Gadolinium modulates gentamicin uptake via an endocytosis-independent pathway in HK-2 human renal proximal tubular cell line

The aim of this study was to characterize the uptake mechanism of gentamicin, an aminoglycoside antibiotic, in human renal proximal tubular cell line HK-2. Sodium-dependent uptake of D-[(3)H]glucose and L-[(3)H]alanine was observed in HK-2 cells, indicating that the cells employed in this study retain functional characteristics of the renal proximal tubular cells. On the other hand, mRNA and protein expression of megalin, an endocytic receptor which is responsible for the internalization of gentamicin into the renal proximal tubular cells, was very faint in HK-2 cells. Various aminoglycoside antibiotics including amikacin and kanamycin inhibited the uptake of [(3)H]gentamicin. Colchicine and cytochalasin D, general endocytosis inhibitors, had no significant effect on [(3)H]gentamicin uptake in HK-2 cells, which was in contrast to the results observed in OK cells, a renal proximal tubular cell line expressing megalin. Furthermore, unlike OK cells, [(3)H]gentamicin uptake in HK-2 cells was not inhibited by N-WASP181-200, a cationic 20-amino acid peptide. Ruthenium red, a nonspecific cation channel blocker, decreased the uptake of [(3)H]gentamicin in HK-2 cells. In contrast, the trivalent cation gadolinium biphasically modulated [(3)H]gentamicin uptake with a maximum increase at 0.3 mM gadolinium. The enhanced effect of gadolinium on [(3)H]gentamicin uptake was independent of gadolinium-induced increase in intracellular calcium concentration. These findings indicate that gentamicin is primarily taken up via an endocytosis-independent pathway in HK-2 cells with very low expression of megalin, and that the uptake of gentamicin is modulated by gadolinium.     

泄浊除痹方总黄酮对小鼠肾小管上皮细胞增殖与尿酸吸收的影响

目的:研究泄浊除痹方总黄酮对小鼠肾小管上皮细胞(RTECs)增殖与尿酸吸收的影响。方法:采用优化后的小鼠RTECs原代培养方法分离该细胞,不同浓度的泄浊除痹方总黄酮孵育48h,MTT法检测细胞活性,配制尿酸吸收培养基,进行尿酸吸收实验,采用尿酸检测试剂盒测定尿酸吸收值。结果:所分离培养的细胞为目的细胞,即小鼠RTECs,具有尿酸吸收功能;高浓度药物(10、7.5、5.0g.L-1)对细胞增殖有不同程度的抑制作用,低浓度药物(2.5、0.5g.L-1)对细胞增殖无显著性影响;在对细胞生长活性无显著性影响的浓度梯度范围内(2.5、2.0、1.5、1.0、0.5g.L-1),药物对尿酸吸收表现出不同程度的抑制作用。结论:泄浊除痹方总黄酮对小鼠RTECs尿酸吸收功能有一定的抑制作用。     

Interference with megalin expression/endocytic function by montelukast mitigates gentamicin nephrotoxicity: Downregulation of ClC-5 expression

Megalin receptor-mediated endocytosis participates a crucial role in gentamicin (GM) uptake, accumulation, and toxicity. In this study, we investigated the potential effects of montelukast (MLK) on megalin expression/endocytic function against GM nephrotoxicity. Male Wistar rats were administered GM (120 mg/kg; i.p.) daily in divided doses along 4 hr; 30 mg/kg/hr; for 7 days. MLK (30 mg/kg/day) was orally administered 7 days before and then concurrently with GM. The protein expressions of megalin and chloride channel-5 (ClC-5); one of the essential regulators of megalin endocytic function; were determined by Western blotting. Besides, the endocytic function of megalin was evaluated by the uptake of bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) into proximal tubular epithelial cells. Moreover, kidney function biomarkers (Cr, BUN, GFR, KIM-1, cystatin-C) and apoptosis markers (p-AKT1, cleaved caspase-3) were estimated. Co-treatment with MLK downregulated ClC-5 expression leading to reduced recycling of megalin to the plasma membrane, reduced expression, and so impaired endocytic function that was evidenced by reduced uptake of FITC-BSA in proximal tubular epithelial cells. The protein expression of the apoptotic executioner cleaved caspase-3 was significantly reduced, while that of the antiapoptotic p-AKT1 was elevated. These results were confirmed by the improvement of kidney functions and histological findings. Our data suggest that MLK could interfere with megalin expression/endocytic function that could be attributed to downregulation of ClC-5 protein expression. That eventually reduces renal cell apoptosis and improves kidney functions after GM administration without affecting the antibacterial activity of GM. Therefore, reduced expression of ClC-5 and interference with megalin expression/endocytic function by MLK could be an effective strategy against GM nephrotoxicity.     

Investigation of biomimetic shear stress on cellular uptake and mechanism of polystyrene nanoparticles in various cancer cell lines

Cancer cells in the tumor microenvironment are affected by fluid shear stress generated by blood flow in the vascular microenvironment and interstitial flows in the tumor microenvironment. Thus, we investigated how fluidic shear stress affects cellular uptake as well as the endocytosis mechanism of nanoparticles using a biomimetic microfluidic system that mimics the human dynamic environment. Positively charged amino-modified polystyrene nanoparticles (PSNs) at 100 μg/mL were delivered to cancer cells under static and biomimetic dynamic conditions (0.5 dyne/cm²). Additionally, the experiment was done in the presence of endocytosis inhibitors specific for one of the endocytosis pathways. To evaluate cellular uptake of cationic PSNs, the fluorescence intensity of cationic PSNs in cancer cells was measured by flow cytometer and fluorescence images were taken using confocal laser scanning microscopy. Cancer cells in dynamic conditions exhibited higher cellular uptake of PSNs and showed different cellular uptake mechanisms compared with those in static conditions. From these results, it suggested that biomimetic dynamic conditions stimulated specific endocytosis and prompted cellular uptake. It was also important to consider fluidic shear stress as one of the critical factors because cellular uptake and drug delivery could play a key role in cancer cells and metastasis.     

The Role of Megalin in the Transport of Gentamicin Across BeWo Cells,an In Vitro Model of?the Human Placenta

Aminoglycosides (AG) are known to readily cross the placenta, although the mechanisms responsible for placental transport have not been characterized. Megalin is expressed in human placenta, and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in placental transport. However, the role of megalin in placental AG uptake has not been established. An in vitro model to study megalin-mediated placental transport has also not been previously described. The objectives of this study, therefore, were to evaluate the human choriocarcinoma (BeWo) cell line as a model to study megalin-mediated placental transport and to assess the uptake kinetics of gentamicin, an AG antibiotic, using this in vitro model. BeWo cells were grown on Transwell® plates, and megalin expression and functional activity were assessed. Uptake of ³H-gentamicin was also evaluated in the presence and absence of megalin inhibitors. Expression of megalin protein and mRNA in BeWo cells were confirmed via immunoblot and qPCR analysis. Uptake of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) (a megalin substrate) was time-, concentration-, and temperature-dependent consistent with a transporter-mediated process. FITC-BSA uptake was also significantly reduced in the presence of unlabeled gentamicin (a megalin substrate) and sodium maleate (to induce megalin shedding) suggesting that megalin is functionally active in BeWo cells. Gentamicin uptake exhibited time and temperature dependence, saturability and Michaelis-Menten kinetics, all of which suggest a transporter-mediated process. Gentamicin uptake was also significantly reduced in the presence of the megalin inhibitors RAP and EDTA suggesting that megalin is likely involved in gentamicin uptake.KEY WORDS: aminoglycosides, gentamicin, megalin, placenta, receptor-mediated endocytosis     

Ligand-modified gene carriers increased uptake in target cells but reduced DNA release and transfection efficiency

DNA delivery to cells can be improved by using particle carriers made from biodegradable polymers such as poly(lactic-co-glycolic)acid (PLGA). It is speculated that addition of targeting moieties to the particle surface to facilitate uptake can further enhance gene expression in specific cells or tissues. Taking advantage of well-known receptor/ligand interactions in intestinal and renal epithelial cells, we formulated PLGA particles with high density of surface-bound bovine serum albumin (BSA; ～768 molecules/particle). BSA-coated particles exhibited significantly higher uptake by cells expressing the albumin receptor, megalin, and resisted degradation in low pH. However, gene expression from BSA-coated particles was 3- to 10-fold lower than that from unmodified particles; this reduction in transfection efficiency was probably due to the slower DNA release rate from modified particles. In this setting, addition of a targeting feature to particles reduced their effectiveness. Our study highlights the importance of the interplay between cell uptake and payload release in the design of polymer drug carriers.From the Clinical EditorDNA delivery to cells can be improved by using particle carriers such as PLGA. Taking advantage of known receptor/ligand interactions in intestinal and renal epithelial cells, PLGA particles with high density surface-bound BSA were formulated. BSA-coated particles exhibited significantly higher uptake; however, gene expression was 3 to 10-fold lower. Unexpectedly, the addition of a targeting feature to these particles reduced their overall effectiveness.     

Fluid shear stress affects the metabolic and toxicological response of the rainbow trout gill cell line RTgill-W1

The Rainbow trout gill cell-line (RTgill-W1) has been accepted by the Organisation for Economic Co-operation and Development (OECD TG249) as a replacement for fish in acute toxicity tests. In these tests cells are exposed under static conditions. In contrast, in vivo, water moves over fish gills generating fluid shear stress (FSS) that alters cell physiology and response to toxicants. The current study uses a specialised 3D printed chamber designed to house inserts and allows for the flow (0.2 dynes cm²) of water over the cells. This system was used to assess RTgill-W1 cell responses to FSS in the absence and presence of copper (Cu) over 24 h. FSS caused increased gene expression of mechanosensitive channel peizo1 and the Cu-transporter atp7a, elevated reactive oxygen species generation and increased expression of superoxidase dismutase. Cell metabolism was unaffected by Cu (0.163 μM to 2.6 μM Cu) under static conditions but significantly reduced by FSS + Cu above 1.3 μM. Differential expression of metallothionein (mt) a and b was observed with increased expression of mta under static conditions and mtb under FSS on exposure to Cu. These findings highlight toxicologically relevant mechanosensory responses by RTgill-W1 to FSS that may influence toxicological responses.     

Up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of renal tubular epithelial cells under high glucose condition

This study aimed to investigate the role and underlying mechanism of miR-135b in high glucose-induced oxidative stress of renal tubular epithelial cells. Here, in vivo experiments found that compared to the control group, miR-135b expression was significantly up-regulated in the diabetes group, whereas BMP7 mRNA and protein levels were down-regulated. In high glucose-treated renal tubular epithelial cells (HK-2) in vitro, oxidative stress was induced, which up-regulated miR-135b expression. In addition, the regulation of miR-135b on BMP7 expression was confirmed in HK-2 cells. Under high glucose conditions, oxidative stress promoted the apoptosis of HK-2 cells through the up-regulation of miR-135b expression. In vivo experiments indicated that interference with miR-135b improved renal function in mice with diabetic nephropathy. In conclusion, these results indicated that the up-regulation of miR-135b expression induced by oxidative stress promotes the apoptosis of HK-2 cells under high glucose conditions.     

Effects of mycophenolic acid–glucosamine conjugates on the base of kidney targeted drug delivery

Mycophenolic acid has played an important role in treating immunosuppression and autoimmune diseases. Nevertheless, the agent needs a high dosage in treatment, following some side effects. To tackle this problem, in this study, mycophenolic acid–glucosamine conjugate (MGC), modified by 2-glucosamine, was synthesized to achieve kidney targeting and improved drug efficacy with a lower dosage. ¹H NMR, ¹³C NMR and HRMS spectroscopy were used to verify the conjugate whose stability was good in vitro. The transport of MGC by human proximal renal tubular epithelial HK-2 cells was temperature-, time-, concentration-dependent and saturable, suggesting the involvement of carrier-mediated uptake. In addition, the cellular uptake of MGC dropped substantially with the inhibition of megalin receptor. The specific tissue distribution indicated the commendable renal-targeting capability of MGC. The concentration of MGC was improved in the kidney except for other tissues, about 6.76 times higher than that of MPA. Further, the bioavailability of MGC in plasma decreased as compared with mycophenolic acid. Moreover, therapeutic effect of MGC was enhanced significantly compared with MPA in the acute kidney injury model. All the findings suggested the potential of mycophenolic acid–glucosamine conjugate in kidney targeted drug delivery.     

雷帕霉素对肾小管上皮细胞增殖和凋亡的影响

目的 探讨雷帕霉素对肾小管上皮细胞增殖和凋亡的影响。方法 体外培养肾小管上皮细胞(HK-2细胞),选择100, 200, 400, 800 ng/ml雷帕霉素作用于HK-2细胞24、48、72h。利用 MTT实验分析雷帕霉素对HK-2细胞增殖的影响,并计算各浓度及不同时间的增殖抑制率优化雷帕霉素作用的浓度和时间;选择最佳的作用浓度和作用时间处理HK-2细胞,通过流式细胞分析技术,分析雷帕霉素对HK-2细胞的凋亡的影响。结果 MTT实验显示不同浓度的雷帕霉素,对增殖有抑制作用,且表现浓度依赖性和时间依赖性;RAPA可以促进HK-2细胞的凋亡。结论 通过HK-2细胞模型研究,发现雷帕霉素可抑制肾小管上皮细胞增殖、促进细胞凋亡。     

Characterization of monocarboxylate transport in human kidney HK-2 cells

The objectives of this study were to characterize the expression and function of monocarboxylate transporters (MCTs) in human kidney HK-2 cells and to compare the expression of MCTs in HK-2 cells to that found in human kidney. mRNA and protein expression of MCTs were determined by RT-PCR and Western analyses, respectively, while immunofluorescence staining was used to determine the membrane localization of MCT1. The driving force, transport kinetics, and inhibition of two MCT substrates, D-lactate and butyrate, were characterized in HK-2 cells. mRNA of MCT1, -2, -3, -4 isoforms were present in HK-2 cells and in human kidney cortex. MCT1 was present predominantly on the basal membranes of HK-2 cells. The cellular uptake of D-lactate and butyrate exhibited pH- and concentration-dependence (D-lactate, Km of 26.5 +/- 2.2 mM and Vmax of 72.0 +/- 14.5 nmol mg-1 min-1; butyrate, Km of 0.8 +/- 0.3 mM, Vmax of 29.3 +/- 2.5 nmol mg-1 min-1, and a diffusional clearance of 2.1 microL mg-1 min-1). The uptake of D-lactate and butyrate by HK-2 cells was inhibited by MCT analogues and the classical MCT inhibitors alpha-cyano-4-hydroxycinnamate, pCMB, and phloretin. The uptake of D-lactate and butyrate by HK-2 cells significantly decreased after transfection with small-interference RNA for MCT1. In summary, MCTs were present in both HK-2 cells and human kidney cortex, and HK-2 cells exhibited polarized MCT expression and pH-dependent transport of D-lactate and butyrate. Our results also support the usefulness of HK-2 cells as an in vitro model for studying monocarboxylate transport in renal proximal tubule cells.     

聚苯乙烯纳米粒对细胞活性氧的影响

目的 观察聚苯乙烯纳米粒对人肾小管上皮细胞(HK-2)内活性氧水平的影响.方法 用不同浓度的PS、氨基聚苯乙烯(PS-NH2)及羧基聚苯乙烯(PS-COOH)纳米粒刺激HK-2细胞0.5h,经DCFH-DA染色后,用流式细胞仪检测其平均荧光强度.结果 0.1 μg·mL-1PS纳米粒可引起细胞内的活性氧增加,2、4、100 μg·mL-1PS纳米粒可引起活性氧降低;4、100 μg·mL-1 PS-NH2纳米粒可引起细胞内活性氧降低;0.1、1、2、4、100 μg· mL-1PS-COOH纳米粒可引起细胞内活性氧降低.结论 低浓度PS纳米粒可增加HK-2细胞内的活性氧水平,高浓度则减少活性氧;PS-COOH纳米粒的活性氧降低效应比PS-NH2强.     

Cyto-genotoxicity and oxidative stress induced by zinc oxide nanoparticle in human lymphocyte cells in vitro and Swiss albino male mice in vivo

ZnO-np has immense potential and application in cosmetic and health care sectors. Hence it was imperative to assess the toxicity/safety of these nanoparticles. In this study, we have evaluated the effects of ZnO-np in human peripheral blood mononuclear cells (PBMCs) in vitro and in Swiss albino male mice in vivo for cyto-genotoxicity and oxidative damage. In vitro results showed that ZnO-nps were weakly genotoxic, induced significant decrease in mitochondrial membrane potential and was capable of ROS generation, leading to apoptosis. In bone marrow cells in vivo, reduction of mitochondrial membrane potential (MMP), increased oxidative stress and G0/G1 cell cycle arrest was observed along with chromosome aberrations and micronuclei formation. In liver cells DNA damage and induction of oxidative stress with concurrent decrease in inhibition of antioxidant enzymes were noted. These in vitro and in vivo results demonstrated that ZnO-np induced genotoxic response and ROS production leading to apoptotic cell death and established a good co-relation between the two biological systems. More importantly, the results stress on the need of multiple endpoint assay-approaches, with an in vitro-in vivo study design to assess nanoparticle toxicology.     

Akebia Saponin D ameliorated kidney injury and exerted anti-inflammatory and anti-apoptotic effects in diabetic nephropathy by activation of NRF2/HO-1 and inhibition of NF-KB pathway

Diabetic nephropathy (DN), a common microvascular complication of type 2 diabetes mellitus (T2DM), causes increasing mortality and morbidity due to its high prevalence and severe consequences. Hence, it is urgent to search for effective agents that provide new insights into novel molecular therapeutic targets for DN. This study was designed to investigate the critical role of Akebia saponin D (ASD) in kidney damage, inflammation and apoptosis of renal tubular cells in DN. To probe the protective effects of ASD on DN in vivo, diabetes mellitus model was established by intraperitoneal (ip) injection of STZ (60 mg/kg) for 5 days consecutively. Besides, HG-induced human renal tubular cells (HK-2) were used to analyze the defined effects and underlying mechanism of ASD on DN in vitro. Blood glucose, insulin, serum creatinine (Scr), blood urea nitrogen (BUN), renal injury, inflammation, oxidative stress and apoptosis of renal tubular cells were respectively measured and evaluated. ASD prevented kidney damage, improved renal function and inflammatory reaction, ameliorated oxidative stress and inhibited apoptosis of renal tubular cells in DN mice via activation of NRF2/HO-1 pathway and inhibition of NF-KB pathway.     

A novel SGLT is expressed in the human kidney

Selective inhibitors of sodium-glucose cotransporter 2 (SGLT2)-mediated reabsorption of glucose in the proximal tubule of the kidney are being developed for the treatment of diabetes. SGLT2 shares high degree of homology with SGLT3; however, very little is known about the expression and functional role of SGLT3 in the human kidney. Indeed, the SGLT2 inhibitors that are currently in clinical trials might affect the expression and/or the activity of SGLT3. Therefore, the present study examined the expression of SGLT3 mRNA and protein in human kidney and in a human proximal tubule HK-2 cell line. The results indicated that human SGLT3 (hSGLT3) message and protein are expressed both in vivo and in vitro. We also studied the activity of hSGLT3 protein following its over-expression in mammalian kidney-derived COS-7 cells and in HK-2 cells treated with the imino sugar deoxynojirimycin (DNJ), a potent agonist of hSGLT3. Over-expression of hSGLT3 in COS-7 cells increased intracellular sodium concentration by 3-fold without affecting glucose transport. Activation of hSGLT3 with DNJ (50μM) increased sodium uptake in HK-2 cells by 5.5 fold and this effect could be completely blocked with SGLT inhibitor phlorizin (50μM). These results suggest that SGLT3 is expressed in human proximal tubular cells where it serves as a novel sodium transporter. Up-regulation of the expression of SGLT3 in the proximal tubule in diabetic patients may contribute to the elevated sodium transport in this segment of the nephron that has been postulated to promote hyperfiltration and renal injury.     

Expression of hsp 27, hsp 60, hsc 70, and hsp 70 by immortalized human proximal tubule cells (HK-2) following exposure to heat shock, sodium arsenite, or cadmium chloride

The expression of hsp 27, hsp 60, hsc 70, and hsp 70 mRNA and protein was determined in immortalized human proximal tubule cells (HK-2) exposed to heat shock, sodium arsenite, or cadmium chloride (CdCl2) under both acute and extended conditions of exposure. It was demonstrated that the HK-2 cells did not exhibit the classic heat-shock response when subjected to an acute physical (heat) or chemical stress (sodium arsenite or CdCl2). Heat stress, elevated temperature at 42.5 degrees C for 1 h, caused a marked increase only in hsp 70 mRNA and protein, but not hsp 27 or hsp 60 mRNA and protein. Similar results were obtained when the cells were subjected to a classic chemical stress of exposure to 100 microM sodium arsenite for 4 h or CdCl2 for 4 h. These findings were in contrast to those found previously with mortal human proximal tubule (HPT) cells, where acute stress by all three stimuli elicited marked increases in hsp 27, hsp 60, and hsp 70 mRNA and protein. It was shown that the basal levels of expression of hsp 27 and hsp 60 in the HK-2 cells were elevated when compared to those found in unstressed HPT cells and that the basal levels were similar to those found in HPT cells under stress conditions. These results suggest that the failure of the HK-2 cells to increase hsp 27 and hsp 60 levels in response to physical and chemical stress is because they already possess elevated basal levels of these proteins. This would indicate that one or more of the genetic events that resulted in the immortalization of the HK-2 cells also elicited a stress response for hsp 27 and hsp 60, but not for hsp 70, stress response family members. Overall, the results suggest that although there are differences in the regulation of the stress response between the immortal HK-2 and mortal HPT cell lines, as long as these differences are recognized, the HK-2 cell line should be a valuable adjunct to study the stress response of the proximal tubule in general and when exposed to environmental pollutants such as cadmium.     

普罗布考对甲基胍诱导纤维化相关因子在HK-2细胞表达的影响

目的研究降脂药普罗布考对小分子尿毒素甲基胍诱导纤维化相关因子在人肾小管上皮细胞表达的影响。方法 HK-2细胞分3组培养,A组（正常对照,不加干预因素）;B组培养基中加入0.5mmol.L-1甲基胍;C组培养基中加入0.5mmol.L-1甲基胍和20μmol.L-1普罗布考。各组细胞培养48h后用免疫细胞化学、RT-PCR检测TGF-β1、CTGF和FN,并用流式细胞仪对以上因子作定量检测,以观察其在HK-2细胞中的表达。结果在甲基胍刺激后,细胞免疫化学染色,HK-2细胞质中着色,显示TGF-β1、CTGF和FN均有表达。正常对照组HK-2细胞质中无着色。测定结果显示甲基胍组TGF-β1、CTGF和FN显著高于正常对照组（P〈0.01）,加入普罗布考后,表达显著下降（P〈0.01）。结论甲基胍能促进纤维化相关因子在肾小管上皮细胞中表达;普罗布考能抑制甲基胍诱导纤维化相关因子在肾小管上皮细胞的表达。     

Nephrotoxicity and Kidney Transport Assessment on 3D Perfused Proximal Tubules

Proximal tubules in the kidney play a crucial role in reabsorbing and eliminating substrates from the body into the urine, leading to high local concentrations of xenobiotics. This makes the proximal tubule a major target for drug toxicity that needs to be evaluated during the drug development process. Here, we describe an advanced in vitro model consisting of fully polarized renal proximal tubular epithelial cells cultured in a microfluidic system. Up to 40 leak-tight tubules were cultured on this platform that provides access to the basolateral as well as the apical side of the epithelial cells. Exposure to the nephrotoxicant cisplatin caused a dose-dependent disruption of the epithelial barrier, a decrease in viability, an increase in effluent LDH activity, and changes in expression of tight-junction marker zona-occludence 1, actin, and DNA-damage marker H2A.X, as detected by immunostaining. Activity and inhibition of the efflux pumps P-glycoprotein (P-gp) and multidrug resistance protein (MRP) were demonstrated using fluorescence-based transporter assays. In addition, the transepithelial transport function from the basolateral to the apical side of the proximal tubule was studied. The apparent permeability of the fluorescent P-gp substrate rhodamine 123 was decreased by 35% by co-incubation with cyclosporin A. Furthermore, the activity of the glucose transporter SGLT2 was demonstrated using the fluorescent glucose analog 6-NBDG which was sensitive to inhibition by phlorizin. Our results demonstrate that we developed a functional 3D perfused proximal tubule model with advanced renal epithelial characteristics that can be used for drug screening studies.     

Berberine protects HK-2 cells from hypoxia/reoxygenation induced apoptosis via inhibiting SPHK1 expression

Renal ischemia reperfusion injury (RIRI) refers to the irreversible damage for renal function when blood perfusion is recovered after ischemia for an extended period, which is common in clinical surgeries and has been regarded as a major risk for acute renal failures (ARF) that is accompanied with unimaginably high morbidity and mortality. Hypoxia during ischemia followed by reoxygenation via reperfusion serves as a major event contributing to cell apoptosis, which has been widely accepted as the vital pathogenesis in RIRI. Preventing apoptosis in renal tubular epithelial cell has been considered as effective method for blocking RIRI. In this paper, we established a hypoxia/reoxygenation (H/R) injury model in human proximal tubular epithelial HK-2 cells. Here, we found increased SPHK1 levels in H/R injured HK-2 cells, which could be significantly down regulated after berberine treatment. Berberine has been reported to exert a protective effect on H/R-induced apoptosis of HK-2 cells. So, in our present study, we planned to investigate whether SPHK1 participated in the anti-apoptosis process of berberine in H/R injured HK-2 cells. Our study confirmed the protective effect of berberine against H/R-induced apoptosis in HK-2 cells through promoting cells viability, inhibiting cells apoptosis, and down-regulating p-P38, caspase-3, caspase-9 as well as SPHK1, while up regulating the ratio of Bcl-2/Bax. However, SPHK1 overexpression in HK-2 cells induced severe apoptosis, which can be significantly ameliorated with additional berberine treatment. We concluded that berberine could remarkably prevent H/R-induced apoptosis in HK-2 cells through down-regulating SPHK1 expression levels, and the mechanisms included the suppression of p38 MAPK activation and mitochondrial stress pathways.     

Reduced tubular degradation of glomerular filtered plasma albumin is a common feature in acute and chronic kidney disease

Tubular epithelial cells take up and degrade plasma albumin filtered by the glomerulus. Tubular damage resulting in reduced albumin uptake or degradation has been suggested as one mechanism contributing to albuminuria in kidney disease. This study investigated whether tubular albumin uptake or degradation is altered in acute and chronic glomerular disease. Mouse models of acute glomerular injury (anti‐GBM disease and LPS‐induced albuminuria) and chronic disease (streptozotocin‐induced diabetes and db/db mice) were examined. Mice were injected intravenously with Alexa‐albumin plus DQ‐albumin and killed 20 minutes later. Tubular uptake of albumin (Alexa‐albumin) and albumin degradation (Dye Quenched (DQ)‐albumin) was assessed in tissue sections via confocal microscopy. Tubular uptake of Alexa‐albumin in the models of diabetic nephropathy was not different to normal mice. However, the fluorescence signal resulting from degradation of DQ‐albumin was significantly reduced in db/db mice, and the ratio of degraded to intact albumin was reduced in both models. The ratio of degraded to intact albumin in tubules was also reduced in the anti‐GBM model. In the LPS model, both tubular uptake and degradation of albumin were significantly reduced, with a substantial reduction in the ratio of degraded to intact albumin in tubules. LPS stimulation of cultured tubular epithelial cells inhibited albumin uptake, indicating a direct role for LPS in modifying tubular handling of albumin. In conclusion, reduced degradation of filtered albumin in the proximal tubule is a common feature of glomerular diseases. This may be a general mechanism whereby tubular dysfunction contributes to the development of albuminuria.