Simoni J, Simoni G, Hartsell A, Feola M
Department of Surgery, Texas Tech University Health Sciences Center, Lubbock 79430, USA.
ASAIO J. 1997 Sep-Oct;43(5):M714-25.
Nephrotoxicity of free hemoglobin (Hb) based blood substitutes still awaits full elucidation. Previous reports attributed Hb passage through the renal glomeruli to a tendency of the Hb tetramer to dissociate into dimers. Now it has become more evident that the Hb tetramer is able to extravasate. It appears that the electrical charge of proteins plays an important role, with electronegativity and a low isoelectric point favoring intravascular persistence. This effect was utilized in the development of an improved blood substitute, comprising Hb reacted with o-ATP and o-adenosine, to form an intra- and intermolecularly cross linked product, which is reduced with glutathione. The modification reagents possess the desired pharmacologic activities and produce an increase in the electronegative charges on the Hb surface. All Hb polymers and chemically modified tetramers present in this solution have a uniform electronegative charge, with a pl of 6.1-6.2. In this present study, unmodified bovine Hb and an improved blood substitute were used for the replacement of 40% of the total blood volume in rats. The nephrotoxic effect was investigated by the determination of urinary output, glomerular filtration rate (GFR), fractional excretion of sodium (FENa), potassium (FEK), and chloride (FECl), urine/plasma osmolality ratio, and urine N-acetyl-beta-D-glucosaminidase (NAG) level. The free Hb and non heme protein contents in the urine were analyzed by using isoelectric focusing and size exclusion liquid chromatography methods. The results indicate that unmodified Hb is nephrotoxic. An initially elevated urinary output was followed by a significant oliguria associated with decreased GFR, FEK, and FECl and elevated FENa and NAG. Severe hemoglobinuria was associated with proteinuria. Analysis of urine from unmodified Hb treated rats revealed the presence of Hb tetramers. Histopathological examination of the kidneys showed cytoplasmic vacuolization of proximal tubular epithelium. On the contrary, an improved blood substitute did not produce any nephrotoxic reactions. It was found that this Hb solution did not pass through the renal glomerular barrier and was not present in urine samples. In conclusion, such a chemical and pharmacological alteration of Hb molecules reduced their interaction with renal glomeruli and suspended nephrotoxicity.
基于游离血红蛋白（Hb）的血液替代品的肾毒性仍有待全面阐明。先前的报道将Hb通过肾小球归因于Hb四聚体解离成二聚体的倾向。现在越来越明显的是，Hb四聚体能够渗出。看来蛋白质的电荷起着重要作用，负电性和低等电点有利于血管内持续存在。这种效应被用于开发一种改进的血液替代品，该替代品包括与邻-ATP和邻-腺苷反应的Hb，形成分子内和分子间交联产物，并用谷胱甘肽还原。修饰试剂具有所需的药理活性，并使Hb表面的负电荷增加。该溶液中存在的所有Hb聚合物和化学修饰的四聚体都具有均匀的负电荷，等电点为6.1-6.2。在本研究中，未修饰的牛Hb和一种改进的血液替代品用于替代大鼠总血容量的40%。通过测定尿量、肾小球滤过率（GFR）、钠（FENa）、钾（FEK）和氯（FECl）的分数排泄、尿/血浆渗透压比以及尿N-乙酰-β-D-氨基葡萄糖苷酶（NAG）水平来研究肾毒性作用。使用等电聚焦和尺寸排阻液相色谱法分析尿液中的游离Hb和非血红素蛋白含量。结果表明，未修饰的Hb具有肾毒性。最初尿量增加，随后出现明显少尿，伴有GFR、FEK和FECl降低以及FENa和NAG升高。严重血红蛋白尿与蛋白尿相关。对未修饰Hb处理的大鼠尿液分析显示存在Hb四聚体。肾脏的组织病理学检查显示近端肾小管上皮细胞胞质空泡化。相反，一种改进的血液替代品未产生任何肾毒性反应。发现这种Hb溶液未通过肾小球屏障，也不存在于尿液样本中。总之，Hb分子的这种化学和药理学改变减少了它们与肾小球的相互作用并消除了肾毒性。
