Effect of hypernatremia on protein reabsorption in renal proximal tubules of the lake frog Pelophylax ridibundus

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Abstract

Protein reabsorption in the kidney proximal tubules occurs simultaneously with the transport of ions and water, but little is known about the dependence of receptor-mediated protein endocytosis on water-salt balance changes. The aim of the study was to investigate tubular reabsorption and intracellular vesicular transport of various proteins in a model of hypernatremia in lake frogs (Pelophylax ridibundus). Frogs were injected with hypertonic sodium chloride solution (0.75 M NaCl) 1 hour before injection of green or yellow fluorescent proteins (GFP or YFP), as well as lysozyme. The method of fluorescent immunohistochemistry was used for detection of lysozyme and endocytic receptor megalin in kidney sections. Specimens were investigated using laser scanning confocal microscopy. The intensity of fluorescent signals of proteins and megalin in proximal tubular cells was determined on the images obtained. To study the dynamics of endocytosis, an automated method for quantifying colocalized protein and megalin signals was used. A statistically significant decrease in the reabsorption of GFP, YFP and lysozyme in the proximal tubules after 0.75 M of NaCl injection was found. The accumulation of proteins in the early endocytic compartment and decrease in their entry into late endosomes and lysosomes are shown, that is considered as evidence of a delay in intracellular vesicular transport in hypernatremia. The data obtained were analyzed in connection with changes in blood parameters and kidney activity during osmoregulation, and also with the role of chloride channels in receptor-mediated protein endocytosis. It can be assumed that increased ion transport in the proximal tubules cells in hypernatremia leads to decreased reabsorption capacity of epitheliocytes and delayed intracellular transport of proteins.

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N. P. Prutskova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Author for correspondence.
Email: natprut@yandex.ru
Russian Federation, St.-Petersburg

E. V. Seliverstova

Sechenov Institute of Evolutionary Physiology and Biochemistry RAS

Email: natprut@yandex.ru
Russian Federation, St.-Petersburg

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2. Fig. 1. Reabsorption of GFP, YFP and lysozyme in megalin-immunopositive renal proximal tubules of marsh frogs. (a–f) – profiles of proximal tubules in control (a–e) and after preliminary administration of NaCl (f). Signals: megalin-Alexa 568 – red, GFP, YFP and lysozyme-Alexa 488 – green and colocalized fluorescence – yellow-orange. The presence of proteins in most megalin-positive tubules (a–c), formation of numerous GFP-containing vesicles (d), vesicular and diffuse fluorescence of lysozyme (e, f) and accumulation of lysozyme in the brush border of epithelial cells (f) are visible. Confocal microscopy, merged images. Calibration: 50 µm.

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3. Fig. 2. Decreased reabsorption of GFP, YFP, and lysozyme in the proximal tubules of frog kidneys after preliminary administration of NaCl. Ordinate axis: fluorescence intensity in individual frogs (arbitrary units), on the left – GFP (circles) and YFP (triangles), on the right – lysozyme (diamonds); light symbols – control, dark symbols – after injection of 0.75 M NaCl; black line – median. Significance of differences compared to the control: * – p < 0.05, ** – p < 0.01 (Mann–Whitney T-test).

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4. Fig. 3. Examples of automated quantification and visualization of colocalized fluorescent signals. (a) GFP (green), megalin (red), and signal colocalization (yellow-orange) in the proximal tubule epithelium of the control. Confocal microscopy, merged images. Calibration: 50 μm. (b) – Dot plot for the image shown in (a). Abscissa and ordinate axes: fluorescence intensity of pixels in the red and green spectral regions, respectively (in arbitrary units). Pixels with overlapping signals are colored yellow-orange; dotted lines are the thresholds separating visible fluorescence from dark pixels. (c) and (d) – the same as (a) and (b) for the variant with the introduction of 0.75 M NaCl. An increase in colocalization of both signals is visible compared to the control.

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5. Fig. 4. Effect of 0.75 M NaCl injections on reabsorption and intracellular transport of GFP, YFP, and lysozyme in the proximal tubules of lake frog kidneys. (a) Evaluation of colocalization of injected proteins with megalin in the tubular epithelium. Ordinate axis: colocalized protein (in % of the total amount of reabsorbed protein) in individual frogs (circles) in the control and after 0.75 M NaCl injection. Black line is the median. (b) Ratio of colocalized protein (dark bars) to non-colocalized protein (light bars) in the same experiments. Data are presented as M ± SEM. Significance of differences in (a) and (b): * – p < 0.05, ** – p < 0.01 compared to the control (Mann–Whitney T-test).

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