通过毛细管电泳测定的碘他拉酸盐是肾微穿刺中放射性标记菊粉的合适替代物。

Capasso Giovambattista, Unwin Robert J, Pica Angelo, Quagliuolo Lucio, Giovane Alfonso

Chair of Nephrology, Department of Biochemistry, Faculty of Medicine, Second University of Naples, Padiglione 17 Policlinico Nuovo, Via Pansini 5, 80131 Naples, Italy.

Kidney Int. 2002 Sep;62(3):1068-74. doi: 10.1046/j.1523-1755.2002.00510.x.

BACKGROUND

Inulin remains the gold standard for measurements of fluid reabsorption (Jv) and single nephron glomerular filtration rate (SNGFR) in micropuncture experiments. However, the method used to measure cold inulin in nanoliter samples is time-consuming, while the use of radiolabeled inulin is disadvantaged by possible radioactive contamination, disposal of radioactive material and cost of the isotope. It has been reported that non-radiolabeled iothalamate may be a suitable alternative for estimation of whole kidney GFR. The present study tested whether iothalamate can be used to measure Jv in microperfusion and free-flow micropuncture experiments.

METHODS

Superficial loops of Henle (LOH) were perfused from late proximal to early distal tubules with an end-like proximal solution. In the first set of experiments, the perfusate contained both iothalamate (1.9 mmol/L) and 3H-methoxy-inulin (50 microCi. mL(-1)). To test if iothalamate was able to detect changes in Jv, two additional sets of experiments were performed: (1) mannitol (61 mmol/L) was added to the perfusate to partially replace NaCl, a condition known to inhibit Jv; (2) LOH of remnant kidneys were perfused, which in previous experiments we showed to have a higher Jv. Lastly, free-flow micropuncture experiments were performed by infusing iothalamate IV at 18.3 mg. h(-1). Iothalamate analysis in nanoliter samples of renal tubular fluid obtained in vivo was performed by capillary electrophoresis (CE).

RESULTS

In the first set of experiments, liquid scintillation counting of 3H-methoxy-inulin versus iothalamate analysis with CE resulted in almost identical calculated perfusion rates (20.4 +/- 0.6 vs. 20.6 +/- 0.7 nL. min(-1), N = 20) and tubular fluid/perfusate ratios (TF/P; 1.35 +/- 0.04 vs. 1.36 +/- 0.04) and thus also Jv (5.17 +/- 0.50 vs. 5.38 +/- 0.59 nL. min(-1)). In the mannitol experiments, iothalamate measurements showed that the addition of mannitol significantly reduced Jv from 4.98 +/- 0.40 (N = 19) to 0.72 +/- 0.58 nL. min(-1) (N = 33; P < 0.0001). Iothalamate determinations by CE were able to detect a significant increase in Jv in LOH of remnant rats perfused at 40 nL. min(-1)[from to 8.40 +/- 0.73 (N = 20) in sham-operated to 17.8 +/- 2.9 nL. min(-1) (N = 6) in remnant animals; P < 0.0001]. In free flow micropuncture experiments the ratio of tubular fluid to plasma iothalamate (TF/P) along the proximal tubule was 1.62 +/- 0.10 (N = 15).

CONCLUSIONS

These data demonstrate that iothalamate can replace inulin to measure Jv in microperfusion and free-flow micropuncture experiments. Since iothalamate analysis by CE technique is a fast, easy and highly reproducible technique, it may become the gold standard method for the detection of fluid reabsorption in microperfused nephron segments.

背景

在微穿刺实验中，菊粉仍是测量液体重吸收（Jv）和单肾单位肾小球滤过率（SNGFR）的金标准。然而，用于测量纳升样本中冷菊粉的方法耗时较长，而使用放射性标记菊粉则存在放射性污染、放射性物质处理及同位素成本等问题。据报道，非放射性标记的碘他拉酸盐可能是估算全肾肾小球滤过率的合适替代物。本研究测试了碘他拉酸盐是否可用于微灌注和自由流微穿刺实验中测量Jv。

方法

从近端小管晚期到远端小管早期，用类似近端的溶液灌注浅表髓袢（LOH）。在第一组实验中，灌注液中同时含有碘他拉酸盐（1.9 mmol/L）和3H-甲氧基菊粉（50微居里·mL⁻¹）。为测试碘他拉酸盐能否检测Jv的变化，进行了另外两组实验：（1）向灌注液中加入甘露醇（61 mmol/L）以部分替代氯化钠，已知该条件可抑制Jv；（2）灌注残余肾的LOH，在先前实验中我们发现其Jv较高。最后，通过以18.3 mg·h⁻¹的速度静脉输注碘他拉酸盐进行自由流微穿刺实验。通过毛细管电泳（CE）对体内获得的肾小管液纳升样本中的碘他拉酸盐进行分析。

结果

在第一组实验中，对3H-甲氧基菊粉进行液体闪烁计数与用CE分析碘他拉酸盐得出的计算灌注率几乎相同（20.4±0.6对20.6±0.7 nL·min⁻¹，N = 20）以及肾小管液/灌注液比率（TF/P；1.35±0.04对1.36±0.04），因此Jv也相同（5.17±0.50对5.38±0.59 nL·min⁻¹）。在甘露醇实验中，碘他拉酸盐测量结果显示，加入甘露醇后Jv从4.98±0.40（N = 19）显著降低至0.72±0.58 nL·min⁻¹（N = 33；P < 0.0001）。通过CE测定碘他拉酸盐能够检测到以40 nL·min⁻¹灌注的残余大鼠LOH中Jv的显著增加[从假手术组的8.40±0.73（N = 20）增加到残余动物组的17.8±2.9 nL·min⁻¹（N = 6）；P < 0.0001]。在自由流微穿刺实验中，沿近端小管的肾小管液与血浆碘他拉酸盐的比率（TF/P）为1.62±0.10（N = 15）。