for Diabetic Kidney Disease: Storied Past and Forward Paths.

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.

Serum amyloid A and inflammation in diabetic kidney disease and podocytes.

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.

Glomerular cell death and inflammation with high-protein diet and diabetes.

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.

Oxidative stress mediates protein kinase C activation and advanced glycation end product formation in a mesangial cell model of diabetes and high protein diet.

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.

Amino acids injure mesangial cells by advanced glycation end products, oxidative stress, and protein kinase C.

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.

Amino acids induce indicators of response to injury in glomerular mesangial cells.

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.

Effects of amino acids and glucose on mesangial cell aminopeptidase a and angiotensin receptors.

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.