Thermoprotection of a functional epithelium: heat stress effects on transepithelial transport by flounder renal tubule in primary monolayer culture.

Primary monolayer cultures of winter flounder renal proximal-tubule cells were used to determine whether transepithelial transport could be protected from the damaging effects of extreme temperature by previous mild heat shock. Renal tubule epithelial cells were enzymatically dispersed and reorganized as confluent monolayer sheets on native rat tail collagen. Transepithelial electrical properties (potential difference, resistance, short-circuit current, and Na(+)-dependent glucose current) and unidirectional [35S]sulfate fluxes were measured in Ussing chambers at 22 degrees C. Examination of transepithelial electrical properties following acute 1-hr elevation of temperature over a range of 22-37 degrees C provided the basis for the "mild" versus "severe" thermal stress protocols. Severe elevation from 22 degrees C to 32 degrees C for 1.5 hr followed by 1.5 hr at 22 degrees C significantly decreased glucose current (7 +/- 0.7 to 3 +/- 0.8 microA/cm2) as well as net sulfate secretion [131 +/- 11 to 33 +/- 11 nmol/(cm2.hr)]. Mild heat shock of 27 degrees C for 6 hr prior to this severe heat shock completely protected both glucose transport (6 +/- 0.7 microA/cm2) and sulfate flux (149 +/- 13 nmol/(cm2.hr)]. Scanning electron microscopy showed that the number of microvilli on the apical (luminal) surface of the epithelium was decreased after a 32 degrees C heat shock. Monolayers exposed to 27 degrees C for 6 hr prior to incubation at 32 degrees C showed no loss of microvilli. SDS/PAGE analysis of protein patterns from the cultures showed that three classes of heat shock proteins were maximally induced at 27 degrees C. Inhibition of protein synthesis by cycloheximide prevented the thermoprotective effect of mild heat shock. This suggests that certain renal transport functions can be protected from sublethal but debilitating thermal stress by prior mild heat shock and that heat shock proteins may play a role in this protection. IMAGES:

本研究采用冬鲽肾近端小管细胞原代单层培养物，探究预先施加温和热激是否可使跨上皮转运功能免受极端温度的损伤。实验中将肾小管上皮细胞经酶解法分散后，在天然大鼠尾胶原基质上重建成致密汇合的单层细胞层。于22℃环境下，借助尤斯灌流室（Ussing chamber）检测跨上皮电生理指标，包括电位差、跨上皮电阻、短路电流以及钠依赖型葡萄糖电流，并同步测定单向[³⁵S]硫酸盐通量。通过在22℃至37℃范围内进行1小时急性升温后检测跨上皮电生理特性，确立了‘温和热激’与‘极端热激’两种应激处理方案的判定标准。将培养物从22℃升温至32℃持续1.5小时，随后恢复至22℃培养1.5小时的极端热激处理，可显著降低葡萄糖电流（从7±0.7 μA/cm²降至3±0.8 μA/cm²）与硫酸盐净分泌量（从131±11降至33±11 nmol/(cm²·h)）。若在该极端热激处理前先行施加27℃、持续6小时的温和热激，则可完全保护葡萄糖转运功能（维持于6±0.7 μA/cm²）与硫酸盐通量（维持于149±13 nmol/(cm²·h)）。扫描电子显微镜观察显示，经32℃热激后，上皮细胞顶（管腔）侧表面的微绒毛数量显著减少；而预先经27℃处理6小时再置于32℃培养的单层细胞，其微绒毛数量未出现丢失。对培养物的蛋白质谱进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS/PAGE）分析后发现，27℃条件下可最大程度诱导三类热休克蛋白的表达。使用放线菌酮（cycloheximide）抑制蛋白质合成，可阻断温和热激带来的热保护效应。上述结果表明，预先施加温和热激可使部分肾脏转运功能免受亚致死性但具有损伤性的热应激影响，且热休克蛋白可能参与了这一保护过程。图像：