Constantinescu A R, Rozental R, Barac-Nieto M
Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Pediatr Nephrol. 1996 Oct;10(5):606-12. doi: 10.1007/s004670050171.
Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in microdissected proximal convoluted tubules (PCT) of < 3-week-old (newborn) and > 12-week-old (adult) rabbits. Tubules were loaded with 10 microM fura-2 AM by incubation for 60 min at 37 degrees C, and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2 (control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245 +/- 10 to 975 +/- 100 nM, P < 0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137 +/- 5 to 165 +/- 5 nM, P < 0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920 +/- 90 nM in tubules from adult and to 396 +/- 16 nM in those from newborn rabbits (P < 0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-dependent uptakes of a glucose analogue (14C-alpha-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of < 3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na(+)-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.
钙（Ca2+）依赖性过程部分介导了缺氧性细胞损伤。这些过程可能解释了某些未成熟和成熟肾细胞对缺氧敏感性的差异。为了解决这个问题，我们研究了缺氧对3周龄以下（新生）和12周龄以上（成年）兔显微解剖的近端曲管（PCT）中胞质游离Ca2+浓度（[Ca2+]i）、细胞完整性和转运功能的影响。将小管在37℃孵育60分钟，用10微摩尔fura-2 AM加载，然后用95%O2-5%CO2（对照组）或95%N2-5%CO2（缺氧组）充气的等渗盐溶液灌注30分钟。通过比率测量法测定[Ca2+]i；通过碘化丙啶（PI）的核结合评估细胞损伤。缺氧导致成年小管中[Ca2+]i增加四倍（从静息值245±10增加到975±100纳摩尔，P<0.001），而新生小管中的升高明显较少（从静息值137±5增加到165±5纳摩尔，缺氧组之间P<0.001）。短暂暴露于100毫摩尔氯化钾使PCT细胞去极化，导致成年小管中[Ca2+]i从基线增加到920±90纳摩尔，新生兔小管中增加到396±16纳摩尔（年龄组之间P<0.001）。暴露于甲状旁腺激素（PTH）和ATP等配体后，新生和成年小管中的[Ca2+]i均增加，但新生小管中的增加幅度较小。两个年龄组对PTH和ATP的反应都是短暂的，[Ca2+]i在2分钟后恢复到基线水平。缺氧后，成年动物的小管在暴露于PI 1分钟后所有细胞核均出现染色，表明细胞发生了严重通透性改变。缺氧的未成熟小管细胞核未被PI染色。缺氧后，新生兔琼脂糖填充小管中葡萄糖类似物（14C-α-甲基葡萄糖苷）和磷酸盐（32Pi）的钠依赖性摄取得以保留，而成年兔小管从缺氧中恢复后，这些溶质的摄取显著减少。总体而言，我们的结果表明：（1）在缺氧期间，与3周龄以下动物相比，成年PCT中细胞Ca2+升高至临界水平；（2）Ca2+内流通过暴露于高[K+]o激活的途径发生，推测是电压敏感性Ca2+通道或Na(+)-Ca2+交换的逆转；（3）与成年兔相比，新生兔近端小管中的这些途径活性较低或数量较少；（4）新生兔而非成年兔的PCT细胞在缺氧后二级主动转运活性和细胞完整性得到良好保留。
