Monocyte/macrophage chemokine receptor CCR2 mediates diabetic renal injury

Monocyte/macrophage recruitment correlates strongly with the progression of renal impairment in diabetic nephropathy (DN). C-C chemokine receptor (CCR)2 regulates monocyte/macrophage migration into injured tissues. However, the direct role of CCR2-mediated monocyte/macrophage recruitment in diabetic kidney disease remains unclear. We report that pharmacological blockade or genetic deficiency of CCR2 confers kidney protection in Ins2(Akita) and streptozotocin (STZ)-induced diabetic kidney disease. Blocking CCR2 using the selective CCR2 antagonist RS504393 for 12 wk in Ins2(Akita) mice significantly attenuated albuminuria, the increase in blood urea nitrogen and plasma creatinine, histological changes, and glomerular macrophage recruitment compared with vehicle. Furthermore, mice lacking CCR2 (CCR2(-/-)) mimicked CCR2 blockade by reducing albuminuria and displaying less fibronectin mRNA expression and inflammatory cytokine production compared with CCR2(+/+) mice, despite comparable blood glucose levels. Bone marrow-derived monocytes from CCR2(+/+) or CCR2(-/-) mice adoptively transferred into CCR2(-/-) mice reversed the renal tissue-protective effect in diabetic CCR2(-/-) mice as evaluated by increased urinary albumin excretion and kidney macrophage recruitment, indicating that CCR2 is not required for monocyte migration from the circulation into diabetic kidneys. These findings provide evidence that CCR2 is necessary for monocyte/macrophage-induced diabetic renal injury and suggest that blocking CCR2 could be a novel therapeutic approach in the treatment of DN.     

KIM-1 mediates fatty acid uptake by renal tubular cells to promote progressive diabetic kidney disease

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HDACs and the senescent phenotype of WI-38 cells

Background  

Normal cells possess a limited proliferative life span after which they enter a state of irreversible growth arrest. This process, known as replicative senescence, is accompanied by changes in gene expression that give rise to a variety of senescence-associated phenotypes. It has been suggested that these gene expression changes result in part from alterations in the histone acetylation machinery. Here we examine the influence of HDAC inhibitors on the expression of senescent markers in pre- and post-senescent WI-38 cells.     

Lipophagy deficiency exacerbates ectopic lipid accumulation and tubular cells injury in diabetic nephropathy

Autophagy-mediated lipotoxicity plays a critical role in the progression of diabetic nephropathy (DN), but the precise mechanism is not fully understood. Whether lipophagy, a selective type of autophagy participates in renal ectopic lipid deposition (ELD) and lipotoxicity in the kidney of DN is unknown. Here, decreased lipophagy, increased ELD and lipotoxcity were observed in tubular cells of patients with DN, which were accompanied with reduced expression of AdipoR1 and p-AMPK. Similar results were found in db/db mice, these changes were reversed by AdipoRon, an adiponectin receptor activator that promotes autophagy. Additionally, a significantly decreased level of lipophagy was observed in HK-2 cells, a human proximal tubular cell line treated with high glucose, which was consistent with increased lipid deposition, apoptosis and fibrosis, while were partially alleviated by AdipoRon. However, these effects were abolished by pretreatment with ULK1 inhibitor SBI-0206965, autophagy inhibitor chloroquine and enhanced by AMPK activator AICAR. These data suggested by the first time that autophagy-mediated lipophagy deficiency plays a critical role in the ELD and lipid-related renal injury of DN.Subject terms: Chronic kidney disease, Diabetes complications     

Decoy receptor 3 inhibits renal mononuclear leukocyte infiltration and apoptosis and prevents progression of IgA nephropathy in mice

The progression of IgA nephropathy (IgAN), the most frequent type of primary glomerulonephritis, is associated with high levels of mononuclear leukocyte infiltration into the kidney. These cells consist mainly of T cells and macrophages. Our previous study showed that a decoy receptor 3 (DCR3) gene therapy can prevent the development of a mouse autoimmune glomerulonephritis model by its potent immune modulating effects (Ka SM, Sytwu HK, Chang DM, Hsieh SL, Tsai PY, Chen A. J Am Soc Nephrol 18: 2473-2485, 2007). Here, we tested the hypothesis that DCR3 might prevent the progression of IgAN, an immune complex-mediated primary glomerulonephritis, by inhibiting T cell activation, renal T cell/macrophage infiltration, and protecting the kidney from apoptosis. We used a progressive IgAN (Prg-IgAN) model in B cell-deficient mice, because the mice are characterized by a dramatic proliferation of activated T cells systemically and progressive NF-κB activation in the kidney. We treated the animals with short-term gene therapy with DCR3 plasmids by hydrodynamics-based gene delivery. When the mice were euthanized on day 21, we found that, compared with empty vector-treated (disease control) Prg-IgAN mice, DCR3 gene therapy resulted in 1) systemic inhibition of T cell activation and proliferation; 2) lower serum levels of proinflammatory cytokines; 3) improved proteinuria, renal function, and renal pathology (inhibiting the development of marked glomerular proliferation, crescent formation, glomerulosclerosis, and interstitial inflammation); 5) suppression of T cell and macrophage infiltration into the periglomerular interstitium of the kidney; and 5) a reduction in apoptotic figures in the kidney. On the basis of these findings, DCR3 might be useful therapeutically in preventing the progression of IgAN.     

Epithelial-mesenchymal transition in renal tubular cells in the pathogenesis of progressive tubulo-interstitial fibrosis

Epithelial-mesenchymal transition (EMT) plays an important role in embryogenesis and organ formation. Over the last 10-15 years it has been established that EMT is a significant mechanism of tumor progression and metastasis formation and also of progressive tissue fibrosis in the kidney, liver and lung. EMT seen in these diverse physiological and pathophysiological contexts shares a number of stages and modules, but also carries distinct, context specific characteristics. EMT in tissue fibrosis is a form of reverse embryogenesis, when highly specialized epithelial cells in the specific organs will respond to injury with loosing their epithelial characteristics and functions and regaining characteristics of the cells from which they originated. EMT in the context of tissue fibrosis can be induced by different forms of injury or a set of humoral factors. The process is regulated by a complex balance of humoral and microenvironmental stimuli, in which cell-cell contacts and interaction of the transitioning cell with the extracellular matrix components is very important. Intense research in this exciting field yielded good understanding of many of the details of this fascinating process, although numerous questions still await proper answers. There is indication that understanding of the molecular mechanisms underlying "fibrotic" EMT may lead to the design of specific and effective therapeutic measures for progressive tissue fibrosis.     

Diabetic nephropathy: the central role of renal proximal tubular cells in tubulointerstitial injury

Diabetic nephropathy is now the commonest cause of end stage renal disease and accounts for 30-40% of all patients requiring renal replacement therapy. Furthermore, the incidence of diabetic nephropathy continues to increase, in part due to the improved survival of type 2 diabetic patients as the cardiovascular mortality in this group declines (Ritz and Stefanski, 1996). Clinically incipient nephropathy is first manifest by the onset of persistent microalbuminuria, after which, overt diabetic nephropathy is heralded by the appearance of persistent proteinuria. Subsequently, there is a progressive decline in glomerular filtration rate (GFR) resulting, within 5 years, in end stage renal disease in 50% of patients (Hasslacher et al., 1989). The pathology of the renal lesions are similar in type I and II diabetes (Taft et al., 1994), although it has been suggested that there is more heterogeneity in type II diabetes (Chihara et al., 1986). Studies analysing structural-functional relationships have demonstrated that the development of proteinuria correlates with the degree of mesangial expansion (Mauer et al., 1984; White and Bilous, 2000). Although diabetic nephropathy was traditionally considered a primarily glomerular disease, it is now widely accepted that the rate of deterioration of function correlates best with the degree of renal tubulointerstitial fibrosis (Mauer et al., 1984, Bohle et al., 1991). This suggests that although in the majority of patients the primary event is a condition manifest by glomerular changes resulting in proteinuria, the long-term outcome is determined by events in the renal interstitium. With the increasing awareness of the importance of these pathological interstitial changes, interest has focused on the role of cells, such as the epithelial cells of the proximal tubule (PTC) or the interstitial myofibroblast, in the initiation of fibrosis. The aim of the present review is to analyse the available data supporting the role for the PTC in orchestrating renal interstitial fibrosis in diabetic nephropathy as a result of glucose-dependent alterations in PTC function. The potential for subsequent effects on PTC-fibroblast cross-talk will also be considered.     

Activation of ERK accelerates repair of renal tubular epithelial cells,whereas it inhibits progression of fibrosis following ischemia/reperfusion injury

Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30 min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1 day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9 days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-β1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.     

Dysfunction of the ER chaperone BiP accelerates the renal tubular injury

Tubular-interstitial injury plays a key role in the progression of chronic kidney disease. Although endoplasmic reticulum (ER) stress plays significant roles in the development of chronic diseases such as neurodegenerative disease, cardiomyopathy and diabetes mellitus, its pathophysiological role in chronic renal tubular cell injury remains unknown. BiP is an essential chaperone molecule that helps with proper protein folding in the ER. Recently, we have produced a knock-in mouse that expresses a mutant-BiP in which the retrieval sequence to the ER is deleted in order to elucidate physiological processes that are sensitive to ER functions in adulthood. The heterozygous mutant-BiP mice showed significant tubular-interstitial lesions with aging. Furthermore, proteinuria induced by chronic protein overload accelerated the tubular-interstitial lesions in the mutant mice, accompanying caspase-12 activation and tubular cell apoptosis. These results suggest that the ER stress pathway is significantly involved in the pathophysiology of chronic renal tubular-interstitial injury in vivo.     

Orai1 contributes to the establishment of an apoptosis-resistant phenotype in prostate cancer cells

The molecular nature of calcium (Ca²⁺)-dependent mechanisms and the ion channels having a major role in the apoptosis of cancer cells remain a subject of debate. Here, we show that the recently identified Orai1 protein represents the major molecular component of endogenous store-operated Ca²⁺ entry (SOCE) in human prostate cancer (PCa) cells, and constitutes the principal source of Ca²⁺ influx used by the cell to trigger apoptosis. The downregulation of Orai1, and consequently SOCE, protects the cells from diverse apoptosis-inducing pathways, such as those induced by thapsigargin (Tg), tumor necrosis factor α, and cisplatin/oxaliplatin. The transfection of functional Orai1 mutants, such as R91W, a selectivity mutant, and L273S, a coiled-coil mutant, into the cells significantly decreased both SOCE and the rate of Tg-induced apoptosis. This suggests that the functional coupling of STIM1 to Orai1, as well as Orai1 Ca²⁺-selectivity as a channel, is required for its pro-apoptotic effects. We have also shown that the apoptosis resistance of androgen-independent PCa cells is associated with the downregulation of Orai1 expression as well as SOCE. Orai1 rescue, following Orai1 transfection of steroid-deprived cells, re-established the store-operated channel current and restored the normal rate of apoptosis. Thus, Orai1 has a pivotal role in the triggering of apoptosis, irrespective of apoptosis-inducing stimuli, and in the establishment of an apoptosis-resistant phenotype in PCa cells.     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

Cannabinoid receptor 1 mediates palmitic acid‐induced apoptosis via endoplasmic reticulum stress in human renal proximal tubular cells

The endocannabinoid system (ECS) is activated at the onset of obesity and diverse metabolic diseases. Endocannabinoids mediate their physiological and behavioral effects by activating specific cannabinoid receptors, mainly cannabinoid receptor 1 (CB₁R). Diabetic nephropathy (DN) is induced by hyperlipidemia, and renal proximal tubule cells are an important site for the onset of DN. However, the pathophysiology of CB₁R, especially in the hyperlipidemia of DN, has not been elucidated. Therefore, we examined the effect of palmitic acid (PA) on CB₁R expression and its related signal pathways in human renal proximal tubular cells (HK‐2 cells). PA significantly increased CB₁R mRNA and protein levels and induced CB₁R internalization. PA‐induced activation of CB₁R is prevented by the treatment of AACOCF₃ (a cPLA₂ inhibitor), indomethacin and NS398 (a COX 2 inhibitors). Indeed, PA increased cPLA₂, and COX‐2 but not COX‐1. We also investigated whether the PA‐induced activation of CB₁R is linked to apoptosis. As a result, AM251 (a CB₁R antagonist) attenuated PA‐mediated apoptosis in a concentration‐dependent manner. Furthermore, PA decreased GRP78 expression and induced increases in the endoplasmic reticulum (ER) stress signaling pathways p‐PERK, p‐eIF2α, p‐ATF4, and CHOP, which were blocked by AM251 treatment. Moreover, PA increased the Bax/Bcl‐2 ratio, cleaved PARP, and caspase‐3 levels. The PA‐induced apoptotic effects were decreased with CB₁R‐specific antagonist (AM251) treatment and CB1 si‐RNA transfection. In conclusion, PA induced apoptosis through ER stress via CB₁R expression in human proximal tubule cells. Our results provide evidence that CB₁R blockade may be a potential anti‐diabetic therapy for the treatment of DN. J. Cell. Physiol. 225: 654–663, 2010. © 2010 Wiley‐Liss, Inc.     

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.     

Sphingosine kinase 1 protects renal tubular epithelial cells from renal fibrosis via induction of autophagy

Autophagy is an important homoeostatic mechanism for the lysosomal degradation of protein aggregates and damaged cytoplasmic components. Recent studies suggest that autophagy which is induced by TGF-β1 suppresses kidney fibrosis in renal tubular epithelial cells (RTECs) of obstructed kidneys. Sphingosine kinase 1(SK1), converting sphingosine into endogenous sphingosine-1-phosphate (S1P), was shown to modulate autophagy and involved in the processes of fibrotic diseases. Since SK1 activity is also up-regulated by TGF-β1, we explored its effect on the induction of autophagy and development of renal fibrosis in this study. In vitro, SK1 expression and activity were markedly increased by TGF-β1 stimulation in a time and concentration dependent manner, and concomitant changes in autophagic response were observed in HK-2 cells. Further, knockdown of SK-1 led to a decrease of autophagy whereas overexpression of SK1 caused a greater induction of autophagy. In addition, overexpression of SK1 resulted in decreased of mature TGF-β levels through autophagic degradation. In vivo, SK1 enzymatic activity and autophagic response were both up-regulated in a mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO); meanwhile, increased of mature TGF-β1 and deposition of extracellular matrix (ECM) were observed in tubulointerstitial areas compared with sham-operated mice. However, aggravation of renal fibrosis was detected when SK1 inhibitor PF-543 was applied to suppress SK1 enzymatic activity in UUO mice. At the same time, autophagy was also inhibited by PF-543. Thus, our findings suggest that SK1 activation is renoprotective via induction of autophagy in the fibrotic process.     

IL-18 mediates proapoptotic signaling in renal tubular cells through a Fas ligand-dependent mechanism

Renal tubular cell apoptosis is a significant component of obstruction-induced renal injury, and it results in a progressive loss in renal parenchymal mass during renal obstruction. Although IL-18 is an important mediator of inflammatory renal disease and renal fibrosis, its role in obstruction-induced renal tubular cell apoptosis remains unclear. To study this, male C57BL6 wild-type mice and C57BL6 mice transgenic for human IL-18-binding protein (IL-18BP Tg) were subjected to renal obstruction vs. sham operation. The kidneys were harvested after 1 or 2 wk and analyzed for IL-18 production, apoptosis, caspase activity, and Fas/Fas Ligand (FasL) expression. HK-2 cells were similarly analyzed for apoptosis and proapoptotic signaling following 3 days of direct exposure to IL-18 vs. control media. Renal obstruction induced a significant increase in IL-18 production, renal tubular cell apoptosis, caspase activation, and FasL expression. IL-18 neutralization, on the other hand, significantly reduced obstruction-induced apoptosis, caspase-8 and caspase-3 activity, and FasL expression. In vitro experiments similarly demonstrate that IL-18 stimulation induces apoptosis, FasL expression, and increases active caspase-8 and caspase-3 expression in a dose-dependent fashion. siRNA knockdown of FasL gene expression, however, significantly reduced IL-18-induced apoptosis. This study reveals that IL-18 is a significant mediator of obstruction-induced tubular cell apoptosis, and it demonstrates that IL-18 stimulates proapoptotic signaling through a FasL-dependent mechanism.     

外源性精胺对糖尿病肾病小鼠肾纤维化的影响及其机制

目的: 观察外源性精胺对糖尿病肾病(DN)肾纤维化的保护作用,并探讨其机制。方法: 24 只雄性 C57 小鼠随机分为正常组(Control)、糖尿病组(T1D)和精胺预处理组(T1D+Sp,每组 n=8)。一次性注射 STZ(60 mg/kg)复制 1 型糖尿病小鼠模型,精胺预处理组在 STZ 注射前两周每天腹腔注射精胺(Sp,5 mg/(kg·d)),随后隔天注射精胺,第 12 周处死小鼠,检测血清肌酐、尿素氮判断肾功能变化,HE、PAS 和 Masson 染色观察肾组织损伤和纤维化水平。Western blot 法检测小鼠肾组织中基质金属蛋白酶(MMP-2、MMP-9)、IV型胶原(Coll-IV)蛋白的表达。结果: 与 Control 相比,T1D 组血糖(5.67±0.22 vs 28.40±0.57 mmol/L)、肌酐(14.33±1.22 vs 30.67±4.73 μmol/L)、尿素氮(6.93±4.94 vs 22.00±1.04 mmol/L)明显升高(P＜0.05),肾组织基底膜增厚,胶原含量明显增加,MMP-2、MMP-9 和 Coll-IV 蛋白表达均升高(分别为 0.57±0.07 vs 1.06±0.20、47.00±0.04 vs 1.29±0.09和0.42±0.16 vs 0.95±0.18,P＜0.05),精胺预处理明显减轻上述变化。结论: 外源性精胺预处理通过调节 MMPs 与胶原的平衡减轻 DN 小鼠的肾纤维化。