scholarly journals Niao Du Kang Mixture Increases the Expression of mir-129-5p to Relieve Renal Fibrosis

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Yan-lin Li ◽  
Lin-na Liu ◽  
Lin Huang ◽  
Hai-wen An ◽  
Jian-lin Jian ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Cell Model ◽  
Rat Kidney ◽  
Kidney Tissue ◽  
Urine Protein ◽  
Renal Interstitial Fibrosis ◽  
Expression Levels ◽  
Qrt Pcr

Objective. To investigate the efficacy of Niao Du Kang (NDK) mixture in renal fibrosis of rats and to explore the mechanism underlying the effect of NDK on renal fibrosis. Methods. Unilateral ureteral obstruction (UUO) was used to replicate a rat renal interstitial fibrosis model. The drug-administered groups were given 20 ml/kg (NDK-H), 10 ml/kg (NDK-M), and 5 ml/kg (NDK-L) NDK mixture once a day for 21 days beginning 48 hours after surgery. The 24-hour urine protein and serum creatinine (CR) levels in the sham group rats, UUO rats, and NDK mixture-treated rats were measured after the last administration. The pathological changes of rat kidney tissue were observed by HE staining. The degree of fibrosis was observed by Masson’s staining and scored. The expression levels of TGF-β, α-SMA mRNA, and mir-129-5p in kidney were detected by qRT-PCR. HK-2 cells were treated with 5 ng/ml TGF-β to induce HK-2 cell fibrosis. The expression levels of TGF-β, α-SMA mRNA, and mir-129-5p in HK-2 cells were detected by qRT-PCR. TargetScan predicted the target gene of mir-129-5p, HK-2 cells were transfected with mir-129-5p mimic, and an overexpressed mir-129-5p HK-2 cell model was constructed. qRT-PCR was used to detect the expression of PDPK1 mRNA. Western blot was used to detect the expression of PDPK1, AKT, and p-AKT in HK-2 cells induced by TGF-β and in UUO rats. Results. NDK mixture significantly reduced the 24-hour urine protein and CR levels of UUO rats. HE staining showed that the NDK mixture group exhibited a significantly reduced degree of renal interstitial fibrosis. NDK mixture also reduced the expression of TGF-β and α-SMA, and the middle-dose group showed a better therapeutic effect. In vitro studies showed that NDK mixture-containing serum increased the expression of mir-129-5p to reduce renal fibrosis. In addition, NDK mixture increased the expression of mir-129-5p in vivo. Further studies indicated that mir-129-5p could target PDPKl to reduce its expression. The NDK-containing serum group also exhibited reduced expression of PDPK1. Conclusion. NDK mixture can significantly improve renal function and improve renal fibrosis in UUO model rats. Furthermore, NDK mixture can inhibit the expression of PDPK1 by upregulating the expression of mir-129-5p and then inhibiting the PI3K/AKT pathway to improve renal fibrosis.

Interleukin-18 deficiency protects against renal interstitial fibrosis in aldosterone/salt-treated mice

2016 ◽  
Vol 130 (19) ◽  
pp. 1727-1739 ◽  
Author(s):  
Akiko Tanino ◽  
Takafumi Okura ◽  
Tomoaki Nagao ◽  
Masayoshi Kukida ◽  
Zuowei Pei ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Renal Fibrosis ◽  
Cardiac Fibrosis ◽  
Interstitial Fibrosis ◽  
Rat Kidney ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Renal Interstitial Fibrosis

Interleukin (IL)-18 is a member of the IL-1 family of cytokines and was described originally as an interferon γ-inducing factor. Aldosterone plays a central role in the regulation of sodium and potassium homoeostasis by binding to the mineralocorticoid receptor and contributes to kidney and cardiovascular damage. Aldosterone has been reported to induce IL-18, resulting in cardiac fibrosis with induced IL-18-mediated osteopontin (OPN). We therefore hypothesized that aldosterone-induced renal fibrosis via OPN may be mediated by IL-18. To verify this hypothesis, we compared mice deficient in IL-18 and wild-type (WT) mice in a model of aldosterone/salt-induced hypertension. IL-18−/− and C57BL/6 WT mice were used for the uninephrectomized aldosterone/salt hypertensive model, whereas NRK-52E cells (rat kidney epithelial cells) were used in an in vitro model. In the present in vivo study, IL-18 protein expression was localized in medullary tubules in the WT mice, whereas in aldosterone-infused WT mice this expression was up-regulated markedly in the proximal tubules, especially in injured and dilated tubules. This renal damage caused by aldosterone was attenuated significantly by IL-18 knockout with down-regulation of OPN expression. In the present in vitro study, aldosterone directly induced IL-18 gene expression in renal tubular epithelial cells in a concentration- and time-dependent manner. These effects were inhibited completely by spironolactone. IL-18 may be a key mediator of aldosterone-induced renal fibrosis by inducing OPN, thereby exacerbating renal interstitial fibrosis. Inhibition of IL-18 may therefore provide a potential target for therapeutic intervention aimed at preventing the progression of renal injury.

LRG1 Mitigates Renal Interstitial Fibrosis through Alleviating Capillary Rarefaction and Inhibiting Inflammatory and Pro-Fibrotic Cytokines

2021 ◽  
pp. 1-11
Author(s):  
Ting-Ting Liu ◽  
Ran Luo ◽  
Yi Yang ◽  
Yi-Chun Cheng ◽  
Dan Chang ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Critical Role ◽  
Tube Formation ◽  
Renal Interstitial Fibrosis ◽  
Genetic Ablation ◽  
Capillary Rarefaction ◽  
Peritubular Capillaries ◽  
Renal Tubular

<b><i>Introduction:</i></b> Increasing evidence has demonstrated that loss of peritubular capillaries plays a critical role in renal interstitial fibrosis. Leucine-rich α2-glycoprotein-1 (LRG1) has been observed promoting angiogenesis in the ocular disease mouse model and myocardial infarction model. We aimed to explore the role of LRG1 in renal interstitial fibrosis. <b><i>Methods:</i></b> We analyzed the expression of LRG1 in the plasma and kidney of CKD patients by ELISA and immunohistochemistry. Relationships between the expression of LRG1 in plasma and kidney and renal fibrosis and inflammation were analyzed. Tube formation assay was used to detect the angiogenesis in the human umbilical vein endothelial cell lines (HUVECs). And real-time PCR was used to detect the mRNA expression of LRG1, inflammatory factors, renal tubular injury indicators, pro-fibrotic cytokines, and CD31. We examined the effects of genetic ablation of LRG1 on renal fibrosis induced by unilateral ureteral obstruction (UUO) mice model at day 7. <b><i>Results:</i></b> We demonstrated that the expression of LRG1 in renal tissues and plasma samples was upregulated in CKD patients. And the expression of LRG1 was elevated in human renal tubular epithelial cell line (HK-2) cells in response to the stimulation of TNF-α in vitro, and in kidney after UUO in vivo. The deficiency of the LRG1 gene aggravated renal fibrosis, inflammatory cells infiltration, and capillary rarefaction after UUO. In vitro, LRG1 promoted the tube formation of HUVEC cells. LRG1 inhibits fibronectin secretion induced by TGF-β1 in HK-2 and overexpression of LRG1 in HK-2 cells decreased fibronectin secretion. <b><i>Conclusion:</i></b> LRG1 may prevent renal fibrosis by inhibiting the secretion of inflammatory and pro-fibrotic cytokines and promoting angiogenesis.

Pterostilbene, a Bioactive Component of Blueberries, Alleviates Renal Interstitial Fibrosis by Inhibiting Macrophage-Myofibroblast Transition

2020 ◽  
Vol 48 (07) ◽  
pp. 1715-1729
Author(s):  
Yanhuan Feng ◽  
Fan Guo ◽  
Hongxia Mai ◽  
Jing Liu ◽  
Zijing Xia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Renal Fibrosis ◽  
Transcriptional Activity ◽  
Interstitial Fibrosis ◽  
Unilateral Ureteral Obstruction ◽  
Rna Seq ◽  
Renal Interstitial Fibrosis ◽  
Bioactive Component

Pterostilbene (PTB) is a derivative of resveratrol present in grapes and blueberries. PTB is structurally similar to resveratrol, possessing properties such as being analgesic, anti-aging, antidiabetic, anti-inflammatory, anti-obesity, anti-oxidation, cholesterol-reductive, and neuroprotective. However, there have not been reports on the effect of PTB on macrophage-myofibroblast transition (MMT) induced fibrosis in kidney. In this study, we investigated the antifibrotic effects of PTB on the in vivo mouse unilateral ureteral obstruction (UUO) model and in vitro MMT cells. Kidneys subjected to UUO with PTB treatment were collected for the investigation of PTB mediating MMT derived renal interstitial fibrosis. We conducted kidney RNA-seq transcriptomes and TGF-[Formula: see text]1-induced bone marrow-derived macrophages assays to determine the mechanisms of PTB. We found that PTB treatment suppressed the interstitial fibrosis in UUO mice. PTB also attenuated the number of MMT cells in vivo and in vitro. The transcriptomic analysis showed that CXCL10 may play a central role in the process of PTB-treated renal fibrosis. The siRNA-mediated CXCL10 knockdown decreased the number of MMT cells in TGF-[Formula: see text]1-induced bone marrow-derived macrophages. Our results suggested that PTB attenuated renal interstitial fibrosis by mediating MMT by regulating transcriptional activity of CXCL10.

scholarly journals SKLB023 hinders renal interstitial fibrosis in obstructive nephropathy by interfering TGF-β1/Smad3 signaling

RSC Advances ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 5891-5896 ◽  
Author(s):  
Yanhuan Feng ◽  
Jun Xu ◽  
Fan Guo ◽  
Rongshuang Huang ◽  
Min Shi ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Therapeutic Strategy ◽  
Interstitial Fibrosis ◽  
Obstructive Nephropathy ◽  
The Novel ◽  
Renal Interstitial Fibrosis ◽  
Molecule Inhibitor ◽  
Tgf Β1

The novel small-molecule inhibitor of iNOS (SKLB023) hindered renal interstitial fibrosis in vivo and in vitro by interfering with TGF-β1/Smad3 signaling, highlighting that SKLB023 has potential in the therapeutic strategy for renal fibrosis.

scholarly journals SO041THE EFFECT AND MECAHNISM OF THE LOX IN AT1R/Β-ARRESTIN BIASED PATHWAY INDUCED RENAL INTERSTITIAL FIBROSIS

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Xiaoqin Zhang ◽  
Chen Yu
Keyword(s):  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Lysyl Oxidase ◽  
Rat Kidney ◽  
Ischemia Reperfusion ◽  
Mek Inhibitor ◽  
Collagen Crosslinking ◽  
Fibrosis Progression

Abstract Background and Aims We studied the downstream and mechanism of β-arrestins signaling in renal fibrosis process and the role of lysyl oxidase (LOX) in the AT1R-β-arrestins pathway. Method The mechanism of β-arrestins signaling was studied in normal rat kidney tubule epithelial cells (NRK-52E) treated with SII in vitro. BAPN or placebo was administered during ischemia reperfusion (IR)-induced fibrosis progression. Collagen crosslinking and fibrosis progression were assessed histologically and biochemically. Results The mRNA and protein levels of β-arrestin-1 and β-arrestin-2 were significantly upregulated in renal fibrosis model both in vitro and in vivo. SII activated the ERK-STAT3 PY705 but not STAT3-Try727 in nucleus of NRK-52E cells, which effects were abolished when transfection of siRNA targeting β-arrestin-1 and β-arrestin-2 or pretreated with PD98059 (MEK inhibitor). LOX was strongly induced in fibrotic kidney and NRK-52E cells treated with SII. Active LOX significantly increased collagen crosslinking. In established IR-28d renal fibrosis, LOX inhibition promoted fibrosis reversal and with a 25% decrease insoluble collagen. Gene silencing of β-arrestin-1 + 2 or STAT3 apparently inhibited SII-induced LOX expression in vitro. Besides, chromatin immunoprecipitation (ChIP) assay clearly demonstrating the interaction between STAT3 and the LOX promoter, which indicated LOX is a direct target gene of SII-β-arrestins-STAT3 signaling. Conclusion The ERK/STAT3 was downstream of AT1R-β-arrestins, ERK entered the nucleus and activated STAT3-PY705. LOX mediates collagen crosslinking and fibrotic matrix stabilization during renal fibrosis via the AT1R-β-arrestins-ERK-STAT3-PY705 signaling. By blocking this profibrotic pathway, therapeutic LOX inhibition attenuates the fibrosis and suggesting target the LOX has significant potency for the treatment of patients with fibrotic kidney disorders.

scholarly journals Exploring the Effect of Dapagliflozin on Alcoholic Kidney Injury and Renal Interstitial Fibrosis in Rats Based on TIMP-1/MMP-24 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Yundou Wu ◽  
Peijun Song ◽  
Xinke Yuan ◽  
Dayong Li
Keyword(s):  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Kidney Tissue ◽  
Kidney Injury ◽  
Inflammatory Factors ◽  
Control Group ◽  
Renal Interstitial Fibrosis ◽  
Alcohol Group ◽  
Losartan Group ◽  
Smad3 Expression

Objective. To establish a rat model of alcoholic kidney injury and detect the expression of TIMP-1/MMP-24 in the kidneys of rats with alcoholic kidney injury at the molecular pathological level, so as to explore the mechanism of alcohol abuse leading to kidney injury and renal interstitial fibrosis as well as the alleviation of alcohol-induced kidney injury and inhibition of renal interstitial fibrosis by dapagliflozin. Methods. 48 male rats were randomly divided into 4 groups: control group, alcohol group, alcohol + dapagliflozin group, and alcohol + losartan group, each with 12 rats. Different drugs were administered by gavage for modeling and treatment. Six days later, the rats were sacrificed, blood was collected from the heart to separate the serum, and the blood creatinine (Scr) and urea nitrogen (BUN) contents were detected biochemically. After blood collection, the kidney tissue was taken and fixed in10% neutral formalin. The expression of renal tissue inflammatory factors (CRP, IL-6, and TNF-α) and renal fibrosis indexes (LN, HA, and TGF-β1) were detected; MMP-24 and TIMP-1 in the kidney tissue of rats in different treatment groups were detected, and Smad3 expression was also detected. Results. After treatment, the general condition of the alcohol + dapagliflozin group and the alcohol + losartan group improved to different degrees. The weight first decreased and then gradually increased over time. There was no statistical difference in the weight change between the two groups; Compared with the control group, the Scr level, BUN content, renal index, inflammatory factors, and renal fibrosis indexes in the alcohol group were significantly increased ( P < 0.05 ); after 6 weeks of treatment, in the alcohol + dapagliflozin group and alcohol + losartan group, Scr level, BUN content, kidney index, inflammatory factors, and renal fibrosis indexes were significantly decreased ( P < 0.05 ); the expression of MMP-24 in the kidney tissue of the control group was upregulated, and the expression of TIMP-1 and Smad3 was downregulated; MMP-24 expression was downregulated, and TIMP-1 and Smad3 expression was significantly upregulated ( P < 0.05 ) in the rats of the alcohol group. After dapagliflozin and losartan treatment, MMP-24 expression gradually increased and TIMP-1 and Smad3 expression gradually decreased ( P < 0.05 ). Conclusion. Long-term large-scale alcohol intake can cause kidney tissue damage and fibrotic lesions. The expression of fibrotic cytokines such as TIMP-1 and Smad3 will increase, and the expression of MMP-24 will be decreased. However, dapagliflozin and losartan have certain therapeutic effects on the abovementioned lesions. The mechanism may be downregulating TIMP-1 and Smad3 and upregulating the expression of MMP-24 and other cytokines in the kidney.

Apamin inhibits renal interstitial inflammation and fibrosis via suppressing TGF-β1 and STAT3 signaling pathway in vivo and in vitro

2020 ◽  
Author(s):  
Mi-Gyoeng Gwon ◽  
Hyun-Jin An ◽  
Hyemin Gu ◽  
Young-Ah Kim ◽  
Sang Mi Han ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Renal Interstitial Fibrosis ◽  
Tubular Atrophy ◽  
Fibroblast Activation ◽  
Stat3 Signaling ◽  
Tgf Β1

Abstract Background Renal fibrosis is a progressive and chronic process that influences kidneys with chronic kidney disease (CKD), irrespective of cause, leading to irreversible failure of renal function and end-stage kidney disease. Among the signaling related to renal fibrosis, transforming growth factor-β1 (TGF-β1) signaling is a major pathway that induces the activation of myofibroblasts and the production of extracellular matrix (ECM) molecules. Apamin, a component of bee venom (BV), has been studied in relation to various diseases. However, the effect of apamin on renal interstitial fibrosis has not been investigated. The aim of this study was to estimate the beneficial effect of apamin in unilateral ureteral obstruction (UUO)-induced renal fibrosis and TGF-β1-induced renal fibroblast activation.Results This study revealed that obstructive kidney injury induced an inflammatory response, tubular atrophy, and ECM accumulation. However, apamin treatment suppressed the increased expression of fibrotic-related genes, including α-SMA, vimentin, and fibronectin. Administration of apamin also attenuated the renal tubular cells injury and tubular atrophy. In addition, apamin attenuated fibroblast activation, ECM synthesis, and inflammatory cytokines such as TNF-α, IL-1β and IL-6 by suppressing the TGF-β1-canonical and non-canonical signaling pathways.Conclusions This study shown that apamin inhibites UUO-induced renal fibrosis in vivo and TGF-β1-induced renal fibroblasts activation in vitro. Apamin inhibited the inflammatory response, tubular atrophy, ECM accumulation, fibroblast activation, and renal interstitial fibrosis through suppression of TGF-β1/Smad2/3 and STAT3 signaling pathways. These results suggest that apamin might be a potential therapeutic agent for renal fibrosis.

scholarly journals Salidroside Ameliorates Renal Interstitial Fibrosis by Inhibiting the TLR4/NF-κB and MAPK Signaling Pathways

2019 ◽  
Vol 20 (5) ◽  
pp. 1103 ◽  
Author(s):  
Rui Li ◽  
Yujuan Guo ◽  
Yiming Zhang ◽  
Xue Zhang ◽  
Lingpeng Zhu ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Inflammatory Cytokines ◽  
Renal Fibrosis ◽  
Interstitial Fibrosis ◽  
Mapk Signaling ◽  
Renal Interstitial Fibrosis ◽  
Mapk Signaling Pathways ◽  
Tgf Β1

Salidroside (Sal) is an active ingredient that is isolated from Rhodiola rosea, which has been reported to have anti-inflammatory activities and a renal protective effect. However, the role of Sal on renal fibrosis has not yet been elucidated. Here, the purpose of the current study is to test the protective effects of Sal against renal interstitial fibrosis (RIF), and to explore the underlying mechanisms using both in vivo and in vitro models. In this study, we establish the unilateral ureteric obstruction (UUO) or folic acid (FA)-induced mice renal interstitial fibrosis in vivo and the transforming growth factor (TGF)-β1-stimulated human proximal tubular epithelial cell (HK-2) model in vitro. The levels of kidney functional parameters and inflammatory cytokines in serum are examined. The degree of renal damage and fibrosis is determined by histological assessment. Immunohistochemistry and western blotting are used to determine the mechanisms of Sal against RIF. Our results show that treatment with Sal can ameliorate tubular injury and deposition of the extracellular matrix (ECM) components (including collagen Ш and collagen I). Furthermore, Sal administration significantly suppresses epithelial-mesenchymal transition (EMT), as evidenced by a decreased expression of α-SMA, vimentin, TGF-β1, snail, slug, and a largely restored expression of E-cadherin. Additionally, Sal also reduces the levels of serum biochemical markers (serum creatinine, Scr; blood urea nitrogen, BUN; and uric acid, UA) and decreases the release of inflammatory cytokines (IL-1β, IL-6, TNF-α). Further study revealed that the effect of Sal on renal interstitial fibrosis is associated with the lower expression of TLR4, p-IκBα, p-NF-κB and mitogen-activated protein kinases (MAPK), both in vivo and in vitro. In conclusion, Sal treatment improves kidney function, ameliorates the deposition of the ECM components and relieves the protein levels of EMT markers in mouse kidneys and HK-2 cells. Furthermore, Sal treatment significantly decreases the release of inflammatory cytokines and inhibits the TLR4/NF-κB and MAPK signaling pathways. Collectively, these results suggest that the administration of Sal could be a novel therapeutic strategy in treating renal fibrosis.

scholarly journals Inhibition of Renal Stellate Cell Activation Reduces Renal Fibrosis

Biomedicines ◽  
2020 ◽  
Vol 8 (10) ◽  
pp. 431
Author(s):  
Jin Joo Cha ◽  
Chanchal Mandal ◽  
Jung Yeon Ghee ◽  
Ji Ae Yoo ◽  
Mi Jin Lee ◽  
...  
Keyword(s):  
Renal Fibrosis ◽  
Cell Activation ◽  
Interstitial Fibrosis ◽  
Stellate Cell ◽  
Rat Kidney ◽  
Stellate Cells ◽  
Retinol Binding Protein ◽  
Therapeutic Effects ◽  
Isolated Cells

Interstitial fibrosis is a common feature of chronic kidney disease, and platelet-derived growth factor receptor-β (PDGFR-β)-positive mesenchymal cells are reportedly the major source of scar-producing myofibroblasts. We had previously demonstrated that albumin and its derivative R-III (a retinol-binding protein-albumin domain III fusion protein) inhibited the transdifferentiation/activation of hepatic stellate cells (HSCs) to myofibroblasts and that R-III administration reduced liver fibrosis. In this study, we isolated cells (referred to as renal stellate cells, RSCs) from rat kidney tissues using the HSC isolation protocol and compared their morphological and biochemical characteristics with those of HSCs. RSCs shared many characteristics with HSCs, such as storage of vitamin A-containing lipid droplets and expression of HSC markers as well as pericyte markers. RSCs underwent spontaneous transdifferentiation into myofibroblasts in in vitro culture, which was inhibited by albumin expression or R-III treatment. We also evaluated the therapeutic effects of R-III in unilateral ureteral obstruction (UUO)-induced renal fibrosis in mice. Injected R-III localized predominantly in cytoglobin/stellate cell activation-associated protein (Cygb/STAP)-positive cells in the kidney and reduced renal fibrosis. These findings suggest that RSCs can be recognized as the renal counterparts of HSCs and that RSCs represent an attractive therapeutic target for anti-fibrotic therapy.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

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