Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.     

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.     

Insulin-like growth factor I in cultured rat astrocytes: expression of the gene, and receptor tyrosine kinase.

Gene expression, receptor binding and growth-promoting activity of insulin-like growth factor I (IGF I) was studied in cultured astrocytes from developing rat brain. Northern blot analysis of poly(A)+ RNAs from astrocytes revealed an IGF I mRNA of 1.9 kb. Competitive binding and receptor labelling techniques revealed two types of IGF receptor in astroglial cells. Type I IGF receptors consist of alpha-subunits (Mr 130,000) which bind IGF I with significantly higher affinity than IGF II, and beta-subunits (Mr 94,000) which show IGF I-sensitive tyrosine kinase activity. Type II IGF receptors are monomers (Mr 250,000) which bind IGF II with three times higher affinity than IGF I. Both types of IGF receptor recognize insulin weakly. DNA synthesis measured by cellular thymidine incorporation was stimulated 2-fold by IGF I and IGF II. IGF I was more potent than IGF II, and both were significantly more potent than insulin. Our findings suggest that IGF I is synthesized in fetal rat astrocytes and acts as a growth promoter for the same cells by activation of the type I IGF receptor tyrosine kinase. We propose that IGF I acts through autocrine or paracrine mechanisms to stimulate astroglial cell growth during normal brain development.     

Kir1.1 expression in embryonic kidney epithelia

K(+) channels may regulate cell cycling, cell volume, and cell proliferation. We have recently shown a role for an inwardly rectifying K(+) channel, Kir6.1/SUR2(B), in the regulation of cell proliferation during early kidney development. Here, we show that the protein of a further K(+) channel, Kir1.1 (ROMK), is also developmentally expressed in prenatal rat kidney epithelia. In the embryonic stage, Kir1.1 protein was localized to the plasma membrane of ureteric buds and collecting ducts, and of nephron stages up to the comma-shaped body. Experimental increase in cAMP upregulated Kir1.1b (ROMK2) mRNA abundance in ureteric buds. Kir1.1 protein was restricted to the distal nephron during later postnatal development and adulthood, as has been reported. In conclusion, we demonstrate redundancy of Kir channel expression in early embryonic kidney which could suggest that Kir1.1 acts in a similar way as Kir6.1/SUR2(B) to promote cell proliferation or other developmental functions.     

生长休止蛋白7在成年大鼠肾脏、心脏和肝脏中的差异表达

目的研究生长休止蛋白7（Gas7）在成年大鼠肾脏、心脏和肝脏的表达。方法成年SD大鼠16只,分别采用逆转录聚合酶链反应（RT-PCR）方法和免疫组织化学方法检测Gas7基因mRNA和蛋白在成年SD大鼠肾脏、心脏和肝脏的表达,并进行图像分析和统计学处理。结果RT—PCR结果显示,Gas7mRNA在肾脏高表达,在心脏的表达弱于肾脏（P〈0．05）,而在肝脏的表达最弱,基本检测不到。免疫组化结果显示,在肾脏中,Gas7免疫阳性产物在近髓肾单位的近曲小管呈强阳性反应,在集合管表达较弱,在肾小球和其余肾小管未见表达;在心脏中,Gas7免疫阳性产物均匀分布于心肌细胞,呈中等强度反应,弱于肾脏（P〈O．05）;在肝脏中,Gas7蛋白未见明显表达,与其mRNA在肝脏的表达相似。结论Gas7在大鼠肾脏、心脏和肝脏表达的不同,尤其在肾脏组织分布的差异性,提示Gas7在成年大鼠肾脏和心脏结构以及功能的维持中可能起着重要作用。     

Characterization of N-ethylmaleimide-sensitive proton pump in the rat kidney. Localization along the nephron

This study is aimed both at characterizing an ATPase activity in rat kidney equivalent to the proton pump described in bovine kidney medulla and at localizing this enzyme along the nephron. Membrane fractions isolated from kidney homogenates by differential and density gradient centrifugations were enriched 7-fold in ATPase activity sensitive to N-ethylmaleimide (NEM). These fractions also displayed ATP-dependent proton transport. ATPase activity and proton transport in vesicles had similar pharmacological properties as both were insensitive to vanadate and ouabain and had similar sensitivities toward NEM (apparent Ki = 20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 50 microM). Proton transport was dependent on chloride availability as chloride addition to the extravesicular medium stimulated proton transport in a dose-dependent fashion (apparent K 1/2 = 7 mM). NEM-sensitive ATPase activity displaying similar pharmacological properties as proton transport in vesicles was also found in single segments of nephron. It was insensitive to vanadate and ouabain, was inhibited by similar concentrations of NEM (apparent Ki = 15-20 microM) and N,N'-dicyclohexylcarbodiimide (apparent Ki = 30 microM), and is therefore likely to be a proton pump. NEM-sensitive ATPase was localized in all the segments of the rat nephron; its activity was highest in proximal convoluted tubules; intermediate in proximal straight tubules, thick ascending limbs, and cortical collecting tubules; and lowest in outer medullary collecting tubules.     

Tissue injury after lithium treatment in human and rat postnatal kidney involves glycogen synthase kinase-3β-positive epithelium

It was hypothesized that lithium causes accelerated and permanent injury to the postnatally developing kidney through entry into epithelial cells of the distal nephron and inhibition of glycogen synthase kinase-3β (GSK-3β). GSK-3β immunoreactivity was associated with glomeruli, the thick ascending limb of Henle's loop, and collecting ducts in the developing and adult human and rat kidney. In rats, the abundance of inactive, phosphorylated GSK-3β (pGSK-3β) protein decreased during postnatal development. After feeding of dams with litters lithium [50 mmol Li/kg chow, postnatal (P) days 7-28], the offspring showed plasma lithium concentration of 1.0 mmol/l. Kidneys from lithium-treated rat pups exhibited dilated distal nephron segments with microcysts. Stereological analysis showed reduced cortex and outer medullary volumes. Lithium increased pGSK-3β and the proliferation marker proliferating cell nuclear antigen (PCNA) protein abundances in the cortex and medulla. After lithium treatment, pGSK-3β-immunopositive cells exhibited restricted distribution and were associated primarily with subsets of cells in dilated and microcystic segments of cortical collecting ducts. After 6 wk of lithium discontinuation, adult rats exhibited attenuated urine concentration capacity and diminished outer medullary volume. Histological sections of two nephrectomy samples and a biopsy from three long-term lithium-treated patients showed multiple cortical microcysts that originated from normally appearing tubules. Microcysts were lined by a cuboidal PCNA-, GSK-3β-, and pGSK-3β-immunopositive epithelium. The postnatal rat kidney may serve as an experimental model for the study of lithium-induced human kidney injury. The data are compatible with a causal relationship between epithelial entry of lithium into cells of the aldosterone-sensitive distal nephron, inactivation of GSK-3β, proliferation, and microcysts.     

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.     

In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells: IFN-gamma and TNF-alpha mediated up-regulation during inflammation.

The reported requirement of functional Toll-like receptor (TLR)4 for resistance to Gram-negative pyelonephritis prompted us to localize the expression of TLR2 and TLR4 mRNA in the kidney at the cellular level by in situ hybridization. The majority of the constitutive TLR2 and TLR4 mRNA expression was found to be strategically located in the renal epithelial cells. Assuming that the TLR mRNA expression is representative of apical protein expression, this suggests that these cells are able to detect and react with bacteria present in the lumen of the tubules. To gain insight in the regulation of TLR expression during inflammation, we used a model for renal inflammation. Renal inflammation evoked by ischemia markedly enhanced synthesis of TLR2 and TLR4 mRNA in the distal tubular epithelium, the thin limb of Henle's loop, and collecting ducts. The increased renal TLR4 mRNA expression was associated with significant elevation of renal TLR4 protein expression as evaluated by Western blotting. Using RT-PCR, the enhanced TLR2 and TLR4 mRNA expression was shown to be completely dependent on the action of IFN-gamma and TNF-alpha. These results indicate a potential mechanism of increased immunosurveillance during inflammation at the site in which ascending bacteria enter the kidney tissue, i.e., the collecting ducts and the distal part of the nephron.     

Histochemical evaluation of mouse and rat kidneys with lectin-horseradish peroxidase conjugates

Paraffin sections of mouse and rat kidney were stained with a battery of ten lectin-horseradish peroxidase conjugates and lectin binding was correlated with the ultrastructural distribution of periodate-reactive sugar residues as determined by the periodic acid-thiocarbohydrazide-silver proteinate technique. Various segments of the uriniferous tubule in both species showed differential affinity for labelled lectins. Significant differences were also evident between comparable tubular segments in mouse and rat kidneys. Neutral glycoconjugates containing terminal beta-galactose and terminal alpha-N-acetylgalactosamine were prevalent on the luminal surface of the proximal convoluted tubule in the rat, but alpha-N-acetylgalactosamine was absent in this site in the mouse. In both species, terminal N-acetylglucosamine was abundant in the brush border of proximal straight tubules but absent in proximal convolutions. Fucose was demonstrated in both proximal and distal segments of mouse kidney tubules but only in the distal nephron and collecting ducts in the rat. Lectin staining revealed striking heterogeneity in the structure and distribution of cellular glycoconjugates. Such cellular heterogeneity was previously unrecognizable with earlier histochemical methods. The marked cellular heterogeneity observed with several lectin-conjugates in distal convoluted tubules and collecting ducts of both species raises a prospect that lectins can provide specific markers for intercalated and principal cells in the mammalian kidney. Glycoconjugates containing terminal sialic acid and penultimate beta-galactose were present on vascular endothelium in both rodent kidneys, as were terminal alpha-galactose residues; but both species lacked reactivity for Ulex europeus I lectin in contrast to human vascular endothelial cells. The constant binding pattern of lectin conjugates allows convenient and precise differentiation of renal tubular segments and should prove valuable in the study of changes in kidney morphology promoted by experimental manipulation or pathologic changes.     

Entry of nephrons into the collecting duct network of the avian kidney: A comparison of chickens and desert quail

The avian kidney contains a population of nephrons with and without loops of Henle. How the collecting ducts of this heterogeneous population of nephrons merge to exit as single ducts from the medullary cones has been uncertain. The results of this study show that the collecting duct tree begins with the coalescence of the distal tubules of pairs of loopless nephrons. These primary collecting ducts receive output from only loopless nephrons. Primary collecting ducts fuse in pairs and become secondary collecting ducts. They receive the distal tubules of transition nephrons. Pairs of secondary collecting ducts fuse and become tertiary collecting ducts. Tertiary collecting ducts receive the distal tubules of looped nephrons. Thus, the fluid from all nephron types comingles as it passes through the medullary cone. The results of this study also show that the anatomical arrangement of medullary cones does not permit the output from one medullary cone to enter a second medullary cone. Thus, all the medullary cones function as parallel units. This anatomical organization of the avian kidney affects its ability to produce a urine hyperosmotic to the plasma. © 1993 Wiley-Liss, Inc.     

Ultrastructural localization of IGF-I in the rat kidney; an immunocytochemical study

Summary We have shown recently by light microscopy that insulin-like growth factor I (IGF-I) immunoreactivity is localized in cells in the collecting ducts and in the thin loop of Henle in the normal rat kidney. In the present study, we have investigated the ultrastructural localisation of IGF-I using preembedding immunocytochemistry.The light microscopical findings were confirmed at the electronmicroscopical level. In collecting ducts as well as in the thin limb of Henle's loop a focal expression of IGF-I immunoreactivity was evident, i.e. distinctly IGF-I positive cells were intermingled with cells lacking IGF-I immunoreactivity. IGF-I immunoreactivity was found to have a diffuse cytoplasmatic distribution in both cell types. No specific association to organelles was found.     

Distribution of saccharide residues in glycoconjugates of the kidney in Gallus domesticus using peroxidase-conjugated lectins

A battery of seven different horseradish-peroxidase labelled lectins (DBA, PNA, SBA, UEA I, WGA, ConA, LTA) was used to study the distribution of sugar residues in the glycoconjugates along the nephron and the collecting duct of the kidney of Gallus domesticus. As far as the glomerular components are concerned, we have demonstrated that the podocytes and, with a lesser extent, the mesangial cells are characterised by the presence of D-mannose, D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. The glomerular capillary wall shows the presence of the disaccharide D-galactose-(beta 1- greater than 3)-N-acetyl-D-galactosamine and sialic acid. With regards to the tubules, the proximal tubule, the descending limb of the loop of Henle, the connecting tubule and the collecting one, are characterised by N-acetyl-D-galactosamine, (1- greater than 6)-alpha-L-fucose, D-mannose, N-acetyl-D-galactosamine and D-galactose-(beta 1- greater than 3)-N-acetyl-D-glucosamine. The cells of the connecting and collecting ducts show the presence of intracellular sialic acid, found also as component of the mucous secretion. The ascending limb of the loop of Henle and the distal tubule contain only three saccharidic residues, i.e. (1- greater than 6)-alpha-L-fucose, D-mannose and N-acetyl-D-glucosamine. Lectin histochemistry was also useful to define the saccharidic components of the mucus, which is normally present within the connecting and collecting ducts of the kidney of the birds. The cellular variability of the connecting and the collecting ducts is similar to that found in the kidney of some mammals. Such a variability seems to suggest a possible cell specialization along a single kidney tubule.     

Immunohistochemical localization of ghrelin in rodent kidneys

Ghrelin is a novel peptide hormone, originally identified in the rat and human stomach that plays various important roles. In the present study, we report the intra-renal localization of ghrelin in laboratory rodents. Kidneys from 3 month-old mice, rats and hamsters of both sexes were analyzed by immunohistochemistry. Positive signals were clearly observed in the epithelium of the distal tubules, whereas other segments of the nephron or interstitial cells, including juxtaglomerular cells, showed negative reactions. Pre-embedding immunoelectron microscopy revealed positive signals exclusively on the basolateral membrane in the distal tubular cells and in the collecting ducts. In addition, prepro-ghrelin gene expression was assessed by RT-PCR, and the expected 329-bp prepro-ghrelin mRNA was clearly detected in the kidney. On Western blot analysis, although a specific band for ghrelin (3 kDa) was not detected in the kidney, the expected band for prepro-ghrelin (13 kDa) was clearly detected in both the stomach and the kidney. This paper clarified the intra-renal localization of ghrelin.     

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.     

Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone- responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co- expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.     

Differential expression of Na+-Cl- cotransporter and Na+-K+-Cl- cotransporter 2 in the distal nephrons of euryhaline and seawater pufferfishes

The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.     

FGF8 is required for cell survival at distinct stages of nephrogenesis and for regulation of gene expression in nascent nephrons

During kidney morphogenesis, the formation of nephrons begins when mesenchymal nephron progenitor cells aggregate and transform into epithelial vesicles that elongate and assume an S-shape. Cells in different regions of the S-shaped body subsequently differentiate into the morphologically and functionally distinct segments of the mature nephron. Here, we have used an allelic series of mutations to determine the role of the secreted signaling molecule FGF8 in nephrogenesis. In the absence of FGF8 signaling, nephron formation is initiated, but the nascent nephrons do not express Wnt4 or Lim1, and nephrogenesis does not progress to the S-shaped body stage. Furthermore, the nephron progenitor cells that reside in the peripheral zone, the outermost region of the developing kidney, are progressively lost. When FGF8 signaling is severely reduced rather than eliminated, mesenchymal cells differentiate into S-shaped bodies. However, the cells within these structures that normally differentiate into the tubular segments of the mature nephron undergo apoptosis, resulting in the formation of kidneys with severely truncated nephrons consisting of renal corpuscles connected to collecting ducts by an abnormally short tubular segment. Thus, unlike other FGF family members, which regulate growth and branching morphogenesis of the collecting duct system, Fgf8 encodes a factor essential for gene regulation and cell survival at distinct steps in nephrogenesis.     

Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+ -K+ ATPase, H+ -ATPase, and Ca2+ -ATPase activities in these nephron segments demonstrated a specific enrichment of Na+ -K+ ATPase in thick ascending limbs, and of H+ -ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.