Ultrastructural observations on the pars descendens of the proximal tubule in the kidney of the male rat

Summary The pars descendens (pars recta) of the proximal tubule in the male rat kidney, consisting of the terminal part of the second proximal segment (P₂) and of the third proximal segment (P₃), was studied with the electron microscope. A technique of tissue orientation and trimming was used which permitted precise topographic definition of the tubules studied in the electron microscope. The terminal descending part of the P₂ showed some minor differences from the convoluted part of this segment, and ultrastructure also changed along the course of the P₃. In the beginning of the latter segment numerous, shallow interdigitations were observed between adjacent cells; along the course of the segment they decreased in number or disappeared. In the initial part of the P₃ mitochondria were more abundant than in the terminal portion of the segment and at least as numerous as in the straight part of the P₂. Also, the dense, acid phosphatase-positive cytoplasmic bodies decreased somewhat in size along the course of the P₃. The smooth surfaced endoplasmic reticulum reached a higher development in the P₃ than anywhere else in the proximal tubules.Investigation supported by grants from: Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — The authors are indebted to Mrs. J. Barslund and Mrs. M. Jacobsen for excellent technical assistance.     

Normal rat kidney proximal tubule cells in primary and multiple subcultures

Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1 to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence, the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture model will be a valuable tool for substrate uptake and nephrotoxicity studies.     

Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells

Characterization of the phosphate transport system across the basolateral membrane of renal proximal tubule has been attempted using isolated proximal tubule cells prepared from chicks. The Pi efflux system is independent of Na+ ions and is not influenced by the nature of the chief anion present in the bathing medium. Pi efflux is not sensitive to DIDS and it is concluded that a generalized anion transporter of band III type is not the chief agent for facilitating Pi exit from the cell across the basolateral membrane. Inhibition of efflux by vanadate is evidence for a specific carrier protein in the membrane. The carrier probably possesses thiol group(s) that are essential for activity. The carrier may effect electroneutral transport of Pi possibly in exchange for OH- ions. The activity of the transport process is not stimulated by depleting the cells of phosphate or inhibited by rearing the chicks on a vitamin D-deficient diet. The system is unlikely to be of great importance for the expression of various regulatory mechanisms that act on the kidney to control the excretion of Pi. The activity declines as the chicks mature however.     

The transepithelial shunt pathway in the renal proximal tubule of newt kidney

The transepithelial shunt pathway of newt proximal tubule was examined with glass micro-electrode and electron microscopic methods. The input resistance of the peritubular (basal) membrane and tubular wall were found to be $\text{4.2 ± 0.1 · 10}{}^6$ ($\text{mean ± S.E.}\text{, n = 16}$) and $\text{11.4 ± 0.2 · 10}{}^4\text{(n = 11)}$, respectively. The input resistance of the peritubular membrane was approximately 40-times larger than that of the tubular wall. When the kidneys were perfused in a lanthanum solution, the lanthanum ions were then observed in the junctional complexes and in the intercellular spaces on both the basal and apical sides. The results indicate that the electrical shunt pathway corresponds to the apical junctional complexes and the intercellular spaces, and that the tight junctions are not truly ‘tight’ for the transepithelial movement of small ions in the proximal tubule of the newt kidney.     

A critical reevaluation of the structure of the rat uriniferous tubule as revealed by scanning electron microscopy

Summary The fine structure of luminal surface of clearly identified portions of uriniferous tubules has been studied by scanning electron microscopy to elucidate some controversies concerning the topography of certain surface formations. The results show a characteristic pattern of the luminal surface in the region of Henle's loop, which was assumed by previous authors, to belong to the collecting tubule. Furthermore it is demonstrated that no cilia are present within the terminal portion of the collecting tubules.     

Megalin-mediated endocytosis of cystatin C in proximal tubule cells

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.     

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

Summary Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.     

Structural heterogeneity of the basement membrane in the rat proximal tubule

Summary The ultrastructure of the basement membrane of the rat proximal tubule was observed by transmission electron microscopy after the use of a cold dehydration technique. The basement membrane of the P1 segment is thick and possesses several structural specializations that are rare in other basement membranes; these include intraepithelial ridges, dense bars, and basement membrane vesicles. The intraepithelial ridges are found in the intercellular spaces between interdigitating processes of the proximal tubule cells. The ridges and the interdigitating processes run circumferentially around the tubule. The dense bars are frequently found in the intraepithelial ridges. They are especially prominent on the concave side of the tubular bends and to a lesser extent near sites where intracellular actin filaments anchor onto the basal cell membranes. The basement membrane vesicles are bounded by unit membranes; they are variable in both their electron density and their size. They are usually found in association with dense bars, and the grade of their accumulation is positively correlated with the development of the dense bars. These three specializations have no topographical relationship with the interstitial structures, such as fibrobalasts and collagen fibrils. The specializations are best developed on the concave side of tubular bends where the circumferential stresses caused by the intraluminal hydraulic pressure are presumably the largest; we therefore propose that they are an adaptation to, or a manifestation of, the increased wall stress in the proximal tubule.     

A freeze-fracture study of tight junctions in the pars convoluta and pars recta of the renal proximal tubule

Summary The morphology of tight junctions of the renal proximal tubule was studied comparing the pars convoluta and pars recta of rat, golden hamster, rabbit, cat, dog and tupaia. Though some interspecies variations were observed, the convoluted portions of the proximal tubules revealed quite uniformly very leaky tight junctions with mainly 1–2 strands.Along the whole proximal tubule of the rabbit kidney including the pars recta only minor differences of the zonulae occludentes were found. By contrast, the tight junctions of the pars recta in other species were much more elaborate, especially in cat and tupaia, having up to 6 strands and an overall depth of more than 150 nm. The implications of these findings are discussed with special regard to the functional differences between the pars convoluta and pars recta of the proximal tubule.This work was supported by the Deutsche Forschungsgemeinschaft     

Aprotinin uptake in the proximal tubules in the rat kidney I. Length of proximal tubular uptake segment

Aprotinin (Ap), a basic polypeptide with a molecular weight of 6500, is filtered at the glomerular membrane without steric restriction and is completely absorbed by the proximal tubule cells. Here Ap is broken down to amino acids, but no breakdown products enter the peritubular circulation during the first 20 min following an intravenous injection. These properties have recently been exploited for measurement of local glomerular filtration rate, based on the assumption that the proximal tubular uptake site is located at the level of the filtering glomerulus. To evaluate that assumption we have now made serial autoradiographs of the rat kidney 20 min after intravenous injection of 2-750 microg of 125I-Aprotinin. With all doses the percent 125I-containing proximal tubular transections were about 50 in the outer and middle cortex and 35 in the inner third. We interpret these numbers to mean that all filtered Ap is taken up in the first two thirds of the proximal convoluted tubular length and does not reach the pars recta. Since the proximal tubule on average is located more superficial than its glomerulus, measurement of local Ap uptake will tend to overestimate glomerular filtration rate in outer layers of the cortex. Quantitative estimate of this "displacement" will be presented in a companion article.     

Studies of the regulation of renal gluconeogenesis in normal and Pi depleted proximal tubule cells

Pi depletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose-2,6-bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pi depletion. Introduction of fructose-2,6-bisphosphate into the cytosol of ATP-permeabilized cells is accompanied by a fall in gluconeogenesis. The presence of external Ca2+ stimulates gluconeogenesis. When cytosolic Ca2+ is raised to 1.8 microM by permeabilization, the resealed cells still require 2.5 mM Ca2+ in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ ions. It is suggested that Pi depletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by protein kinase C.     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

Regulation of gluconeogenesis in swine kidney proximal tubule cells

Summary Proximal tubule cells were isolated from swine kidney and cultured for periods of more than 30 days. The cells formed confluent monolayers after plating on a collagen surface and they were passaged more than 5 times on this matrix. The cells maintain several metabolic functions of proximal tubule cells, including gluconeogenesis and the ability to respond to epinephrine and parathyroid hormone. Gluconeogenesis, a principal metabolic pathway in proximal tubule cells, was examined as a function of days in culture. The isolated cells showed a nearly constant rate of gluconeogenesis from ¹⁴C-lactate, ¹⁴C-alkaine and 14C-glycerol with no significant loss of activity for at least 30 days in culture. Likewise, the activities of several cytosolic and membrane associated enzymes including, alkaline phosphatase, -glutamyltransferase, fructose-1,6-bisphosphatase and phosphofructokinase were nearly constant over the same time period.The cells responded to treatment with epinephrine and parathyroid hormone, and the rate of gluconeogenesis from ¹⁴C-lactate doubled in the presence of these hormones. The morphological and biochemical evidence obtained in these studies show that the proximal tubule cells isolated from swine kidney provide an excellent well defined system for studying the hormonal regulation of carbohydrate metabolism in this tissue.Abbreviations PTH Parathyroid Hormone - cAMP cyclic 3,5-adenosine Monophosphate     

Magnesium influx enhanced by nitric oxide in hypertensive rat proximal tubule cells

An abnormal handling of renal magnesium has been suggested to cause salt-sensitive hypertension. The filtered magnesium is first reabsorbed in the proximal tubule. Amiloride has been shown to enhance renal magnesium conservation, but the regulatory mechanisms are unknown yet. High-salt (8% NaCl) diet decreased serum magnesium concentration, while increased urinary magnesium in Dahl salt-sensitive (DS) rat. Furthermore, the expression of nitric oxide synthase type 3 and nitric oxide (NO) content were decreased in high-salt loaded DS rat. In isolated proximal tubule cells, amiloride (0.1 mM) increased intracellular free magnesium concentration ([Mg(2+)](i)). However, the net [Mg(2+)](i) increase in the high-salt loaded DS rat was smaller than other groups. NOR1 (0.1 mM), a NO donor, restored the increase of [Mg(2+)](i) to the same level of other groups. On the contrary, L-NMMA (0.1 mM), an inhibitor of NO production, inhibited the increase of [Mg(2+)](i) in all groups. These results suggest that intracellular NO has an important role to up-regulate amiloride-elicited magnesium influx.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Adenosine deamination to inosine in isolated basolateral membrane from kidney proximal tubule: Implications for modulation of the membrane-associated protein kinase A

In this work, the metabolism of adenosine by isolated BLM associated-enzymes and the implications of this process for the cAMP-signaling pathway are investigated. Inosine was identified as the major metabolic product, suggesting the presence of adenosine deaminase (ADA) activity in the BLM. This was confirmed by immunoblotting and ADA-specific enzyme assay. Implications for the enzymatic deamination of adenosine on the receptor-modulated cAMP-signaling pathway were also investigated. We observed that inosine induced a 2-fold increase in [³⁵S] GTPγS binding to the BLM and it was inhibited by 10⁻⁶ M DPCPX, an A₁ receptor-selective antagonist. Inosine (10⁻⁷ M) inhibited protein kinase A activity in a DPCPX-sensitive manner. Molecular association between ADA and G_αi-3 protein-coupled A₁ receptor was demonstrated by co-immunoprecipitation assay. These data show that adenosine is deaminated by A₁ receptor-associated ADA to inosine, which in turn modulates PKA in the BLM through A₁ receptor-mediated inhibition of adenylyl cyclase.     

Innervation of the renal proximal convoluted tubule of the rat

Experimental data suggest the proximal tubule as a major site of neurogenic influence on tubular function. The functional and anatomical axial heterogeneity of the proximal tubule prompted this study of the distribution of innervation sites along the early, mid, and late proximal convoluted tubule (PCT) of the rat. Serial section autoradiograms, with tritiated norepinephrine serving as a marker for monoaminergic nerves, were used in this study. Freehand clay models and graphic reconstructions of proximal tubules permitted a rough estimation of the location of the innervation sites along the PCT. In the subcapsular nephrons, the early PCT (first third) was devoid of innervation sites with most of the innervation occurring in the mid (middle third) and in the late (last third) PCT. Innervation sites were found in the early PCT in nephrons located deeper in the cortex. In juxtamedullary nephrons, innervation sites could be observed on the PCT as it left the glomerulus. This gradient of PCT innervation can be explained by the different tubulovascular relationships of nephrons at different levels of the cortex. The absence of innervation sites in the early PCT of subcapsular nephrons suggests that any influence of the renal nerves on the early PCT might be due to an effect of neurotransmitter released from renal nerves reaching the early PCT via the interstitium and/or capillaries.     

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD⁺/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.     

Aprotinin uptake in the proximal tubules in the rat kidney. II. Uptake site relative to glomerulus

Glomerular filtration rates in whole kidney and in outer, middle and inner cortical zones have previously been estimated by measuring the amount of iodinated Aprotinin, filtered and taken up in the first two thirds of the proximal convoluted tubules, in part positioned more superficial than the parent glomerulus. Thus, an appreciable amount of the absorbed Aprotinin may be located superficial to its filtration site and lead to an underestimate of glomerular filtration in deep cortical layers. Therefore, in this study we have measured the distance from the glomerulus to the center of proximal convoluted tubular ball and the site of Aprotinin uptake. Measurements were made on photos of Microfil-injected tubules and on camera lucida drawings of tubular transections from autoradiographs of nephrons containing both Microfil and iodinated Aprotinin. Both techniques showed that the center of the tubular ball was localized more superficial in all cortical layers. The average distance, in percent of cortical thickness, from all proximal convoluted tubular transections to the parent glomerulus was 9% in deep and 13% in middle and superficial cortex. Corresponding distances for tubular transections containing Aprotinin were 7 and 12%. Grain density in five reconstructed proximal convoluted tubules showed a continuous and exponential fall of Aprotinin along the uptake segment. The results may be used to estimate single nephron filtration rate from Aprotinin uptake and glomerular density in outer, middle, and inner cortex.     

Incorporation of 3H-oleic acid by the proximal convoluted tubule cells of the chick (Gallus domesticus)

Summary Lipid metabolism in the cells of the renal proximal convoluted tubules (PCT) was investigated in healthy fowls and in fowls with the Fatty Liver and Kidney Syndrome (FLKS). The tissue was fixed at 10–25 min intervals after intravenous injection of ³H-oleic acid. The distribution of autoradiographic grains was analysed by the circle method. In normal cells most of the silver grains were associated with the cytoplasmic organelles. Lipid droplets and Golgi elements had the highest specific activity relative to the nuclear activity, which was little above background level. Lysosome-like bodies and mitochondria had lower values. In the cells of the FLKS-affected birds a large proportion of the grains was located over the lipid droplets, which are abundant in this condition. The specific activity of the cytoplasmic organelles was barely 2-fold higher than the nuclear activity. The results suggest that there is a diminished incorporation of esterified fatty acids by the organelles of these cells and that the excess is transferred to the lipid droplets. The identity of low electron density particles observed in the PCT cells of severely affected birds is discussed.