Localization of inward rectifier potassium channel Kir7.1 in the basolateral membrane of distal nephron and collecting duct

Inward rectifier potassium channels (Kir) play an important role in the K(+) secretion from the kidney. Recently, a new subfamily of Kir, Kir7.1, has been cloned and shown to be present in the kidney as well as in the brain, choroid plexus, thyroid, and intestine. Its cellular and subcellular localization was examined along the renal tubule. Western blot from the kidney cortex showed a single band for Kir7.1 at 52 kD, which was also observed in microdissected segments from the thick ascending limb of Henle, distal convoluted tubule (DCT), connecting tubule, and cortical and medullary collecting ducts. Kir7.1 immunoreactivity was detected predominantly in the DCT, connecting tubule, and cortical collecting duct, with lesser expression in the thick ascending limb of Henle and in the medullary collecting duct. Kir7.1 was detected by electron microscopic immunocytochemistry on the basolateral membrane of the DCT and the principal cells of cortical collecting duct, but neither type A nor type B intercalated cells were stained. The message levels and immunoreactivity were decreased under low-K diet and reversed by low-K diet supplemented with 4% KCl. By the double-labeling immunogold method, both Kir7.1 and Na(+), K(+)-ATPase were independently located on the basolateral membrane. In conclusion, the novel Kir7.1 potassium channel is located predominantly in the basolateral membrane of the distal nephron and collecting duct where it could function together with Na(+), K(+)-ATPase and contribute to cell ion homeostasis and tubular K(+) secretion.     

Distribution of N-acetyl-beta-D-glucosaminidase isoenzymes along the rabbit nephron

Total N-acetyl-beta-D-glucosaminidase (NAG) activity has been measured in microdissected glomeruli (G) and tubular segments [proximal convoluted tubule (PCT), pars recta (PR), medullary thick ascending limb (MAL), and cortical collecting tubule (CCT)] of the rabbit kidney, by a fluorimetric method using synthetic substrate. Selective activity of the isoenzyme NAG B was also determined. Isoenzyme profiles of NAG were obtained by electrofocusing on each segment. Characterization of the isoenzymes was performed by chromatofocusing and thermosensitivity experiments on PCT. Total NAG activity, mainly composed of NAG A, was low in glomeruli and two and one-half to four times higher in PCT than in other segments, in which comparable activities were found. NAG B was detectable all along the nephron. It represented a very small fraction of total NAG, except in PCT where it was more abundant (20 to 30%). Electrofocusing revealed the presence of a minor form (NAG I) all along the nephron. Chromatofocusing and thermosensitivity studies indicated that NAG I could represent imperfectly solubilized NAG A rather than a well defined entity. From these results, it could be suggested that the reported increase in urinary excretion of NAG B after renal injury may reflect the intensity of proximal tubular lesions.     

IL-18 is expressed in the intercalated cell of human kidney

We determined the cellular location of interleukin-18 (IL-18) and caspase-1 and the purinergic receptor P2X7, two proteins necessary for its activation and secretion. The mRNA and protein of IL-18 were detectable in normal human kidney by means of polymerase chain reaction (PCR), in situ hybridization, and Western blot. Immunohistochemistry located IL-18 to nephron segments containing calbinbin-D28k or aquaporin-2 that suggest location in the distal convoluted and the connecting tubule and to parts of the collecting duct. IL-18 was not detected in the thick ascending limb of Henle. Confocal microscopy showed that IL-18 was expressed in cells negative for calbindin-D28k and for aquaporin-2 but positive for the vacuolar H(+)-ATPase. This demonstrates that the intercalated cells produce IL-18. These segments were also positive for caspase-1 and P2X7 that are essential for IL-18 secretion. Our results show that IL-18 is constitutively expressed by intercalated cells of the late distal convoluted tubule, the connecting tubule, and the collecting duct of the healthy human kidney. Since IL-18 is an early component of the inflammatory cytokine cascade, its location suggests that renal intercalated cells may contribute to immediate immune response of the kidney.     

Enzyme activity in obstructive uropathy: basis for salt wastage and the acidification defect

Unilateral ureteral obstruction results in marked changes in renal function throughout the nephron, including impaired acid and potassium secretion and salt wastage. The nephron site believed responsible for the acidification defect is the collecting duct. It has been presumed, although not demonstrated, that the cellular mechanism for the acidification defect is both a decrease in transepithelial voltage and a decrease in activity of the proton pump located at the luminal membrane. The mechanism for the abnormalities in sodium handling are thought due to alterations in Na-K ATPase activity. Our laboratory has recently mapped the profile of the N-ethylmaleimide (NEM)-sensitive ATPase and Na-K ATPase in microdissected rat nephron, documenting their presence throughout much of the nephron. In animals with acute unilateral ureteral obstruction for 18 to 24 hours, we measured NEM-sensitive ATPase and Na-K ATPase activities in several nephron sites. In all nephron segments Na-K ATPase activity was markedly decreased. In the medullary collecting duct, NEM-sensitive ATPase activity was also markedly reduced in animals with acute ureteral obstruction; in the cortical collecting duct, activity fell significantly, but to a lesser degree than was observed in the medullary collecting duct. NEM-sensitive ATPase activity was unchanged from control in the proximal convoluted tubule and in the medullary thick ascending limb; in the cortical thick ascending limb enzyme activity increased. These results demonstrate a change in both Na-K ATPase and NEM-sensitive ATPase activities as a direct consequence of a defect known to result in salt wastage and an acidification defect in humans and animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of aldosterone on NEM-sensitive ATPase in rabbit nephron segments

Aldosterone (aldo) treatment of animals stimulates the rate of H+ secretion in the collecting duct, a process which may involve an H+-ATPase sensitive to inhibition by NEM (N-ethylmaleimide). Therefore, we determined NEM-sensitive ATPase activity in distal nephron segments from three groups of adrenalectomized (adx) rabbits maintained on different doses of aldo (in an osmotic minipump) for seven days. Group 1 was given 1.5 micrograms aldo/100 g body wt/day, whereas groups 2 and 3 were maintained on 5 micrograms and 50 micrograms of aldo/100 g body wt/day, respectively. Aldo concentrations in the plasma of groups 1, 2 and 3 were 10.4 +/- 0.8, 70 +/- 7 and 408 +/- 133 ng/dl, respectively. There was a significant increase in NEM-sensitive ATPase activity in connecting tubule (CNT) and cortical, outer and inner medullary duct segments (CCD, OMCD and IMCD) but not in cortical thick ascending limb (CTAL) and distal convoluted tubule (DCT) in group 2 as compared to group 1. A further increase in plasma concentration of aldo (group 3) did not produce any more increase in NEM-sensitive ATPase activity in the CNT, CCD, OMCD and IMCD, but did increase the enzyme activity in the DCT. These results are consistent with the hypothesis that aldo increases H+ secretion in the connecting tubule and collecting duct segments by increasing the activity of NEM-sensitive H+-ATPase activity in these segments.     

Prostaglandin-E2 receptors in the rat kidney: biochemical characterization and localization

A radiohistochemical technique yielding data on the distribution and characteristics of PGE2-receptor binding in tissue sections is described. The binding of tritiated PGE2 (3H-PGE2) to slide-mounted tissue sections had all the characteristics associated with ligand-receptor interactions: it was saturable, of high affinity and displayed high specificity for PGE2 binding. From the binding curves a Hill coefficient of 1.1 was calculated which suggests the presence of a homogeneous receptor population. Pretreatment with indomethacin for four days resulted in a 66% increase in maximal binding capacity (Bmax) without any change in affinity. The distribution of receptor was mapped in rats with and without indomethacin pretreatment and compared to the distribution of Tamm-Horsfall glycoprotein, a specific marker for the thick ascending limb of Henle. In both groups the PGE2 receptor showed striking regional variation. In the untreated group the distribution of PGE2 receptors was similar to that of the thick ascending limb of Henle, with maximal density in the outer medullary zone. After indomethacin pretreatment, however, a striking increase in inner medullary binding was observed together with increased binding in the cortex. Thus, in accordance with the main action of PGE2 being regulation of renal water and sodium excretion, we found the highest receptor density in areas of the kidney dominated by the thick ascending limb of Henle and collecting tubules, and much less binding to glomeruli and cortical vessels. In order to investigate characteristics and distribution of PGE2 receptor binding, however, it was mandatory that endogenous prostanoid synthesis is blocked.     

Distribution of postsynaptic density proteins in rat kidney: relationship to neuronal nitric oxide synthase

BACKGROUND: Neuronal nitric oxide synthase (nNOS) is expressed in skeletal muscle beneath the sarcolemma associated with dystrophin complex. In brain, nNOS is anchored to synaptic membranes by specific postsynaptic density proteins (PSD)-95 and PSD-93. We have investigated the cellular and subcellular localization of these PSD proteins in the kidney and their relationship to nNOS and the cell membrane. METHODS: Kidneys from male Sprague-Dawley rats were processed for light and electron microscopic immunohistochemistry with polyclonal antibodies against PSD and nNOS proteins. RESULTS: Western blot analysis of rat kidney revealed a specific band for PSD-93 at the molecular weight of 103 kDa. Immunostaining for PSD-93 was located in the thick ascending limb of the loop of Henle, macula densa cells, distal convoluted tubules, cortical collecting ducts, outer and inner medullary collecting duct, glomerular epithelium, and Bowman's capsule. A pre-embedding electron microscopic immunoperoxidase procedure localized PSD-93 to the basolateral membrane of these tubular cells. Using different sized immunogold particles, a portion of nNOS in the macula densa colocalized with PSD-93 adjacent to cytoplasmic vesicles and the basolateral membrane. In contrast, PSD-95 protein was detected only weakly in the cortex by Western blot. Immunostaining for PSD-95 was located only faintly in the apical membrane of the thick ascending limb, macula densa, distal convoluted tubule and cortical collecting duct cells. CONCLUSION: PSD-93 is the predominant PSD expressed in the rat kidney. It is located primarily in the basolateral membranes of distal nephron and colocalizes with a pool of nNOS in cytoplasmic vesicles and basolateral membranes of macula densa cells.     

Effects of hydrochlorothiazide on Na-K-ATPase activity along the rat nephron

Na-K-ATPase activity was determined in seven nephron segments of five-week-old, spontaneously hypertensive rats (SHR) with or without continuous hydrochlorothiazide (HCTZ) treatment for seven days. For comparison, the effects of HCTZ treatment on Na-K-ATPase activity in the nephron segments of age-matched normotensive Wistar-Kyoto rats (WKY) were also determined. Na-K-ATPase activity in proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), distal convoluted tubule (DCT) and cortical collecting duct (CCD) was significantly lower in HCTZ-treated SHR compared to control (untreated) SHR. However, there was no significant difference in Na-K-ATPase activity in proximal straight tubule (PST) and medullary collecting duct (MCD) between HCTZ-treated and control SHR. HCTZ treatment also produced a significant decrease in blood pressure (BP) and creatinine clearance (CCr) in SHR. On the other hand, HCTZ treatment did not produce a significant change in Na-K-ATPase activity in PCT, PST, MTAL, CTAL and MCD, in BP or in CCr in WKY. However, HCTZ treatment produced a decrease in the enzyme activity in the DCT and an increase in the enzyme activity in the CCD in WKY. The decrease in Na-K-ATPase activity in almost all nephron segments from SHR may be due to a significant decrease in CCr produced by HCTZ. On the other hand, a decrease in Na-K-ATPase activity in the DCT with an increase in the enzyme activity in the CCD from WKY suggest that renal compensation to the natriuretic effect of HCTZ occurs by an increase in Na+ reabsorption in the CCD.     

Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in the human kidney

The secosteroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a vital role in calcium metabolism, tissue differentiation, and normal bone growth. Biosynthesis of 1,25(OH)2D3 is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although activity of this enzyme has been described in several tissues, the kidneys are recognized to be the principal site of 1,25(OH)2D3 production. To date, enzyme activity studies using vitamin D-deficient animals have suggested that 1alpha-hydroxylase is expressed exclusively in proximal convoluted tubules. With the recent cloning of 1alpha-hydroxylase, specific cRNA probes and in-house polyclonal antiserum have been used to determine the distribution of 1alpha-hydroxylase along the human nephron. Immunohistochemistry and in situ hybridization studies indicated strong expression of 1alpha-hydroxylase protein and mRNA in the distal convoluted tubule, the cortical and medullary part of the collecting ducts, and the papillary epithelia. Lower expression was observed along the thick ascending limb of the loop of Henle and Bowman's capsule. Weaker and more variable expression of 1alpha-hydroxylase protein and mRNA was seen in proximal convoluted tubules, and no expression was observed in glomeruli or vascular structures. These data show for the first time the distribution of alpha1-hydroxylase expression in normal human kidney. In contrast to earlier enzyme activity studies conducted in vitamin D-deficient animals, our data indicate that the distal nephron is the predominant site of 1alpha-hydroxylase expression under conditions of vitamin D sufficiency.     

Glomerulonephritis and sodium retention: enhancement of Na+/K+-ATPase activity in the collecting duct is shared by rats with puromycin induced nephrotic syndrome and mice with spontaneous lupus-like glomerulonephritis.

BACKGROUND: In rats with puromycin aminoglucoside-induced (PAN) nephrotic syndrome, micropuncture studies have localized the site of sodium retention to the collecting duct. We have confirmed this finding by demonstrating a two-fold increase in Na+/K+-ATPase activity specifically limited to the cortical collecting duct in PAN rats. To further define whether this phenomenon was dependent on the chemical induction of the nephrotic syndrome or was a general phenomenon observed in glomerulonephritis, we measured Na+/K+-ATPase activity in nephron segments from mice with spontaneous lupus-like nephritis. METHODS: Hydrolytic activity of Na+/K+-ATPase was measured in three isolated nephron segments: proximal convoluted tubule, thick ascending limb and cortical collecting duct. The Na+/K+-ATPase activities were measured in PAN rats, sham-injected controls, and in (MRL x BXSB) F1 male mice which develop a well established spontaneous lupus-like glomerulonephritis by 4 months of age and their controls. Control mice have the same genetic background, but lack the Yaa mutant gene responsible for autoimmune acceleration and are free of glomerular lesions at 4 months of age. RESULTS: In (MRL x BXSB) F1 male mice, Na+/K+-ATPase was similar to control mice in the proximal convoluted tubule and the thick ascending limb. In contrast, cortical collecting duct Na+/K+-ATPase activity was two times higher in (MRL x BXSB) F1 mice than controls. These results were identical to those observed in PAN rats compared to their sham-injected controls studied 7 days after an intraperitoneal injection of puromycin or isotonic saline, respectively. CONCLUSIONS: Enhancement of Na+/K+-ATPase activity localized to the cortical collecting duct is a general characteristic of glomerulonephritis independent of its mode of induction, i.e. chemical versus autoimmune. Therefore, the experimental model of PAN is suitable to study the underlying mechanisms leading to Na+/K+-ATPase dysfunction.     

Immunocytochemical localization of vitamin D-dependent calcium-binding protein in renal tubules of rabbit, rat, and chick

Vitamin D-dependent calcium-binding protein (CaBP) was localized in tissue sections of kidneys from rabbits, rats, and chicks using antiserum specific for chick intestinal CaBP. In rabbit kidney, CaBP was present in all cells of the distal convoluted tubule and most cells of the connecting tubule. Fewer, but still a majority, of the cells of cortical collecting ducts contained CaBP. The intensity of immunochemical staining and the number of stained cells decreased markedly in medullary collecting ducts, and only a few collecting duct cells contained CaBP at the junction of the inner and outer medulla. In the rat kidney, CaBP was present in all distal convoluted tubule cells, but the immunochemical staining was less intense than in the rabbit. The protein also was found in most connecting tubule cells of the rat; however, only a few collecting duct cells in the superficial corte of the rat contained CaBP. CaBP was essentially absent from mid- to deep-cortical collecting duct cells, while a very few collecting duct cells always contained CaBP at the junction of the inner and outer stripes of the outer medulla. In the chick, CaBP was present in distal convoluted tubule cells as the distal convoluted tubule coursed adjacent to the central vein. CaBP was absent from chick collecting duct cells. In all three species CaBP was not detected in the other portions of the nephron.     

Branched-chain amino acid aminotransferase along the rabbit and rat nephron

The activity of branched-chain amino acid aminotransferase (EC 2.6.1.42) is reported for four or five different segments of the rat and rabbit nephron as well as for patches from the papilla. In the rat the levels ranged 40-fold, from a high in the thick ascending limb of Henle to a low in the proximal convoluted tubule. The peak activity is far above that reported for most other parts of the body. Maximum activity was located also in the thick ascending limb in the rabbit, but the level was only one-third as high as in the rat. It is postulated that ammonia liberated by this amino transferase, in cooperation with glutamate dehydrogenase, could diffuse readily into the adjacent proximal straight tubule where all of the renal glutamine synthase and the highest level of alanine aminotransferase are located. Thus alanine and glutamine could be produced when the ammonia was not needed to neutralize excess acidity.