Placental growth factor deficiency initiates obesity- and aging-associated metabolic syndrome.

Obesity often leads to inadequate angiogenesis in expanding adipose tissue, resulting in inflammation and insulin resistance. We explored the role of placental growth factor (PlGF) in metabolic syndrome (MS) using mice models of type 2 diabetes, high-fat diet, or aging. Reduced serum PlGF levels were associated with decreased insulin sensitivity and development of MS features. PlGF was localized within endothelial cells and pericytes of adipose tissue. In vitro, low PlGF levels in hypoxic conditions worsened oxidative stress, apoptosis, and reduced autophagy. This was associated with a reduction in expression of vascular endothelial growth factor (VEGF)-A/VEGF-R1/-R2, which was influenced by a decrease and increase in PlGF/pAMPK/PI3K-pAkt/PLCγ1-iCa++/eNOS and PTEN/GSK3ß axes, respectively. PlGF-knockout mice exhibited MS traits through alterations in the same signaling pathways, and these changes were mitigated by recombinant PlGF and metformin. These enhanced angiogenesis and lipid metabolism, underscoring PlGF's role in age-related MS and its potential as a therapeutic target.

Phosphodiesterase inhibitor ameliorates senescent changes of renal interstitial pericytes in aging kidney.

Pericytes are mesenchymal cells that surround endothelial cells, playing a crucial role in angiogenesis and vessel maturation. Additionally, they are associated with interstitial fibrosis as a major contributor to renal myofibroblasts. In this study, we aim to investigate whether the phosphodiesterase inhibitor, pentoxifylline (PTX), can ameliorate aging-related functional and histological deterioration in the kidney. We subjected aging C57BL/6 mice, dividing into young, aging, and PTX-treated aging groups. Renal function, albuminuria, and histological changes were assessed. Interstitial pericytes were assessed by immunohistochemistry analysis. We examined changes in pericytes in elderly patients using human kidney tissue obtained from healthy kidney donors for kidney transplantation. In vitro experiments with human pericytes and endothelial cells were performed. Aging mice exhibited declined renal function, increased albuminuria, and aging-related histological changes including mesangial expansion and tubulointerstitial fibrosis. Notably, number of pericytes declined in aging kidneys, and myofibroblasts increased. PTX treatment ameliorated albuminuria, histological alterations, and microvascular rarefaction, as well as modulated angiopoietin expression. In vitro experiments showed PTX reduced cellular senescence and inflammation. Human kidney analysis confirmed similar pericyte changes in aging kidneys. The phosphodiesterase inhibitor, PTX preserved microvascular density and improved renal interstitial fibrosis and inflammation in aging mice kidneys. These protective effects were suggested to be associated with the amelioration of pericytes reduction and the transition to myofibroblasts. Additionally, the upregulation of angiopoietin-1 expression may exert potential impacts. To the best of our knowledge, this is the first report on the changes in renal interstitial pericytes in aging human kidneys.

Extracellular Superoxide Dismutase Attenuates Hepatic Oxidative Stress in Nonalcoholic Fatty Liver Disease through the Adenosine Monophosphate-Activated Protein Kinase Activation.

Oxidative stress is key in type 2 diabetes-associated nonalcoholic fatty liver disease (NAFLD). We explored whether extracellular superoxide dismutase (EC-SOD) activates adenosine monophosphate-activated protein kinase (AMPK) to enhance antioxidant synthesis and lipid metabolism in NAFLD. Human recombinant EC-SOD (hEC-SOD) was administered to 8-week-old male C57BLKS/J db/db mice through intraperitoneal injection once a week for 8 weeks. Target molecules involved in oxidative stress and lipid metabolism were investigated. hEC-SOD improved insulin resistance and systemic and hepatic oxidative stress characterized by increases in urinary 8-hydroxy-deoxyguanosine and 8-isoprostane levels in db/db mice and a decrease in DHE expression in the liver, respectively. Hepatic SOD3 expression in db/db mice was reversed by hEC-SOD, which improved hepatic steatosis, inflammation with M2 polarization, apoptosis, autophagy, fibrosis and lipid metabolism in db/db mice, as reflected by the changes in serum and hepatic markers, monocyte chemoattractant protein-1, tumor necrosis factor-α, TUNEL-positive cells, Bcl-2/BAX ratio, beclin1 and LC3-II/LC3-1. At the molecular level, hEC-SOD increased phosphorylated-AMPK related to CaMKKß, activation of peroxisome proliferative-activated receptor-gamma coactivator (PGC)-1α and dephosphorylation of forkhead box O (FoxO)1 and their subsequent downstream signaling. In HepG2Cs cells using AMPKα1 and AMPKα2 siRNA, hEC-SOD demonstrated a protective effect via the direct activation of both AMPK-PGC-1α and AMPK-FoxO1. EC-SOD might be a potential therapeutic agent for NAFLD through the activation of AMPK-PGC-1α and AMPK-FoxO1 signaling in hepatocytes, which modulates lipid metabolism, leading to anti-inflammatory, antioxidative and antiapoptotic effects and improving autophagy in the liver.

Adiponectin receptor agonist ameliorates cardiac lipotoxicity via enhancing ceramide metabolism in type 2 diabetic mice.

Accumulation of lipids and their metabolites induces lipotoxicity in diabetic cardiomyopathy. Lowering ceramide concentration could reduce the impact of metabolic damage to target organs. Adiponectin improves lipotoxicity through its receptors (AdiopRs), which have sequence homology with ceramidase enzymes. Therefore, cardioprotective role of AdipoR agonism by AdipoRon was investigated. Sixteen-week-old male db/m and db/db mice were fed a diet containing AdipoRon for four weeks. Phenotypic and metabolic profiles with associated cellular signaling pathways involved in lipid metabolism were investigated in the mice heart and human cardiomyocytes to establish treatment effect of AdipoRon. AdipoRon ameliorated insulin resistance, fibrosis, M1-dominant inflammation, and apoptosis in association with reduced accumulations of free fatty acid, triglycerides, and TLR4-related ceramide in the heart. This resulted in overall reduction in the level of oxidative stress which ameliorated cardiac hypertrophy and its function. AdipoRon increased the expression of AdipoR1 and AdipoR2 via pAMPK/FoxO1-induced Akt phosphorylation resulting from a decrease in PP2A level. It also increased acid ceramidase activity which reduced ceramide and increased sphingosine-1 phosphate levels in the heart of db/db mice and cultured human cardiomyocytes. Consistent upregulation of AdipoRs and their downstream regulatory pathways involving pAMPK/PPARα/PGC-1α levels led to lipid metabolism enhancement, thereby improving lipotoxicity-induced peroxisome biogenesis and oxidative stress. AdipoRon might control oxidative stress, inflammation, and apoptosis in the heart through increased AdipoR expression, acid ceramidase activity, and activation of AMPK-PPARα/PGC-1α and related downstream pathways, collectively improving cardiac lipid metabolism, hypertrophy, and functional parameters.

Role of Aberrantly Activated Lysophosphatidic Acid Receptor 1 Signaling Mediated Inflammation in Renal Aging.

The increasing load of senescent cells is a source of aging, and chronic inflammation plays a pivotal role in cellular senescence. In addition, senescent renal tubular epithelial cells are closely associated with renal aging. Lysophosphatidic acid (LPA) is a bioactive lipid mainly produced by the catalytic action of autotaxin (ATX), and its ligation to LPA receptor-1 (LPAR1) is associated with chronic inflammation and renal fibrosis; however, its role in renal aging is unclear. Male 2-, 12-, and 24-month-old C57BL/6 mice and Human renal proximal tubular epithelial cells (HRPTEpiC) were used in the present study. DNA damage and oxidative stress-induced senescence were simulated using doxorubicin (DOXO) and H2O2, respectively. The aged kidney showed decreased renal function, increased fractional mesangial area, and tubulointerstitial fibrosis. Both aged kidney and senescent cells showed increased levels of LPAR1, Nuclear factor κB (NF-κB), and inflammatory cytokines. In addition, LPAR1-knockdown reduced NF-κB and subsequent inflammatory cytokine induction, and NF-κB-knockdown resulted in decreased LPAR1 expression. Our study revealed a positive feedback loop between LPAR1 and NF-κB, which reinforces the role of inflammatory response, suggesting that blocking of aberrantly activated LPAR1 may reduce excessive inflammation, thereby providing a new possible therapeutic strategy to attenuate renal aging.

Effects of periostin deficiency on kidney aging and lipid metabolism.

Periostin plays a crucial role in fibrosis, which is involved in kidney aging. A few studies have shown that lipid metabolism is involved in kidney aging. We investigated the role of periostin in lipid metabolism during kidney aging. Renal function, fibrosis, and inflammatory markers were studied using urine, blood, and tissue samples from wild-type (WT) C57BL/6 mice and Postn-null mice of 2 and 24 months of age. Lipids were quantitatively profiled using liquid chromatography-tandem mass spectrometry in the multiple reaction monitoring mode. Renal function was worse and tubular atrophy/interstitial fibrosis, periostin expression, and inflammatory and fibrotic markers were more severe in aged WT mice than in young WT mice. In aged Postn-null mice, these changes were mitigated. Thirty-five differentially regulated lipids were identified. Phosphatidylcholines, cholesteryl ester, cholesterol, ceramide-1-phosphate, and CCL5 expression were significantly higher in aged WT mice than in aged Postn-null mice. Particularly, linoleic acid, linolenic acid, arachidonic acid, and docosahexaenoic acid differed strongly between the two groups. Lysophosphatidylcholine acyltransferase 2, which converts lysophosphatidylcholine to phosphatidylcholine, was significantly higher in aged WT mice than in aged Postn-null mice. Periostin expression in the kidneys increased with age, and periostin ablation delayed aging. Changes in lipids and their metabolism were found in Postn-null mice. Further research on the precise mechanisms of and relationships between lipid expression and metabolism, kidney aging, and periostin expression is warranted.

Inhibition of lymphatic proliferation by the selective VEGFR-3 inhibitor SAR131675 ameliorates diabetic nephropathy in db/db mice.

Recent studies have demonstrated that chronic inflammation-induced lymphangiogenesis plays a crucial role in the progression of various renal diseases, including diabetic nephropathy. SAR131675 is a selective vascular endothelial cell growth factor receptor-3 (VEGFR-3)-tyrosine kinase inhibitor that acts as a ligand for VEGF-C and VEGF-D to inhibit lymphangiogenesis. In this study, we evaluated the effect of SAR131675 on renal lymphangiogenesis in a mouse model of type 2 diabetes. Male C57BLKS/J db/m and db/db mice were fed either a regular chow diet or a diet containing SAR131675 for 12 weeks from 8 weeks of age. In addition, we studied palmitate-induced lymphangiogenesis in human kidney-2 (HK2) cells and RAW264.7 monocytes/macrophages, which play a major role in lymphangiogenesis in the kidneys. SAR131475 ameliorated dyslipidemia, albuminuria, and lipid accumulation in the kidneys of db/db mice, with no significant changes in glucose and creatinine levels and body weight. Diabetes-induced systemic inflammation as evidenced by increased systemic monocyte chemoattractant protein-1 and tumor necrosis factor-α level was decreased by SAR131475. SAR131475 ameliorated the accumulation of triglycerides and free fatty acids and reduced inflammation in relation to decreased chemokine expression and pro-inflammatory M1 macrophage infiltration in the kidneys. Downregulation of VEGF-C and VEGFR-3 by SAR131475 inhibited lymphatic growth as demonstrated by decreased expression of LYVE-1 and podoplanin that was further accompanied by reduced tubulointerstitial fibrosis, and inflammation in relation to improvement in oxidative stress and apoptosis. Treatment with SAR131475 improved palmitate-induced increase in the expression of VEGF-C, VEGFR-3, and LYVE-1, along with improvement in cytosolic and mitochondrial oxidative stress in RAW264.7 and HK2 cells. Moreover, the enhanced expression of M1 phenotypes in RAW264.7 cells under palmitate stress was reduced by SAR131475 treatment. The results suggest that modulation of lymphatic proliferation in the kidneys is a new treatment approach for type 2 diabetic nephropathy and that SAR131675 is a promising therapy to ameliorate renal damage by reducing lipotoxicity-induced lymphangiogenesis.

Attenuated Lymphatic Proliferation Ameliorates Diabetic Nephropathy and High-Fat Diet-Induced Renal Lipotoxicity.

Lymphangiogenesis occurs in response to renal injury and is correlated with interstitial fibrosis. Diabetes- and high-fat diet (HFD)-induced intrarenal lipotoxicity and their relationships with lymphangiogenesis are not established. We used PPARα agonist, fenofibrate, to unravel the linkage between lipotoxicity and lymphangiogenesis. Eight-week-old male C57BLKS/J db/db mice and HFD Spontaneously hypertensive rats (SHRs) were fed fenofibrate for 12 weeks. HK-2 and RAW264.7 cells were used to investigate their lymphangiogenic capacity in relation to lipotoxicity. Fenofibrate improved intrarenal lipotoxicity by increasing expression of PPARα and phosphorylation of AMPK. Lymphatic proliferation was attenuated; expression of lymphatic endothelial hyaluronan receptor-1 (LYVE-1), podoplanin, vascular endothelial growth factor-C (VEGF-C), and vascular endothelial growth factor receptor-3 (VEGFR-3) was decreased. In parallel, extent of tubulointerstitial fibrosis, apoptosis and inflammatory cell infiltration was reduced. In HK2 cells, palmitate- and high glucose-induced over expression of lymphatic makers was diminished by fenofibrate via activation of PPARα-AMPK-pACC signaling. Enhanced expression of M1 phenotype in RAW264.7 cells correlated with increased lymphatic growth. A causal relationship between lipotoxicity and lymphatic proliferation with a cellular link to macrophage activation can be speculated; pro-inflammatory M1 type macrophage is involved in the development of lymphangiogenesis through stimulation of VEGF-C and by its transdifferentiation into lymphatic endothelial cells.

Calcimimetic restores diabetic peripheral neuropathy by ameliorating apoptosis and improving autophagy.

Decreased AMPK-eNOS bioavailability mediates the development of diabetic peripheral neuropathy (DPN) through increased apoptosis and decreased autophagy activity in relation to oxidative stress. Schwann cells are responsible for maintaining structural and functional integrity of neurons and for repairing damaged nerves. We evaluated the neuro-protective effect of cinacalcet on DPN by activating the AMPK-eNOS pathway using db/db mice and human Schwann cells (HSCs). Sciatic nerve of db/db mice was characterized by disorganized myelin, axonal shrinkage, and degeneration that were accompanied by marked fibrosis, inflammation, and apoptosis. These phenotypical alterations were significantly improved by cinacalcet treatment along with improvement in sensorimotor functional parameters. Cinacalcet demonstrated favorable effects through increased expression and activation of calcium-sensing receptor (CaSR)-CaMKKß and phosphorylation of AMPK-eNOS signaling in diabetic sciatic nerve. Cinacalcet decreased apoptosis and increased autophagy activity in relation to decreased oxidative stress in HSCs cultured in high-glucose medium as well. This was accompanied by increased expression of the CaSR, intracellular Ca++ ([Ca++]i) levels, and CaMKKß-LKB1-AMPK signaling pathway, resulting in the net effect of increased eNOS phosphorylation, NOx concentration, Bcl-2/Bax ratio, beclin 1, and LC3-II/LC3-I ratio. These results demonstrated that cinacalcet treatment ameliorates inflammation, apoptosis, and autophagy through increased expression of the CaSR, [Ca++]i levels and subsequent activation of CaMKKß-LKB-1-AMPK-eNOS pathway in the sciatic nerve and HSCs under diabetic condition. Therefore, cinacalcet may play an important role in the restoration and amelioration of DPN by ameliorating apoptosis and improving autophagy.

Effects of Resveratrol on the Renin-Angiotensin System in the Aging Kidney.

The renin-angiotensin system (RAS), especially the angiotensin II (Ang II)/angiotensin II type 1 receptor (AT1R) axis, plays an important role in the aging process of the kidney, through increased tissue reactive oxygen species production and progressively increased oxidative stress. In contrast, the angiotensin 1-7 (Ang 1-7)/Mas receptor (MasR) axis, which counteracts the effects of Ang II, is protective for end-organ damage. To evaluate the ability of resveratrol (RSV) to modulate the RAS in aging kidneys, eighteen-month-old male C57BL/6 mice were divided into two groups that received either normal mouse chow or chow containing resveratrol, for six months. Renal expressions of RAS components, as well as pro- and antioxidant enzymes, were measured and mouse kidneys were isolated for histopathology. Resveratrol-treated mice demonstrated better renal function and reduced albuminuria, with improved renal histologic findings. Resveratrol suppressed the Ang II/AT1R axis and enhanced the AT2R/Ang 1-7/MasR axis. Additionally, the expression of nicotinamide adenine dinucleotide phosphate oxidase 4, 8-hydroxy-2'-deoxyguanosine, 3-nitrotyrosine, collagen IV, and fibronectin was decreased, while the expression of endothelial nitric oxide synthase and superoxide dismutase 2 was increased by resveratrol treatment. These findings demonstrate that resveratrol exerts protective effects on aging kidneys by reducing oxidative stress, inflammation, and fibrosis, through Ang II suppression and MasR activation.

The protective effect of resveratrol on vascular aging by modulation of the renin-angiotensin system.

BACKGROUND AND AIMS: This study evaluated the effects of resveratrol on arterial aging and the renin-angiotensin system (RAS) in mice and vascular smooth muscle cells (VSMCs). METHODS: Aging mice were divided into control and resveratrol groups. Histological changes, inflammation, oxidative stress, RAS components, and the expression of AMP-activated protein kinase (AMPK), silent information regulator T1 (SIRT1), peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α), and anti-oxidative enzymes was measured in thoracic aortas of 24-month-old mice. The effect of resveratrol on fibrosis, cell senescence, and RAS components was also investigated in VSMCs stimulated by angiotensin (Ang) II. RESULTS: Aorta media thickness, inflammation, fibrosis, and oxidative stress were significantly lower in the resveratrol group than in the control group. Resveratrol treatment decreased serum Ang II level and the aortic expression of prorenin receptor (PRR) and angiotensin converting enzyme (ACE), and increased serum Ang-(1-7) level and the expression of ACE2, Ang II type 2 receptor (AT2R), and Mas receptor (MasR). Resveratrol increased the expression of phosphorylated AMPK, SIRT1, PGC-1α, phosphorylated endothelial nitric oxide synthase and superoxide dismutase 1 and 2, and decreased that of NADPH oxidase 2 and 4. In Ang II-stimulated VSMCs, resveratrol treatment markedly decreased the number of senescence associated ß-galactosidase stained cells and pro-fibrotic protein expression and increased the expression of AT2R and MasR. CONCLUSIONS: Resveratrol protects against arterial aging and this effect is associated with reduced activity of the PRR-ACE-Ang II axis and stimulation of the ACE2-Ang-(1-7)-ATR2-MasR axis.

Adiponectin receptor agonist AdipoRon decreased ceramide, and lipotoxicity, and ameliorated diabetic nephropathy.

BACKGROUND: Adiponectin is known to take part in the regulation of energy metabolism. AdipoRon, an orally-active synthetic adiponectin agonist, binds to both adiponectin receptors (AdipoR)1/R2 and ameliorates diabetic complications. Among the lipid metabolites, the ceramide subspecies of sphingolipids have been linked to features of lipotoxicity, including inflammation, cell death, and insulin resistance. We investigated the role of AdipoRon in the prevention and development of type 2 diabetic nephropathy. METHODS: AdipoRon (30 mg/kg) was mixed into the standard chow diet and provided to db/db mice (db + AdipoRon, n = 8) and age-matched male db/m mice (dm + AdipoRon, n = 8) from 17 weeks of age for 4 weeks. Control db/db (db cont, n = 8) and db/m mice (dm cont, n = 8) were fed a normal diet of mouse chow. RESULTS: AdipoRon-fed db/db mice showed a decreased amount of albuminuria and lipid accumulation in the kidney with no significant changes in serum adiponectin, glucose, and body weight. Restoring expression of adiponectin receptor-1 and -2 in the renal cortex was observed in db/db mice with AdipoRon administration. Consistent up-regulation of phospho-Thr172 AMP-dependent kinase (AMPK), peroxisome proliferative-activated receptor α (PPARα), phospho-Thr473 Akt, phospho-Ser79Acetyl-CoA carboxylase (ACC), and phospho-Ser1177 endothelial NO synthase (eNOS), and down-regulation of protein phosphatase 2A (PP2A), sterol regulatory element-binding protein-1c (SREBP-1c), and inducible nitric oxide synthase (iNOS) were associated within the same group. AdipoRon lowered cellular ceramide levels by activation of acid ceramidase, which normalized ceramide to sphingosine-1 phosphate (S1P) ratio. In glomerular endothelial cells (GECs) and podocytes, AdipoRon treatment markedly decreased palmitate-induced lipotoxicity, which ultimately ameliorated oxidative stress and apoptosis. CONCLUSIONS: AdipoRon may prevent lipotoxicity in the kidney particularly in both GECs and podocytes through an improvement in lipid metabolism, as shown by the ratio of ceramide to sphingosines, and further contribute to prevent deterioration of renal function, independent of the systemic effects of adiponectin. The reduction in oxidative stress and apoptosis by AdipoRon provides protection against renal damage, thereby ameliorating endothelial dysfunction in type 2 diabetic nephropathy.

Cinacalcet-mediated activation of the CaMKKß-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy.

Apoptosis and autophagy are harmoniously regulated biological processes for maintaining tissue homeostasis. AMP-activated protein kinase (AMPK) functions as a metabolic sensor to coordinate cellular survival and function in various organs, including the kidney. We investigated the renoprotective effects of cinacalcet in high-glucose treated human glomerular endothelial cells (HGECs), murine podocytes and C57BLKS/J-db/db mice. In cultured HGECs and podocytes, cinacalcet decreased oxidative stress and apoptosis and increased autophagy that were attributed to the increment of intracellular Ca2+ concentration and the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinaseß (CaMKKß)-Liver kinase B1 (LKB1)-AMPK and their downstream signals including the phosphorylation of endothelial nitric oxide synthase (eNOS) and increases in superoxide dismutases and B cell leukemia/lymphoma 2/BCL-2-associated X protein expression. Interestingly, intracellular chelator BAPTA-AM reversed cinacalcet-induced CaMKKß elevation and LKB1 phosphorylation. Cinacalcet reduced albuminuria without influencing either blood glucose or Ca2+ concentration and ameliorated diabetes-induced renal damage, which were related to the increased expression of calcium-sensing receptor and the phosphorylation of CaMKKß-LKB1. Subsequent activation of AMPK was followed by the activation of peroxisome proliferator-activated receptor γ coactivator-1α and phospho-Ser1177eNOS-nitric oxide, resulting in a decrease in apoptosis and oxidative stress as well as an increase in autophagy.Our results suggest that cinacalcet increases intracellular Ca2+ followed by an activation of CaMKKß-LKB1-AMPK signaling in GECs and podocytes in the kidney, which provides a novel therapeutic means for type 2 diabetic nephropathy by modulation of apoptosis and autophagy.

The Adiponectin Receptor Agonist AdipoRon Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes.

Adiponectin exerts renoprotective effects against diabetic nephropathy (DN) by activating the AMP-activated protein kinase (AMPK)/peroxisome proliferative-activated receptor-α (PPARα) pathway through adiponectin receptors (AdipoRs). AdipoRon is an orally active synthetic adiponectin receptor agonist. We investigated the expression of AdipoRs and the associated intracellular pathways in 27 patients with type 2 diabetes and examined the effects of AdipoRon on DN development in male C57BLKS/J db/db mice, glomerular endothelial cells (GECs), and podocytes. The extent of glomerulosclerosis and tubulointerstitial fibrosis correlated with renal function deterioration in human kidneys. Expression of AdipoR1, AdipoR2, and Ca2+/calmodulin-dependent protein kinase kinase-ß (CaMKKß) and numbers of phosphorylated liver kinase B1 (LKB1)- and AMPK-positive cells significantly decreased in the glomeruli of early stage human DN. AdipoRon treatment restored diabetes-induced renal alterations in db/db mice. AdipoRon exerted renoprotective effects by directly activating intrarenal AdipoR1 and AdipoR2, which increased CaMKKß, phosphorylated Ser431LKB1, phosphorylated Thr172AMPK, and PPARα expression independently of the systemic effects of adiponectin. AdipoRon-induced improvement in diabetes-induced oxidative stress and inhibition of apoptosis in the kidneys ameliorated relevant intracellular pathways associated with lipid accumulation and endothelial dysfunction. In high-glucose-treated human GECs and murine podocytes, AdipoRon increased intracellular Ca2+ levels that activated a CaMKKß/phosphorylated Ser431LKB1/phosphorylated Thr172AMPK/PPARα pathway and downstream signaling, thus decreasing high-glucose-induced oxidative stress and apoptosis and improving endothelial dysfunction. AdipoRon further produced cardioprotective effects through the same pathway demonstrated in the kidney. Our results show that AdipoRon ameliorates GEC and podocyte injury by activating the intracellular Ca2+/LKB1-AMPK/PPARα pathway, suggesting its efficacy for treating type 2 diabetes-associated DN.

Resveratrol, an Nrf2 activator, ameliorates aging-related progressive renal injury.

BACKGROUND: Two important issues in the aging kidney are mitochondrial dysfunction and oxidative stress. An Nrf2 activator, resveratrol, is known to have various effects. Resveratrol may prevent inflammation and oxidative stress by activating Nrf2 and SIRT1 signaling. We examined whether resveratrol could potentially ameliorate the cellular condition, such as renal injury due to cellular oxidative stress and mitochondrial dysfunction caused by aging. METHODS: Male 18-month-old C57BL/6 mice were used. Resveratrol (40 mg/kg) was administered to aged mice for 6 months. We compared histological changes, oxidative stress, and aging-related protein expression in the kidney between the resveratrol-treated group (RSV) and the control group (cont). We performed experiments using small-interfering RNAs (siRNAs) for Nrf2 and SIRT1 in cultured HK2 cells. RESULTS: Resveratrol improved renal function, proteinuria, histological changes and inflammation in aging mice. Also, expression of Nrf2-HO-1-NOQ-1 signaling and SIRT1-AMPK-PGC-1α signaling was increased in the RSV group. Transfection with Nrf2 and SIRT1 siRNA prevented resveratrol-induced anti-oxidative effect in HK2 cells in media treated with H2O2. CONCLUSIONS: Activation of the Nrf2 and SIRT1 signaling pathways ameliorated oxidative stress and mitochondrial dysfunction. Pharmacological targeting of Nrf2 signaling molecules may reduce the pathologic changes of aging in the kidney.

Age-Associated Changes in the Vascular Renin-Angiotensin System in Mice.

Background. This study evaluated whether the change in the renin-angiotensin system (RAS) is associated with arterial aging in mice. Methods. Histologic changes and expressions of transforming growth factor-ß (TGF-ß), collagen IV, fibronectin, angiotensin II (Ang II), angiotensin-converting enzyme (ACE), angiotensin-converting enzyme 2 (ACE2), angiotensin II type 1 receptor (AT1R), angiotensin II type 2 receptor (AT2R), prorenin receptor (PRR), Mas receptor (MasR), endothelial nitric oxide synthase (eNOS), NADPH oxidase 2 and oxidase 4 (Nox2 and Nox4), 8-hydroxy-2'-deoxyguanosine (8-OHdG), 3-nitrotyrosine, and superoxide dismutase 1 and dismutase 2 (SOD1 and SOD2) were measured in the thoracic aortas from 2-month-old, 12-month-old, and 24-month-old C57/BL6 mice. Results. Twenty-four-month-old mice showed significantly increased aortic media thickness and expressions of TGF-ß, collagen IV, and fibronectin, compared to 2-month-old and 12-month-old mice. The expressions of PRR, ACE, and Ang II, and AT1R-positive area significantly increased, whereas expressions of ACE2 and MasR and AT2R-positive area decreased with age. The expressions of phosphorylated serine(1177)-eNOS, SOD1, and SOD2 decreased, and the 8-OHdG-positive area and the 3-nitrotyrosine-positive area increased with age. The expression of Nox2 significantly increased with age, but that of Nox4 did not change. Conclusions. The enhanced PRR-ACE-Ang II-AT1R axis and reduced ACE2-MasR axis were associated with arterial aging in mice.

PPARα agonist, fenofibrate, ameliorates age-related renal injury.

The kidney ages quickly compared with other organs. Expression of senescence markers reflects changes in the energy metabolism in the kidney. Two important issues in aging are mitochondrial dysfunction and oxidative stress. Peroxisome proliferator-activated receptor α (PPARα) is a member of the ligand-activated nuclear receptor superfamily. PPARα plays a major role as a transcription factor that regulates the expression of genes involved in various processes. In this study, 18-month-old male C57BL/6 mice were divided into two groups, the control group (n=7) and the fenofibrate-treated group (n=7) was fed the normal chow plus fenofibrate for 6months. The PPARα agonist, fenofibrate, improved renal function, proteinuria, histological change (glomerulosclerosis and tubular interstitial fibrosis), inflammation, and apoptosis in aging mice. This protective effect against age-related renal injury occurred through the activation of AMPK and SIRT1 signaling. The activation of AMPK and SIRT1 allowed for the concurrent deacetylation and phosphorylation of their target molecules and decreased the kidney's susceptibility to age-related changes. Activation of the AMPK-FOXO3a and AMPK-PGC-1α signaling pathways ameliorated oxidative stress and mitochondrial dysfunction. Our results suggest that activation of PPARα and AMPK-SIRT1 signaling may have protective effects against age-related renal injury. Pharmacological targeting of PPARα and AMPK-SIRT1 signaling molecules may prevent or attenuate age-related pathological changes in the kidney.

The protective effect of neutralizing high-mobility group box1 against chronic cyclosporine nephrotoxicity in mice.

BACKGROUND: High-mobility group box1 (HMGB1) is known to be involved in innate immune response through interaction with receptor for advanced glycation end products (RAGE) and toll-like receptors (TLRs), besides its proper role within the nucleus. Immunological pathways, including TLR signaling, are also involved in chronic cyclosporine (CsA) nephrotoxicity. This study was designed to determine whether neutralizing HMGB1 prevents chronic CsA nephrotoxicity. METHODS: Chronic CsA nephrotoxicity was induced by CsA subcutaneous injection daily for 4weeks under salt-depletion in mice. Anti-HMGB1 neutralizing antibody for HMGB1 blockade (600mcg/mouse) was administered weekly to mice in the anti-HMGB1 treatment group. The effects of HMGB1 neutralization were evaluated in terms of renal function as well as histological and immunopathological examination. RESULTS: Anti-HMGB1 administration prevented the increases in serum creatinine and 24h albuminuria and the decrease in creatinine clearance associated with CsA treatment. Increased tubulointerstitial fibrosis and transforming growth factor (TGF)-ß immunohistochemical staining associated with CsA treatment were also prevented by anti-HMGB1 administration. Anti-HMGB1 administration prevented the activation of the TLR4 signaling pathway, which resulted in the reduction of nuclear factor kappa B (NF-κB) expression. In cultured tubular cells, anti-HMGB1 pretreatment also prevented the increases in fibronectin and collagen IV levels associated with CsA treatment. CONCLUSIONS: Neutralizing HMGB1 with an anti-HMGB1 antibody ameliorated chronic CsA nephrotoxicity via inhibition of the TLR4 signaling pathway. Our study suggests that HMGB1 blockade can be beneficial for increasing allograft survival in renal transplant recipients by protecting against calcineurin inhibitor-induced nephrotoxicity.

Delayed treatment with oleanolic acid attenuates tubulointerstitial fibrosis in chronic cyclosporine nephropathy through Nrf2/HO-1 signaling.

BACKGROUND: Nuclear factor erythroid-2-related factor-2 (Nrf2) is known to protect against tissue injury by orchestrating antioxidant and detoxification responses to oxidative stress. This study investigated whether upregulation of Nrf2-dependent signaling by oleanolic acid (OA), which is known to activate Nrf2, could attenuate renal inflammation and fibrosis in cyclosporine (CsA)-induced kidney injury. METHODS: Male ICR mice were divided into four treatment groups: Vehicle (VH, n = 6), VH + OA (n = 6), CsA (n = 8), and CsA + OA (n = 8). For the OA-treated groups, OA (25 mg/kg/day) was administered by intraperitoneal injection for the final week of the 4-week experimental period. Renal function, morphologies and signaling were evaluated at the end of the study. RESULTS: Treatment with CsA resulted in decreased kidney function and urine osmolality and increased urine volume and urinary albumin levels. The CsA-induced changes were improved by OA treatment. Specifically, administration of OA decreased tubulointerstitial fibrosis and inflammation scores that were increased in CsA-treated mice. Furthermore, OA treatment decreased urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-epi-prostaglandin F2α (8-iso-PGF2α) levels. The beneficial effects of OA were attributed to an increased ratio of nuclear/total Nrf2 and subsequently enhanced expression of heme oxygenase (HO)-1, as well as a stable level of Kelch-like ECH-associated protein 1 (Keap1) expression, indicating that OA enhanced nuclear translocation of Nrf2. Increased apoptotic cell death and a high ratio of B cell leukaemia/lymphoma 2 (Bcl-2)-associated X protein (Bax) to Bcl-2 in CsA-treated mice were also significantly ameliorated by OA treatment. CONCLUSION: Our results suggest that OA activates Nrf2/HO-1 signaling in chronic CsA nephropathy, which may have beneficial effects on inflammation and oxidative stress.

Age-associated molecular changes in the kidney in aged mice.

BACKGROUND: Aging is a multifactorial process characterized by a progressive decline in physiological function. Decreased kidney function is associated with cardiovascular disease and mortality. Therefore, increasing our insight into kidney aging by understanding the anatomic, physiologic, and pathologic changes of aging in the kidney is important to prevent disastrous outcomes in elderly people. METHODS: Male two-, 12-, and 24-month-old C57/BL6 mice were used in this study. We measured histological change, oxidative stress, and aging-related protein expression in the kidneys. RESULTS: Twenty-four-month-old mice displayed increased albuminuria. Creatinine clearance decreased with aging, although this was not statistically significant. There were increases in mesangial volume and tubulointerstitial fibrosis in 24-month-old mice. There were also increases in F4/80 expression and in apoptosis detected by TUNEL assay. Urine isoprostane excretion increased with aging and SOD1 and SOD2 were decreased in 24-month-old mice. Oxidative stress may be mediated by a decrease in Sirt1, PGC-1α, ERR-1α, and PPARα expression. Klotho expression also decreased. CONCLUSIONS: Our results demonstrate that Sirt1 was decreased with aging and may relate to changed target molecules including PGC-1α/ERR-1α signaling and PPARα. Klotho can also induce oxidative stress. Pharmacologically targeting these signaling molecules may reduce the pathologic changes of aging in the kidney.