Zhibin Ye

Soluble uric acid increases NALP3 inflammasome and interleukin-1β expression in human primary renal proximal tubule epithelial cells through the Toll-like receptor 4-mediated pathway

Urate crystals activate innate immunity through Toll like receptor 4 (TLR4) activation, leading to the formation of the NACHT, LRR and PYD domains-containing protein 3 [NALP3; also known as NOD-like receptor family, pyrin domain containing 3 (NALP3) and cryopyrin] inflammasome, caspase-1 activation and interleukin (IL)-1β expression in gout. However, whether elevated serum uric acid (UA) levels are associated with the development and progression of renal diseases without renal urate crystal deposition remains unknown. In the present study, human primary renal proximal tubule epithelial cells were incubated with soluble UA (100 µg/ml) with or without the TLR4 inhibitor, TAK242 (1 µM). The gene expression and protein synthesis of TLR4, NALP3, caspase-1, IL-1β and intercellular adhesion molecule-1 (ICAM-1) were detected by real-time PCR, ELISA, western blot analysis and fluorescence-activated cell sorting (FACS), respectively. Soluble UA significantly enhanced TLR4, NALP3, caspase-1, IL-1β and ICAM-1 expression in the human primary renal proximal tubule epithelial cells. The TLR4 inhibitor, TAK242 effectively blocked the soluble UA-induced upregulation of TLR4, NALP3, caspase-1, IL-1β and ICAM-1 expression in the human primary renal proximal tubule epithelial cells. Our findings indicate that soluble UA enhances NALP3 expression, caspase-1 activation, IL-1β and ICAM-1 production in renal proximal tubule epithelial cells in a TLR4-dependent manner, suggesting the activation of innate immunity in human primary renal proximal tubule epithelial cells by soluble UA.

Soluble monosodium urate, but not its crystal, induces toll like receptor 4-dependent immune activation in renal mesangial cells

BACKGROUND Uric acid has emerged as a novel and potential modifiable risk factor for the incidence and progression of kidney diseases, however, the deteriorate effect of uric acid on renal mesangial cells remains unclear. The present study is to examine the immune activation of soluble and crystal forms of uric acid in human mesangial cells. METHODS We stimulated primary human mesangial cells (HMCs) with increasing concentrations (from 50 to 200 μg/ml) of soluble monosodium urate (MSU) or MSU crystals. We examined interleukin (IL)-1β protein expression levels in cell culture by ELISA. The stimulated HMCs were further stimulated with soluble MSU or MSU crystals at 200 μg/ml with or without the pre-incubation of toll like receptor (TLR) 4 inhibitor TAK242 (1μM). TLR4, nod-like receptor protein (NLRP3, also known as NALP3), IL-1β, human leukocyte antigen (HLA)-DR and CD40 were examined by Realtime-PCR, Western blot and ELISA, respectively. RESULTS We found that both soluble MSU and MSU crystals increased IL-1β protein expression levels in dose-dependent fashion. Soluble MSU significantly enhanced the expression of TLR4, NLRP3, IL-1β, HLA-DR and CD40 while MSU crystals only upregulated the expression of TLR4 and IL-1β. TLR4 inhibitor TAK242 significantly blocked the up-regulation of NLRP3, IL-1β, HLA-DR and CD40 induced by soluble MSU while no TAK242 suppression effect on MSU crystals induced IL-1β up-regulation was found. CONCLUSIONS Our results suggested that soluble MSU, but not MSU crystals, induce NLRP3, IL-1β, HLA-DR and CD40 upregulation in a TLR4-dependent manner. These findings indicate that soluble MSU may play a pathological role in hyperuricemia induced renal mesangial injury.

Peroxisome proliferator‑activated receptor γ prevents the production of NOD-like receptor family, pyrin domain containing 3 inflammasome and interleukin 1β in HK‑2 renal tubular epithelial cells stimulated by monosodium urate crystals

Recent evidence showed that peroxisome proliferator‑activated receptor γ (PPARγ) ameliorates a variety of inflammatory conditions. The present study aimed to investigate the role of PPARγ in regulating NOD-like receptor family, pyrin domain containing 3 (NALP3) inflammasome and interleukin (IL)‑1β levels during monosodium urate (MSU) crystal‑induced inflammation. HK‑2 cells were incubated with or without 200 µg/ml MSU crystals, and mRNA and protein levels of PPARγ were determined using reverse transcription quantitative polymerase chain reaction and western blot analysis, respectively. To verify the role of PPARγ, HK‑2 cells were pre‑treated with PPARγ agonist pioglitazone, and the levels of NALP3 inflammasome and IL‑1β were detected by western blot analysis and ELISA. The results showed that MSU crystals increased PPARγ expression in HK‑2 cells at 24 h, while the expression decreased to normal levels at 48 h. It was also demonstrated that although the PPARγ agonist pioglitazone did not alter the mRNA and protein levels of PPARγ, it significantly reduced the MSU crystal‑induced production of NALP3 inflammasome and IL‑1β in HK‑2 cells, possibly by increasing the level of PPARγ activity. In conclusion, the results of the present study indicated that PPARγ prevented NALP3 inflammasome formation and IL‑1β production in HK‑2 cells stimulated by MSU crystals, which indicated that PPARγ may represent a novel target for the treatment of hyperuricemic nephropathy.

Ischemic preconditioning attenuates ischemia/reperfusion-induced kidney injury by activating autophagy via the SGK1 signaling pathway.

Ischemic preconditioning (IPC) has a strong renoprotective effect during renal ischemia/reperfusion (I/R) injury that is thought to relate to autophagy. However, the role of autophagy during IPC-afforded renoprotection and the precise mechanisms involved are unknown. In this study, an in vitro hypoxia/reoxygenation (H/R) model was established in which oxygen and glucose deprivation (OGD) was applied to renal cells for 15 h followed by reoxygenation under normal conditions for 30 min, 2 h or 6 h; transient OGD and subsequent reoxygenation were implemented before prolonged H/R injury to achieve hypoxic preconditioning (HPC). 3-Methyladenine (3-MA) was used to inhibit autophagy. In a renal I/R injury model, rats were subjected to 40 min of renal ischemia followed by 6 h, 12 h or 24 h of reperfusion. IPC was produced by four cycles of ischemia (8 min each) followed by 5 min of reperfusion prior to sustained ischemia. We found that IPC increased LC3II and Beclin-1 levels and decreased SQSTM/p62 and cleaved caspase-3 levels in a time-dependent manner during renal I/R injury, as well as increased the number of intracellular double-membrane vesicles in injured renal cells. IPC-induced renal protection was efficiently attenuated by pretreatment with 5 mM 3-MA. Pretreatment with IPC also dynamically affected the expression of SGK1/FOXO3a/HIF-1α signaling components. Moreover, knocking down SGK1 expression significantly downregulated phosphorylated-FOXO3a (p-FOXO3a)/FOXO3 and HIF-1α, suppressed LC3II and Beclin-1 levels, increased SQSTM/p62 and cleaved caspase-3 levels, and abolished the protective effect of IPC against I/R-induced renal damage. SGK1 overexpression efficiently increased p-FOXO3a/FOXO3 and HIF-1α levels, promoted the autophagy flux and enhanced the protective effect mediated by HPC. Furthermore, FOXO3a overexpression decreased HIF-1α protein levels, inhibited HIF-1α transcriptional activity and reduced the protective effect of IPC. Our study indicates that IPC can ameliorate renal I/R injury by promoting autophagy through the SGK1 pathway.

Serum uric acid is associated with lumbar spine bone mineral density in healthy Chinese males older than 50 years

Objectives We aimed to investigate the association of serum uric acid (UA) levels with bone mineral density (BMD) at all skeletal sites in healthy Chinese males >50 years of age. Methods A cross-sectional study of 385 Chinese males >50 years of age who underwent health checkup in Huadong Hospital Affiliated to Fudan University in Shanghai, China, was conducted. Clinical and bone characteristics were compared in different UA tertiles (UA1: UA <4.7 mg/dL, UA2: 4.7 mg/dL ≤ UA <6 mg/dL and UA3: UA ≥6 mg/dL). Pearson correlation and multiple regression analysis were used to study the correlation of UA with BMD at various skeletal sites. Results Serum UA levels were positively associated with higher BMD and T-values at the lumbar spine, but not at other skeletal sites, after adjusting for multiple confounding factors. Lumbar spine BMD; the T- and Z-values at the lumbar spine, total hip and femoral neck; as well as intact parathyroid hormone (iPTH) levels are higher in the highest tertile of UA than in the second tertile of UA. Conclusion Our results provide epidemiological evidence in Chinese Han males aged >50 years that serum UA levels are positively correlated with lumbar spine BMD and T-values, suggesting that UA may exert protective effect on bone density at the lumbar spine in Chinese males >50 years of age.

Impaired Na+−K+-ATPase signaling in renal proximal tubule contributes to hyperuricemia-induced renal tubular injury

Hyperuricemia contributes to renal inflammation. We aimed to investigate the role of Na+–K+–ATPase (NKA) in hyperuricemia-induced renal tubular injury. Human primary proximal tubular epithelial cells (PTECs) were incubated with uric acid (UA) at increasing doses or for increasing lengths of time. PTECs were then stimulated by pre-incubation with an NKA α1 expression vector or small interfering RNA before UA (100 μg ml−1, 48 h) stimulation. Hyperuricemic rats were induced by gastric oxonic acid and treated with febuxostat (Feb). ATP levels, the activity of NKA and expression of its α1 subunit, Src, NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and interleukin 1β (IL-1β) were measured both in vitro and in vivo. Beginning at concentrations of 100 μg ml−1, UA started to dose-dependently reduce NKA activity. UA at a concentration of 100 μg ml−1 time-dependently affected the NKA activity, with the maximal increased NKA activity at 24 h, but the activity started to decrease after 48 h. This inhibitory effect of UA on NKA activity at 48 h was in addition to a decrease in NKA α1 expression in the cell membrane, but an increase in lysosomes. This process also involved the subsequent activation of Src kinase and NLRP3, promoting IL-1β processing. In hyperuricemic rats, renal cortex NKA activity and its α1 expression were upregulated at the 7th week and both decreased at the 10th week, accompanied with increased renal cortex expression of Src, NLRP3 and IL-1β. The UA levels were reduced and renal tubular injuries in hyperuricemic rats were alleviated in the Feb group. Our data suggested that the impairment of NKA and its consequent regulation of Src, NLRP3 and IL-1β in the renal proximal tubule contributed to hyperuricemia-induced renal tubular injury.

Ischemic Preconditioning Promotes Autophagy and Alleviates Renal Ischemia/Reperfusion Injury

Autophagy is important for cellular survival during renal ischemia/reperfusion (I/R) injury. Ischemic preconditioning (IPC) has a strong renoprotective effect during renal I/R. Our study here aimed to explore the effect of IPC on autophagy during renal I/R injury. Rats were subjected to unilateral renal ischemia with or without prior IPC. Hypoxia/reoxygenation (H/R) injury was induced in HK-2 cells with or without prior hypoxic preconditioning (HPC). Autophagy and apoptosis were detected after reperfusion or reoxygenation for different time. The results showed that the levels of LC3II, Beclin-1, SQSTM1/p62, and cleaved caspase-3 were altered in a time-dependent manner during renal I/R. IPC further induced autophagy as indicated by increased levels of LC3II and Beclin-1, decreased level of SQSTM1/p62, and accumulation of autophagosomes compared to I/R groups at corresponding reperfusion time. In addition, IPC reduced the expression of cleaved caspase-3 and alleviated renal cell injury, as evaluated by the levels of serum creatinine (Scr), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) in renal tissues. In conclusion, autophagy and apoptosis are dynamically altered during renal I/R. IPC protects against renal I/R injury and upregulates autophagic flux, thus increasing the possibility for a novel therapy to alleviate I/R-induced acute kidney injury (AKI).

Uric acid upregulates the adiponectin‑adiponectin receptor 1 pathway in renal proximal tubule epithelial cells

Adiponectin (APN) is a protein hormone that is primarily derived from adipocytes. It can also be secreted by renal cells. Hypoadiponectinemia has been documented in patients with hyperuricemia, however, whether soluble uric acid (SUA) regulates the expression of APN and APN receptor 1 (AdipoR1) in renal proximal tubule epithelial cells (PTECs) remains to be elucidated. The present study investigated the expression of APN and AdipoR1 in cultured PTECs that were exposed to SUA through immunofluorescence and western blot analysis. In addition, Sprague-Dawley rats with oxonic acid-induced hyperuricemia (HUA) with or without febuxostat treatment were employed as an animal model to measure 24 h urine protein, serum creatinine, urea nitrogen, uric acid and homeostasis model assessment of insulin resistance. Renal pathology was evaluated using hematoxylin and eosin and immunohistochemical staining. APN and AdipoR1 expression in the renal cortex were evaluated by western blotting. The results demonstrated that, in PTECs, the expression of APN and AdipoR1 was constant and increased upon SUA exposure. Similar observations were made within the proximal renal tubules of rats, and the oxonic acid-induced increases in APN and AdipoR1 were offset by febuxostat treatment. Furthermore, SUA-treated PTECs exhibited an increase in the expression of NLR family pyrin domain-containing (NLRP) 3, which was dose-dependent. NLRP3 expression was also significantly increased in the renal cortex of HUA rats compared with control and febuxostat-treated rats. In conclusion, SUA enhanced the expression of APN and AdipoR1 in PTECs, which was associated with an increase in NLRP3 expression. The APN-AdipoR1 pathway was demonstrated to have an important role in in vitro and in vivo models of renal proximal tubule inflammatory injury. Therefore, this pathway may be a potential therapy target in urate nephropathy.

The Association of Urinary Sodium and Potassium with Renal Uric Acid Excretion in Patients with Chronic Kidney Disease

Background/Aims: Hypertension and hyperuricemia are closely associated with an intermingled cause and effect relationship. Additionally, urinary sodium and potassium excretion is related to blood pressure. Whether or not it is associated with urinary uric acid excretion is not clear. Therefore, we aim to study the association of urinary sodium and potassium with renal uric acid excretion in patients with CKD. Methods: A cross-sectional study of 428 patients with CKD recruited from our department was conducted. All patients were divided into hypertension and non-hypertension group. In these two groups, Spearman correlation and multiple linear regression analysis were used to study the correlation of urinary sodium and potassium with renal handling of uric acid. Results: According to multiple linear regression analysis, in hypertension group, fractional excretion of sodium (FEna) was negatively correlated with 24 hour urinary uric acid (24-hUur) and uric acid clearance rate (Cur) (beta coefficients [B]=-0.066, -0.182, respectively; both P< 0.05), and positively correlated with fractional excretion of uric acid (FEur) (B=1.641, P< 0.001). Additionally, fractional excretion of potassium (FEk) was positively correlated with FEur (B=0.576, P< 0.001), but not related to 24-hUur and Cur (both P>0.05). And urinary sodium/potassium ratio (Una/k) was negatively related to 24-h Uur and Cur (B=-0.047, -0.159, both P< 0.05), and positively related to FEur (B=0.578, P< 0.05). Furthermore, FEna and FEk was still positively related to FEur in the lowest tertile of eGFR groups (both P< 0.05), but not related in the second and highest tertile of eGFR groups (all P> 0.05). In non-hypertension group, FEna was negatively correlated with 24-hUur (B=-0.589, P< 0.05), but not related to Cur and FEur (both P> 0.05). both FEk and Una/k was not related to 24-h Uur, Cur and FEur (all P> 0.05). Moreover, FEna and FEk was still not correlated with FEur in all tertiles of eGFR groups (all P> 0.05). Conclusion: We found that in patients with CKD, urinary sodium and potassium excretion is closely correlated to renal handling of uric acid, which was pronounced in hypertensive patients with low eGFR. This phenomenon may be one of the mechanisms of the relationship between hypertension and hyperuricemia. Further research is needed to confirm it. It is expected to manage hyperuricemia in terms of controlling the diet of sodium and potassium.

Toll-like Receptor 4 Signaling Pathway in the Protective Effect of Pioglitazone on Experimental Immunoglobulin A Nephropathy

Background: In vitro experiments have revealed that toll-like receptor 4 (TLR4) pathway is involved in the progression of immunoglobulin A nephropathy (IgAN) by induction of proinflammatory cytokines. Evidence showed that, in other disease models, peroxisome proliferator-activated receptor-&ggr; (PPAR-&ggr;) agonists have been shown to exert anti-inflammatory effects through suppression of the expression and activity of TLR4. However, the interaction between PPAR-&ggr; and TLR4 in IgAN has not been fully studied both in vitro and in vivo. In this study, we explored whether TLR4 pathway attributed to the progression of IgAN in experimental rats. Methods: Bovine gamma globulin was used to establish IgAN model. Fifty-four Lewis rats were randomly divided into six groups: ControlTAK242, IgANTAK242, toll-like receptor 4 inhibitor (TAK242) groups (rats were administrated with TLR4 inhibitor, TAK242) and ControlPio, IgANPio, Pio groups (rats were administrated with PPAR-&ggr; agonist, pioglitazone). Urinary albumin-to-creatinine ratio (ACR), serum creatinine, and blood urea nitrogen were detected by automatic biochemical analyzer. Renal histopathological changes were observed after hematoxylin-eosin staining, and the IgA deposition in glomeruli was measured by immunofluorescence staining. Real-time polymerase chain reaction and Western blotting were used to detect TLR4 and interleukin-1 beta (IL-1&bgr;) message ribonucleic acid (mRNA) and protein expression in renal tissues. Results were presented as mean ± standard deviation. Differences between groups were analyzed by one-way analysis of variance. Results: Compared to normal rats, experimental rats showed higher ACR (4.45 ± 1.33 mg/mmol vs. 2.89 ± 0.96 mg/mmol, P < 0.05), obvious IgA deposition with mesangial hypercellularity, hyperplasia of mesangial matrix accompanied by increased serum IL-1&bgr; (48.28 ± 13.49 pg/ml vs. 35.56 ± 7.41pg/ml, P < 0.05), and renal expression of IL-1&bgr; and TLR4. The biochemical parameters and renal pathological injury were relieved in both TAK242 group and Pio group. The expressions of renal tissue TLR4, IL-1&bgr;, and serum IL-1&bgr; were decreased in rats treated with TAK242, and the expression of TLR4 mRNA and protein was significantly reduced in Pio group compared to IgANPio group (1.22 ± 0.28 vs. 1.72 ± 0.45, P < 0.01, and 0.12 ± 0.03 vs. 0.21 ± 0.05, P < 0.01). Conclusions: Our study proves that inflammation mediated by TLR4 signaling pathway is involved in the progression of IgAN in rat models. Moreover, pioglitazone can inhibit the expression of TLR4 in IgAN.