Uptake of nephrotoxic S-conjugates by isolated rat renal proximal tubular cells

Freshly isolated, rat renal proximal tubular cells have been widely used to study the biochemical mechanisms of cysteine and homocysteine S-conjugate-induced cytotoxicity. Because cellular transport may be an important determinant of S-conjugate-induced nephrotoxicity, the characteristics of the transport of nephrotoxic cysteine S-conjugates and analogs were studied in rat renal proximal tubular cells. Time- and concentration-dependent uptake of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) into cells were observed; higher concentrations were accumulated in the presence of aminooxyacetic acid (AOAA), an inhibitor of cysteine conjugate beta-lyase, indicating that AOAA does not inhibit transport. Saturable sodium-dependent and sodium-independent transport processes were identified; approximately 50% of DCVC uptake, but only 30% of DCVHC uptake, was sodium-dependent. Competition studies indicated that the probenecid-sensitive organic anion transport system, which is sodium-dependent, and system L, which is sodium-independent, participate in the renal proximal tubular uptake of both S-conjugates. In addition, DCVHC uptake occurs by the sodium-dependent system ASC and system A. The greater degree of sodium independence of DCVHC uptake as compared to that of DCVC indicates that system L plays a greater role in DCVHC uptake than it does in DCVC uptake. Both sodium-dependent and sodium-independent uptake of the nonmetabolizable and nontoxic alpha-methyl analogs, S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine and S-(1,2-dichlorovinyl)-DL-alpha-methylhomocysteine, were observed at rates comparable to those of DCVC and DCVHC.(ABSTRACT TRUNCATED AT 250 WORDS)     

小鼠肾脏近端小管上皮细胞原代培养条件优化及功能测试

背景:好的肾脏近端小管上皮细胞原代培养方法能够为药物筛选、机制研究等提供模型基础.目的:探索小鼠肾脏近端小管上皮细胞原代培养的最佳处理条件,并对其进行尿酸吸收功能测试.方法:无菌取小鼠肾脏,去除外膜及脂肪组织,切取近端皮质部分,机械破碎,Ⅰ型胶原酶消化,依次过50目,100目筛网过滤,Percoll密度梯度离心法分离肾小管节段,接种至一次性细胞培养瓶.制备细胞爬片,SP法染色鉴定细胞.在1 500 μmol/L尿酸浓度下,以不同浓度梯度的丙磺舒和苯溴马隆作为指示药物,检测细胞尿酸吸收及作为药物作用体外模型功能.结果与结论:经机械破碎的肾脏近端皮质组织在400 U胶原酶处理20 min时,肾小管节段贴壁率达到最高为54.5%;首次换液最佳时间为72 h;细胞在原代培养第5天生长达到对数期,在第10天状态开始有所下降,折光率低,出现部分漂浮死亡细胞.在1 500 pmol/L尿酸浓度的吸收培养基中,吸收实验进行30 min时,其对尿酸吸收速率达到最大.指示药物丙磺舒和苯溴马隆在不同的浓度F,对于尿酸吸收的抑制率不同,其中苯溴马隆的抑制率要高于丙磺舒.提示在上述分离培养条件下能够培养出数量多、活性高的目的细胞,并且具有良好的尿酸吸收功能,在一定的尿酸和药物浓度下,能够作为研究药物作用的体外细胞模型.     

Proteomic analysis of alternative protein tyrosine phosphorylation in 1,2-dichlorovinyl-cysteine-induced cytotoxicity in primary cultured rat renal proximal tubular cells

Toxicant exposure affects the activity of various protein tyrosine kinases. Using phosphotyrosine proteomics, we identified proteins that were differentially phosphorylated before renal cell detachment and apoptosis. Treatment of primary cultured rat proximal tubular epithelial cells with the model nephrotoxicant S-(1,2-dichlorovinyl)-L-cysteine (DCVC) resulted in early reorganization of F-actin stress fibers and formation of lamellipodia, which was followed by cell detachment from the matrix and apoptosis. This was prevented by genistein-mediated inhibition of protein tyrosine kinases and enhanced by inhibition of protein tyrosine phosphatases using vanadate. Phosphotyrosine proteomics revealed that DCVC-induced renal cell apoptosis was preceded by changes in the tyrosine phosphorylation status of a subset of proteins, as identified by matrix-assisted laser desorption ionization/time of flight-mass spectrometry (MS)/MS including actin-related protein 2 (Arp2), cytokeratin 8, t-complex protein 1 (TCP-1), chaperone containing TCP-1, and gelsolin precursor. The major differentially tyrosine-phosphorylated protein was Arp2, whereas phosphorylation of Arp3 was not affected. Arp2 was located in the lamellipodia that were formed before the onset of apoptosis. Because DCVC-induced cell detachment and apoptosis is regulated by tyrosine kinases, we propose that alterations in tyrosine phosphorylation of a subset of proteins, including Arp2, play a role in the regulation of the F-actin reorganization and lamellipodia formation that precede renal cell apoptosis caused by nephrotoxicants.     

Interactions of free and liposomal amphotericin B with renal proximal tubular cells in primary culture

We studied in vitro the renal toxicity of amphotericin B (AMB) and liposomal AMB using primary cultures of rabbit proximal tubular cells grown to confluence in serum-free medium. Toxicity was assessed by changes 1) in the Na(+)-dependent uptakes of P1 and alpha-methylglucopyranoside (MGP), characteristic functions of proximal tubular cells; 2) K+ release into the supernatant, dependent upon membrane permeability; and 3) lactic dehydrogenase release as a marker for cellular death. Cells were exposed for 1 hr to AMB, alone or intercalated in small unilamellar vesicles prepared with one of the following phospholipids: dipalmitoylphosphatidyl choline, distearoylphosphatidyl choline or dimyristoylphosphatidyl choline. Although AMB concentrations of 20 microM or less did not increase lactic dehydrogenase release, P1 and MGP uptakes were significantly reduced (50% inhibition) by 2.5 and 5 microM AMB, respectively. AMB toxicity was dose-dependent, up to 20 microM. Analysis of P1 and MGP uptake kinetics, after treatment of the cells with 10 microM AMB, showed that inhibition occurred through a decrease in Vmax (66 and 57% inhibition for P1 and MGP, respectively) without affecting Km value. K+ release appeared for 2.5 microM AMB and increased with higher concentrations. Alteration of Na(+)-dependent uptakes by AMB, which parallels K+ release, may result from an alteration of the sodium gradient. Na(+)-dependent uptakes and K+ release were unaffected by liposomal AMB, even at the highest concentration tested (80 microM). The protective effect of liposomes was the same regardless of the phospholipid used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha adrenoceptor agonist stimulation of oxygen consumption in rat proximal and distal nephrons

Selective alpha-1 and alpha-2 adrenergic agonists were used to test the hypothesis that both receptor subtypes increase transcellular Na+ transport in the nephron. Oxygen consumption (QO2) was used as an index of transcellular transport and provided a continuous dynamic record of the tubules' response to an agonist. Both alpha-1 and alpha-2 adrenoceptor agonists produced a linear dose-related increase in QO2 at a steeper slope than the control in proximal and distal nephron segments. Stimulation of QO2 by the adrenergic agonists did not occur in the presence of ouabain and did not exceed the maximal respiratory rate achieved with nystatin. The response was determined to be a receptor-mediated increase in the ouabain-sensitive component of respiration. An inactive stereoisomer of epinephrine produced no effect, and adrenergic antagonists inhibited the stimulation by the respective agonists. Adrenergic agonists stimulated QO2 in proximal segments to a much greater degree than observed with suspensions of distal segments. These results are consistent with the density of adrenoceptors on nephrons reported in radioligand binding and autoradiographic studies. The alpha-1 agonists, cirazoline and phenylephrine, had similar dose-response curves and stimulated proximal tubules more than distal tubules. The alpha-2 agonists, guanabenz, UK 14,304 and B-HT 933, equivalently stimulated QO2 in distal tubules, but a spectrum of enhanced oxygen consumption was observed in proximal nephrons. Although both alpha adrenoceptor subtypes increased QO2 in proximal and distal nephrons, the mechanisms are likely to be different.     

Roles of necrosis,Apoptosis, and mitochondrial dysfunction in S-(1,2-dichlorovinyl)-L-cysteine sulfoxide-induced cytotoxicity in primary cultures of human renal proximal tubular cells

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate beta-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time- and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular glutathione (GSH) status. Confluent hPT cells incubated with as little as 10 microM DCVCS for 24 h exhibited morphological changes, although at least 100 microM DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 h with at least 200 microM DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 h with as little as 10 microM DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 microM, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (>50%) of cellular GSH content was only observed with 500 microM DCVCS. These results, combined with previous studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells.     

Endotoxin-tobramycin additive toxicity on renal proximal tubular cells in culture.

Aminoglycoside-induced renal damage is enhanced in animals with Escherichia coli pyelonephritis. Bacterial endotoxin is liberated during antibiotic therapy. The toxic effect of endotoxin and tobramycin, alone or in combination, was investigated in primary cultures of rabbit proximal tubular cells grown to confluence in serum-free medium. Sodium-dependent uptakes of Pi and alpha-methylglucopyranoside (MGP) and enzymatic activities (lactate dehydrogenase [LDH] released as a marker of cell necrosis and gamma-glutamyltransferase [GGT] and N-acetyl-beta-D-glucosaminidase [NAG] present in the homogenate as markers of brush border membrane and lysosome integrity) were measured. Cells were exposed to (i) endotoxin (20 mg/liter), tobramycin (1 mM), or endotoxin plus tobramycin for 48 h, or (ii) endotoxin (100 mg/liter), tobramycin (4 mM), or endotoxin plus tobramycin for 72 h. Endotoxin alone did not alter Pi uptake, but tobramycin inhibited Pi uptake through a decrease in Vmax. The effect was not enhanced by the combination of endotoxin and tobramycin. Endotoxin and tobramycin alone exerted no significant effect upon MGP uptake, but strong inhibition of the Vmax was observed after exposure to a combination of endotoxin plus tobramycin, without alteration of the Km. Endotoxin decreased residual GGT activity in the cell homogenate. Tobramycin increased LDH release in the medium and NAG activity in the homogenate. Endotoxin plus tobramycin resulted in an additive effect upon LDH and NAG activities. In conclusion, by disturbing apical membrane integrity, endotoxin increased tobramycin toxicity in vitro in the absence of serum hormonal mediator.     

Prostaglandin and thromboxane synthesis by highly enriched rabbit proximal tubular cells in culture

Prostaglandin synthetic profiles were studied in monolayers of highly enriched rabbit renal proximal tubular cells cultured in serum-free, hormone-supplemented, defined media. The cultures were initiated from glomeruli-free cortical suspensions. Cells in culture demonstrated morphologic and functional characteristics highly suggestive of proximal tubular cells. The basal and stimulated synthesis of immunoassayable prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 in response to various agonists, as well as the effect of two cyclooxygenase inhibitors, was assessed. Under both basal and stimulated conditions, PGE2 was the major product synthesized. PGF2 alpha and 6-keto-PGF1 alpha were synthesized to a lesser extent, and TxB2 was undetectable. The basal synthesis of PGE2 and PGF2 alpha in cultured cells was found to be higher than in isolated proximal tubular fragments by sevenfold and fivefold, respectively. Exogenous arachidonate, angiotensin II, and the divalent cation ionophore A23187 stimulated all three immunoassayable prostaglandins in a dose-dependent manner. Arginine vasopressin (10(-5) mol/L) had no stimulatory effect. In Ca++-free media or in the presence of 10(-5) mol/L Ca++ channel blocker, verapamil, the stimulatory effects of angiotensin II and A23187 were ameliorated. The stimulatory effect of angiotensin II was inhibited by saralasin (10(-5) mol/L), indicating that receptor binding could mediate PGE2 synthesis. Both indomethacin and sulindac sulfide (10(-5) mol/L) reversibly inhibited PGE2 synthesis.     

Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney

The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of gamma-glutamyltransferase, gamma-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.     

Regulation of dedifferentiation and redifferentiation in renal proximal tubular cells by the epidermal growth factor receptor

Repair of injured renal epithelium is thought to be mediated by surviving renal proximal tubular cells (RPTC) that must dedifferentiate to allow the proliferation and migration necessary for epithelial regeneration. RPTC then redifferentiate to restore tubular structure and function. Current models suggest that epidermal growth factor receptor (EGFR) activation is required for dedifferentiation characterized by enhanced vimentin expression, decreased N-cadherin expression, spindle morphology, and loss of apical-basal polarity after injury. Because an in vitro model of RPTC redifferentiation has not been reported, and the mechanism(s) of redifferentiation has not been determined, we used rabbit RPTC in primary cultures to address these issues. H2O2 induced the dedifferentiated phenotype that persisted >48 h; redifferentiation occurred spontaneously in the absence of exogenous growth factors after 72 to 120 h. Phosphorylation of two tyrosine residues of EGFR increased 12 to 24 h, peaked at 24 h, and declined to basal levels by 48 h after injury. EGFR inhibition during dedifferentiation restored epithelial morphology and apical-basal polarity, and it decreased vimentin expression to control levels 24 h later. In contrast, exogenous epidermal growth factor addition increased vimentin expression and potentiated spindle morphology. p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta receptor inhibitors did not affect redifferentiation after H2O2 injury. Similar results were observed in a mechanical injury model. These experiments represent a new model for the investigation of RPTC redifferentiation after acute injury and identify a key regulator of redifferentiation: EGFR, independent of p38 MAPK and the TGF-beta receptor.     

Mercuric chloride-induced cytotoxicity and compensatory hypertrophy in rat kidney proximal tubular cells.

Previous work showed that uninephrectomized (NPX) rats are more susceptible to the nephropathy induced by some doses of HgCl2 than sham-operated (SHAM) rats. The aim of the present study was to investigate the cytotoxic effects of HgCl2 in proximal tubular (PT) cells isolated from the kidney(s) of both NPX and SHAM rats. The study was designed to test if isolated PT cells that have undergone compensatory hypertrophy in vivo are more sensitive to the cytotoxic effects of HgCl2 in vitro than PT cells isolated from the kidneys of control animals. PT cells were purified from suspensions of renal cortical cells by Percoll density gradient centrifugation. The cellular content of protein (mg/10(6) cells) was 68% higher and the cellular specific activity (mU/10(6) cells) of lactate dehydrogenase was 56% higher in PT cells isolated from NPX rats than in PT cells isolated from SHAM rats. The cytotoxicity of HgCl2, as assessed by inhibition of lactate dehydrogenase activity, exhibited a steep concentration dependence. In the presence of bovine serum albumin, PT cells from both NPX and SHAM rats displayed no signs of cellular injury at concentrations of HgCl2 less than or equal to 0.1 mM. At concentrations of HgCl2 greater than or equal to 0.25 mM, cellular necrosis occurred rapidly in all PT cells. In the absence of bovine serum albumin, cellular injury and death occurred at concentrations of HgCl2 as low as 0.01 mM. PT cells from NPX rats exhibited signs of cellular injury at lower concentrations of HgCl2 than PT cells from SHAM rats when BSA was absent from the extracellular medium. Preincubation of PT cells from both NPX and SHAM rats with glutathione provided the cells with concentration-dependent protection from the cytotoxic effects of HgCl2. Preincubation of PT cells from both NPX and SHAM rats with the chelating agent 2,3-dimercapto-1-propane sulfonate provided cells with complete protection from the cytotoxic effects of HgCl2 when the concentration of 2,3-dimercapto-1-propane sulfonate was slightly below or higher than the concentration of HgCl2. Results of this study demonstrate the usefulness of freshly isolated PT cells from NPX rats as an in vitro model system to investigate biochemical mechanisms by which compensatory renal growth alters susceptibility to chemical-induced nephrotoxicity.     

Depression of proximal tubular sodium reabsorption in the dog in response to renal beta adrenergic stimulation by isoproterenol

Water diuresis was produced in anesthetized hypophysectomized, cortisone-treated dogs by infusion of 2.5% dextrose. Alpha adrenergic blockade of the left kidney produced by infusion of phenoxybenzamine in the left renal artery was associated with a significantly (P < 0.05) greater rate of urine flow (V) and free water excretion (C(H2O)) in the left kidney than in the right despite similar glomerular filtration rates (GFR) (17 +/- 1.3 ml/min, left; 18 +/-0.9 ml/min, right). Sodium excretion (U(Na)V) was similar in the two kidneys (3 and 5 muEq/min).When beta adrenergic stimulation of the left kidney was superimposed on alpha blockade by the addition of isoproterenol to the left renal artery infusate, GFR remained unchanged and similar in the two kidneys, as V and C(H2O) increased significantly (P < 0.01) in the left kidney but not in the right. When isoproterenol was discontinued, V and C(H2O) returned towards control in the left kidney and remained unchanged in the right. The ratios of the left kidney to the right during control, isoproterenol, and postcontrol were 1.22, 1.65, and 1.35, respectively, for V and 1.36, 1.90, and 1.44, respectively, for C(H2O). Sodium excretion remained unchanged and similar in the two kidneys throughout the study.The results indicate that blockade of alpha adrenergic activity inhibits the increased proximal tubular sodium reabsorption which anesthesia induces in the dog.Beta adrenergic stimulation appears to decrease proximal tubular sodium reabsorption but does not prevent virtually complete reabsorption of the increased quantity of delivered sodium by the ascending limb of the loop of Henle and the distal tubule. These changes in sodium reabsorption presumably are not associated with changes in colloid osmotic pressure or hydrostatic pressure in the peritubular capillary inasmuch as cortical and non-cortical plasma flow, filtration fraction, and mean arterial pressure in the left kidney were unchanged. Thus, isoproterenol probably produced its effects through a direct action on the renal tubule, possibly through the mediation of the adenyl cyclase system.     

Metabolism of the aminoterminal propeptide of type III procollagen in cultures of human proximal tubular cells.

Degradation of the intact form of the aminoterminal propeptide of type III procollagen (PIIINP) has been established in the liver, whereas the col 1 domain of PIIINP is extracted by the kidneys. We used native human PIIINP and col 1 domain of PIIINP to investigate the degradation of PIIINP in cultures of human proximal tubular cells. Normal renal tissue was obtained from the healthy part of kidneys surgically removed and from biopsies from a total of 10 patients. The degradation was characterized by incubation of [125I]-PIIINP followed by gel filtration. We found that in physiological concentrations (4.4 micrograms l-1 and 11.9 micrograms l-1 intact PIIINP was almost totally degraded, but not col 1 domain. High concentrations of PIIINP (20-50 micrograms l-1) had a non-linear, non-monoexponential degradation over time, which suggests several steps. Gel filtration of [125I]-PIIINP after 1 h, 3 h, 6 h and 24 h of incubation confirmed the observation by showing the rapid formation of a high-molecular-weight fraction, followed by the slower formation of a low-molecular-weight fraction. The high-molecular-weight fraction was PIIINP immunoreactive, but not the low-molecular-weight fraction. We conclude that cultures of human proximal tubular cells degrade intact human PIIINP by the formation of high- and low-molecular-weight fractions. Earlier findings that extraction of the PIIINP col 1 domain takes place in the kidneys, cannot be explained by degradation by the proximal tubular cells.     

Levofloxacin inhibits rhinovirus infection in primary cultures of human tracheal epithelial cells

Respiratory virus infections, including infections with rhinoviruses (RVs), are related to exacerbations of chronic obstructive pulmonary disease (COPD). A new quinolone antibiotic, levofloxacin (LVFX), has been used to treat bacterial infections that cause COPD exacerbations as well as bacterial infections that are secondary to viral infection in COPD patients. However, the inhibitory effects of LVFX on RV infection and RV infection-induced airway inflammation have not been studied. We examined the effects of LVFX on type 14 rhinovirus (RV14) (a major human RV) infection of human tracheal epithelial cells pretreated with LVFX. LVFX pretreatment reduced the RV14 titer, the level of cytokines in the supernatant, the amount of RV14 RNA in the cells after RV14 infection, and the cells' susceptibility to RV14 infection. LVFX pretreatment decreased the mRNA level of intercellular adhesion molecule 1 (ICAM-1), a receptor for RV14, in the cells and the concentration of the soluble form of ICAM-1 in the supernatant before RV14 infection. LVFX pretreatment also decreased the number and the fluorescence intensity of the acidic endosomes from which RV14 RNA enters the cytoplasm. LVFX pretreatment inhibited the activation of nuclear factor κB proteins, including p50 and p65, in nuclear extracts. LVFX pretreatment did not reduce the titers of RV2 (a minor human RV) but reduced the titers of RV15 (a major human RV). These results suggest that LVFX inhibits major-group rhinovirus infections in part by reducing ICAM-1 expression levels and the number of acidic endosomes. LVFX may also modulate airway inflammation in rhinoviral infections.     

人近端肾小管上皮细胞上表达黏附分子CD146与细胞的增殖生长状态

目的:观察正常人近端肾小管上皮细胞(HK2)是否表达黏附分子CD146,初步探讨CD146与肾小管上皮细胞的关系及其生理意义。方法:实验于2005-05/2006-02在上海交通大学医学院临床检验系实验室完成。①实验材料:人近端肾小管上皮细胞株(HK2,由中国科学院上海生物化学与细胞生物学研究所惠赠)。②实验干预:体外培养的HK2连续观察72h。③实验评估:采用倒置显微镜、光学显微镜下观察肾小管上皮细胞的形态;用反转录-聚合酶链反应方法检测CD146 mRNA的表达;流式细胞仪和免疫荧光法测定CD146蛋白质的表达及其定位;进一步在培养细胞的上清液中检测CD146的可溶性形式(sCD146)。结果:①肾小管上皮细胞的形态学观察和鉴定:传代培养的HK2三四天融合后呈铺路石样铺于培养瓶底,相关抗原检测抗人keratin阳性,抗Ⅷ因子相关抗原阴性。②CD146 mRNA水平的表达:HK2在培养早期(24h)即表达CD146 mRNA(0.092±0.012),但延长细胞的培养时间似乎并没有进一步改变CD146 mRNA的表达水平(0.097±0.005,0.113±0.015,P>0.05)。③CD146蛋白质水平的表达和定位:CD146不仅位于肾小管上皮细胞膜上,而且在细胞核和胞浆中也有表达。体外培养的HK2相互融合时,位于细胞膜上的CD146表达增强,呈线性、持续性地表达于细胞-细胞间的连接部位,同时胞内的CD146标记也相应增强。④细胞上清液中sCD146的检测:HK2上清液中存在CD146的可溶性形式(sCD146)。培养细胞观察24～72h,sCD146水平在48h升高[(18.00±0.80)μg/L],到72h达到高峰[(29.33±1.22)μg/L],与24h[(13.87±0.46)μg/L]比较,差异均有显著性意义(P<0.05)。结论:人近端肾小管上皮细胞组成性地表达CD146,是肾小管上皮细胞新的生物学标志;CD146在细胞内外的表达强度有赖于细胞间联系的建立和细胞增殖的程度,CD146在促进细胞生长和维持组织完整性等方面发挥重要作用。肾小管上皮细胞上清液中存在CD146的可溶性形式,sCD146在一定程度上也反映了细胞的生长和增殖状况。     

Effect of acute hypercapnia on renal and proximal tubular total carbon dioxide reabsorption in the acetazolamide-treated rat.

The present study evaluates the effect of acute hypercapnia on renal total CO2 (tCO2) reabsorption after inhibition of renal carbonic anhydrase. Simultaneous renal clearance studies and free-flow micropuncture studies of the superficial proximal tubule were performed on plasma-repleted Sprague-Dawley rats treated with acetazolamide, 50 mg/kg body weight. Acute hypercapnia (arterial PCO2, 120 mmHg; blood pH, 7.02) was induced by ventilation with a 10% CO2-90% O2 gas mixture. Control rats (PCO2, 49.5 mmHg, pH 7.34) were ventilated with room air. The renal fractional excretion of tCO2 was approximately 20% lower in the hypercapnic group compared with the rats given acetazolamide alone. Acute hypercapnia reduced the fractional delivery of tCO2 to the late proximal tubule by a comparable amount. The absolute proximal reabsorption of tCO2 was increased by hypercapnia to 410 +/- 47 vs. 170 +/- 74 pmol X min-1, P less than 0.05. The single nephron glomerular filtration rate was 32.6 +/- 0.7 nl X min-1 in the hypercapnic group and 43.8 +/- 1.7 nl X min-1 in the rats given acetazolamide only, P less than 0.01. Acute hypercapnia enhances renal sympathetic nerve activity. To eliminate this effect, additional experiments were performed in which the experimental kidney was denervated before study. Denervation prevented the change in the single nephron filtration rate during acute hypercapnia, but absolute and fractional proximal tCO2 reabsorption remained elevated in comparison to denervated controls. The concentration of H2CO3 in the late proximal tubule, calculated from the measured luminal pH and bicarbonate concentration and the estimated cortical PCO2, was higher in the hypercapnic group, which was a finding compatible with H2CO3 cycling from lumen into proximal tubular cell, which provided a source of hydrogen ions for secretion.     

Glomerular filtration is required for transfection of proximal tubular cells in the rat kidney following injection of DNA complexes into the renal artery

Gene transfer to the kidney can be achieved with various DNA vectors, resulting in transgene expression in glomerular or tubular districts. Controlling transgene destination is desirable for targeting defined renal cells for specific therapeutic purposes. We previously showed that injection of polyplexes into the rat renal artery resulted in transfection of proximal tubular cells. To investigate whether this process involves glomerular filtration of the DNA-containing particles, fluorescent polyethylenimine polyplexes were prepared, containing fluoresceinated poly-L-lysine. This allowed visualization of the route of the particles into the kidney. Our polyplexes were filtered through the glomerulus, since fluorescent proximal tubuli were observed. Conversely, fluorescent lipopolyplexes containing the cationic lipid DOTAP were never observed in tubular cells. Size measurements by laser light scattering showed that the mean diameter of polyplexes (93 nm) was smaller than that of lipopolyplexes (160 nm). The size of the transfecting particles is therefore a key parameter in this process, as expected by the constraints imposed by the glomerular filtration barrier. This information is relevant, in view of modulating the physico-chemical properties of DNA complexes for optimal transgene expression in tubular cells. Gene Therapy (2000) 7, 279-285.     

Succinate ameliorates energy deficits and prevents dysfunction of complex I in injured renal proximal tubular cells

We previously reported that mitochondrial function, intracellular ATP levels, and complex I activity are decreased in renal proximal tubular cells (RPTC) after oxidant (tert-butyl hydroperoxide; TBHP)-induced injury. This study examined the hypothesis that succinate supplementation decreases mitochondrial dysfunction, ameliorates energy deficits, and increases viability in TBHP-injured RPTC. Basal and uncoupled respirations in injured RPTC decreased 33 and 35%, respectively, but remained unchanged in injured RPTC supplemented with 10 mM succinate (electron donor to respiratory complex II). State 3 respiration supported by electron donors to complex I decreased 40% in injured RPTC but improved significantly by succinate supplements. The activity of mitochondrial complex I in TBHP-injured RPTC decreased 48%, whereas complex II activity remained unchanged. Succinate supplementation prevented decreases in complex I activity. ATP levels decreased 43% in injured RPTC but were maintained in injured cells supplemented with succinate. Lipid peroxidation increased 19-fold in injured RPTC but only 9-fold in injured cells supplemented with succinate. Exposure of primary cultures of RPTC to TBHP produced 24% cell injury and lysis but no apoptosis. In contrast, no cell lysis was found in RPTC supplemented with succinate. We conclude that mitochondrial dysfunction and energy deficits in oxidant-injured RPTC are ameliorated by succinate, and we propose that succinate supplementation may prove therapeutically valuable. Succinate 1) uses an alternate pathway of mitochondrial energy metabolism, 2) improves activity of complex I and oxidation of substrates through complex I, and 3) decreases oxidative stress and cell lysis in oxidant-injured RPTC.