Norcantharidin, a protective therapeutic agent in renal tubulointerstitial fibrosis

Progressive renal tubulointerstitial fibrosis is a common final pathway of nearly all forms of chronic kidney disease. Many efforts have been done to arrest or prevent renal tubulointerstitial fibrosis but with little progress. Nowadays, few therapeutic agents are available in clinical use. Norcantharidin (NCTD) is of great benefit in anticancer treatment, by inducing cell apoptosis, inhibiting cell proliferation, in addition, blocking tumor metastasis and angiogenesis in cancer, whereas little attention is given to its relationship with other diseases. Our recent studies demonstrated that NCTD was protective against renal tubulointerstitial fibrosis both in vivo and in vitro. The underlying mechanisms may include modulation of TGF-β1/Smad signal cascade, inhibition of protein serine/threonine phosphatases (PPP) as well as NF-κB. NCTD may be a promising therapeutic agent for renal tubulointerstitial fibrosis. In the present article, we will review the action of NCTD in renal tubulointerstitial fibrosis and discuss its possible mechanisms.     

miR-200b precursor can ameliorate renal tubulointerstitial fibrosis

Members of the miR-200 family of micro RNAs (miRNAs) have been shown to inhibit epithelial-mesenchymal transition (EMT). EMT of tubular epithelial cells is the mechanism by which renal fibroblasts are generated. Here we show that miR-200 family members inhibit transforming growth factor-beta (TGF-beta)-induced EMT of tubular cells. Unilateral ureter obstruction (UUO) is a common model of EMT of tubular cells and subsequent tubulointerstitial fibrosis. In order to examine the role of miR-200 family members in tubulointerstitial fibrosis, their expression was investigated in the kidneys of UUO mice. The expression of miR-200 family miRNAs was increased in a time-dependent manner, with induction of miR-200b most pronounced. To clarify the effect of miR-200b on tubulointerstitial fibrosis, we injected miR-200b precursor intravenously. A single injection of 0.5 nM miR-200b precursor was sufficient to inhibit the increase of collagen types I, III and fibronectin in obstructed kidneys, and amelioration of fibrosis was confirmed by observation of the kidneys with Azan staining. miR-200 family members have been previously shown to inhibit EMT by reducing the expression of ZEB-1 and ZEB-2 which are known repressors of E-cadherin. We demonstrated that expression of ZEB-1 and ZEB-2 was increased after ureter obstruction and that administration of the miR-200b precursor reversed this effect. In summary, these results indicate that miR-200 family is up-regulated after ureter obstruction, miR-200b being strongly induced, and that miR-200b ameliorates tubulointerstitial fibrosis in obstructed kidneys. We suggest that members of the miR-200 family, and miR-200b specifically, might constitute novel therapeutic targets in kidney disease.     

Mefunidone ameliorates renal inflammation and tubulointerstitial fibrosis via suppression of IKKβ phosphorylation

Mefunidone is a new pyridone agent that attenuates renal tubulointerstitial fibrosis. However, the signaling pathways involved in the effect of mefunidone on renal tubulointerstitial fibrosis have not been well explained. Inflammatory response initiates and promotes renal tubulointerstitial fibrosis, and the inhibitor of nuclear factor kappa-B kinase beta (IKKβ) is a master regulator of inflammation. This study is determined to clarify the influence of mefunidone on renal inflammation and the phosphorylation of IKKβ. Experimental renal tubulointerstitial fibrosis was induced by unilateral ureteral obstruction (UUO) for 3, 7 and 14 days in sprague dawley rat. Treatment with mefunidone was conducted simultaneously. Obstructed kidneys were harvested for the assessment. Our results showed that treatment with mefunidone ameliorated renal inflammatory injury, renal tubular lesions and interstitial fibrosis. Further studies indicated that treatment with mefunidone mitigated the expressions of tumor necrosis factorα (TNFα) and interleukin-1β (IL-1β) in the kidney. The phosphorylation of IKKβ and inhibitor of kappa-B (IκB) and the expression of NOD-like receptor family, pyrin domain containing 3 (NALP3) were also reduced in vivo after treatment with mefunidone. In vitro, peritoneal macrophages were incubated with necrotic cells or lipopolysaccharide in the presence or absence of mefunidone. Mefunidone markedly decreased necrotic cell or LPS induced IL-1β production and LPS induced TNFα production in primary peritoneal macrophages. Furthermore, mefunidone significantly inhibited the phosphorylation of IKKβ/IκB and nuclear transition of NF-κB p65 in peritoneal macrophages stimulated by necrotic cell or lipopolysaccharide. In conclusion, mefunidone serves as a novel anti-inflammatory agent that attenuates UUO-induced renal interstitial inflammation and fibrosis, possibly through suppressing IKKβ phosphorylation.     

Progressive renal dysfunction and macrophage infiltration in interstitial fibrosis in an adenine-induced tubulointerstitial nephritis mouse model

Adenine phosphoribosyltransferase deficiency in mice or an excessive oral intake of adenine leads to the accumulation of 2,8-dihydroxyadenine (DHA) in renal tubules and that causes progressive renal dysfunction accompanied by interstitial fibrosis. However, the precise mechanism responsible for DHA-induced progressive fibrosis is not fully understood. The present study investigates the possible involvement of monocytes/macrophages in the progressive fibrosis induced by feeding adenine to mice. Urinary calculi were deposited in tubules on day 7 after the initiation of adenine feeding. Elevation of the serum creatinine level and loss of body weight were observed in a time-dependent manner, suggesting the development of typical renal dysfunction induced by the adenine feeding. In renal tissue, mRNA expression of MCP-1, MIP-1α, RANTES, IL-1β, CCR2, TGF-β, α-smooth muscle actin (α-SMA) and collagen 1a1 was increased in parallel. Along with the increased expression of these genes, a remarkable infiltration of macrophages into the tubulointerstitial area was observed in a time-dependent manner. In addition, in the tubulointerstitial area, α-SMA positive fibroblasts were increased in parallel with collagen deposition. These results suggest that the excessive consumption of adenine leads to progressive renal dysfunction in mice. We speculate that the accumulation of DHA in tubules might stimulate epithelium to produce MCP-1 and that profibrogenic TGF-β produced by infiltrated macrophages might stimulate interstitial fibroblasts to produce collagen. These results indicate that macrophage infiltration is one of the triggers that initiates interstitial fibroblast activation and collagen deposition followed by renal dysfunction.     

肾脏免疫区室化与肾小管间质损伤

免疫系统区室化(compartmentalization)是近年提出的一个新观念,为人们从整体和局部两方面进一步了解免疫系统提供了新的视角,且有助于对临床疾病免疫机制的阐释。最近肾脏免疫系统区室化现象也已引起人们重视。肾小管损伤和肾间质纤维化是各类肾脏疾病发展到终末期肾衰竭的重要原因和共同通路,也与肾小管间质免疫区室的局部微环境调控密切相关,并涉及区域内树突状细胞等专职免疫细胞,以及具有免疫特性肾小管上皮细胞的共同作用及相互调节,由此影响着肾脏疾病的发生、发展及预后。因此,从肾脏免疫区室化角度进一步探讨肾小管间质损伤机制,有助于深入分析肾脏疾病的病变过程,并可为相关研究及临床诊治提供新的思路。     

Dual effect of chemokine CCL7/MCP-3 in the development of renal tubulointerstitial fibrosis

Most end-stage renal disease kidneys display accumulation of extracellular matrix (ECM) in the renal tubular compartment (tubular interstitial fibrosis – TIF) which is strongly correlated with the future loss of renal function. Although inflammation is a key event in the development of TIF, it can also have a beneficial anti-fibrotic role depending in particular on the stage of the pathology. Chemokines play an important role in monocyte extravasation in the inflammatory process. CCL2 has already been shown to be involved in the development of TIF but CCL7, a close relative of CCL2 and able to bind to similar receptors, has not been studied in renal disease. We therefore studied chemokine CCL7 in a model of unilateral ureteral obstruction (UUO)-induced TIF. We observed that the role of CCL7 differs depending on the stage of the pathology. In early stages (0–8 days), CCL7 deficient (CCL7-KO) mice displayed attenuated TIF potentially involving two mechanisms: an early (0–3 days) decrease of inflammatory cell infiltration followed (3–8 days) by a decrease in tubular ECM production independent of inflammation. In contrast, during later stages of obstruction (10–14 days), CCL7-KO mice displayed increased TIF which was again associated with reduced inflammation. Interestingly, the switch between this anti- to profibrotic effect was accompanied by an increased influx of immunosuppressive regulatory T cells. In conclusion, these results highlight for the first time a dual role for CCL7 in the development of renal TIF, deleterious in early stages but beneficial during later stages.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

Although recent major advances have developed a much better understanding of the pathophysiological pathways, tubulointerstitial fibrosis (TIF) is still currently incurable. Therefore, early detection may mean that the condition is more manageable than it was in the past. C-type natriuretic peptide (CNP) has been found to be a potent vasodilator but a weak natriuretic factor. In addition, CNP has also been believed to be produced in tubular cells and presented as a local modulator with anti-inflammatory and anti-proliferative effects. Elimination of CNP occurs by three main mechanisms, neutral endopeptidase, natriuretic peptide receptor-C and urinary excretion. Among them, the status of urinary CNP excretion in nephropathies is not yet fully elucidated. In the present study, subgroups of rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and observed for 24 h to 3 months. Urinary CNP excretion was significantly enhanced in UUO rats from 24 h to 1 month post-ligation compared to sham-operated rats. Urinary CNP excretion was also markedly higher than CNP concentrations both in abdominal aorta and in renal vein, and almost identical concentrations in these two vessels excluded major renal extraction of circulating CNP of systemic origin. Urinary CNP excretion was negatively correlated with urinary protein concentration, blood urea nitrogen and creatinine, while positively correlated with albumin. In conclusion, the increased urinary CNP excretion is strongly associated with TIF progression, and may serve as an early marker of TIF.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.     

Response gene to complement 32 is essential for fibroblast activation in renal fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.     

AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice

Renal tubulointerstitial fibrosis was a crucial pathological feature of diabetic nephropathy (DN), and renal tubular injury might associate with abnormal mitophagy. In this study, we investigated the effects and molecular mechanisms of AMPK agonist metformin on mitophagy and cellular injury in renal tubular cell under diabetic condition. The high fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetic mice model and HK-2 cells were used in this study. Metformin was administered in the drinking water (200 mg/kg/d) for 24 weeks. Renal tubulointerstitial lesions, oxidative stress and some indicators of mitophagy (e.g., LC3II, Pink1, and Parkin) were examined both in renal tissue and HK-2 cells. Additionally, compound C (an AMPK inhibitor) and Pink1 siRNA were applied to explore the molecular regulation mechanism of metformin on mitophagy. We found that the expression of p-AMPK, Pink1, Parkin, LC3II, and Atg5 in renal tissue of diabetic mice was decreased obviously. Metformin reduced the levels of serum creatinine, urine protein, and attenuated renal oxidative injury and fibrosis in HFD/STZ induced diabetic mice. In addition, Metformin reversed mitophagy dysfunction and the over-expression of NLRP3. In vitro pretreatment of HK-2 cells with AMPK inhibitor compound C or Pink1 siRNA negated the beneficial effects of metformin. Furthermore, we noted that metformin activated p-AMPK and promoted the translocation of Pink1 from the cytoplasm to mitochondria, then promoted the occurrence of mitophagy in HK-2 cells under HG/HFA ambience. Our results suggested for the first time that AMPK agonist metformin ameliorated renal oxidative stress and tubulointerstitial fibrosis in HFD/STZ-induced diabetic mice via activating mitophagy through a p-AMPK-Pink1-Parkin pathway.Subject terms: Diabetes complications, End-stage renal disease, Experimental models of disease     

Dojuksan ameliorates tubulointerstitial fibrosis through irisin-mediated muscle-kidney crosstalk

BackgroundSarcopenia progresses in chronic kidney disease (CKD) and is positively correlated with mortality in end-stage kidney disease patients. Circulating irisin, an exercise-induced myokine, gradually decreases during CKD stage progression. Irisin inhibits the progression of kidney fibrosis, which is the final common outcome of CKD. Our preliminary study with C2C12 cells showed that Dojuksan, a herbal decoction, increases the expression of PGC1α (a regulator of irisin) and FNDC5 (a precursor of irisin).HypothesisDojuksan may increase circulating irisin and prevent the progression of kidney fibrosis.Study Design and MethodsUnilateral ureteral obstruction (UUO) was performed on seven-week-old male C57BL/6 mice to induce kidney tubulointerstitial fibrosis. Dojuksan (50, 100, or 200 mg/kg/day) or losartan (1.5 mg/kg/day), a standard clinical treatment for CKD, was administered orally one day prior to surgery and continued for seven days thereafter. To determine the role of irisin released from muscles, TGFβ-stimulated murine proximal tubular epithelial cells (mProx24 cells) were treated with conditioned media (CM) from Dojuksan-treated C2C12 muscle cells transfected with FNDC5 siRNA.ResultsUUO mice exhibited muscle wasting along with progressive kidney injury. Similar to losartan, Dojuksan ameliorated kidney inflammation and fibrosis in UUO mice. Dojuksan, but not losartan, increased plasma irisin concentration in UUO mice. Dojuksan significantly increased basal FNDC5 expression and inhibited TNFα-induced and indoxyl sulfate-induced FNDC5 down-regulation in C2C12 cells. The TGFβ-induced collagen I (COL1) up-regulation in mProx24 cells was effectively inhibited by CM from C2C12 cells after Dojuksan treatment. Moreover, irisin inhibited TGFβ-induced COL1 in mProx24 cells, which was not affected by CM from C2C12 cells transfected with FNDC5 siRNA.ConclusionDojuksan ameliorates kidney fibrosis through irisin-mediated muscle-kidney crosstalk, suggesting that Dojuksan may be used as an alternative therapeutic agent against CKD.     

Over-nutrition contributes to tubulointerstitial fibrosis by targeting nutrient-sensing kinases

Comment on: Whaley-Connell A, et al. Am J Nephrol 2011; 34:115-25.     

The role of EMT in renal fibrosis

It is clear that the well-described phenomenon of epithelial–mesenchymal transition (EMT) plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. EMTs have been classified into three subtypes based on the functional consequences and biomarker context in which they are encountered. This review will highlight findings on type II EMT as a direct contributor to the kidney myofibroblast population in the development of renal fibrosis, specifically in diabetic nephropathy, the signalling molecules and the pathways involved in type II EMT and changes in the expression of specific miRNA with the EMT process. These findings have provided new insights into the activation and development of EMT during disease processes and may lead to possible therapeutic interventions to suppress EMTs and potentially reverse organ fibrosis.     

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis

BackgroundIn chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure.PurposeWe isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis.Study designUnilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-β1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms.MethodsWestern blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA.ResultsPAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect.ConclusionPAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.     

Role of prostaglandins in fibroblast activation and fibrosis

Fibroblasts release prostaglandins and express a range of prostanoid receptors. However the importance of prostaglandins in fibroblast biology have not been fully explored. Our studies showed that the prostaglandin metabolite PGI₂ blocks the activation of fibroblasts, antagonising the induction of Ras/MEK/ERK signalling by TGFβ. Endogenous PGI₂ acts so as to limit the activation of fibroblasts following tissue injury. By contrast PGE₂ induced in injured tissues or disease states may promote recruitment of inflammatory cells and lead to secondary activation of fibroblasts. The effects of PGI₂ on cell signaling could be manipulated to inhibit fibrosis in patients.     

The metabolic fate of lactate in renal cortical tubules

1. Isolated kidney cortex tubules prepared from fed rats and incubated with near-physiological concentrations of [¹⁴C]lactate decrease the specific radioactivity of the added lactate. This effect may be attributable to at least two mechanisms; formation of lactate from endogenous precursors, or entry of unlabelled carbon into the lactate pool as a result of substrate cycling, via phosphoenolpyruvate, pyruvate and oxaloacetate, together with equilibration of the oxaloacetate pool with malate and fumarate. Such substrate cycling could occur within a single cell, or between two populations of different cells, one glycolytic and the other gluconeogenic. These possibilities have been investigated by using metabolic inhibitors or alternative metabolic substrates. 2. Tubules from fed rats produced a fall in specific radioactivity of 14.4% when incubated for 40min with 2mm-lactate alone. A mathematical treatment of this result is presented, which allows the rate of fall in specific radioactivity to be expressed as the addition of unlabelled lactate to the pool. This corresponds to a rate of formation of unlabelled lactate of 121±22μmol/h per g dry wt., a rate close to that of gluconeogenesis. In tubules from fasting rats, there was no reduction of the specific radioactivity of lactate, indicating that fasting for 24h suppresses production of unlabelled-lactate carbon. 3. Addition of 2mm-fumarate resulted in a significantly greater decrease in the specific radioactivity of lactate, but aspartate (2mm), malate (2mm) and glucose (5mm) were without effect. Total inhibition of gluconeogenesis with 3-mercaptopicolinate did not prevent the fall in specific radioactivity of lactate observed in tubules from fed-rat kidney, thereby excluding significant activity of the substrate cycle pyruvate→oxaloacetate→phosphoenolpyruvate→pyruvate. 4. The capacity of pyruvate kinase under the test conditions in tubules prepared from kidneys of fed or starved rats was at least ten times higher than the observed rate of production of lactate, so that failure to observe recycling of lactate in starved-rat tubules indicates suppression of pyruvate kinase activity. 5. The endogenous glycogen and glucose content of isolated renal cortex tubules is too low to account for the dilution of label of lactate. Endogenous concentrations of glycerol and amino acids were also very low. As for glycogen, the possibility that very rapid turnover of these metabolites, in fed rats but not in starved rats, may account for formation of unlabelled lactate cannot be excluded. 6. It is concluded that substrate cycling via phosphoenolpyruvate does not occur to any significant extent in either fed or starved-rat kidney. In fed rats recycling of lactate carbon does occur and the rate of this reaction is similar to the rate of gluconeogenesis at physiological concentrations of lactate. The present results favour participation of oxaloacetate decarboxylase rather than `malic' enzyme in this cycle.