Quantitative microphotometric succinate dehydrogenase histochemistry in human nephron

A simple method for microphotometric evaluation of cryostat sections from human renal tissue routinely stained for succinate dehydrogenase activity by means of tetranitro-blue tetrazolium chloride is described and tested for validity. Manual absorbance measurement within single nephron segments from the same section allows to directly visualize the distribution pattern of this enzyme along the nephron. Photometric data can be expressed in relative enzyme activities by using the cortical collecting ducts within the same section as reference. This allows to compare measurements of different kidney sections stained by various incubation procedures. The agreement found between relative succinate dehydrogenase activities and recently published morphometric data on mitochondrial inner membranes along the rat nephron suggests that quantitative succinate dehydrogenase microphotometry is a useful histochemical tool for the assessment of renal mitochondrial cristae membranes.     

Stromal progenitors are important for patterning epithelial and mesenchymal cell types in the embryonic kidney

Growth and expansion of the embryonic kidney is driven in large part by continuous branching morphogenesis and nephron induction that occurs in a restricted domain beneath the renal capsule called the nephrogenic zone. Here, new ureteric bud branches and nephron aggregates form surrounded by a layer of cortical stromal cell progenitors. The boundaries and inductive activities of the nephrogenic zone are maintained as the kidney grows. As new ureteric bud branches and nephrogenic aggregates form, older generations of ureteric bud branches, renal vesicles and stromal progenitors are displaced from the nephrogenic zone and undergo further differentiation surrounded by medullary stroma, a different population of stromal cells. Recent studies suggest that cortical and medullary stromal progenitors may be an important source of signals that maintain outer and inner zones of differentiation in the embryonic kidney, and regulate distinct events important for differentiation of nephrons and the collecting duct system.     

Expression and localization of aquaporins in the kidney of the musk shrew (Suncus murinus).

Expression and localization of members of the aquaporin (AQP) family (AQP1, 2, 3, 4, and 5) in the kidney of the musk shrew (Suncus murinus) was examined by immunohistochemistry. AQP1 was expressed in the proximal tubules and in the thin limb of the loops of Henle. AQP1 was the only water channel expressed in the proximal nephron examined, indicating that AQP1 may be an independent water transporter in the proximal nephron. AQP2 and AQP5 were localized to the apical cytoplasm of the cortical to medullary collecting duct (CD) cells and AQP3 and AQP4 were localized to the basal aspect of the cortical to medullary CD cells. AQP3 expression was weaker in the cortical cells compared with the medullary cells, whereas AQP4 was strongly positive throughout the CD. These indicate that the CD is the main water reabsorption segment of the nephron and is regulated by AQPs. Indeed, apical water transport of CD cells of the musk shrew may be controlled by both AQP2 and AQP5. The characteristic expression pattern of the AQPs in this animal provides a novel animal model for elucidating the regulation of water reabsorption by AQPs in the mammalian kidney.     

Regulation by vasopressin of NaCl absorption in the renal collecting duct

In the kidney, the fine control of NaCl absorption takes place in the distal nephron and is controlled by aldosterone and vasopressin. This review summarizes the effects of vasopressin on Na+ transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in immortalized or primary cultured cortical collecting duct cells, expressing either the wild-type ENaC subunits, or mutations, or deletions of the PY domain of the beta- or gamma-ENaC subunits responsible for Liddle's syndrome, an inherited form of hypertension due to excessive salt absorption.     

Effects of low-potassium diet on N-ethylmaleimide-sensitive ATPase in the distal nephron segments.

The present study was undertaken to investigate whether or not potassium deficiency influences N-ethylmaleimide (NEM)-sensitive ATPase in the distal nephron segments of the rat. One group of animals was fed a low-K diet, whereas the normal K-group was given the same diet after supplementation with KCl. The nephron segments examined were: the medullary and cortical thick ascending limbs, the distal convoluted tubule, and the cortical, outer and inner medullary collecting ducts. NEM-sensitive ATPase activity in microdissected segments was measured by a fluorometric microassay. The plasma K+ concentration in the low-K group was 3.1 +/- 0.3 mEq/l compared with 4.2 +/- 0.1 mEq/l in the normal-K group. NEM-sensitive ATPase activity in the outer medullary collecting duct of low-K diet animals was significantly greater than in normal-K animals. There was no significant difference in NEM-sensitive ATPase activity between the two groups of animals in the other nephron segments examined. It is suggested that NEM-sensitive H-ATPase activity in the outer medullary collecting duct is modulated by the potassium status of the animal.     

Developing nephrons in adolescent dogfish, Scyliorhinus caniculus (L.), with reference to ultrastructure of early stages, histogenesis of the renal countercurrent system, and nephron segmentation in marine elasmobranchs

Light and electron microscopy of the excretory kidney of adolescent dogfish, Scyliorhinus caniculus (L.), revealed immature and mature nephrons as well as four developmental stages of nephrons. At stage I the nephron was characterized by a condensed mass of mesenchymal cells in the center of several concentric layers of connective tissue. At stage II of the nephron, the S-shaped body was an elongate cyst with a high prismatic epithelium that was connected by a developing collecting tubule with the collecting duct system. At stage III, the developing nephrons already possess the essential features of the mature nephron but lack complete differentiation. Developing renal corpuscles had one afferent arteriole and two efferent vessels. Developing tubules ran four times between the lateral bundle zone and the mesial tissue zone before they joined the collecting duct system. A continuous sheath of flat cells, encompassing the collecting duct system, extended around the developing lateral bundle. A rudimentary central vessel ran from the developing lateral bundle to the venous sinusoid capillaries between the mesial convolutions. Developmental stage IV was similar to the mature nephron, however, renal corpuscles and tubular segments were smaller than those of mature nephrons. Conclusive evidence for morphological homology of elasmobranch nephron segments and collecting tubule-collecting duct system with those of other vertebrates is provided. The origin and nature of the central vessel and the bundle sheath is clarified. These specific structures of marine elasmobranch kidney supposedly are of great functional relevance for the renal countercurrent system that in turn is essential for ion- and osmo-regulation.     

Rat urinary kallikrein localization in kidney: Effects of fixation

Summary The influence of fixation on the immunocytochemical localization of tissue kallikrein in the kidney has been evaluated using both monoclonal and polyclonal antibodies. These studies have provided several results relevant to kallikrein localization in kidney: (1) the intensity and distribution of immunostaining with both polyclonal and monoclonal anti-kallikrein antibodies is fixation-dependent; (2) the most intense and consistent localizations of kallikrein are in the connecting tubule and the cortical collecting duct of the nephron; (3) kallikrein-like immunoreactivity is seen in proximal tubules with polyclonal but not with non-cross-reactive monoclonal antibodies; and (4) fixatives which disrupt membranes reveal a kallikrein-like antigen in straight tubules of the outer medulla. However, immunostaining with monoclonal antibodies indicates that much of the observed immunostaining at this site probably represents cross-reactivity with another member of the kallikrein family of enzymes.     

Changes in the pool and spectrum of adenine nucleotides in the cortex of the solitary kidney in chronic denervation

Chronic energy-++-deficient condition of nephron cortical portions manifested in a two ware pool decrease and spectrum impairment of the adenyl system components was revealed as a result of the long-term studies in tissue contents of ATP, ADP, AMP in the rat renal cortex under conditions of experimental combination of compensatory hypertrophy and chronic nervous decentralization. A negative trophic effect of chronically overloaded cortex tissue de-mediation is responsible for energy metabolism at the early phase while at the later stage, beginning two months after the experiment, the progressing hypoperfusion of the cortical tubular system of a single denervated kidney is.     

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.     

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.     

Focal expression of insulin-like growth factor I in rat kidney collecting duct

To address the question of insulin-like growth factor (IGF) I localization and synthesis in kidney, we used two complementary experimental approaches: immunohistochemistry of fixed paraffin-embedded rat kidney sections; and measurement of IGF I mRNA in isolated components of the rat nephron, using a highly sensitive and specific solution hybridization assay. Immunostainable IGF I was localized exclusively to principal cells of cortical and medullary collecting ducts. Administration of growth hormone to hypophysectomized rats for 8 d resulted in enhanced immunohistochemical staining of IGF I within collecting ducts, but no detectable IGF I in other portions of the nephron. The abundance of IGF I mRNA was 7-12-fold higher in isolated papillary collecting ducts than in proximal tubules or glomeruli, and was enriched 10-fold compared with whole kidney. Our data demonstrate colocalization of IGF I and IGF I mRNA in the collecting duct, consistent with focal expression of the IGF I gene at this site.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

Summary The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells segment of the nephron preceeding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. the close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Recapitulation in the development of the components of the counterflow system in the vertebrate metanephros

The investigation has been performed on 107 renal preparations obtained from persons of various age (from 5-month-old fetuses up to 45 years of age), certain representatives of other classes of the Vertebrata are also included: fish, amphibia, reptile and mammalia at various stages of pre- and postnatal periods of ontogenesis by means of preparing graphic and plastic reconstructive models, histological investigation and microdissection. The complexity of the intrarenal branching of derivatives of the mesonephric duct diverticulum, development and structure of the canalicular part in nephrons directly depend on the phylogenetic position of the animal. Complexity of the nephron architectonics occurs along the progressive line of taxonomic groups of higher Vertebrata. The nephron loop becomes longer, thin segment of the nephron canalicular part increases in its length and, at last, in mammalia a cone-shaped fasciculus appears as a structural-functional unit of the osmoregulating apparatus of the constant kidney. In the comparative anatomical and comparative embryological aspects recapitulation is observed concerning certain morphological signs of derivatives of the metanephric duct and nephron.     

Expression and function of arginine-producing and consuming-enzymes in the kidney

The kidney plays a key role in arginine metabolism. Arginine production is controlled by argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) which metabolize citrulline and aspartate to arginine and fumarate whereas arginine consumption is dependent on arginine:glycine amidinotransferase (GAT), which mediates creatine and ornithine synthesis. Histological and biochemical techniques have been used to study the distribution and activity of these enzymes in anatomically dissected segments, in isolated fragments of tubules and in whole tissues. ASS and ASL mRNAs and proteins are expressed in the proximal tubule. Within this nephron segment, the proximal convoluted tubule has a higher arginine synthesis capacity than the proximal straight tubules. Furthermore, this arginine-synthesizing portion of the nephron matches perfectly with the site of citrulline reabsorption from the glomerular filtrate. The kidney itself can produce citrulline from methylated arginine, but this capacity is limited. Therefore, intestinal citrulline synthesis is required for renal arginine production. Although the proximal convoluted tubule also expresses a significant amount of GAT, only 10% of renal arginine synthesis is metabolized to guanidinoacetic acid, possibly because GAT has a mitochondrial localization. Kidney arginase (AII) is expressed in the cortical and outer medullary proximal straight tubules and does not degrade significant amounts of newly synthesized arginine. The data presented in this review identify the proximal convoluted tubule as the main site of endogenous arginine biosynthesis.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

Development of the nephrons of the mesonephros of chicken embryo]

The number of nephron populations in the postinduction period was established in 6- and 8-days chicken embryos and the development of an individual nephron and its parts was studied. The investigation by microdissection method has shownand the number of nephrons is different along the length of the kidney. Only two layers ofthe nephrons were found in the cranial portion, while in the caudal direction their number increased up to 4-6 populations which distinguished from one another by the glomerule position, the length of the nephron and its segments. All the populations of the ventral nephrons enter immediately into the mesonephritic (Wolffian) duct, while the dorsal nephrons have a system ofcollecting tubes by which they are connected with the mesonephric duct. The development of mesonephros was accompained by the increase of the absolute length of the nephrons of all populationsand their segments.Laboratory of Individual Development, Institute of Physiology, Czechoslovakian Academy of Sciences, Prague, and Laboratory of the Evolution of the Kidney and Water-Salt Exchange, Sechenov Institue of Evolutionary Physiologyand Biochemistry, Leningrad.     

Regional expression and histological localization of cysteine sulfinate decarboxylase mRNA in the rat kidney.

Cysteine sulfinate decarboxylase (CSD) is the rate-limiting biosynthetic enzyme of the pathway that forms taurine, a putative osmolyte in the kidney, which was previously localized in various segments of the nephron. Although CSD is known to be expressed in whole kidney extracts, no information on CSD mRNA regional expression and histological localization is yet available. Western blotting and Northern blotting were performed in four dissected regions of the kidney using an antiserum against recombinant CSD and a [(32)P]-dCTP-labeled CSD cDNA probe, respectively. In situ hybridization was carried out using a [(35)S]-CTP-labeled CSD RNA probe. A single protein (53 kD) and a single mRNA (2.5 kb) were detected, both of which appeared to be most enriched in the outer stripe of the outer medulla. In situ hybridization of CSD mRNA showed strong labeling of the thick tubules in the outer stripe of the outer medulla and in cortical medullary rays that corresponded to the proximal straight tubules. The significance of this restricted expression of CSD is discussed in relationship to the data previously reported on the location of taurine and the location of the taurine transporter along the nephron.