Expression of Aquaporin-6 in Intercalated Cell subtypes in Rat and Mouse Kidney / 대한해부학회지

Aquaporin-6 (AQP6) is a water channel protein located in intracellular vesicles of the proximal tubules and in intercalated cells (ICs) in the collecting duct (CD) of the rat kidney. The function of AQP6 is unknown. However, it colocalizes with vacuolar H+-ATPase in type-A ICs, indicating that it may be important for the function of proton pumps in these cells. The aims of this study were to compare the expression of AQP6 between rat and mouse kidneys, and to establish which types of IC express AQP6. Kidneys of adult male rats and mice were processed for immunohistochemistry using antibodies against AQP6, H+-ATPase, and anion exchanger 1 (AE1). AQP6 was expressed in the S1, S2, and S3 segments of the proximal tubule and in ICs of the CD and connecting tubule (CNT) in both rats and mice. In the rat proximal tubule, AQP6 immunoreactivity was present in intracellular vesicles, whereas in the mouse proximal tubule it was present in the brush border as well as in intracellular vesicles. Triple immunostaining for AQP6, AE1, and H+-ATPase revealed that AQP6 was expressed only in type-A ICs in the CDs and CNTs of both rats and mice, and that the staining was diffuse throughout. There was no AQP6 labeling of type-B ICs, non-A-non-B ICs, or principal cells. The functional significance of the expression of AQP6 in proximal tubule cells and type-A ICs remains to be established. We propose that AQP6 is involved in the retrieval and maintenance of H+-ATPasecontaining vesicles in acid-secreting epithelial cells in the kidney.

Immunocytochemical Study of Calbindin D(28k) Positive Cells in the Developing Rat Kidney / 대한해부학회지

Calbindin D(28k),a calcium binding protein,is found in various tissues,including some cells in the distal nephron.It plays an important role in the regulation of calcium reabsorption. We previously reported the expression of calbindin D(28k) in adult rat kidney.However,the exact time of expression during differentiation in the embryonic kidney is not known.During development,intercalated cells are deleted from the medullary collecting duct by two distinct mechanisms.However,the reason for the different modes of cell death is not known.As calbindin is reported to protect cells against apoptosis,we examined the expression of calbindin D(28k) in the developing rat kidney.Kidneys from 16-,17-,18-and 20-day-old fetuses and 1-,3-,5-,7-,14-and 21-day-old pups and adult Sprague awley rats were processed for immunohistochemistry using a monoclonal antibody against calbindin D(28k) .Intercalated cells were identified by immunostaining for H+ -ATPase and by electron microscopy.Calbindin D(28k) immunoreactivity first appeared in subpopulations of cells in the connecting tubule and medullary collecting duct in the 17-day-old fetus.In the connecting tubule,calbindin D(28k) was expressed only in H+ -ATPase negative connecting tubule cells,and there was no labeling of intercalated cells.In the medullary collecting duct,calbindin D(28k) immunostaining was observed in a few cells with apical H+ -ATPase,characteristic of type A intercalated cells.The numbers of calbindin D(28k) -positive type A intercalated cells increased from day 18 of gestation.In contrast,there was little or no calbindin D(28k) immunoreactivity in the type B intercalated cells or principal cells.During the first two weeks after birth,calbindin D(28k) -positive type A intercalated cells were lost from the terminal part of the medullary collecting duct by simple extrusion. After two weeks,calbindin D(28k) immunostaining decreased in the type A intercalated cells throughout the medullary collecting duct.However,the immunoreactivity of calbindin D(28k) in the cortical collecting duct was increased in some of the type A intercalated cells and the adult pattern was observed in 21-day-old pups.Thus,we propose that the different expression of calbindin D(28k) in type A and type B intercalated cells may be responsible-at least partly-for the different modes of cell death demonstrated in these cells during kidney development.

Ultrastructural Localization of the Ammonia Transporter Protein, RhCG, in Rat Kidney Collecting Duct / 대한해부학회지

Ammonia excretion in the renal collecting duct is critical in the regulation of the acid-base homeostasis. A novel family of ammonium transporter protein, Rh C Glycoprotein (RhCG) was recently identified in the mouse and rat kidney collecting duct. The purpose of this study was to examine the ultrastructural localization of RhCG in the collecting duct. Rat kidneys were processed for light and electron microscope immunocytochemistry using anti-RhCG rabbit polyclonal antibody. Strong RhCG immunolabeling was observed in the apical domain of AE1-positive type A intercalated cells in the collecting duct. Transmission electron microscopy revealed that a high level of RhCG was expressed in the apical plasma membrane and in subapical vesicles of type A intercalated cell. Interestingly, type A cells also expressed weak immunolabel in the basolateral plasma membrane. Type B cells had weak apical plasma membrane and subapical vesicle immunolabel, and very weak basolateral plasma membrane immunolabel. Principal cells had very weak apical plasma membrane immunolabel without detectable subapical or basolateral immunolabel These results demonstrate that RhCG is mainly expressed in the apical plasma membrane and in subapical vesicles of acid-secreting type A cells and may mediate ammonia excretion in the collecting duct.

Ultrastructural Localization of the Ammonium Transporter Protein, RhBG, in the Intercalated Cell of Rat Kidney Collecting Duct / 대한해부학회지

Ammonia excretion in the renal collecting duct is critical in the regulation of the acid-base homeostasis. A novel family of ammonium transporter protein, Rh B Glycoprotein (RhBG) was recently identified in the mouse and rat kidney collecting duct. The purpose of this was to examine the ultrastructural localization of RhBG in the collecting duct. Rat kidneys were processed for light and electron microscope immunocytochemistry using anti-RhBG rabbit polyclonal antibody. Strong RhBG immunolabeling was observed in the basolateral plasma membrane of type A intercalated cells in the collecting duct. In contrast, RhBG labeling was very weak or negative in type B intercalated cells and principal cells. Transmission electron microscopy confirmed that RhBG immunostaining was located mainly in the basolateral plasma membrane and infoldings of type A intercalated cells, but very weak in type B cells. RhBG labeling was not observed in the apical plasma membrane both in type A and B cells. These results demonstrate that RhBG is a basolateral transporter in acid-secreting type A cells and may mediate ammonia excretion in the collecting duct.

Expression of an Anion Exchanger Pendrin in Human Kidney / 대한해부학회지

It has been reported that new apical anion exchanger perndrin, encoded by the pendred syndrome (PDS/pds, Slc26A4) gene, was expressed in the AE1-negative intercalated cells of rat and mouse kidneys. The purpose of this study was performed that expression of pendrin in the subtypes of intercalated cells in human kidney. The normal human renal tissues obtained from nephrotomized kidneys for renal cell carcinoma were fixed in periodate-lysine-paraformalde-hyde, and processed for immunohistochemistry. Subtypes of intercalated cells were identified by using antibodies for H(+)-ATPase and AE1, and connecting tubule cells and principal cells of collecting duct were identified using antibodies for calbindin D28K and AQP2, respectively. In human kidney, pendrin was expressed in the apical domain of AE1-negative intercalated cells including type B cells with diffuse and/or basolateal H(+)-ATPase, non A-non B (non -A/B) type intercalated cells with apical H(+)-ATPase and bipolar type of intercalated cells with apical and basolateral H(+)-ATPase. The AQP2-positive principal cells of cortical collecting duct were also had apical pendrin immunoreactivity. However, there was no pendrin immunoreactivity in AE1-positive type A intercalated cells, calbindin D28K-positive connecting tubule cells, and AQP2-positive medullary collecting duct. These results suggest that pendrin is an apical anion exchanger not only in the AE1-negative intercalated cells (type B, non-A/B and bipolar cells) but also in the principal cells of cortical collecting duct, and has an essential role in HCO3-secretion in human kidney.

Morphological Adaptive Responses of Intercalated Cell of Renal Collecting Duct to Chronic Changes of Potassium Diet / 대한신장학회잡지

Potassium(K+) balance is achieved by the control of urinary K+ excretion and by the control of K+ absorption from the digestive tract. It has been established that chronic potassium depletion is associated with a remarkable hypertrophy of the outer medullary collecting duct of the kidney. But, there are no morphological studies regarding the intercalated cells during the chronic changes of potassium diet. Electron microscopy was performed to observe the morphological alterations of the intercalated cell of the entire collecting duct in response to chronic changes of potassium diet in rat kidney. By electron microscopy, the characteristic features of normal type A intercalated cell of the cortical collecting duct included numerous micro-projections of the apical plasma membrane, complicated basal infolding, apical cytoplasmic tubulovesicles, evenly distributed mitochondia, and centrally located nucleus. In potasium-depleted type A intercalated cell, microprojections of the apical plasma membrane were increased in length and number, basal infoldings were uncomplicated, tubulovesicles were almostly disappeared, and mitochondria were increased in number. Type A intercalated cell of potassium-loading after restriction was found to be almost normal except longer microprojections and increased mitochondria. The characteristic features of normal intercalated cell of the outer medullarycollecting duct(OMCD) included relatively short micro-projections of the apical plasma membrane, uncomplicated basal infoldings, apical cytoplasmic tubulovesicles, and apically distributed mitochondia. In comparison with normal, potassium-depleted intercalated cell of OMCD was hypertrophy, microprojections of the apical plasma membrane were increased in length and number, basal infoldings were complicated, tubulovesicles were almost disappeared, mitochondria were increased in number, and several lysosomes were appeared. Intercalated cell of OMCD of potassium-loading after restriction was found to be almost normal except increased cell size, longer microprojections, and increased mitochondria and lysosomes compared to control. The characteristic features of normal intercalated cell of the inner medullary collecting duct (IMCD) included very short and scant microprojections of the apical plasma membrane, uncomplicated basal infoldings,apica cytoplasmic tubulovesicles, evenly distributed mitochondia, and some lysosomes. In potasium-depleted intercalated cell of IMCD, cell size was prominently increased, microprojections of the apical plasma membrane were increased in length and number, basal infoldings were complicated, tubulovesicles were almostly disappeared, and mitochondria were increased in number. Intercalated cell of IMCD of potassium-loading after restriction was found to be almost normal except increased cell size and increased microprojections in number and length compared to control. These results suggest that intercalated cells adapt through morphological changes to preserve potassium balance during chronic changes of potassium diet.

Effect of acute respiratory acidosis and alkalosis on the subtypes of intercalated cells in rat kidney / 대한해부학회지

Intercalated cells play a major role in proton and bicarbonate secretion in the collecting duct of kidney. A third type of intercalated cell (non A-non B cell), besides type A and B intercalated cells, and a bipolar cell are known to exist in the kidneys of the rat or the mouse. The third type cell has H(+)-ATPase in the apical membrane like the type A intercalated cell, but has no Cl(-)-HCO(3)- exchanger (AE1) on the basolateral membrane. The bipolar cell was shown to express H(+)-ATPase on both the apical and basolateral membranes. The functions of these cells, however, are not determined yet. This study was intended to know the immunohistochemical changes of the intercalated cell subtypes in the acute respiratory acidosis and alkalosis. After midline tracheostomy, respiratory acidosis and alkalosis were induced and maintained for 4 hours in the Sprague-Dawley rats (450~500 g) using a Rodent Ventilator. The kidneys were preserved for immunohistochemical studies by in vivo perfusion fixation with periodate-lysine-paraformaldehyde solution through the abdominal aorta. To identify the subtypes of intercalated cells and the tubule segments in which they are located, a triple immunolabeling procedure was used. Distal convoluted tubule cells and principal cells in the collecting duct were identified using antibody to thiazide sensitive Na(+)Cl(-) cotransporter and antibody to aquaporin-2, respectively. Antibodies to H(+)-ATPase and AE1 were used to identify subpopulation of intercalated cells. Type A cells were activated in respiratory acidosis with enhanced AE1 activity on the basolateral membrane and H(+)-ATPase reactivity moved to the apical membrane, whereas inactivated in respiratory alkalosis with decreased AE1 reactivity and H(+)-ATPase reactivity moved to the supranuclear cytoplasm. The change in reactivity of type A cells in respiratory acidosis or alkalosis was shown to differ depending on the tubular segments: most of the intercalated cells were activated in the outer medullary collecting duct while only a portion of the type A cells activated in the distal convoluted tubule, connecting tubule and cortical collecting duct. No changes were observed in type B cells in respiratory acidosis and alkalosis. In non A-non B cell which was increased in size in respiratory acidosis, H(+)-ATPase reactivity was seen on the apical membrane in respiratory acidosis, while seen in the supranuclear cytoplasm in respiratory alkalosis. These findings indicated that the renal compensation for respiratory acid-base imbalance was mediated mainly by type A cells rather than by type B or non A-non B cells. Among type A cells, more of those of outer medullary collec-ting duct were thought to be recruited compared with those of the cortical collecting duct and connecting tubule.

Immunocytochemical Study of Calcium Binding Protein in the Distal Nephron of Rat Kidney / 대한해부학회지

Calbindin-D28k, a vitamin D-dependent calcium binding protein, plays a cardinal role in transport of calcium in kidney. Previous studies have demonstrated calbindin-D28k immunoreactivity in the distal nephron of mammalian kidney. However, it is well known that in most species including rat and human, there is a gradual transition from the distal convoluted tubule to the cortical collecting duct, and that the connecting segment do no tclearly demarcated, because of intermingling of distal convoluted tubule cells, connecting tubule cells, principal cells and at least two configurations of intercalated cells. In this study, to identify the cell types of calbindin-D28k-positive cells in distal nephron of rat kidney, we used double immunostaining with an antibody against calbindin-D28k and antibodies against thiazide sensitive Na+/Cl- cotransporter for distal convoluted tubule or H+-ATPase for intercalated cells. In the distal convoluted tubule, most of the distal convoluted tuble cells were calbindin-D28k-positive, whereas the intercalated cells were calbindin-D28k-negative. In the connecting tubule, 68% of the cells were calbindin-D28k-positive, and about 97% of the positive cells were connecting tubule cells and only 3% of them were intercalated cells. In the cortical collecting duct, and outer medullary collecting duct of outer stripe and inner stripe, only 8.6%, 11.8% and 4.4% of cells were weak positive for calbindin-D28k respectively. These weak positive cells in the collecting duct are mainly identified as intercalated cells. These findings indicate that calbindin-D28k is involved in not only transcellular transport of calcium but also processes regulating intracellular calcium in rat kidney.