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This study aimed to investigate the metabolic changes in the kidneys in a murine adenine–diet model of chronic kidney disease (CKD). Kidney fibrosis is the common pathological manifestation across CKD aetiologies. Sustained inflammation and fibrosis cause changes in preferred energy metabolic pathways in the cells of the kidney. Kidney cortical tissue from mice receiving a control or adenine‐supplemented diet for 8 weeks (late inflammation and fibrosis) and 12 weeks (8 weeks of treatment followed by 4 weeks recovery) were analysed by 2D‐correlated nuclear magnetic resonance spectroscopy and compared with histopathology and biomarkers of kidney damage. Tissue metabolite and lipid levels were assessed using the MestreNova software. Expression of genes related to inflammation, fibrosis, and metabolism were measured using quantitative polymerase chain reaction. Animals showed indicators of severely impaired kidney function at 8 and 12 weeks. Significantly increased fibrosis was present at 8 weeks but not in the recovery group suggesting some reversal of fibrosis and amelioration of inflammation. At 8 weeks, metabolites associated with glycolysis were increased, while lipid signatures were decreased. Genes involved in fatty acid oxidation were decreased at 8 weeks but not 12 weeks while genes associated with glycolysis were significantly increased at 8 weeks but not at 12 weeks. In this murine model of CKD, kidney fibrosis was associated with the accumulation of triglyceride and free lactate. There was an up‐regulation of glycolytic enzymes and down‐regulation of lipolytic enzymes. These metabolic changes reflect the energy demands associated with progressive kidney disease where there is a switch from fatty acid oxidation to that of glycolysis. [ABSTRACT FROM AUTHOR]

Background. People receiving hemodialysis (HD) treatment have higher cardiovascular morbidity and mortality, ascribed to an increased prevalence of traditional cardiovascular risk factors. However, the role of nontraditional risk factors, such as inflammation, has become increasingly recognized. The origin of this inflammation remains elusive and one putative cause is elevated levels of circulating bacterial endotoxin. Methods. In this study, serum concentrations of endotoxin and inflammatory biomarkers, including high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), interleukin-1β (IL1β), ferritin and tumor necrosis factor (TNF), were measured in 30 adults receiving HD and 10 healthy individuals without kidney disease. In people receiving HD, samples were collected immediately before dialysis (preHD), after dialysis (postHD), and 48 hours after (postHD48hrs). Results. Endotoxin was detectable in only 1 of 90 samples analyzed. There were no significant differences in serum hsCRP, IL1β, and IL6 levels, before and after dialysis. Serum TNF levels decreased significantly from 30.9 (8.0, 39.5) pg/mL preHD to 13.9 (8.5, 17.3) pg/mL post-HD (p = 0.002) and then increased back to 27.37 (14.5, 35) pg/mL 2 days later (p < 0.001). Ferritin increased from 1153 ng/mL (782, 1458) preHD to 1313 ng/mL (657, 1638) post HD (p < 0.001) and then decreased back to 1186 ng/mL (754, 1597) (p = 0.66) postHD48hrs. Compared to controls, people receiving HD had significantly elevated levels of hsCRP [6.16 mg/L (2.1, 16.8) vs. 1.1 mg/L (0.81, 3.63) p = 0.015 ], IL1β [1.5 pg/mL (0.05, 2.51) vs. 0.5 pg/mL (1.81, 2.95) p ≤ 0.001 ], and ferritin [1153 (782, 1458) vs. 132.9 (111, 257) ng/mL p ≤ 0.001 ], but comparable levels of in IL6 [6.15 pg/mL (4.82, 9.12) vs. 7.49 pg/mL (4.56, 10.39), p = 0.77 ] and TNF [27.35 pg/mL ± 17.48 vs. 17.87 pg/mL ± 12.28, p < 0.12 ]. In conclusion, people on HD have elevated levels of inflammatory biomarkers, which are not associated with endotoxemia (which is rare) or the dialysis procedure. [ABSTRACT FROM AUTHOR]

Aim: Protease‐activated receptor 2 (PAR2) has been implicated in the development of renal inflammation and fibrosis. In particular, activation of PAR2 in cultured tubular epithelial cells induces extracellular signal‐regulated kinase signalling and secretion of fibronectin, C–C Motif Chemokine Ligand 2 (CCL2) and transforming growth factor‐β1 (TGF‐β1), suggesting a role in tubulointerstitial inflammation and fibrosis. We tested this hypothesis in unilateral ureteric obstruction (UUO) in which ongoing tubular epithelial cell damage drives tubulointerstitial inflammation and fibrosis. Methods: Unilateral ureteric obstruction surgery was performed in groups (n = 9/10) of Par2−/− and wild type (WT) littermate mice which were killed 7 days later. Non‐experimental mice were controls. Results: Wild type mice exhibited a 5‐fold increase in Par2 messenger RNA (mRNA) levels in the UUO kidney. In situ hybridization localized Par2 mRNA expression to tubular epithelial cells in normal kidney, with a marked increase in Par2 mRNA expression by tubular cells, including damaged tubular cells, in WT UUO kidney. Tubular damage (tubular dilation, increased KIM‐1 and decreased α‐Klotho expression) and tubular signalling (extracellular signal‐regulated kinase phosphorylation) seen in WT UUO were not altered in Par2−/− UUO. In addition, macrophage infiltration, up‐regulation of M1 (NOS2) and M2 (CD206) macrophage markers, and up‐regulation of pro‐inflammatory molecules (tumour necrosis factor, CCL2, interleukin‐36α) in WT UUO kidney were unchanged in Par2−/− UUO. Finally, the accumulation of α‐SMA+ myofibroblasts, deposition of collagen IV and expression of pro‐fibrotic factors (CTGF, TGF‐β1) were not different between WT and Par2−/− UUO mice. Conclusion: Protease‐activated receptor 2 expression is substantially up‐regulated in tubular epithelial cells in the obstructed kidney, but this does not contribute to the development of tubular damage, renal inflammation or fibrosis. SUMMARY AT A GLANCE: This study reported that protease‐activated receptor 2 (PAR2) did not contribute to the renal tubular inflammation and fibrosis in a 7‐day model of unilateral ureteric obstruction using PAR2 knockout mice. [ABSTRACT FROM AUTHOR]

Summary: Glomerular crescent formation is a hallmark of rapidly progressive forms of glomerulonephritis. Thrombosis and macrophage infiltration are features of crescent formation in human and experimental kidney disease. Protease‐activated receptor‐2 (PAR‐2) is a G‐protein coupled receptor that links coagulation and inflammation. This study investigated whether pharmacological inhibition of PAR‐2 can suppress glomerular crescent formation in rat nephrotoxic serum nephritis (NTN). Disease was induced in Wistar Kyoto rats by immunisation with sheep IgG followed by administration of sheep nephrotoxic serum. Rats (n = 8/group) received the PAR‐2 antagonist (GB88, 10 mg/kg/p.o.), vehicle or no treatment starting 3 days before nephrotoxic serum injection and continuing until day 14. Vehicle and untreated rats developed thrombosis and macrophage infiltration in the glomerular tuft and Bowman's space in conjunction with prominent crescent formation. Activation of JNK signalling and proliferation in parietal epithelial cells was associated with crescent formation. GB88 treatment significantly reduced crescent formation with a substantial reduction in glomerular thrombosis, reduced macrophage infiltration in Bowman's space, and reduced activation of parietal epithelial cells. However, GB88 did not protect against the development of proteinuria, renal function impairment, inflammation or tubular cell damage in the NTN model. In conclusion, PAR‐2 plays a specific role in glomerular crescent formation by promoting glomerular thrombosis, macrophage accumulation in Bowman's space and activation of parietal epithelial cells. [ABSTRACT FROM AUTHOR]

In the last decade, chronic kidney disease ( CKD), defined as reduced renal function (glomerular filtration rate ( GFR) < 60 mL/min per 1.73 m2) and/or evidence of kidney damage (typically manifested as albuminuria) for at least 3 months, has become one of the fastest-growing public health concerns worldwide. CKD is characterized by reduced clearance and increased serum accumulation of metabolic waste products (uremic retention solutes). At least 152 uremic retention solutes have been reported. This review focuses on indoxyl sulphate ( IS), a protein-bound, tryptophan-derived metabolite that is generated by intestinal micro-organisms (microbiota). Animal studies have demonstrated an association between IS accumulation and increased fibrosis, and oxidative stress. This has been mirrored by in vitro studies, many of which report cytotoxic effects in kidney proximal tubular cells following IS exposure. Clinical studies have associated IS accumulation with deleterious effects, such as kidney functional decline and adverse cardiovascular events, although causality has not been conclusively established. The aims of this review are to: (i) establish factors associated with increased serum accumulation of IS; (ii) report effects of IS accumulation in clinical studies; (iii) critique the reported effects of IS in the kidney, when administered both in vivo and in vitro; and (iv) summarize both established and hypothetical therapeutic options for reducing serum IS or antagonizing its reported downstream effects in the kidney. [ABSTRACT FROM AUTHOR]

ABSTRACT Thimerosal is an ethyl mercury-containing compound used mainly in vaccines as a bactericide. Although the kidney is a key target for mercury toxicity, thimerosal nephrotoxicity has not received the same attention as other mercury species. The aim of this study was to determine the potential cytotoxic mechanisms of thimerosal on human kidney cells. Human kidney proximal tubular epithelial (HK2) cells were exposed for 24 h to thimerosal (0-2 µM), and assessed for cell viability, apoptosis, and cell proliferation; expression of proteins Bax, nuclear factor-κB subunits, and transforming growth factor-β1 (TGFβ1); mitochondrial health (JC-1, MitoTracker Red CMXRos); and fibronectin levels (enzyme-linked immunosorbent assay). Thimerosal diminished HK2 cell viability and mitosis, promoted apoptosis, impaired the mitochondrial permeability transition, enhanced Bax and TGFβ1 expression, and augmented fibronectin secretion. This is the first report about kidney cell death and pro-fibrotic mechanisms promoted by thimerosal. Collectively, these in vitro results demonstrate that (1) thimerosal induces kidney epithelial cell apoptosis via upregulating Bax and the mitochondrial apoptotic pathway, and (2) thimerosal is a potential pro-fibrotic agent in human kidney cells. We suggest that new evidence on toxicity as well as continuous surveillance in terms of fibrogenesis is required concerning thimerosal use. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1423-1433, 2015. [ABSTRACT FROM AUTHOR]

Multiple serine proteases exert proinflammatory actions by signaling through protease-activated receptor-2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti-inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis-like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2-induced intracellular Ca2+ release in human monocyte-derived macrophages, being 1000 times more potent than a control compound, ENMD-1068 (IC50 1.60±5 µM vs. 1.20±4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F55%, Tmax 4 h , Cmax 1.7 µM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ-carrageenan or PAR2 agonists 2-furoyl-LIGRLO-NH2 or mast cell β-tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen-induced model of arthritis in rats, GB88 (10 mg/kg) was disease modifying and ameliorated pathological and histopathological changes (edema, pannus formation, synovial hyperplasia, collagen degradation, macrophage invasion, mast cell degranulation) compared to untreated arthritic controls. The results suggest that an orally active PAR2 antagonist is effective in treating chronic arthritis in rats through inhibiting macrophage infiltration, mast cell degranulation, and β-tryptase-PAR2 signaling in joint inflammation. [ABSTRACT FROM AUTHOR]

OBJECTIVES To investigate if the feeding of silibinin (an anticancer flavonoid) to mice inhibits in vivo renal cell carcinoma (RCC) growth via changes in insulin-like growth factor binding protein-3 (IGFBP-3) levels. MATERIALS AND METHODS Male severe combined immunodeficiency disease (SCID) mice (7 weeks old), with left kidneys injected with 1 million SN12K1 cells, were fed a silibinin-containing diet (0.1%, 0.2% and 0.4% w/w) or control AIN-93G diet for 39 days from 1 day after tumour engraftment. RESULTS There was a reduction in tumour deposits and tumour kidney weight in SCID mice fed with a 0.4% silibinin-containing diet compared to those fed the control diet. Mice with tumour injection (silibinin or control-diet group) had constant total body weight and food consumption. The mean plasma and tumourous kidney silibinin concentrations, as measured by high-pressure liquid chromatography-tandem mass spectrometry, increased with escalating doses of silibinin. Using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, the mean tissue IGFBP-3 mRNA (in SN12K1-implanted kidney) and plasma IGFBP-3 levels increased in mice fed with 0.1% silibinin (tumour IGFBP-3 mRNA levels, 156% higher vs control-diet group, P = 0.007; and plasma IGFBP-3 levels, 61% higher vs control-diet group, P = 0.002) but not in mice fed with the higher silibinin pellet strengths. CONCLUSION Oral administration of silibinin suppressed local and metastatic tumour growth in vivo in an orthotopic xenograft model of RCC. This anti-neoplastic action of silibinin might involve IGFBP-3. The exact mechanism through which IGFBP-3 promotes silibinin’s anticancer effects warrants further investigation. [ABSTRACT FROM AUTHOR]

Proximal tubule cells (PTC) are the major cell type in the cortical tubulointerstitium. Because PTC play a central role in tubulointerstitial pathophysiology, it is essential to prepare pure PTC from kidney tissue to explore the mechanisms of tubulointerstitial pathology. The authors have successfully refined and characterized primary cultures of human PTC using Percoll density gradient centrifugation as a key PTC enrichment step. The cells obtained by this method retain morphological and functional properties of PTC and are minimally contaminated by other renal cells. In particular, the primary isolates have characteristics of epithelial cells with uniform polarized morphology, tight junction and well-formed apical microvilli. Cytokeratin is uniformly and strongly expressed in the isolates. Brush border enzyme activities and PTC transport properties are retained in the isolates. This method therefore provides an excellent in vitro model for the physiologic study of the human proximal tubule. [ABSTRACT FROM AUTHOR]

OBJECTIVE To investigate whether silibinin, a flavonoid antioxidant with anticancer properties that inhibits cellular growth via the up-regulation of insulin-like growth factor binding protein 3 (IGFBP-3), inhibits renal cell carcinoma (RCC) growth via the IGFBP-3 pathway. MATERIALS AND METHODS Cell morphology, DNA synthesis by thymidine incorporation, viability assessed by 3,4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assays, trypan blue exclusion, and lactate dehydrogenase (LDH) release, apoptosis and IGFBP-3 protein (Western blotting) were evaluated in silibinin-treated SN12K1 cells (a cell line derived from metastatic RCC) in the presence or absence of IGFBP-3 immunoneutralization. RESULTS Silibinin suppressed SN12K1 DNA synthesis at 24 and 48 h incubation by a mean (sd) of at least 68 (10)% of the control values at ≥2 µmol/L ( P < 0.05). At high concentrations (>80 µmol/L) cell viability was compromised, as shown by decreased MTT uptake of 62 (10)% of control values ( P < 0.001), reduced cell counts of ≥77 (9)% of control values ( P < 0.05), elevated LDH release of 212 (49)% of control ( P < 0.001), and the presence of necrotic and apoptotic cells on immunofluorescence staining. Removing endogenous IGFBP-3 by neutralizing with anti-IGFBP-3 antibody increased DNA synthesis by 240 (35)% of control for 24 h, ( P < 0.001). However, on Western blot analysis of IGFBP-3 levels in conditioned media incubated in the presence of silibinin there was down-regulation of protein expression. CONCLUSION Silibinin suppresses SN12K1 cell growth and, at high doses, increases cell death. These effects are independent of IGFBP-3. [ABSTRACT FROM AUTHOR]

The protease-activated receptor-2 (PAR-2), the second of four members of a unique subfamily of G-protein coupled receptors, is abundantly expressed in the kidney. In a similar manner to other PAR cleavage of its extracellular N-terminus exposes a tethered ligand, SLIGKV in humans, which acts as an intramolecular ligand to activate itself. In the kidney, PAR-2 expression has been variably reported in collecting duct cells, mesangial cells, interstitial fibroblasts, vascular endothelial cells, vascular smooth muscle cells and proximal tubular cells. Despite this renal expression data, the function of PAR-2 in the kidney remains unknown. More than 15 different mammalian serine proteases have been shown to activate PAR-2 in an in vitro setting, but it is still unclear which of these are physiologically relevant activators of PAR-2 in specific tissues. Their identification could provide novel therapeutic targets. PAR-2 activates a number of down-stream signalling molecules that include protein kinase C, extracellular signal regulated kinase and nuclear factor kappa-B. Proteases that can activate PAR-2 are generated and released from cells during injury, inflammation and malignancy and can thus signal to cells under these conditions. Potential physiological and pathophysiological roles for PAR-2 in the kidney include the regulation of inflammation, blood flow, and ion transport and tissue protection, repair and fibrosis. In this review the potential roles of PAR-2 in the kidney are highlighted and discussed. [ABSTRACT FROM AUTHOR]

Erythropoietin (EPO) has been used widely for the treatment of anaemia associated with chronic kidney disease and cancer chemotherapy for nearly 20 years. More recently, EPO has been found to interact with its receptor (EPO-R) expressed in a large variety of non-haematopoietic tissues to induce a range of cytoprotective cellular responses, including mitogenesis, angiogenesis, inhibition of apoptosis and promotion of vascular repair through mobilization of endothelial progenitor cells from the bone marrow. Administration of EPO or its analogue, darbepoetin, promotes impressive renoprotection in experimental ischaemic and toxic acute renal failure, as evidenced by suppressed tubular epithelial apoptosis, enhanced tubular epithelial proliferation and hastened functional recovery. This effect is still apparent when administration is delayed up to 6 h after the onset of injury and can be dissociated from its haematological effects. Based on these highly encouraging results, at least one large randomized controlled trial of EPO therapy in ischaemic acute renal failure is currently underway. Preliminary experimental and clinical evidence also indicates that EPO may be renoprotective in chronic kidney disease. The purpose of the present article is to review the renoprotective benefits of different protocols of EPO therapy in the settings of acute and chronic kidney failure and the potential mechanisms underpinning these renoprotective actions. Gaining further insight into the pleiotropic actions of EPO will hopefully eventuate in much-needed, novel therapeutic strategies for patients with kidney disease. [ABSTRACT FROM AUTHOR]

Interleukin (IL)-1β, a pro-inflammatory macrophage-derived cytokine, is implicated as a key mediator of interstitial fibrosis and tubular loss or injury in progressive renal insufficiency. This study investigates some of the mechanisms of action of IL-1β on the proximal tubule.Confluent cultures of primary human proximal tubule cells (PTC) were incubated in serum-free media supplemented with either IL-1β (0–4 ng/mL), phorbol-12-myristate 13-acetate (PMA, protein kinase C activator) (6.25–100 nmol/L), or vehicle (control), together with a non-specific protein kinase C inhibitor (H7), a specific protein kinase C inhibitor (BIM-1), an anti-oxidant (NAC) or a NADPH oxidase inhibitor (AEBSF).Interleukin-1β-treated PTC exhibited time-dependent increases in fibronectin secretion (ELISA), cell injury (LDH release) and reactive nitrogen species (RNS) release (Griess assay). Proximal tubule cell DNA synthesis (thymidine incorporation) was also significantly suppressed. The effects of IL-1β, which were reproduced by incubation of PTC with PMA (6.25–100 nmol/L), were blocked by H7 but not by BIM-1. The anti-oxidant (4 mmol/L) partially blocked IL-1β-induced fibronectin secretion by PTC, but did not affect IL-1β-induced LDH release, RNS release or growth inhibition. The NADPH oxidase inhibitor (AEBSF) significantly attenuated all observed deleterious effects of IL-1β on PTC.Interleukin-1β directly induces proximal tubule injury, extracellular matrix production and impaired growth. The anti-oxidant, NAC, appears to ameliorate part of the fibrogenic effect of IL-1β on PTC through mechanisms that do not significantly involve protein kinase C activation or nitric oxide release. [ABSTRACT FROM AUTHOR]

The purpose of the present paper was to describe the pattern of expression of insulin-like growth factor (IGF-I) and its regulatory binding proteins (IGFBP) in renal cell carcinoma (RCC).The expressions of mRNA and protein for various IGF members were assessed in 24 paired normal and malignant human renal tissues (16 clear cell and 8 papillary RCC) using semiquantitative reverse transcription–polymerase chain reaction and immunohistochemistry. Paired tissue samples were also obtained from six patients with oncocytoma in order to compare the specificity of changes in IGF/IGFBP expression between tumors derived from proximal (RCC) and distal (oncocytoma) tubular epithelium.Clear cell RCC were characterized by significant increases in the mRNA expression of IGF-I, IGFBP-3 and IGFBP-6 while papillary RCC exhibited down-regulated expression of IGF-I, IGFBP-4 and IGFBP-5. The IGFBP-2, IGFBP-4 and IGFBP-5 mRNA were down-regulated in oncocytomas. Semiquantitative assessment of immunohistochemical staining demonstrated significant increases in epithelial associated IGF-I and IGFBP-3 in clear cell RCC, increased IGFBP-5 protein in papillary RCC and no significant changes in IGF/IGFBP protein expression in oncocytoma.The expression of IGF-I and certain IGFBP is significantly altered in RCC compared with normal renal tissue and oncocytomas. This altered expression is differentially regulated according to the histologic subtype of RCC, and suggests that the IGF/IGFBP axis may play an important role in determining the malignant phenotype of RCC. [ABSTRACT FROM AUTHOR]