Distribution of Subgingival Bacteria in Chronic Periodontitis Patients Correlated with IgA Nephropathy.

BACKGROUND: Several studies indicated that chronic periodontitis (CP) and its subgingival bacteria correlated with IgA nephropathy (IgAN). Previous research has shown that prevalence of IgAN in chronic periodontitis patients is significantly higher than that in non CP patients in Xinjiang especially in ethnic Uyghur. The aim of this study is to investigate the distribution of plaque bacterial microbes in CP and IgAN patients and to find correlation between CP and IgAN. METHODS: All of the subgingival plaque samples including 7 healthy controls (N group), 8 CP patients, 14 IgAN patients, and 14 CP with IgAN patients were obtained from ethnic Uyghur people. To investigate the distribution of plaque microbe in Uyghur CP and IgAN patients, the 16s rRNA sequencing and comparative analysis of subgingival bacteria were performed. RESULTS: There were no statistically differences in the community richness estimator (Chao) and the diversity estimator (Shannon index) among four groups. The abundance of Burkholderiales (order), Ottowia (genus) in the plaque microbes were significantly higher in CP with IgAN patients than CP patients. The abundance of Eubacterium (genus) was significantly higher in CP with IgAN patients than IgAN patients. The abundance of Veillonella (genus) was significantly higher while Streptococcus (genus), Tannerella (genus) were significantly lower in CP patients than healthy volunteers. CONCLUSIONS: The composition and abundance of subgingival plaque microbes in Uyghur CP and IgAN patients were significantly different at several levels. Which suggested that abundance of subgingival bacteria is correlated to CP and IgAN.

M-Type Phospholipase A2 Receptor Autoantibody Measured by ELISA has Diagnostic Value in Patients with Idiopathic Membranous Nephropathy.

BACKGROUND: This study investigated the diagnosis value of phospholipase A2 receptor autoantibodies (PLA2R-AB) for idiopathic membranous nephropathy (IMN). METHODS: Patients with IMN, lupus nephritis, hepatitis B virus-associated nephropathy and IgA nephropathy and healthy participants were included. Receiver operating characteristics (ROC) curve was plotted for PLA2R-AB to diagnose IMN. RESULT: The level of serum PLA2R-AB was significantly higher in patients with IMN than with other MN, and was positively correlated to urine albumin-creatinine ratio and proteinuria in patients with IMN. The area under the ROC curve displaying the performance of PLA2R-AB to diagnose IMN was 0.907 with a sensitivity and specificity of 94.3% and 82.1%, respectively. CONCLUSIONS: PLA2R-AB is a reliable biomarker to diagnose Chinese patients with IMN.

Indoxyl sulfate induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3-mediated uptake and activation of AhR/NF-κB pathway.

BACKGROUND/AIMS: Interleukin-6 (IL-6) is one of the inflammation biomarkers with highest predictive value for outcome in chronic kidney disease (CKD) patients. The present study aimed to determine the effects of indoxyl sulfate (IS) on IL-6 expression in vascular cells. METHODS: IS was administered to normo- and hypertensive rats. Human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) were incubated with or without IS. RESULTS: Immunohistochemistry revealed that IS-administered rats showed increased expression of IL-6 in the aortic tissues. IS increased IL-6 expression in HUVECs and HASMCs in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3) using small interfering RNA (siRNA) inhibited IS-induced expression of IL-6 in HUVECs and HASMCs. IS induced activation of aryl hydrocarbon receptor (AhR) and nuclear factor-κB (NF-κB) subunit p65 in HUVECs and HASMCs. Both AhR siRNA and p65 siRNA inhibited IS-induced expression of IL-6. AhR siRNA inhibited IS-induced phosphorylation and nuclear translocation of p65 without change in total p65 level. However, p65 siRNA did not inhibit IS-induced nuclear translocation of AhR. Thus, AhR is responsible for IS-induced p65 signaling transduction. CONCLUSION: IS induces IL-6 expression in vascular endothelial and smooth muscle cells through OAT3/AhR/NF-κB pathway.

Indoxyl sulfate-induced activation of (pro)renin receptor promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

UNLABELLED: Chronic kidney disease (CKD) is associated with an increased risk of cardiovascular disease (CVD). (Pro)renin receptor (PRR) is activated in the kidney of CKD. The present study aimed to determine the role of indoxyl sulfate (IS), a uremic toxin, in PRR activation in rat aorta and human aortic smooth muscle cells (HASMCs). We examined the expression of PRR and renin/prorenin in rat aorta using immunohistochemistry. Both CKD rats and IS-administrated rats showed elevated expression of PRR and renin/prorenin in aorta compared with normal rats. IS upregulated the expression of PRR and prorenin in HASMCs. N-acetylcysteine, an antioxidant, and diphenyleneiodonium, an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase, suppressed IS-induced expression of PRR and prorenin in HASMCs. Knock down of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR) and nuclear factor-κB p65 (NF-κB p65) with small interfering RNAs inhibited IS-induced expression of PRR and prorenin in HASMCs. Knock down of PRR inhibited cell proliferation and tissue factor expression induced by not only prorenin but also IS in HASMCs. CONCLUSION: IS stimulates aortic expression of PRR and renin/prorenin through OAT3-mediated uptake, production of reactive oxygen species, and activation of AhR and NF-κB p65 in vascular smooth muscle cells. IS-induced activation of PRR promotes cell proliferation and tissue factor expression in vascular smooth muscle cells.

Indoxyl sulfate downregulates expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells.

UNLABELLED: Renin-angiotensin system (RAS) plays a pivotal role in chronic kidney disease (CKD). Angiotensin converting enzyme-related carboxypeptidase 2 (ACE2)/angiotensin (Ang)-(1-7)/Mas receptor axis counteracts the deleterious actions of Ang II. ACE2 exerts its actions by cleaving Ang II into Ang-(1-7) which activates Mas receptor. This study aimed to determine if the expression of Mas receptor is altered in the kidneys of CKD rats, and if indoxyl sulfate (IS), a uremic toxin, affects the expression of Mas receptor in rat kidneys and cultured human proximal tubular cells (HK-2 cells). The expression of Mas receptor was examined in the kidneys of CKD and AST-120-treated CKD rats using immunohistochemistry. Further, the effects of IS on Mas receptor expression in the kidneys of normotensive and hypertensive rats were examined. The effects of IS on the expression of Mas receptor and phosphorylation of endothelial nitric oxide synthase (eNOS) in HK-2 cells were examined using immunoblotting. CKD rats showed reduced renal expression of Mas receptor, while AST-120 restored its expression. Administration of IS downregulated Mas receptor expression in the kidneys of normotensive and hypertensive rats. IS downregulated Mas receptor expression in HK-2 cells in a time- and dose-dependent manner. Knockdown of organic anion transporter 3 (OAT3), aryl hydrocarbon receptor (AhR), and signal transducer and activator of transcription 3 (Stat3) inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. N-acetylcysteine, an antioxidant, also inhibited IS-induced downregulation of Mas receptor and phosphorylated eNOS. Ang-(1-7) attenuated IS-induced transforming growth factor-ß1 (TGF-ß1) expression. CONCLUSION: Mas receptor expression is reduced in the kidneys of CKD rats. IS downregulates renal expression of Mas receptor via OAT3/AhR/Stat3 pathway in proximal tubular cells. IS-induced downregulation of Mas receptor might be involved in upregulation of TGF-ß1 in proximal tubular cells.

Indoxyl sulfate counteracts endothelial effects of erythropoietin through suppression of Akt phosphorylation.

BACKGROUND: Erythropoietin (EPO) is used to treat anemia in patients with chronic kidney disease (CKD). A wide variation in individual response to EPO, however, is often observed, causing EPO resistance. EPO exhibits not only hematopoietic but also extra-hematopoietic functions such as endothelial effects. Indoxyl sulfate, a uremic toxin, is involved in endothelial dysfunction, and consequently, the pathogenesis of CKD-associated cardiovascular disease. The aim of the present study was to determine the effect of indoxyl sulfate on the extra-hematopoietic functions of EPO in human umbilical vein endothelial cells (HUVECs). METHODS AND RESULTS: HUVECs were incubated with or without indoxyl sulfate or an Akt inhibitor, and then stimulated with or without EPO. Indoxyl sulfate suppressed EPO-induced survival/proliferation, anti-apoptosis function, phosphorylation of endothelial nitric oxide synthase, and the expression of thrombospondin-1, an erythroid-stimulating factor, in HUVECs. Although EPO induced phosphorylation of both Akt and extracellular signal-regulated kinases (ERK) in HUVECs, indoxyl sulfate suppressed phosphorylation of Akt but not ERK. An Akt kinase inhibitor or Akt small interfering RNA suppressed all the EPO-induced cellular effects in HUVECs. As a site of action of indoxyl sulfate on EPO signaling, indoxyl sulfate attenuated EPO-induced tyrosine phosphorylation of EPO receptor (EPOR) in HUVECs. CONCLUSIONS: Indoxyl sulfate negatively regulates the EPOR-Akt pathway in endothelial cells, and might contribute to EPO resistance and endothelial dysfunction in patients with CKD.

Indoxyl sulfate induces endothelial cell senescence by increasing reactive oxygen species production and p53 activity.

BACKGROUND/AIM: We have reported that indoxyl sulfate (IS), a uremic toxin, accelerates proximal tubular cell senescence. Asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase, has been reported to induce endothelial cell senescence. This study aimed to determine whether IS induces endothelial cell senescence in comparison with ADMA, and to investigate its molecular mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) were incubated with IS (250 µM) and/or ADMA (10 µM). These concentrations were comparable with their mean serum levels in hemodialysis patients. Cell senescence was evaluated by measuring senescence-associated beta-galactosidase (SA-ß-gal) activity. N-acetylcysteine, an antioxidant, and pifithrin alpha p-nitro, a p53 inhibitor, were used to determine the role of reactive oxygen species (ROS) and p53 in the induction of cell senescence. RESULTS: Both IS and ADMA significantly increased SA-ß-gal activity in HUVECs. Further, some additional increase in SA-ß-gal activity was observed when IS and ADMA were co-incubated. Preincubation of N-acetylcysteine or pifithrin alpha p-nitro significantly inhibited SA-ß-gal activity induced by IS and ADMA in HUVECs. Thus, both IS and ADMA induced endothelial senescence through ROS and p53. CONCLUSION: IS induces endothelial cell senescence by increasing ROS production and p53 activity, like ADMA.

Indoxyl sulfate downregulates renal expression of Klotho through production of ROS and activation of nuclear factor-ĸB.

BACKGROUND/AIM: Klotho, an anti-aging gene, is expressed in the kidneys, and its renal expression is decreased in chronic kidney disease (CKD). The present study aimed to examine whether renal expression of Klotho is regulated by indoxyl sulfate, a uremic toxin, using rat kidneys and human proximal tubular cells (HK-2). METHODS: The effect of indoxyl sulfate on renal expression of Klotho was examined using (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN+IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH+IS). The effects of indoxyl sulfate, inhibitors of nuclear factor-κB (NF-κB) and an antioxidant on the expression of Klotho in HK-2 cells were examined. RESULTS: DH+IS and DN+IS rats showed decreased expression of Klotho mRNA in the kidneys as compared with DH and DN rats, respectively. Indoxyl sulfate suppressed the expression of Klotho mRNA and protein in HK-2 cells, whereas an antioxidant, N-acetylcysteine, and NF-κB inhibitors, pyrrolidine dithiocarbamate and isohelenin, alleviated these effects. CONCLUSIONS: Indoxyl sulfate downregulates Klotho expression in kidneys through production of reactive oxygen species and activation of NF-κB in proximal tubular cells. Indoxyl sulfate may be involved in reduced renal expression of Klotho in CKD.