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The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.

Madin-Darby canine kidney (MDCK) cells, when seeded into collagen gel, from polarized, spherical, epithelial cysts, which grow by a process involving fluid secretion and cell proliferation. These cysts are a useful model for understanding the dynamics of cyst enlargement in renal cystic disease. The hypothesis that MDCK cyst fluid secretion depends upon chloride secretion was tested, and a cell model for this process is presented here. Lumen and epithelial cell volumes were measured by video microscopy in acute experiments. Fluid absorption (-0.073 +/- 0.007 microliters.h-1.cm-2; N = 8) was observed when cysts were superfused with unsupplemented Dublecco's modified Eagle's medium at 36 to 37 degrees C. Fluid secretion (0.221 +/- 0.0016 microliters.h-1.cm-2; N = 25) was seen when 1 mM dibutyryl cAMP plus 0.1 mM 3-isobutyl-1-methylxanthine were added to the superfusate. cAMP-induced fluid secretion was significantly inhibited by basolateral 1 mM ouabain, 0.1 mM furosemide, or 1 mM amiloride. It was not significantly affected by 1 mM chlorothiazide, 0.01 mM bumetanide, or 0.1 mM acetazolamide in the presence of normal bicarbonate/CO2. In the nominal absence of bicarbonate/CO2 fluid secretion was 18% of control. Vasopressin-induced fluid secretion was significantly inhibited by pretreatment of cysts with 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Cyst cell shrinkage in isosmotic chloride-free Ringer's solution (chloride replaced by gluconate) was inhibited by 0.1 mM basolateral DIDS. The results suggest that chloride-bicarbonate exchange in the basolateral membrane of MDCK cyst epithelial cells plays a critical role in cyst fluid secretion.

This study examined the effects on proximal tubule morphology of blocking single nephrons with paraffin wax for one day, one week, or one month in the rat. Proximal tubule lumens were blocked with a short column of wax using micropuncture. Chronically blocked and control (normal) tubules were fixed by either intravascular or intraluminal perfusion of glutaraldehyde solution. Proximal tubule segments downstream to the wax block were examined by light and transmission electron microscopy. Intraluminal Alcian blue dye, serial sectioning, and nephron microdissection techniques were used to identify nephrons. One day after obstruction, all proximal tubule cells downstream to the block were injured. Some recovery was seen. S1 and S2 segments showed more severe damage than S3 segments. Alcian blue, which normally is excluded from cells, entered the cytoplasm of some damaged S1-S2 cells. After one week of obstruction, the tubule appeared to have reconstituted itself, but cells were less differentiated than normal. One month after obstruction, blocked tubules were atrophied. Tubule cells were simplified and were surrounded by a thickened basement membrane. The results suggest that prolonged proximal tubule blockade produces injury and atrophy of the proximal tubule probably due to ischemia and interruption of normal reabsorptive activity.