CHIP28 water channels are localized in constitutively water-permeable segments of the nephron

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.     

Lysosomal sulfoglycolipid storage in the kidneys of mice deficient for arylsulfatase A (ASA) and of double-knockout mice deficient for ASA and galactosylceramide synthase

The inherited deficiency of arylsulfatase A (ASA) causes lysosomal accumulation of sulfoglycolipids (mainly sulfo-galactosylceramide, S-GalCer ) and leads to metachromatic leukodystrophy in humans. Among visceral organs, kidneys are particularly affected. In the present study, the regional distribution and temporal development of sulfoglycolipid storage in kidneys of ASA-/- mice was investigated histochemically (alcian blue) and ultrastructurally. Furthermore, the sulfoglycolipid storage was examined in kidneys of double-knockout mice, which are incapable of: (a) degrading any sulfolipids (ASA-/-) and (b) synthesizing the major sulfolipid S-GalCer because of deficiency for galactosylceramide synthase (CGT), with the aim to search for additional ASA substrates. In ASA-/- mice, the nephron segments could be ranged in the order of decreasing sulfolipid storage: thin limbs of long-looped nephrons approximately thick ascending limbs > distal convoluted tubules > collecting ducts approximately short thin limbs. Macula densa and proximal tubules were unaffected. In ASA-/-/CGT-/- mice, the long thin limbs and distal convoluted tubules resembled those of ASA-/-/CGT+/+ mice, while the other segments showed less storage. The results suggest that the turnover of sulfolipids in general is highest in the distal nephron except macula densa, and that long thin limbs and distal convoluted tubules are the main sites for turnover of a minor sulfolipid species, which is known to be synthesized in the kidney of CGT-/- mice.     

Carbonic anhydrase activity in kidney and lower intestine of the European starling

A histochemical investigation of kidney and lower intestine of the European starling (Sturnus vulgaris) shows no carbonic anhydrase activity in proximal convoluted tubules, although activity is seen in similarly prepared sections of rat proximal tubules. Early distal tubule cells in the starling are stained throughout the cytoplasm and at the apical and highly infolded basolateral membranes. Late distal tubules lose apical activity and have reduced basolateral infolding, resulting in less intense staining. Darkly stained intercalated cells appear in the connecting tubules and cortical collecting ducts. Both of these segments also show intense basolateral staining. Medullary cones of the starling are highly organized, with central zones containing unstained thin descending limbs of loops of Henle, surrounded by both medullary collecting ducts with only scattered cells staining for enzyme, and by thick ascending limb segments. The latter contain many uniformly stained cells intermingled with occasional unstained cells. Scattered cells of the starling colonic villi demonstrate intense apical brush border membrane staining as well as cytoplasmic staining. Cells lining the cloaca stain less intensely. A biochemical assay for carbonic anhydrase was used to quantify enzyme activity in these tissues. Starling kidney contained 1.96 ± 0.33 (mean ± SEM) enzyme units/mg protein, less than half the activity seen in rat kidney. Stripped colonic epithelium contained 0.66 ± 0.15 enzyme units/mg protein. These quantitative results correlate well with the interpretations derived from the histochemical observations. The lack of proximal tubule carbonic anhydrase activity suggests that the avian kidney relies more on distal nephron segments to achieve net acidification of the urine.     

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.     

Immunocytochemical localization of Na+, K+-ATPase in the rat kidney

To determine if rat kidney Na+, K+-ATPase can be localized by immunoperoxidase staining after fixation and embedding, we prepared rabbit antiserum to purified lamb kidney medulla Na+, K+-ATPase. When sodium dodecylsulfate polyacrylamide electrophoretic gels of purified lamb kidney Na+, K+-ATPase and rat kidney microsomes were treated with antiserum (1:200), followed by [125I]-Protein A and autoradiography, the rat kidney microsomes showed a prominent radioactive band coincident with the alpha-subunit of the purified lamb kidney enzyme and a fainter radioactive band which corresponded to the beta-subunit. When the Na+, K+-ATPase antiserum was used for immunoperoxidase staining of paraffin and plastic sections of rat kidney fixed with Bouin's, glutaraldehyde, or paraformaldehyde, intense immunoreactive staining was present in the distal convoluted tubules, subcapsular collecting tubules, thick ascending limb of the loops of Henle, and papillary collecting ducts. Proximal convoluted tubules stained faintly, and the thin portions of the loops of Henle, straight descending portions of proximal tubules, and outer medullary collecting ducts did not stain. Staining was confined to basolateral surfaces of tubular epithelial cells. No staining was obtained with preimmune serum or primary antiserum absorbed with purified lamb kidney Na+, K+-ATPase, or with osmium tetroxide postfixation. We conclude that the basolateral membranes of the distal convoluted tubules and ascending thick limb of the loops of Henle are the major sites of immunoreactive Na+, K+-ATPase concentration in the rat kidney.     

Localization by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques of Tamm–Horsfall glycoprotein in adult hamster kidney

1. Tamm-Horsfall glycoprotein was isolated from hamster urine and antiserum against it was produced in rabbits. Immunoglobulin G was isolated from the antiserum. 2. Indirect methods of immunofluorescence staining were applied to kidney sections previously fixed by both perfusion and immersion methods. Tamm-Horsfall glycoprotein was identified associated with only the cells of the ascending limb of the loop of Henle and the distal convoluted tubule. Maculae densae were free of the glycoprotein. 3. Indirect immunoperoxidase procedures with light microscopy were applied to kidney sections. The results extended those found by immunofluorescence by showing that the glycoprotein is largely associated with the plasma membrane of the cells. Macula densa cells were shown to be free of the glycoprotein, although the luminal surface of the remaining cells in the transverse section of the nephron at that region was shown to contain it. 4. A variety of immuno-electron-microscopic techniques were applied to sections previously fixed in a number of ways. Providing periodate/lysine/paraformaldehyde was used as the fixative, the glycoprotein was often seen to be present not only on the luminal surface of the cells of the thick ascending limb of the loop of Henle and of the distal convoluted tubule, but also on the basal plasma membrane, including the infoldings. 5. It is generally accepted that the hyperosmolarity in the medulla of the kidney results from passage of Cl(-) ions with their accompanying Na(+) ions across the single cell layer of the lumen of the thick ascending limb of the loop of Henle, a region of the nephron with relatively high impermeability to water. We suggest that Tamm-Horsfall glycoprotein operates as a barrier to decrease the passage of water molecules by trapping the latter at the membrane of the cells. Our hypothesis requires the glycoprotein on the basal plasma membrane also.     

Cellular basis of an avian countercurrent multiplier system

A kidney from the budgerigar (budgie, parakeet; Melopsittacus undulatus) is composed of cortical reptilian-type nephrons (without loops of Henle) and mammalian-type nephrons (with loops) grouped together in medullary cones. The loop of the mammalian-type nephrons has a descending segment composed of thin and highly interdigitated cells. These thin limb cells have few mitochondria (15% of cell volume), undetectable Na+,K(+)-ATPase activity, and virtually no basolateral surface amplification. Prior to the hairpin turn, the descending limb thickens, but the cells continue to lack basolateral amplification. Cells just prior to and within the hairpin turn resemble cells of the entire ascending limb. These cells are thick (there is no thin ascending segment in the avian loop), with extensive infoldings of the basolateral membrane surrounding numerous mitochondria (45% of cell volume). The area of basolateral membrane is 25 times that of the apical membrane. The basolateral membrane (but not the apical membrane) is enriched in Na+,K(+)-ATPase activity. The structure of the avian mammalian-type nephron (as epitomized by the budgie nephron) and the fact that NaCl accounts for over 90% of the osmotic activity of avian urine leads to the conclusion that the countercurrent multiplier of the avian kidney functions by active NaCl transport from the entire ascending limb. No explanation is offered for the transport specializations found in the thick descending segment of the loop, just prior to the hairpin turn.     

ELECTRON MICROSCOPE STUDIES ON THE SURFACE COAT OF THE NEPHRON

Attempts to make visible the carbohydrate coat at the free cell surface of glomeruli as well as the tubules of rabbit kidney were undertaken. The ruthenium red procedure was performed, according to Luft, at various pH values. Moreover, the colloidal iron and the colloidal thorium methods were used. Neuraminidase digestion was also performed. In the ruthenium red procedure the luminal face of the epithelial cells of the nephron was coated distinctly with reaction product. The results obtained revealed that some of the differences at various levels of the nephron depended on the pH values. In glomeruli and proximal convoluted tubules the optimum pH value was 7.4; in the ascending limb of Henle loops and distal convoluted tubules the optimum pH value was 6.8. The ruthenium red-positive surface coat was either closely connected with, or appeared as a part of, the outer leaflet of the unit membrane. The slit pores of glomeruli were also covered by a coat continuous with the surface coat of the adjacent foot processes. The coat lining the microvilli of proximal convoluted tubules completely filled the intervillous spaces. Also, both the colloidal iron method and the colloidal thorium method evidenced the presence of surface coat. Pre-treatment with neuraminidase abolished the effect of the Hale reaction. These results may indicate that the surface coat of the epithelia of the nephron shows the presence of glycoproteins containing siliac acid residues.     

Stem cell marker TRA-1-60 is expressed in foetal and adult kidney and upregulated in tubulo-interstitial disease

The kidney has an intrinsic ability to repair itself when injured. Epithelial cells of distal tubules may participate in regeneration. Stem cell marker, TRA-1-60 is linked to pluripotency in human embryonic stem cells and is lost upon differentiation. TRA-1-60 expression was mapped and quantified in serial sections of human foetal, adult and diseased kidneys. In 8- to 10-week human foetal kidney, the epitope was abundantly expressed on ureteric bud and structures derived therefrom including collecting duct epithelium. In adult kidney inner medulla/papilla, comparisons with reactivity to epithelial membrane antigen, aquaporin-2 and Tamm–Horsfall protein, confirmed extensive expression of TRA-1-60 in cells lining collecting ducts and thin limb of the loop of Henle, which may be significant since the papillae were proposed to harbour slow cycling cells involved in kidney homeostasis and repair. In the outer medulla and cortex there was rare, sporadic expression in tubular cells of the collecting ducts and nephron, with positive cells confined to the thin limb and thick ascending limb and distal convoluted tubules. Remarkably, in cortex displaying tubulo-interstitial injury, there was a dramatic increase in number of TRA-1-60 expressing individual cells and in small groups of cells in distal tubules. Dual staining showed that TRA-1-60 positive cells co-expressed Pax-2 and Ki-67, markers of tubular regeneration. Given the localization in foetal kidney and the distribution patterns in adults, it is tempting to speculate that TRA-1-60 may identify a population of cells contributing to repair of distal tubules in adult kidney.     

Expression of epidermal growth factor in the rat kidney. An immunocytochemical and in situ hybridization study

The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Expression of epidermal growth factor in the rat kidney

Summary The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.     

Specialized expression of simple O-glycans along the rat kidney nephron.

Glycosyltransferases can exhibit tissue-specific expression. By histochemistry glycosyltransferases and their products can be localized to specific cell types in organs of complex cellular composition. We have applied the lectin Amaranthin, having a nominal specificity for Galbeta1,3GalNAcR and Neu5Ac2,3Galbeta1, 3GalNAcalpha-R, and a monoclonal antibody raised against Galbeta1, 3GalNAcalphaR to examine the distribution of these simple O-glycans in adult rat kidney. The monoclonal antibody stained ascending thin limbs of Henle, distal convoluted tubules, and collecting ducts of cortex and outer medulla. Remarkably, the ascending thick limb of Henle, located between ascending thin limb and distal convoluted tubules, was unreactive. However, Amaranthin staining was detectable in ascending thick limbs of Henle, in addition to the structures positive with the monoclonal antibody. In kidney extracts, two bands of approximately 160 kDa and >210 kDa were reactive with both Amaranthin and the monoclonal antibody. One band at approximately 200 kDa, and a smear at approximately 100 kDa, were reactive only with Amaranthin. Our data show that in rat kidney simple O-linked glycans are expressed in a highly specialized manner along the renal tubule and can be detected only on a few glycoproteins. This may reflect a cell-type-specific expression of the corresponding glycosyltransferases.     

Expression of the membrane-associated carbonic anhydrase isozyme XII in the human kidney and renal tumors.

Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von Hippel-Lindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von Hippel-Lindau carcinogenesis.     

Renal synthesis of epidermal growth factor is unchanged by vitamin D deficiency.

In mammalian kidney, epidermal growth factor (EGF) is produced as a small internal domain of an abundant high molecular weight peptide associated with the luminal membrane of the thick ascending limb of Henle's loop and distal convoluted tubule. At present, there is no evidence to indicate a mitogenic function for the EGF-containing molecule in kidney; consideration of its molecular structure suggests the possibility of a membrane-associated physiologic role. In this study, we examine regulation of renal EGF synthesis during induction of vitamin D deficiency in mice. Despite evidence of marked hyperparathyroidism, urinary excretion of EGF was equivalent in control (2.54 +/- 0.72 micrograms/mg creatinine) and vitamin D deficient (2.13 +/- 0.97 micrograms/mg creatinine) animals. Similarly, EGF mRNA levels in kidney were comparable in the two groups. These data indicate that parathyroid hormone has no effect on renal EGF regulation, although it is known to stimulate calcium reabsorption in distal nephron segments.     

New insights into sodium transport regulation in the distal nephron:Role of G-protein coupled receptors

The renal handling of Na~+ balance is a major determinant of the blood pressure(BP) level. The inability of the kidney to excrete the daily load of Na+ represents the primary cause of chronic hypertension. Among the different segments that constitute the nephron, those present in the distal part(i.e., the cortical thick ascending limb, the distal convoluted tubule, the connecting and collecting tubules) play a central role in the fine-tuning of renal Na~+ excretion and are the target of many different regulatory processes that modulate Na~+ retention more or less efficiently. G-protein coupled receptors(GPCRs) are crucially involved in this regulation and could represent efficient pharmacological targets to control BP levels. In this review, we describe both classical and novel GPCR-dependent regulatory systems that have been shown to modulate renal Na~+ absorption in the distal nephron. In addition to the multiplicity of the GPCR that regulate Na~+ excretion, this review also highlights the complexity of these different pathways, and the connections between them.     

Ultrastructural evaluation of the effect of endosulfan on mice kidney

In this study, the effect of the endosulfan on mice kidney was investigated at ultrastructural level. Moreover, biochemical analyses (G6PD, CAT, SOD, GSH and MDA) were determined in supernatant of kidney tissue. Endosulfan (13mg/kg/day body weight) was administered orally to mices via intragastric-during 10 days. The presence of mitochondrial degeneration in cytoplasm of proximal convoluted tubule cells were a striking feature. Furthermore, there was lipofuscin granules and membranous structures in some of proximal convoluted tubule cells. In some glomeruli, ultrastructural changes such as fusion in pedicels and focal thickening at glomerular basal membrane were seen. There were cytoplasmic bulges in some distal convoluted tubule cells. The biochemical results of the experimental group were significant when compared to the control. The effect of the endosulfan was mainly on the proximal convoluted tubule cells. Morever, the other parts of the nephron were effected. Thus, this degeneration in kidney may be thought that oxidative stress may play a role to the mediator in changing configuration of cell membrane and seem to account for the morphologic alteration of kidney.     

Expression and nephron segment-specific distribution of major renal aquaporins (AQP1-4) in Equus caballus, the domestic horse

Aquaporins (AQPs) play fundamental roles in water and osmolyte homeostasis by facilitating water and small solute movement across plasma membranes of epithelial, endothelial, and other tissues. AQP proteins are abundantly expressed in the mammalian kidney, where they have been shown to play essential roles in fluid balance and urine concentration. Thus far, the majority of studies on renal AQPs have been carried out in laboratory rodents and sheep; no data have been published on the expression of AQPs in kidneys of equines or other large mammals. The aim of this comparative study was to determine the expression and nephron segment localization of AQP1-4 in Equus caballus by immunoblotting and immunohistochemistry with custom-designed rabbit polyclonal antisera. AQP1 was found in apical and basolateral membranes of the proximal convoluted tubules and thin descending limbs of the loop of Henle. AQP2 expression was specifically detected in apical membranes of cortical, medullary, and papillary collecting ducts. AQP3 was expressed in basolateral membranes of cortical, medullary, and papillary collecting ducts. Immunohistochemistry also confirmed AQP4 expression in basolateral membranes of cells lining the distal convoluted and connecting tubules. Western blots revealed high expression of AQP1-4 in the equine kidney. These observations confirm that AQPs are expressed in the equine kidney and are found in similar nephron locations to mouse, rat, and human kidney. Equine renal AQP proteins are likely to be involved in acute and chronic regulation of body fluid composition and may be implicated in water balance disorders brought about by colic and endotoxemia.