Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy

Weinlich, M.; Capasso, Giovambattista; Kinne, Rolf

doi:10.1007/bf00375081

Cited by 18 publications

(19 citation statements)

References 32 publications

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“…Only one tubule was studied from each rat. Calibration of pH i was performed in the same tubule at the end of each experiment using the high-potassium nigericin technique (25,30).…”

Section: Methodsmentioning

confidence: 99%

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

Walstead

Yip

2004

American Journal of Physiology-Regulatory, Integrative and Comp

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Walstead, Christopher, and Kay-Pong Yip. Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR. Am J Physiol Regul Integr Comp Physiol 286: R726-R733, 2004. First published December 24, 2003 10.1152/ ajpregu.00352.2003.-The effect of acute arterial hypertension on proximal tubular fluid reabsorption was investigated in SpragueDawley rats and spontaneously hypertensive rats (SHR) by measuring proximal tubular flow with a nonobstructive optical method. Under control conditions, spontaneous tubular flow was oscillating at 0.02-0.03 Hz in Sprague-Dawley rats. Acute hypertension induced an immediate increase of mean tubular flow (50% increase after 20 min of hypertension) and augmentation of oscillatory amplitude. Acute hypertension did not alter single-nephron blood flow as measured by laser-Doppler velocimetry (n ϭ 12), suggesting that the increase of tubular flow was due to inhibition of reabsorption but not increase of filtration. By contrast, spontaneous tubular flow was fluctuating aperiodically in SHR. Acute hypertension did not induce a continuous increase of tubular flow or an increase in amplitude of fluctuations (n ϭ 15). When apical Na ϩ /H ϩ exchanger activity of proximal tubule was monitored, acute hypertension did not alter the activity in SHR (n ϭ 8), while similar procedures had been shown to inhibit apical Na ϩ /H ϩ exchanger activity of proximal tubules by more than 40% in Sprague-Dawley rats. These observations suggest that acute hypertension inhibits proximal tubular fluid reabsorption by inhibiting apical Na ϩ /H ϩ exchanger activity in Sprague-Dawley rats and that this mechanism is impaired in SHR.laser-Doppler velocimetry; sodium/hydrogen exchangers; oscillations; tubuloglomerular feedback ACUTE ARTERIAL HYPERTENSION induced by increasing total peripheral resistance with aortic clamps triggers a reversible inhibition of apical Na ϩ /H ϩ exchange activity in proximal tubules of Sprague-Dawley rats (32). Apical Na ϩ /H ϩ exchangers are responsible for 90% of NaCl transcellular reabsorption and 70% of NaHCO 3 reabsorption in the proximal tubule (1). Inhibition of apical Na ϩ /H ϩ exchange activity by acute hypertension is likely to increase proximal tubular flow because of the reduction in NaCl and NaHCO 3 reabsorption. However, the conventional methods of measuring tubular flow by collecting tubular fluid interrupts ambient tubular flow, which might increase glomerular filtration via tubuloglomerular feedback. To circumvent this complication, a nonobstructive optical method was used in the present study to monitor directly the changes of spontaneous proximal tubular flow during the induction of acute hypertension. Moreover, single-nephron blood flow on the efferent arteriole was monitored with a noninvasive laser-Doppler velocimetry device (23, 29) to delineate whether the increase of tubular flow induced by acute hypertension is due to the inhibition of tubular fluid reabsorption or the increase of glomerular filtration.Acute hypertension-...

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“…Only one tubule was studied from each rat. Calibration of pH i was performed in the same tubule at the end of each experiment using the high-potassium nigericin technique (25,30).…”

Section: Methodsmentioning

confidence: 99%

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

Walstead

Yip

2004

American Journal of Physiology-Regulatory, Integrative and Comp

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“…The relative intensity measured at 488 nm excitation plotted against the distance of the focus from the bead yielded a bell-shaped curve [16]. The depth of focus (∆Z) is the distance of the focus from the bead at half maximal intensity.…”

Section: Determination Of the Depth Of Fieldmentioning

confidence: 99%

“…An approximation of the depth of field ∆Z can be calculated by using the equation ∆Z = 0.89 · n · λ/(NA) 2 · ∆Z is the distance of the focus at half maximum intensity, n is the refractive index, λ is the excitation wavelength and NA is the numerical aperture of the objective [16].…”

Section: Determination Of the Depth Of Fieldmentioning

confidence: 99%

“…Excitation with two lasers was provided to measure ratio values. A second 442 nm He-Cd laser (Liconix, Santa Clara, Calif., USA) was adapted to the pre-existing 488-nm Argon ion laser (Ion laser technology, Utah) [16]. A ×20 objective (Nikon, Japan) with a numerical aperture of 0.75 was used.…”

Section: Confocal Laser Scan Microscopy With Two Excitation Wavelengthsmentioning

confidence: 99%

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Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF

Weinlich¹,

Heydasch

Mooren

et al. 1998

Res. Exp. Med.

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The combination of confocal laser scan microscopy and the pH-sensitive fluorescent dye BCECF allowed us to record simultaneously intracellular pH, cell viability and relative cell volume. pH was measured by using the pH-sensitive excitation wavelength at 488 nm and the pH-independent excitation wavelength at 442 nm to obtain ratio images. Cell volume was traced by measuring fluorescence dye concentration at 442 nm. Isolated villus tip rat duodenal enterocytes were exposed to 20 mM NH4Cl, sodium free, or 1 mM amiloride buffer. Sodium free buffer and amiloride buffer acidified the cells. Cell volume did not change in sodium free buffer, or NH4Cl exposure, but amiloride led to an increase in cell volume of 20%. After acidification of the duodenal cells, amiloride buffer increased cell volume by almost 50%. These studies revealed that cell volume regulation during pH changes in short-living cells could easily be detected by confocal microscopy and BCECF.

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“…There has been little attempt, or limited progress, in adapting this technique for use in vivo. Epifluorescence and confocal fluorescence microscopy has been applied to studies of tubular cell intracellular pH in vivo [3]and in vitro [4, 5]. …”

Section: Introductionmentioning

confidence: 99%

Conventional and Confocal Epi-Reflection and Fluorescence Microscopy of the Rat Kidney in vivo

Boyde

Capasso²,

Unwin³

1998

Nephron Exp Nephrol

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To visualize superficial and accessible renal tubule cells functioning in situ and to relate what we can ‘see’ to what we know of their function from more invasive in vivo or less direct in vitro studies means applying and adapting recent advances in epifluorescence and confocal microscopy to improve image resolution and to combine this with the use of fluorescent labels to monitor the handling of specific molecules by the proximal and distal renal tubule cells in vivo. Doing this in living tissue is novel, especially in the kidney. Application of confocal microscopy to the imaging of living tissue, as opposed to isolated cells, has not been widely reported. The kidney surface has been imaged before using the confocal microscope and in preliminary studies we have extended this by using a different confocal system with and without fluorescence. While the studies published up to now have been morphological, comparing standard renal (structural) histology of surface glomeruli and renal tubules with the corresponding in vivo confocal images, more dynamic, real-time studies have been limited. Individual red blood cells can be seen flowing around the peritubule capillary network and nucleated white blood cells can also be distinguished. Tubule cells, endothelial cells, the proximal tubule cell brush border and cell mitochondria can be visualized. Filtration and secretion can be observed, and the early and late parts of the proximal tubule distinguished, and the distal tubule recognized. Localization of fluorescently labeled insulin to the luminal brush border and progressive uptake of label and distribution within proximal tubule cells toward the basolateral (blood side) membrane can be demonstrated. The possibility of monitoring hemodynamic changes and tracking the filtration, uptake, secretion and absorption of fluorescently tagged molecules, as well as intracellular fluorescence, e.g. calcium or pH, is an exciting prospect and is ripe for detailed exploration.

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Intracellular pH in renal tubules in situ: single-cell measurements by confocal laserscan microscopy

Cited by 18 publications

References 32 publications

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

Acute arterial hypertension inhibits proximal tubular fluid reabsorption in normotensive rat but not in SHR

Simultaneous detection of cell volume and intracellular pH in isolated rat duodenal cells by confocal microscopy and BCECF

Conventional and Confocal Epi-Reflection and Fluorescence Microscopy of the Rat Kidney in vivo

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