应用MTS方法检测生物人工肾小管的细胞活性

目的:探讨应用MTS方法在生物人工肾小管(RAD)构建过程中测定血液滤器中肾小管上皮细胞活性的可行性.方法:采用人肾近曲小管上皮细胞株(HK2)体外培养,然后按不同浓度植入6孔板,应用MTS方法测定490 nm吸光度(OD)值,判断其测定值与细胞数量之间的关系.然后将培养的HK2细胞植入FH66血液滤过器内腔继续培养构建RAD,在培养过程中每隔3 d用MTS方法测定一次OD值,并与无细胞植入的空白滤器作对照.结果:HK2细胞数在105～106范围内,与OD值呈正相关关系,相关系数(r)为0.985,P<0.01.各时间点RAD组MTS测定均值与对照组相比均有统计学意义(P<0.01,n＝5).RAD组MTS测定值随培养时间的延长而增加,提示RAD在培养过程中细胞增殖.结论:MTS方法能较好地反映RAD内的细胞活性,能在RAD培养过程中作为一种简便有效的手段监测滤器内的细胞活性.     

生物人工肾小管对β_2微球蛋白重吸收功能的研究

目的：探讨应用人肾近曲小管上皮细胞株（HK2）构建的生物人工肾小管辅助装置（RAD）体外对β2微球蛋白（β2-MG）的清除作用及机制。方法：A组以HK2体外培养后植入F60血液滤过器内腔构建的RAD后用含β2-MG的置换液体外模拟液体滤过,B组以上述条件构建的RAD用不含β2-MG的置换液模拟液体滤过,C组用含β2-MG的置换液对无细胞植入的F60滤器模拟液体滤过,对各组测定不同时间点的β2-MG滤过率,并应用免疫荧光法观察滤过后A、B组HK2细胞Megalin与β2-MG的蛋白表达。结果：A组β2-MG滤过率在各时间点均明显高于C组（P〈0.05）;C组β2-MG滤过率在第4h较同组前期各时间点明显降低（P〈0.05）。免疫荧光显示A组Megalin与β2-MG同步表达,B组则仅见Megalin表达。结论：以HK2细胞构建的RAD可显著提高β2-MG的滤过率,其功能在4小时内可稳定维持,作用机制可能与Megalin的内吞作用相关。     

生物人工肾小管对多器官功能衰竭细胞因子及存活时间的影响

目的研究用细胞株(LLC-PK1)构建的生物人工肾小管装置(RAD)对多器官功能衰竭(MOF)猪白介素(IL)-10、肿瘤坏死因子(TNF)α及存活时间的影响。方法ARF并MOF猪随机分为3组:(1)RAD组(A组,n=5):将RAD与血滤器相连,行连续静脉静脉血液滤过(CVVH)治疗24h;(2)假RAD组(B组,n=5):将RAD换为无细胞的假RAD,余与A组同;(3)未治疗组(C组,n=6)。观察血压、肝肾功能、血气、血IL-10、TNF-α(用特异性猪IL-10、TNF-α抗体检测)和生存时间。结果(1)A组治疗24h低血压状况显著改善;4～20h精神状态明显好转。(2)血IL-10(pg/ml)的峰值A组明显高于B、C组(241.40±86.64比106.30±9.69、102.59±10.21,P<0.05)。(3)血TNF-α(pg/ml)A组治疗后较治疗前明显降低(415.30±34.83比526.67±40.08,P<0.05);B、C两组无明显变化。(4)A组生存时间为(110.25±18.69)h,明显长于B组(81.20±11.76)h和C组(74.96±23.00)h(P<0.05),A组比B、C组分别延长了35.8%、47.1%。结论用RAD治疗明显改善了MOF猪低血压,升高血IL-10、降低TNF-α及延长存活时间。     

含黄芪腹透液对高腹膜转运CAPD患者超滤功能的影响

目的:观察含黄芪腹透液对高腹膜转运CAPD患者腹膜超滤功能的影响。方法:高腹膜转运CAPD患者34例,治疗组(含黄芪腹透液)与对照组(市售腹透液)各17例,观察治疗后透析超滤量(UF)、腹膜溶质转运性能(D/P Cr、D/P urea、MTAC urea、MTAC Cr)及葡萄糖吸收率的变化。结果:治疗组治疗后第1透析周期超滤量和24h总超滤量明显增加,治疗观察期高糖透析液使用量减少,葡萄糖吸收率有降低趋势,并能一定程度地提高腹膜对溶质的转运,总有效率76.5%,优于对照组(P<0.05)。结论:腹透液中加入黄芪注射液可有效地提高透析超滤量,对高腹膜转运CAPD患者腹膜的超滤功能具有较好的保护作用。     

腹膜溶质转运特性对腹膜透析患者营养指标的影响

目的探讨腹膜溶质转运特性对腹膜透析患者营养指标的影响.方法按照腹膜平衡试验(PET)计算结果,将68例稳定的连续性不卧床性腹膜透析(CAPD)患者分为高转运组(37例)和低转运组(31例).检测患者血浆和腹透透出液中的总蛋白、白蛋白及氨基酸量,同步计算蛋白质摄入量(DPI)和蛋白质分解率(nPCR).比较两组营养状态及进行相关因素分析.结果两组的残余肾功能、CAPD治疗时间、每日透析液剂量、超滤量、葡萄糖吸收量、血糖、尿素氮(BUN)、血清肌酐(Scr)和尿素清除指数(Kt/V)比较,差异无显著性意义(均P>0.05).高转运组每日经腹透透出液丢失的总蛋白质和多种氨基酸量显著高于低转运组(均P<0.05);各项营养指标均低于低转运组;两组体重及血清白蛋白比较,差异有显著性意义(均P<0.05).每日经腹透液丢失的Alb量和TAA与D/P4Scr呈正相关关系(均P<0.05).结论腹膜透析高转运患者营养指标较低转运者差,应加强对高转运患者的营养护理和指导;每日透析液量及超滤量能达到TCcr与DPI平衡即可,不宜盲目增加腹透液的剂量、浓度和存留时间,导致过度透析,引起蛋白质和氨基酸经腹透液大量丢失及吸收大量的葡萄糖,加重患者蛋白质缺乏性营养不良.     

氨基酸腹膜透析液与传统腹膜透析液对腹膜间皮细胞功能的影响

目的通过体外实验对照观察了含氨基酸腹膜透析液(1.1%Nutrineal)和传统葡萄糖腹膜透析液(1.5%Dianeal)对正常人腹膜间皮细胞(HPMC)的生物相容性及功能的影响。方法用1.1%Nutrineal和1.5%Dianeal进行腹膜透析,4h后引流。观察两种不同透出液及不同时间对体外培养的HPMC功能的影响。采用四甲基偶氮多胍(MTT)和细胞直接计数法测定HPMC增殖程度,以测定乳酸脱氢酶(LDH)水平显示细胞损伤程度。采用ELISA法测定白细胞介素6(IL-6)水平。结果与5%葡萄糖对照组相比,1.5%Dianeal透出液可显著降低HPMC细胞增殖能力、细胞总蛋白合成、IL-6分泌水平和细胞黏附能力(P<0.001、P<0.001、P<0.0001、P<0.0001),而细胞培养液上清中的LDH分泌水平明显提高(P<0.001)。1.1%Nutrineal透出液可降低HPMC细胞增殖能力(P<0.001)和明显增加IL-6的分泌水平(P<0.0001),但对HPMC的LDH分泌水平,细胞黏附能力,蛋白总合成量无明显影响。结论与传统的葡萄糖腹膜透析液相比,氨基酸腹膜透析液能更好地维持HPMC在细胞存活能力、细胞黏附功能及蛋白合成等方面的功能。氨基酸腹膜透析液可诱导HPMC分泌IL-6水平显著升高,其机制有待进一步探讨。     

达格列净联合胰激肽原酶治疗糖尿病肾病的效果及对HbA1c、尿蛋白水平的影响

<正>糖尿病肾病(diabetic nephropathy,DN)是糖尿病中多见的并发症之一,肾功能检测显示患者出现大量蛋白尿,且肾小球滤过率降低,患者可出现水肿,还常伴有糖尿病视网膜病变等其他微血管并发症^[1]。达格列净是一种新型抗糖尿病的药物,属于钠-葡萄糖转运蛋白2(sodium-dependent glucose transporters 2,SGLT-2)抑制剂,其具有独特的降糖机制,通过抑制葡萄糖与钠离子的重吸收而起到降糖降压的作用^[2]。胰激肽原酶是血管扩张药,主要通过改善微循环,     

严重烧伤早期心肌收缩性与钙转运功能变化

目的研究烧伤早期心肌肌浆网(SR)钙转运功能变化,探讨其在烧伤后心肌收缩功能下降发病中的作用。方法采用30％TBSAⅢ度烧伤大鼠模型,离体心脏灌流,测定伤前及伤后心肌室内压最大变化速率(±dp/dtmax)变化,制备心肌肌浆网,应用微孔滤膜过滤技术测定心肌SR~(45)Ca~(2 )转运功能改变。结果与对照组相比,烧伤组左心室±dp/dtmax明显降低(P<0.01),心肌SRCa~(2 )-ATPase活性及SR~(45)Ca~(2 )摄取初速度、摄取容量均明显降低(P<0.01),偶联率比对照值大幅度下降。结论烧伤后早期心肌肌浆网Ca(2 )转运功能严重障碍,其是烧伤后心肌收缩功能降低的重要因素之一。     

在线血液透析滤过的临床疗效观察

目的 观察血液透析滤过(HDF)和血液透析(HD)对尿毒症患者不同溶质的清除和常见并发症的发生率.方法 45例规律透析患者前6个月行HD治疗为自身对照组.后6个月间歇行HDF治疗为实验组.对照组采用F6聚砜膜透析器,每周透析2～3次,每次4小时;实验组采用F60s聚砜膜透析器,每周透析2～3次,每次4小时,每2周HDF 1次.观察两组透析前后溶质:血尿素氮(Bun)、肌酐(Cr)、β2微球蛋白(β2-MG)清除及透析相关并发症的发生率.结果 两组透析前后Bun、Cr、均明显下降(P<0.01).HDF组透后β2-MG明显低于透前(P<0.01),HD组透析前后β2-MG差异无显著性(P>0.05).HDF组并发症的发生率较HD组明显降低(P<0.05).结论 HDF较HD能明显清除中分子物质,能明显降低透析相关并发症.     

高容血液稀释联合控制性降压对腰椎手术病人肾小球功能和内环境的影响

目的 探讨术前急性高容血液稀释联合控制性降压对腰椎手术病人肾小球功能和内环境的影响。方法 将30例腰椎骨折椎板减压、切复内固定病人随机分为两组,术前急性高容血液稀释联合术中硝普钠控制性降压组(联合组)和单纯硝普钠控制性降压组(对照组),每组15例。测定联合组插管后稀释前(T0)、稀释后降压前(T1)、降压后30min(T2)和停降压后30min(T3)四个时间点,对照组插管后降压前(T1)、降压后30 min(T2)和停降压后30 min(T3)三个时间点动脉血K 、Na 、Ca2 、Cl-浓度和血气分析以及血浆β2-微球蛋白(β2-MG)的变化。结果 pH值:联合组T1-3较T0降低(P<0.01),而且低于对照组(P<0.05),T1、T2和T3之间无明显改变(P>0.05)。两组K 、Na 、Ca2 、Cl-和BE的变化比较无统计学意义(P>0.05)。联合组β2-MGT1低于T0(P<0.05);对照组T2较T1升高(P<0.05),而且高于联合组(P<0.05)。结论 硝普钠控制性降压可降低肾小球滤过率,术前急性高容血液稀释可以改善术中控制性降压对肾小球滤过率的影响,对内环境无明显影响。     

The bioartificial kidney in the treatment of acute renal failure

The application of cell therapy to the successful substitution process of hemofiltration may improve the poor prognosis of patients with acute renal failure (ARF) in the intensive care unit. An extracorporeal bioartificial kidney consisting of a conventional hemofilter followed in series with a renal tubule assist device (RAD) has been developed. The RAD is a hemofiltration cartridge containing 109 human renal tubule cells grown as monolayers along the inner surface of the hollow fibers. The fibers provide a porous scaffold that is immunoprotective. The ultrafiltrate from the hemofilter is delivered to the luminal compartment of the RAD, and the postfiltered blood is delivered to the extracapillary space of the RAD. The RAD has been shown to possess multiple differentiated transport, metabolic, and endocrinologic activities of renal epithelium. These activities have been demonstrated to occur when the RAD is placed in the extracorporeal circuit of the bioartificial kidney in uremic animals. This approach may improve the current therapies used to treat patients with ARF because of the RAD's ability to restore lost metabolic renal function and cytokine balance in these desperately ill patients. In this regard, the RAD was able to ameliorate endotoxin and bacteremic shock in uremic animals by altering cytokine levels, improve mean arterial blood pressure, and maintain better cardiac output. With these supportive preclinical data, an FDA-approved phase I/II clinical trial has been initiated and early results are encouraging.     

Tissue engineering of a bioartificial renal tubule assist device: in vitro transport and metabolic characteristics.

BACKGROUND: Current renal substitution therapy for acute or chronic renal failure with hemodialysis or hemofiltration is life sustaining, but continues to have unacceptably high morbidity and mortality rates. This therapy is not complete renal replacement therapy because it does not provide active transport nor metabolic and endocrinologic functions of the kidney, which are located predominantly in the tubular elements of the kidney. METHODS: To optimize renal substitution therapy, a bioartificial renal tubule assist device (RAD) was developed and tested in vitro for a variety of differentiated tubular functions. High-flux hollow-fiber hemofiltration cartridges with membrane surface areas of 97 cm2 or 0. 4 m2 were used as tubular scaffolds. Porcine renal proximal tubule cells were seeded into the intraluminal spaces of the hollow fibers, which were pretreated with a synthetic extracellular matrix protein. Attached cells were expanded in the cartridge as a bioreactor system to produce confluent monolayers containing up to 1.5 x 109 cells (3. 5 x 105 cells/cm2). Near confluency was achieved along the entire membrane surface, with recovery rates for perfused inulin exceeding 97 and 95% in the smaller and larger units, respectively, compared with less than 60% recovery in noncell units. RESULTS: A single-pass perfusion system was used to assess transport characteristics of the RADs. Vectorial fluid transport from intraluminal space to antiluminal space was demonstrated and was significantly increased with the addition of albumin to the antiluminal side and inhibited by the addition of ouabain, a specific inhibitor of Na+,K+-ATPase. Other transport activities were also observed in these devices and included active bicarbonate transport, which was decreased with acetazolamide, a carbonic anhydrase inhibitor, active glucose transport, which was suppressed with phlorizin, a specific inhibitor of the sodium-dependent glucose transporters, and para-aminohippurate (PAH) secretion, which was diminished with the anion transport inhibitor probenecid. A variety of differentiated metabolic functions was also demonstrated in the RAD. Intraluminal glutathione breakdown and its constituent amino acid uptake were suppressed with the irreversible inhibitor of gamma-glutamyl transpeptidase acivicin; ammonia production was present and incremented with declines in perfusion pH. Finally, endocrinological activity with conversion of 25-hydroxy(OH)-vitamin D3 to 1,25-(OH)2 vitD3 was demonstrated in the RAD. This conversion activity was up-regulated with parathyroid hormone and down-regulated with increasing inorganic phosphate levels, which are well-defined physiological regulators of this process in vivo. CONCLUSIONS: These results clearly demonstrate the successful tissue engineering of a bioartificial RAD that possesses critical differentiated transport, and improves metabolic and endocrinological functions of the kidney. This device, when placed in series with conventional hemofiltration therapy, may provide incremental renal replacement support and potentially may decrease the high morbidity and mortality rates observed in patients with renal failure.     

Efficacy and safety of renal tubule cell therapy for acute renal failure

The mortality rate for patients with acute renal failure (ARF) remains unacceptably high. Although dialysis removes waste products and corrects fluid imbalance, it does not perform the absorptive, metabolic, endocrine, and immunologic functions of normal renal tubule cells. The renal tubule assist device (RAD) is composed of a conventional hemofilter lined by monolayers of renal cells. For testing whether short-term (up to 72 h) treatment with the RAD would improve survival in patients with ARF compared with conventional continuous renal replacement therapy (CRRT), a Phase II, multicenter, randomized, controlled, open-label trial involving 58 patients who had ARF and required CRRT was performed. Forty patients received continuous venovenous hemofiltration + RAD, and 18 received CRRT alone. The primary efficacy end point was all-cause mortality at 28 d; additional end points included all-cause mortality at 90 and 180 d, time to recovery of renal function, time to intensive care unit and hospital discharge, and safety. At day 28, the mortality rate was 33% in the RAD group and 61% in the CRRT group. Kaplan-Meier analysis revealed that survival through day 180 was significantly improved in the RAD group, and Cox proportional hazards models suggested that the risk for death was approximately 50% of that observed in the CRRT-alone group. RAD therapy was also associated with more rapid recovery of kidney function, was well tolerated, and had the expected adverse event profile for critically ill patients with ARF.     

Role of rBAT Gene Products in Cystinuria

To investigate whether rBAT gene products function as a crystine transporter component or as a transport activator, we microinjected several C–terminal deletion mutants of rBAT cRNA into Xenopus oocytes, and measured transport activity for arginine, leucine and cystine in the presence and absence of sodium. Wild type rBAT significantly stimulated the uptake of all 3 amino acids 10–20 fold compared to control mutants. On the other hand, no mutant, except a Δ511–685 mutant, stimulated the uptake of these amino acids. However, the Δ511–685 mutant significantly increased the uptake of arginine. In the presence of sodium, the Δ511–685 mutant also increased the uptake of leucine. The Δ511–685 mutant did not stimulate crystine uptake in the presence and absence of sodium. Furthermore, inhibition of L–arginine uptake by L–homoserine was seen only in the presence of sodium. These results suggest that mutant rBAT stimulates the endogenous amino acid transport system y⁺ in oocytes. Finally, rBAT gene products, as the primary cause of cystinuria, may function as activators of the amino acid transport system in renal brush border membrane.     

Evaluation of long-term transport ability of a bioartificial renal tubule device using LLC-PK1 cells.

BACKGROUND: Haemodialysis therapy does not provide renal tubule function, such as active fluid and solute transport, nor metabolic or endocrine action. Moreover, this treatment is usually associated with serious complications and high mortality. We constructed a bioartificial renal tubule device by using renal tubule epithelial cells in an artificial membrane, and evaluated transport properties of the device for 2 weeks. METHODS: A renal epithelial cell line, LLC-PK(1) (Lewis-lung cancer porcine kidney), was seeded on polysulfone hollow fibres in small and large modules. We studied perfusion and leakage of urea nitrogen (UN) and creatinine (Cr), as well as reabsorption of water, glucose and sodium for a period of 2 weeks. RESULTS: Cell-lined hollow fibre membranes significantly reduced the leakage of UN and Cr throughout the 2 week period. Reabsorption of water, glucose and sodium were adequate from days 3 to 10 and gradually decreased thereafter. LLC-PK(1) cells actively transported these substances. Scanning electron microscopy revealed that cells in the hollow fibres on day 8 became completely confluent. However, they became multi-layered and almost obstructed the hollow fibres on day 13. CONCLUSIONS: This bioartificial renal tubule device functioned to reabsorb water, glucose and sodium for approximately 10 days. This is the first report of successful long-term evaluation of a bioartificial renal tubule device. This device, in combination with continuous haemofiltration, may provide treatment to prevent complications of dialysis and raise the quality of life in chronic renal failure patients.     

Effects of a rice meal on renal hemodynamics and excretory functions in normal subjects

The nature of renal hemodynamic response to a large carbohydrate-rich meal and the associated renal excretory functions were examined. Seven normal subjects were studied after ingestion of 300 g of rice meal. Five healthy men served as a time control. After eating the test meal, the mean creatinine clearance began to rise and became significantly higher in the 2nd hour compared with the control (118 +/- 7 vs. 91 +/- 3 ml/min/1.73 m2; p less than 0.01). Urinary excretion rates of sodium and chloride after the meal were significantly higher compared with those in the premeal period. Blood glucose concentrations rose significantly following the meal. The postprandial plasma levels of amino acids did not vary significantly compared with the premeal values. These results demonstrate that rice meal ingestion increased glomerular filtration rate via a mechanism(s) different from the protein-induced glomerular hyperfiltration.     

Renal function following acute renal failure in childhood: a long term follow-up study

We measured glomerular and tubular function in 10 children, aged 7 to 19 years, 7 to 12 years after apparent recovery from their episodes of acute renal failure. Although glomerular filtration rate was normal in all, filtration fraction (inulin clearance/PAH clearance) was elevated in six of eight patients. Segmental tubular sodium transport was assessed using clearance techniques during hypotonic saline diuresis; both proximal and distal sodium reabsorption were normal when compared with age-appropriate normal standards. Tubular reabsorption of phosphate, glucose and amino acids were also normal, as were urinary concentrating and diluting capacity and distal tubular hydrogen ion secretion. We conclude that, despite normal glomerular filtration rate, glomerular function is abnormal in a significant number of children who have apparently recovered completely from ARF, probably due to destruction of a proportion of the total nephron population, predominantly those located in the superficial layers of the cortex.