The nephron microanatomy of the kidney in the lamprey Lampetra fluviatilis L. before and after metamorphosis

Isolated nephrons from premetamorphic lamprey larvae and adult animals were obtained using microdissection and kidney collagenase treatment methods. Significant differences in the architectonics of kidney tubules were found in larvae as compared with adults. The position of larval kidney was cranial while that in adult lampreys was caudal. The absence of nephron loop was noted in larval kidney. Distal and proximal tubules were shifted one from another in mediolateral direction. Distal tubule was significantly larger than proximal one. Mesonephric duct was located laterally near the glomus. Kidney tubules morphogenesis and nephron population number increase was observed at premetamorphic stage of larval kidney development.     

Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice

To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more fluid than wild-type mice and had lower urine osmolality (505 +/- 40 vs. 1081 +/- 68 milliosmolar). Transepithelial osmotic water permeability (Pf) in isolated microperfused S2 segments of proximal tubule from AQP1 knockout [-/-] mice was 0.033 +/- 0.005 cm/s (SE, n = 6 mice, 37 degreesC), much lower than that of 0.15 +/- 0.03 cm/s (n = 8) in tubules from wild-type [+/+] mice (P < 0.01). In the presence of isosmolar luminal perfusate and bath solutions, spontaneous fluid absorption rates (nl/min/mm tubule length) were 0.31 +/- 0.12 (-/-, n = 5) and 0.64 +/- 0.15 (+/+, n = 8). As determined by free-flow micropuncture, the ratios of tubular fluid-to-plasma concentrations of an impermeant marker TF/P in end proximal tubule fluid were 1.36 +/- 0. 05 (-/-, n = 8 mice [53 tubules]) and 1.95 +/- 0.09 (+/+, n = 7 mice [40 tubules]) (P < 0.001), corresponding to 26 +/- 3% [-/-] and 48 +/- 2% [+/+] absorption of the filtered fluid load. In collections of distal tubule fluid, TF/P were 2.8 +/- 0.3 [-/-] and 4.4 +/- 0.5 [+/+], corresponding to 62 +/- 4% [-/-] and 76 +/- 3% [+/+] absorption (P < 0.02). These data indicate that AQP1 deletion in mice results in decreased transepithelial proximal tubule water permeability and defective fluid absorption. Thus, the high water permeability in proximal tubule of wild-type mice is primarily transcellular, mediated by AQP1 water channels, and required for efficient near-isosmolar fluid absorption.     

The differentiation of proximal and distal tubules in the male rat kidney: the appearance of aldolase isozymes, aminopeptidase and alkaline phosphatase during ontogeny

1. The specific activities of aminopeptidase, alkaline phosphatase and aldolase isozymes were measured in homogenates of kidneys taken at different stages of ontogeny. The cellular localization of these enzymes was studied in cryostat tissue sections using substrate linked assays for aminopeptidase and alkaline phosphatase and the mixed aggregation immuno-cytochemical technique for aldolase isozymes; local enzyme concentrations were estimated photometrically. 2. The presence of both aldolase-A and aldolase-B was demonstrated in all metanephrogenic cells (and at still higher concentrations in collecting tubule cells) of the rat fetus 16 days after conception and in the undifferentiated cells of the neogenetic zone of kidney up to 8 days after birth; no aminopeptidase or alkaline phosphatase could be found in these cells. 3. Measurements made on stained tissue sections show that the shift towards aldolase-B, seen in homogenate analyses, is due to a change in the relative amounts of proximal tubules. No evidence was seen for repression in the synthesis of aldolase-A or aldolase-B monomers in the different kidney cells during ontogeny. 4. Two transitions in the mode of nephron differentiation were observed: one was shortly after birth, the other followed weaning. Before the first transition the concentrations of the enzymes increased to different degrees, such that the enzymes reached concentrations comparable with those as in the cells of adult rats by 2 to 4 days post partum. After the second transition proximal tubule size and specific activity of brush border membrane enzymes increased 3 fold. In contrast, the distal tubules did not increase significantly in size, but their aldolase-A concentration increased 3 fold. 5. Evidence based on enzyme quantification and morphometry in kidney sections is presented to demonstrate that the proximal tubule cells show functional adaptation by two independent mechanisms: specific amplification of gene expression and hypertrophy. In contrast, the distal tubule shows functional adaptation only by specific amplification of gene expression.     

Immunohistochemical localization of transforming growth factor-alpha and epidermal growth factor-receptor in the mesonephros and metanephros of the chicken

Transforming growth factor-alpha (TGF-alpha) is a polypeptide related to epidermal growth factor (EGF). Both bind to EGF-receptor (EGF-R) to carry out their function in a variety of tissues and cell lines. Several studies have shown their presence in mammalian kidney, however, nothing has to date been stated concerning their existence in avian kidney. Expression of TGF-alpha and EGF-R is reported here for the first time during the development of the chicken kidney. Using immunohistochemical techniques, we identified a TGF-alpha (but not EGF) in mesonephric distal tubule cells from day 8 to day 20 of embryonic development and in metanephric distal tubule cells from day 14 of embryonic development to the adult. The histochemical characteristics of these cells and their histological localization suggest that they may be the "principal cells" of the distal tubules. Similarly, EGF-R was found in mesonephric proximal tubule cells from day 7 to day 18 of embryonic development and in metanephric proximal tubule cells from day 13 of embryonic development up to adult stages. The coexistence of both TGF-alpha and EGF-R from the onset of development of mesonephros and metanephros supports their possible role in mechanisms of proliferation and differentiation of the cells of these organs.     

Copper binding to mouse liver S-adenosylhomocysteine hydrolase and the effects of copper on its levels

The dissociation constant and stoichiometry of copper binding to mouse liver S-adenosylhomocysteine hydrolase (SAHH) was determined as part of characterizing the possible roles of SAHH in copper metabolism. Copper (64Cu(II)) binding was measured by an ultrafiltration method in the presence of EDTA as a competing ligand. The KD was 3.9 +/- 0.7 x 10(-16) M, and the stoichiometry was one g atom of copper per 48-kDa subunit. Western blots indicated that the liver contains approximately 12 times more SAHH than the kidney, which in turn contains approximately 5 times more SAHH than the brain. The high concentration and copper affinity of SAHH in the liver may contribute to the liver's ability to preferentially accumulate copper, and the low levels of SAHH in the brain may contribute to the sensitivity of the brain to copper deficiency. The effects of genetic defects of copper metabolism and copper deficiency on SAHH were also determined. Normal SAHH levels were detected in brindled mouse liver, kidney, and brain. However, SAHH from brindled mouse liver eluted abnormally from phenyl Superose columns implying an effect of the brindled mouse defect on SAHH protein structure. Hepatic cytosols from the toxic milk mouse contained approximately 42% the amount of SAHH detected in controls, and hepatic levels of SAHH were also decreased by approximately 45% in copper-deficient mice. The binding properties of SAHH and the effects of abnormal states of copper metabolism on its levels are consistent with significant roles for SAHH in normal and abnormal copper metabolism. SAHH may have roles in regulating tissue copper levels and the distribution of intracellular copper.     

Immunologic characterization of plasma membranes from the renal proximal tubule of the dog

Plasma membrane fractions from the brush border (BBM) and antiluminal (ALM) surfaces of the dog's renal proximal tubule cell were separated using free-flow electrophoresis. Rabbits immunized with BBM rapidly produced antibody, but rabbits immunized with ALM did not respond. Indirect immunofluorescence and immunoferritin studies showed that the antibody reacts with the brush border of the proximal tubules in the normal kidney of the adult dog. It also reacts with the surface membranes of certain other absorptive and secretory epithelia, such as gall bladder, small intestine, epididymis, and lacrimal gland. The antibody has affinity for the membrane maltase without affecting its catalytic activity, but does not appear to have affinity for the membrane alkaline phosphatase or the high affinity binding site for phlorizin present in the BBM. Polyacrylamide electrophoresis of solubilized BBM showed approximately 37 protein bands and four glycoproteins. We conclude that the proximal tubule cell is immunologically polarized with respect to the distribution of antigenic proteins, and that the BBM is highly antigenic. The antigenic components appear to be high molecular weight glycoproteins present in the glycocalyx.     

Use of digitalis glycosides to identify the mechanisms of amantadine transport by renal tubules

The mechanism(s) for uptake of organic cations by renal cortical tubules was (were) examined further. Renal cortical tubules were purified from rat kidneys by a Percoll gradient centrifugation technique. Bicarbonate buffer (Krebs-Henseleit, KHS) conditions were altered, and chemical modulators were used which affect the activity of the basolateral Na+/K+-ATPase. Renal tubule uptake of the achiral organic cation amantadine was determined. The cardiac glycosides digoxin and acetylstrophanthidin and ouabagenin did not alter amantadine uptake by either proximal or distal tubule fragments in KHS. However, ouabain inhibited proximal tubule amantadine uptake in a dose-dependent manner with lower potency than distal tubule amantadine uptake in KHS. Ouabain did not inhibit amantadine tubule uptake in phosphate buffer. However, inhibition of amantadine uptake by ouabain returned in a time-dependent manner upon addition of bicarbonate to the phosphate buffer. Low extracellular sodium or potassium did not alter amantadine uptake by proximal tubules. Hypokalemic and hypokalemic/ hyponatremic conditions decreased the inhibitory potency of ouabain for amantadine uptake by proximal tubules. For distal tubules, both hyponatremic and hypokalemic conditions, alone and together, decreased the inhibitory potency of ouabain, but did not affect amantadine uptake in the absence of ouabain. Hypochloremic conditions decreased affinity for amantadine uptake by distal, but not proximal tubules. No change in maximal transport capacity for amantadine uptake was observed under hypochloremic conditions for either tubule fragment. These studies challenge the widely accepted concept of Na+/ K+-adenosine triphosphatase activity and maintenance of the basolateral membrane potential as rate-limiting steps for the energy-dependent renal tubule uptake of organic cations. Furthermore, these studies suggest a mechanism for ouabain inhibition of organic cation renal tubule uptake that may not involve the Na+/K+-adenosine triphosphatase and may be possibly bicarbonate-dependent.     

Induction of rabbit renal mixed-function oxidases by phenobarbital: cell specific ultrastructural changes in the proximal tubule

Administration of phenobarbital (60 mg/kg) daily for 4 days to male rabbits resulted in induction of renal cytochrome P-450 (3.5-fold) and a corresponding increase in ethoxycoumarin-O-deethylase and benzphetamine-N-demethylase activity (17- and 4-fold, respectively). Kidney weight to body weight ratio and renal ethoxyresorufin-O-deethylase were not affected by phenobarbital pretreatment. Numerous focal areas of proliferation of smooth endoplasmic reticulum (SER) were evident in proximal tubule cells from phenobarbital treated rabbits while proximal tubular cells from control rabbits had only small and sparcely located aggregates of SER. Phenobarbital-induced SER proliferation was specifically localized to the S3 segment of the proximal tubule. Proliferation was not observed in S2 cells of the proximal tubule, cells of Henle's loop, distal tubules, or collecting tubules in rabbits pretreated with phenobarbital. These data demonstrate the biochemical heterogeneity of cell types within the proximal tubules of rabbits. Furthermore, induction of mixed-function oxidases specifically in S3 cells of the proximal tubule may be of toxicological significance in the metabolic activation of certain nephrotoxicants.     

Immunohistochemical localization of peroxisomal enzymes in developing rat kidney tissues

We studied the developmental changes in the localization of peroxisome-specific enzymes in rat kidney tissues from embryonic Day 16 to postnatal Week 10 by immunoblot analysis and immunohistochemistry, using antibodies for the peroxisomal enzymes catalase, d-amino acid oxidase, l-alpha-hydroxyacid oxidase (isozyme B), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein. Peroxisomal enzymes were detected in the neonatal kidney by immunoblot analysis and their amount increased with kidney development. By light microscopic immunohistochemistry, they were first localized in a few proximal tubules in the juxtamedullary cortex of 18-day embryos. The distribution of proximal tubules positive for them expanded towards the superficial cortex with development. The full thickness of the cortex became positive for the staining by 14 days after birth. Peroxisomes could be detected by electron microscopy in structurally immature proximal tubules in 18-day embryos. Their size increased and the ultrastructure of subcompartments became clear with continuing development of proximal tubules. These results show that peroxisomal enzymes appear in the immature proximal tubules in the kidney of embryos and that the ultrastructure of the peroxisomes and localization of the peroxisomal enzymes develop along with the maturation of proximal tubules and kidney tissues.     

Microcephaly with chorioretinopathy. A report of two dominant families and three sporadic cases

This study was designed to assess the effect of paraquat on the rat kidney. The experimental animals used were Wistar rats, a strain selected and maintained at the Institut d'hygiène alimentaire. Their diet was well defined and their state of health monitored. Female animals with mean weight of 200 g were used in this study. Doses of 20, 30 and 50 mg/kg were administered via the gastrointestinal route. In this experimentation, performed on 12 rats plus 3 controls, the dose considered to be sublethal was 30 mg/kg administered by gavage. These animals were sacrificed after 24 h, 48 h, 4, 8, 15, 30 and 60 day, selecting those animals with the most severely altered state, at each time. Tubular lesions started to appear by the 24th hour; the proximal tubule was the most sensitive. Lesions of the distal tubule were observed slightly later, from the 48th hour. Lesions became more intense from the 4th day onwards and reached a maximum on the 8th day. The first signs of repair of the proximal tubule and distal tubule were observed on the 15th day, but were less marked for the proximal tubule. This repair was slow and progressive. Persistent lesions of the proximal or distal tubules were still observed after two months. The glomeruli presented several alterations, which were always only moderate. Overall, paraquat induces serious life-threatening lesions of acute tubular necrosis in the absence of adequate intensive care.     

In progressive nephropathies, overload of tubular cells with filtered proteins translates glomerular permeability dysfunction into cellular signals of interstitial inflammation

Progression to end-stage renal failure is the final common pathway of many forms of glomerular disease, independent of the type of initial insult. Progressive glomerulopathies have in common persistently high levels of urinary protein excretion and tubulointerstitial lesions at biopsy. Among the cellular mechanisms that may determine progression regardless of etiology, the traffic of excess proteins filtered from glomerulus in renal tubule may have functional importance by initiating interstitial inflammation in the early phase of parenchymal injury. This study analyzes the time course and sites of protein accumulation and interstitial cellular infiltration in two different models of proteinuric nephropathies. In remnant kidneys after 5/6 renal mass ablation, albumin and IgG accumulation by proximal tubular cells was visualized in the early stage, preceding interstitial infiltration of MHC-II-positive cells and macrophages. By double-staining, infiltrates developed at or near tubules containing intracellular IgG or luminal casts. This relationship persisted thereafter despite more irregular distribution of infiltrate. Similar patterns were found in an immune model (passive Heymann nephritis), indicating that the interstitial inflammatory reaction develops at the sites of protein overload, regardless of the type of glomerular injury. Osteopontin was detectable in cells of proximal tubules congested with protein in both models at sites of interstitial infiltration, and by virtue of its chemoattractive action this is likely mediator of a proximal tubule-dependent inflammatory pathway in response to protein load. Protein overload of tubules is a key candidate process translating glomerular protein leakage into cellular signals of interstitial inflammation. Mechanisms underlying the proinflammatory response of tubular cells to protein challenge in diseased kidney should be explored, as well as ways of limiting protein reabsorption/deposition to prevent consequent inflammation and progressive disease.     

Comparative study of the teratogenicity of phenobarbitone, diphenylhydantoin and carbamazepine in mice

Tendrils have been reported to radiate from luminal surfaces of proximal tubules in rat kidneys by Andrews and Porter ('74) using scanning microscopy, but they were not seen by Bulger et al. ('74). In this study, the perfusion procedure of Andrews and Porter was re-investigated. Tendrils were found only in regions which had characteristics of poor fixation. They were especially prominent if the perfusion pressure was purposely allowed to fall and subsequently increased to re-expand the tubules. The tendrils could then be seen in all portions of the proximal convoluted tubule and not exclusively in the initial portion as previously reported.     

Acupuncture and blood studies in sickle-cell anemia

The effect of testosterone on the activity of ornithine decarboxylase (ODC), its protein level and immunocytochemical distribution were examined in the mouse kidney. Male BALB C mice at 8 weeks of age were used throughout. Fourteen hours before death, they received a subcutaneous injection of testosterone (1 mg/animal) or solvent to measure renal ODC activity or to detect the distribution of ODC immunoreactivity in the kidney. Renal ODC activity and the content of the enzyme were markedly increased after testosterone treatment. Histologically, few cells that were obviously immunoreactive to ODC were observed in the control animals and in the testosterone-treated animals a marked increase in ODC immunoreactivity was observed only in the cortex. ODC immunoreactive cells were located diffusely in the proximal tubule. In the pars recta, cells were stained weakly and homogeneously, while in the pars convoluta, the luminal surface of the cells showed stronger immunoreactivity. Moreover, many granule-like particles that were strongly ODC immunoreactive were observed inside the lumen of the pars convoluta. These results show that testosterone treatment induces an increase in ODC content in certain cells located in the proximal tubule of the cortex.     

Localization of the ROMK potassium channel to the apical membrane of distal nephron in rat kidney

BACKGROUND: The apical potassium (K+) channels mediate K+ recycling in thick ascending limb (TAL) and K+ secretion in cortical collecting duct (CCD). Recently, the cDNAs for a family of renal K+ channels, ROMK1, -2 and -3, were identified. Based on the biophysical properties and mRNA distribution, it is believed that these ROMK cDNAs encode the apical K+ channels of TAL and CCD. However, the information for cellular and subcellular localization of the ROMK proteins in these tubules is still not available. METHODS: Paraffin or frozen kidney sections from adult Sprague-Dawley rats were stained by polyclonal antibodies against the N- and C-terminal domain of ROMK. Immunoreactive staining was visualized by color development from horseradish peroxidase reaction. Membrane homogenates from kidney were analyzed by Western blot analysis. RESULTS: The polyclonal antibodies against cytoplasmic epitope of ROMK recognized a approximately 42 kD protein in the membrane homogenates from kidney, but not from liver. Staining by immunocytochemistry revealed that ROMK channels were localized to the apical membranes of the distal nephron in cortex and outer medulla, including thick ascending limb and collecting tubule. ROMK staining was absent in glomerulus, proximal tubule and inner medulla. Double staining of the tissue section with both ROMK-specific and H+-ATPase-specific antibodies revealed labeling of ROMK in the principal cells of the collecting tubules. CONCLUSIONS: These results further strengthen the idea that ROMK channels play important roles in the recycling of K+ in TAL and the secretion of K+ in CCD.     

D-Glucose enhancement of water reabsorption in proximal tubule of the rat kidney

Water reabsorption in the proximal convoluted tubule of the rat kidney was examined by in vivo microperfusion techniques in order to examine the effect of D-glucose within the tubular lumen. When tubules were perfused with a balanced artificial solution containing Na, K, Cl, HCO3, urea, and D-glucose, absolute reabsorption averaged 4.01 +/- 0.24 nl/min per mm. Addition of D-glucose to the NaCl perfusate enhanced water reabsorption to values similar to those obtained with the balanced artificial perfusate. The enhanced water reabsorption consequent to the addition of D-glucose to the NaCl perfusion solution was completely inhibited by addition of phloridzin to the perfusate. The addition of an unabsorbed hexose, 2-deoxy-D-glucose, to the NaCl perfusate failed to enhance water reabsorption, whereas the addition of an incompletely reabsorbed sugar that is not metabolized, 3-O-methyl-D-glucose, resulted in partial enhancement of theabsolute rate of water reabsorption. These studies demonstrate that D-glucose has the specific effect of augmenting water reabsorption in the proximal tubule of the rat kidney.     

Anatomy and ultrastructure of the excretory system of the lizard, Sceloporus cyanogenys

The anatomy and ultrastructure of the lizard kidney (Sceloporus cyanogenys) have been studied by light and electron microscopy. The number of glomeruli was counted in serial sections and estimated to be 2,000 (in the two kidneys). Beginning with the glomerulus and Bowman's capsule the nephron segments are sequentially: (a) proximal tubule; (b) intermediate ciliated segment consisting of a proximal and distal part; (c) distal tubule, which can be divided into two segments, followed by (d) connecting tubule and (e) initial collecting duct. The initial collecting ducts from several nephrons open into the collecting duct. Tubular epithelium in this lizard has similarities to that of other reptiles. The lateral borders do not overlap like in mammals, but interdigitate by fingerlike projections. The length of the nephron segments was measured in disected tubules and the diameter was measured on light and electron micrographs. From these measurements estimates of inner tubular surface area were made. Together with data from physiological studies (Stolte et al., '76; Schmidt-Nielsen, '76) the estimated surface area was used to calculate transport rates per unit area across the epithelium. Comparisons of structure and transport rates per unit area across the epithelium. Comparisons of structure and transport rates were made between S. cyanogenys and other reptiles and mammals.     

The ultrastructure of the protonephridial tubules of the freshwater planarian Bdellocephala brunnea

A systematic electron microscopic investigation was undertaken to elucidate the regional differentiation of the protonephridial tubules of the freshwater planarian Bdellocephala brunnea. The excretory system consists of many independent protonephridia, each made up of the repeatedly branching "proximal tubules," and highly tortuous "distal tubule." The proximal tubules are composed of a simple ciliated epithelium except in the trunk which lacks ciliation. The cytoplasmic features of the ciliated cell closely resemble those of the flame cell, indicating highly active protein reabsorption by pinocytosis. Pinocytosis seems also active in the trunk of the proximal tubules and in the distal tubule, resulting in cytoplasmic vesicles of typical appearance. Salient features of the distal tubule cell are the increased density of the cytoplasmic matrix and the presence of many basal compartments of cytoplasm with mitochondria, a pattern common to many epithelia with osmoregulatory function. The morphology and possible functions of the planarian protonephridium are discussed in comparison with the vertebrate nephron.     

Mechanism of fluid secretion common to aglomerular and glomerular kidneys

Isolated renal proximal tubules of sea water fish net secrete fluid in vitro. The principal electrolytes in secreted fluid are Na, Cl, Mg and S. Transepithelial voltages may be lumen-negative or -positive by a few millivolts, and transepithelial resistances are low partly due to high paracellular Na and Cl permeabilities. Transepithelial electrochemical potentials indicate secretion of Mg into the tubule lumen by active transport. As Mg concentration in secreted fluid rises, Na concentration falls. Surprisingly, these observations of fluid secretion are made in glomerular and aglomerular proximal tubules, suggesting a fundamental mechanism common to both. Central to this commonality appears to be their behavior as open Donnan systems. Mg actively secreted into the tubule lumen from which it cannot diffuse back into the peritubular medium causes the transepithelial secretion of diffusible Na and Cl. Water follows by osmosis. Since there is flow out of the distal end of the tubule Donnan equilibrium is not attained. Instead, a dynamic Donnan system is maintained, driven by active transport of Mg. A mathematical model of tubular electrolyte and fluid secretion confirms the operation of this open, dynamic Donnan system in aglomerular and glomerular proximal tubules.     

Copper-metallothionein in the kidney of macular mice: a model for Menkes disease

Menkes disease is an X-linked disorder of copper metabolism. Excess amounts of copper in the kidney of Macular mice, a model for this disease, were found as copper-metallothionein (Cu-MT) from kidney of the mice. Histochemical studies of Cu-MT based on its autofluorescent emission properties showed that the protein was predominant in the proximal convoluted tubule (PCT) cells of the cortex. PCT cells are known to be the primary site of the nephrotoxicity caused by heavy metals. MT mRNA was also observed in the cortex, indicating that the protein was biosynthesized in this region. On the basis of these results, we suggest that biosynthesis and degradation of Cu-MT occur repeatedly in the PCT cells of the cortex. We also compared the histochemical localization of Cu-MT in Macular mice and Long-Evans cinnamon rats, a model for Wilson's disease. The significance of this comparison is discussed.