Transient Receptor Potential Channel 6 (TRPC6) Protects Podocytes during Complement-mediated Glomerular Disease

Gain-of-function mutations in the calcium channel TRPC6 lead to autosomal dominant focal segmental glomerulosclerosis and podocyte expression of TRPC6 is increased in some acquired human glomerular diseases, particularly in membranous nephropathy. These observations led to the hypothesis that TRPC6 overactivation is deleterious to podocytes through pathological calcium signaling, both in genetic and acquired diseases. Here, we show that the effects of TRPC6 on podocyte function are context-dependent. Overexpression of TRPC6 alone did not directly affect podocyte morphology and cytoskeletal structure. Unexpectedly, however, overexpression of TRPC6 protected podocytes from complement-mediated injury, whereas genetic or pharmacological TRPC6 inactivation increased podocyte susceptibility to complement. Mechanistically, this effect was mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation. Podocyte-specific TRPC6 transgenic mice showed stronger CaMKII activation, reduced podocyte foot process effacement and reduced levels of proteinuria during nephrotoxic serum nephritis, whereas TRPC6 null mice exhibited reduced CaMKII activation and higher levels of proteinuria compared with wild type littermates. Human membranous nephropathy biopsy samples showed podocyte staining for active CaMKII, which correlated with the degree of TRPC6 expression. Together, these data suggest a dual and context dependent role of TRPC6 in podocytes where acute activation protects from complement-mediated damage, but chronic overactivation leads to focal segmental glomerulosclerosis.     

贝那普利联合肾炎四味片对慢性肾小球肾炎患者临床疗效的影响及机制研究

目的:探讨贝那普利联合肾炎四味片对慢性肾小球肾炎患者临床疗效的影响及其作用机制。方法:收集我院收治的慢性肾小球肾炎患者120例,随机分为观察组和对照组,每组各60例。对照组给予肾炎四味片治疗;观察组在对照组的基础上给予贝那普利片。治疗结束后,比较两组患者白细胞诱素1(Lkn-1)、尿酸、TNF-α、收缩压、舒张压、24小时蛋白尿水平的变化及临床总有效率。结果:与治疗前相比,两组患者治疗后Lkn-1、尿酸、TNF-α、收缩压、24小时蛋白尿水平均显著降低(P0.05);与对照组比较,观察组患者Lkn-1、尿酸、TNF-α、收缩压、24小时蛋白尿水平较低(P0.05),临床总有效率较高(P0.05)。结论:贝那普利联合肾炎四味片可有效治疗慢性肾小球肾炎,推测其机制与降低Lkn-1、尿酸、TNF-α、收缩压、24小时蛋白尿水平有关。     

双歧杆菌对PHN保护作用的研究

被动型Ｈｅｙｍａｎｎ肾炎（ＰａｓｉｖｅＨｅｙｍａｎｎＮｅｐｈｒｉｔｉｓ,ＰＨＮ）是一种在临床生化及病理学改变方面均酷似人类膜性肾病的实验动物模型,是研究人类膜性肾病的理想模型。本文在成功复制ＰＨＮ模型的基础上,应用双歧杆菌经口饲喂和腹腔内注射两种途径治疗ＰＨＮ大鼠,同时以生理盐水作为治疗对照。结果表明腹腔内注射双歧杆菌可以明显降低２４小时尿蛋白（与经口饲喂和治疗对照组相比Ｐ＜００５）,而经口饲喂双歧杆菌组,作用不明显,推测双歧杆菌全身应用可以通过提高机体细胞免疫能力降低该病的发生     

胰岛素样生长因子-1、转化生长因子-beta1 水平变化在过敏性紫癜患儿肾损害的临床意义

目的：探讨胰岛素样生长因子-1（IGF-1）、转化生长因子-beta1（TGF-beta1）水平变化在过敏性紫癜患儿肾损害发病机制中作用。 方法：选取HSP患儿30 例、HSPN患儿30 例和30 例健康儿童为对照组,分别采血,用化学发光法检测各组标本IGF-1 浓度,采用 双抗体一步夹心法酶联免疫吸附试验检测各组TGF-茁1 浓度;同时用全自动生化仪检测各组患儿血清Cys C、BUN、SCr 水平。结 果：血IGF-1、TGF-beta1 的水平HSPN组、HSP组与对照组患儿比较,差异均有统计学意义（P<0.05）。血清Cys C 水平对照组、HSP 组与HSPN 组患儿比较,差异有统计学意义（P<0.05）。采用直线相关分析HSPN 组的血清Cys C 水平与血IGF-1、TGF-beta1 水平 比较,存在正相关（r=0.759、r=0.802,均P<0.05）,血IGF-1 与TGF-beta1 也存在正相关（r=0.850,P<0.05）。结论：血IGF-1、TGF-beta1 共同 参与紫癜性肾炎发病,其水平变化与病理损害程度相关。     

Sublytic C5b-9 complexes induce apoptosis of glomerular mesangial cells in rats with Thy-1 nephritis through role of interferon regulatory factor-1-dependent caspase 8 activation

The apoptosis of glomerular mesangial cells (GMC) in rat Thy-1 nephritis (Thy-1N), a model of human mesangioproliferative glomerulonephritis, is accompanied by sublytic C5b-9 deposition, but the mechanism of sublytic C5b-9-mediated GMC apoptosis has not been elucidated. In the present study, the gene expression profiles both in the GMC stimulated by sublytic C5b-9 and the rat renal tissue of Thy-1N were detected using microarrays. Among the co-up-regulated genes, the up-regulation of interferon regulatory factor-1 (IRF-1) was further confirmed. Increased caspase 8 and caspase 3 expression and caspase 8 promoter activity in the GMC were also identified. Meanwhile, overexpression or knockdown of IRF-1 not only enhanced or inhibited GMC apoptosis and caspase 8 and 3 induction but also increased or decreased caspase 8 promoter activity, respectively. The element of IRF-1 binding to the caspase 8 promoter was first revealed. Furthermore, silencing IRF-1 or repressing the activation of caspases 8 and 3 significantly reduced GMC apoptosis, including other pathologic changes of Thy-1N. These novel findings indicate that GMC apoptosis of Thy-1N is associated with the IRF-1-activated caspase 8 pathway.     

肾炎康复片对慢性肾小球肾炎患者血清FN、IL-2及胱抑素C 水平的影响

目的:探讨肾炎康复片对慢性肾小球肾炎患者血清粘连蛋白(fibronectin,FN)、白介素-2(interleukin-2,IL-2)及胱抑素C水平影响。方法:收集我院慢性肾小球肾炎患者80例,随机分为实验组和对照组。两组患者均予低盐优质蛋白饮食、控制血压、预防感染等常规治疗。对照组予以贝那普利治疗,实验组在对照组基础上加用肾炎康复片。两组患者分别于治疗前、后各进行一次血清FN、IL-2、胱抑素C水平测定;血压,肾功能血清肌酐(SCr)、尿素氮(BUN),24 h尿蛋白测定。结果:治疗后两组患者血清FN、IL-2水平均上升,胱抑素C水平均下降(P0.05),与对照组比较,实验组三项指标均变化更明显(P0.05);治疗后两组患者血压、血清肌酐、尿素氮、24 h尿蛋白水平均下降(P0.05),与对照组比较,实验组四项指标均较低(P0.05)。结论:肾炎康复片可更显著升高慢性肾小球肾炎患者血清FN、IL-2水平,降低胱抑素C水平,对患者血压控制,肾功能修复及减少尿蛋白有重要作用。     

Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines

Background

A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes.

Methods

Human CD14+ monocytes were incubated with interferon alpha (IFNα), interferon gamma (IFNγ), interleukin-6 (IL6), interleukin-1 beta (IL1β), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFα). Hepcidin expression was examined by real-time PCR and enzyme immunoassay.

Results

Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 h of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNα or IL6. One hour of incubation with IFNα or IL6 increased hepcidin mRNA 20 and 80-fold, respectively; by 24 h the mRNA remained 5- and 2.4-fold higher than baseline. IL1β, IFNγ, and MCP-1 did not affect monocyte hepcidin expression. TNFα inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 h of treatment with IFNα or IL6, immunoreactive hepcidin production by monocytes increased 3- and 2.6-fold, respectively.

Conclusion

Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNα and IL6.

General significance

The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.     

Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.     

Role of extracellular matrix renal tubulo-interstitial nephritis antigen (TINag) in cell survival utilizing integrin (alpha)vbeta3/focal adhesion kinase (FAK)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B-serine/threonine kinase (AKT) signaling pathway

Tubulo-interstitial nephritis antigen (TINag) is an extracellular matrix protein expressed in tubular basement membranes. Combined mutations in TINag and nephrocystin-1 genes lead to nephronophthisis with reduced cell survival. Because certain extracellular matrix proteins are known to modulate cell survival, studies were initiated in Lewis rats lacking TINag to assess if they are more susceptible to cisplatin-induced injury. Cisplatin induced a higher degree of tubular cell damage and apoptosis in regions where TINag is expressed in a parental Wistar strain. This was accompanied by an accentuated increase in serum creatinine and Kim-1 RNA and renal expression of Bax, p53, and its nuclear accumulation, mtDNA fragmentation, and a decrease of Bcl-2. Cisplatin induced fulminant apoptosis of HK-2 cells with increased caspase3/7 activity, mtDNA fragmentation, and a reduced cell survival. These effects were partially reversed in cells maintained on TINag substratum. Far Western/solid phase assays established TINag binding with integrin αvβ3 comparable with vitronectin. Transfection of cells with αv-siRNA accentuated cisplatin-induced apoptosis, aberrant translocation of cytochrome c and Bax, and reduced cell survival. The αv-siRNA decreased expression of integrin-recruited focal adhesion kinase (FAK) and p-FAK, while increasing the expression of p53 and p-p53. Similarly, p-AKT was reduced although ILK was unaffected. Inhibition of PI3K had similar adverse cellular effects. These effects were ameliorated in cells on TINag substratum. In vivo, a higher degree of decrease in the expression of p-FAK and pAKT was observed in Lewis rats following cisplatin treatment. These in vivo and in vitro studies demonstrate an essential role of TINag in cellular survival to maintain proper tubular homeostasis utilizing integrin αvβ3 and downstream effectors.