A key role for membrane transporter NKCC1 in mediating chondrocyte volume increase in the mammalian growth plate

Abstract

The mechanisms that underlie growth plate chondrocyte volume increase and hence bone lengthening are poorly understood. Many cell types activate the Na-K-Cl cotransporter (NKCC) to bring about volume increase. We hypothesised that NKCC may be responsible for the volume expansion of hypertrophic chondrocytes. Metatarsals/metacarpals from 16 rat pups (P(7)) were incubated in the presence/absence of the specific NKCC inhibitor bumetanide and measurement of whole-bone lengths and histologic analysis of the growth plate were done after 24 hours. Fluorescent NKCC immunohistochemistry was visualised using a confocal laser scanning microscopy on seven rat tibial growth plates (P(7)). Microarray analysis was performed on mRNA isolated from proliferative and hypertrophic zone cells of tibial growth plates from five rats of each of three ages (P(49/53/58)). Exposure to bumetanide resulted in approximately 35% reduction (paired Student's t test, p < .05) of bone growth in a dose-dependent manner; histologic analysis showed that a reduction in hypertrophic zone height was responsible. Quantification of fluorescence immunohistochemistry revealed a significant (paired Student's t test, p < .05) change in NKCC from the intracellular space of proliferative cells to the cytosolic membrane of hypertrophic zone cells. Further, microarray analysis illustrated an increase in NKCC1 mRNA between proliferative and hypertrophic cells. The increase in NKCC1 mRNA in hypertrophic zone cells, its cellular localization, and reduced bone growth in the presence of the NKCC inhibitor bumetanide implicate NKCC in growth plate hypertrophic chondrocyte volume increase. Further investigation is warranted to determine the regulatory control of NKCC in the mammalian growth plate and the possible detrimental effect on bone growth with chronic exposure to loop diuretics.

Fig. 1

The effect of NKCC inhibition on rat metatarsal bone rudiment growth by the NKCC inhibitor bumetanide. Each data point contains paired metatarsals, and metacarpals from each animal (main graph) or metatarsals alone (inset) were cultured for 24 hours and measured as described in “Materials and Methods.” There was a dose-dependent relationship for bumetanide inhibition; individual points were significantly different from control (no bumetanide, open circle) with bumetanide concentrations of 12.5, 100, and 200 µM (^ap < .05; ^bp < .01, paired t test). The inset chart shows the significant reduction (^cp < .002, paired t test) of bone lengthening by 100 µM of bumetanide (pattern fill) on metatarsals alone (n = 19). Data expressed as means with errors bars representing SEM.

Fig. 2

Light micrograph of rat metatarsal growth plate in the absence (A) and presence (B) of 100 µM of bumetanide. The irregular horizontal lines were traced by eye along the beginning of late HZCs that exhibited an unambiguous volume enlargement and the bottom at the zone of calcification. The black-and-white vertical bars indicate the average height of the late hypertrophic and remaining growth plate, respectively. Scale bar = 100 µm.

Fig. 3

Growth plate NKCC distribution. Low-power (×10 objective) confocal microscope images (projected) of proximal tibial growth plate stained for NKCC (T4 antibody). Rat proximal tibias were removed and fixed, and transverse sections were prepared for immunohistochemistry as described in “Materials and Methods.” (A) AlexaFluor 488 secondary antibody fluorescence, (B) transmitted light image of the same field of view, and (C) as for panel A but with primary antibody omitted. The proliferative zone showed some fluorescence that appeared to reduce to the upper edge of the HZ. Cells in the HZ exhibited strong fluorescence, which even at this low magnification appeared to be localized to the plasma membrane. Scale bar = 200 µm.

Fig. 4

Growth plate chondrocyte NKCC localization in typical proliferative (A–C) and hypertrophic (D–F) zone chondrocytes. Rat proximal tibias were prepared for and subjected to immunohistochemical procedures, and then images were acquired as described in “Materials and Methods.” NKCC immunofluorescence from a single optical section is shown in panels A and D, with the corresponding transmitted light images (B) and (E) for PZCs and HZCs, respectively. The intensity profile along a line drawn through the center of a cell (gray line in A and D) is shown (C, F). The overlaid transparent vertical bars in C and F indicate the approximate position of the cytoplasmic membrane determined from the transmitted light image (B, E). Fluorescence intensity is in arbitrary units (AUs). For all panels, scale bar = 5 µm.

Fig. 5

Quantification of cellular NKCC distribution along the growth plate. The fluorescence associated with the plasma membrane or intracellular space was obtained from images of cells along the entire length of the growth plate (see “Materials and Methods” and Fig. 4). Owing to the variability in section position, angle, and inherent differences in growth plate dimensions, cell positions within the growth plate were expressed in eight equidistant sections; sections 1 to 3 PZC and 5 to 8 HZC. Significant differences (^ap < .05, Student's t test) between the fluorescent signal of the intracellular space (solid line, □) and membrane-associated fluorescence (broken line, ○). Similarly, significant differences (p < .05, Student's t test) between PZC and HZC intracellular fluorescence (b) and between PZC and HZC membrane-associated fluorescence (c) are shown. An overlay (gray line, ▵) of cell volumes (right axis) has been included to highlight the appearance of NKCC in the plasma membrane immediately preceding a volume increase (data replotted from ref. (2)). Data points are for the mean of a minimum of four animals (421 cells) with bars denoting SEM.

Fig. 6

The effect of the AE inhibitor DIDS (in the presence of 100 µM of bumetanide) on rat metatarsal/metacarpal bone rudiment growth. Each data point represents a metatarsal and metacarpal from of each of at least four animals, cultured for 24 hours, and measured as described in “Materials and Methods.” There was a dose-dependent relationship of DIDS inhibition, significantly reduced (^ap < .05, t test) from control at DIDS concentrations of 175 µM or greater with maximal inhibition at 500 µM. Data points are for the mean of a minimum of four animals with bars denoting SEM.