Proximal Convoluted Tubule Segments Research Articles

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

STRATEGY FOR THE DEVELOPMENT OF A MATCHED SET OF TRANSPORT-COMPETENT, ANGIOTENSIN RECEPTOR–DEFICIENT PROXIMAL TUBULE CELL LINES

In the proximal convoluted tubule (PCT) angiotensin II (Ang II) modulates fluid and electrolyte transport through at least two pharmacologically distinct receptor subtypes: AT(1) and AT(2). Development of cell lines that lack these receptors are potentially useful models to probe the complex cellular details of Ang II regulation. To this end, angiotensin receptor- deficient mice were bred with an Immortomouse(R), which harbors a thermolabile SV40 large-T antigen (Tag). S1 PCT segments from kidneys of F(2) mice were microdissected, placed in culture, and maintained under conditions that enhanced cell growth, i.e., promoted Tag expression and thermostability. Three different types of angiotensin receptor-deficient cell lines, (AT(1A) [-/-], Tag [+/-]), (AT(1B) [-/-], Tag [+/-]), and (AT(1A) [-/-], AT(1B) [-/-], Tag [+/+]), as well as wild type cell lines were generated. Screening and characterization, which were conducted under culture conditions that promoted cellular differentiation, included: measurements of transepithelial transport, such as basal monolayer short-circuit current (Isc; -3 to 3 microA/cm2), basal monolayer conductance (G, 2 to 10 mS/cm2), Na3(+)-phosphate cotransport (DeltaIsc of 2 to 3 microA/cm(2) at 1 mM), and Na(3)(+)-succinate cotransport (DeltaIsc of 1 to 9 microA/cm(2) at 2 mM). Morphology of cell monolayers showed an extensive brush border, well-defined tight junctions, and primary cilia. Receptor functionality was assessed by Ang II-stimulated beta-arrestin 2 translocation and showed an Ang II-mediated response in wild type but not (AT(1A) [-/ -], AT(1B) [-/-]) cells. Cell lines were amplified, yielding a virtually unlimited supply of highly differentiated, transport-competent, angiotensin receptor-deficient PCT cell lines.

Age-Related Changes in Renal Function, Membrane Protein Metabolism, and Na,K-ATPase Activity and Abundance in Hypokalemic F344 × BNF1 Rats

Background: Potassium depletion is a common electrolyte abnormality in elderly humans, usually as a consequence of diuretic use or poor oral intake. Hypokalemia is associated with a number of changes in renal function and an increase in some renal membrane transporters; its growth-promoting effect in young animals is well known. With aging, the renal adaptation to a number of challenges is often diminished. We hypothesized that aging is related to decreases in renal function, renal membrane protein metabolism, as well as Na,K-ATPase protein abundance and activity in both control animals as well as in those with potassium depletion. Objective: We examined the effects of dietary-induced hypokalemia in true-aged nonobese rats (30 months old) on renal function, cortical brush border membrane (BBM) and basolateral membrane (BLM) protein metabolism, and Na,K-ATPase protein abundance and activity. We compared the results obtained to those seen in their 4-month-old counterparts similarly treated. Methods: Young (4-month-old) and senescent (30-month-old) male Fisher 344 × Brown-Norway F₁ rats (F344 × BNF₁) were fed either a normal or potassium-deficient diet for 7 days. At 24 h, the U-¹⁴C-leucine incorporation was measured for determination of protein metabolism in renal BBM and BLM. Cortical BLM vesicle and microdissected proximal convoluted tubule (PCT) Na,K-ATPase activities were determined along with Western blot analysis of the cortical BLM α₁ subunit of Na,K-ATPase. Metabolic and renal function parameters were also examined. Results: Hypokalemia caused hyperbicarbonatemia, hyperglycemia, and azotemia, but only in the senescent animals. The aged control rats had a higher basal level of urine volume, ammonium excretion, and fractional excretion of chloride. By contrast, aging in the F344 × BNF₁ rats was associated with a decrease in plasma aldosterone (by 35%) and phosphate (by 40%) levels as compared with their young controls. Hypokalemia resulted in a significant reduction of plasma aldosterone and a rise in muscle sodium concentration in both age groups; it significantly increased renal BBM and BLM protein concentrations in the young group, while these parameters remained unchanged in the senescent rats. The aged potassium-depleted animals showed a 14% decrease in BBM protein biosynthesis, but there were no changes in the young hypokalemic rats. Both potassium-depleted elderly and young rats had a significant reduction (by 33%) in BLM protein biosynthesis. Hypokalemia significantly increased the Na,K-ATPase activity in both cortical BLM vesicles and in microdissected PCT. The percentage increase in microdissected PCT segments (Na,K-ATPase activity) in elderly potassium-depleted animals was significantly less than that seen in hypokalemic young ones. Aging, per se, was associated with decreased basal microdissected PCT Na,K-ATPase activity in control animals. Hypokalemia had no effect on cortical BLM α₁ subunit Na,K-ATPase protein abundance in either age group. Conclusions: The present study provides the first evidence in nonobese aged rats as to the metabolic parameters, renal function, renal cortical membrane protein metabolism, and transporter Na,K-ATPase activity and abundance during potassium depletion. The aged nonobese F344 × BNF₁ rats responded differently from their young nonobese counterparts following potassium depletion. These differences may contribute substantially to the effects often encountered in elderly humans receiving diuretics or having a poor dietary potassium intake.

Heterogeneous expression of Gal alpha1-3Gal xenoantigen in pig kidney: a lectin and immunogold electron microscopic study.

The Gal alpha1-3Gal antigen (Gal alpha) is the primary target for human natural anti-pig xenoantibodies. The presence of Gal alpha has been shown in porcine endothelial cells (ECs) using light microscopy, whereas the expression of Gal alpha in other cell structures in the porcine kidney is only partially characterized. Immunogold electron microscopy of pig kidney cryosections was performed using Griffonia simplicifolia isolectin B4 and affinity isolated human anti-Gal alpha1-3Gal antibodies. The most intense expression of Gal alpha was found on the apical and basolateral portions of the plasma membrane of the proximal convoluted tubule segments 1 and 2 cells, whereas segment 3 and 4 cells were negative. A strong staining was found in peritubular capillary ECs and in the inner medullary and papillary collecting duct cells. Moderate labeling of ECs and subendothelium was observed in large blood vessels, whereas glomerular ECs reacted weakly. Additionally, glomerular parietal epithelial cells, connecting tubule cells, and some cortical collecting duct cells were labeled. Among interstitial cells, a part of type-1 cells and all type-2 cells were labeled, whereas others were negative. By immune electron microscopy, a detailed information of the Gal alpha antigen distribution in porcine nephrons and blood vessels has been revealed, which clarifies conflicting data obtained by light microscopy. In addition, expression of the Gal alpha antigen in the renal interstitial cells was documented for the first time. These data are of importance for the understanding of xenoantibody-mediated hyperacute rejection, for interpretation of pig kidney xenograft biopsies, and for generating transgenic pigs lacking the Gal alpha epitope.

Cholinergic agonists increase phosphoinositide metabolism and cell calcium in isolated rat renal proximal tubule.

The intracellular signaling involved in cholinergic modulation of renal proximal tubule functions has not been addressed. We report that acetylcholine and carbachol increase the production of inositol phosphates and the intracellular calcium concentration in rat proximal tubule. Muscarinic M3 receptors are involved, given the inhibitory effects of selective antagonists (4-diphenylacetoxy-N-methylpiperidine > > pirenzepine > methoctramine). The muscarinic response is absent in the early part of proximal straight tubule. The response is smaller and more variable in proximal convoluted tubule segments selected at random from cortical tubule suspensions than in the early part of proximal convoluted tubule. This contrasts with the norepinephrine response, which has almost the same magnitude all along the proximal tubule. We conclude that cholinergic agonists activate muscarinic M3 receptors in proximal tubule and that there is a decreasing response gradient from the early convoluted tubule to the early straight tubule.

Clonidine inhibits fluid absorption in the rabbit proximal convoluted renal tubule

Previous studies have shown that norepinephrine (NE) and the beta-adrenoceptor agonist, isoproterenol (I), enhance fluid absorption (JV) in isolated, perfused proximal convoluted tubule segments (PCT). Pretreatment of PCT with the beta-adrenoceptor antagonist, propranolol, inhibited the action of NE and produced a significant decline in JV, suggesting modulation of JV by both alpha- and beta-adrenoceptors. The present studies further characterize the alpha-adrenoceptor control of JV in isolated perfused PCT using specific agonists and antagonists. Basal JV declined significantly with the addition of the alpha 2-adrenoceptor agonist, clonidine (10(-4) M), to the bath; however, it was unchanged with the addition of the alpha 1-adrenoceptor agonist, methoxamine (10(-6) or 10(-4) M). With the addition of 10(-6) M isoproterenol JV increased significantly, and returned to control values with the subsequent addition of clonidine (10(-6) or 10(-4) M). Pretreatment of PCT with the alpha 2-adrenoceptor antagonist, yohimbine (10(-5) M), or with pertussis toxin (100 ng/ml) did not interfere with the stimulation of JV by isoproterenol, but abolished the inhibition of isoproterenol-stimulated JV by clonidine. Thus, clonidine inhibits JV in PCT via an alpha 2-adrenoceptor. This effect is mediated by a pertussis toxin inhibitable GTP-binding protein, but not one that is coupled to adenylyl cyclase.

Regulation of Na+-K+-ATPase activity in kidney proximal tubules: involvement of GTP binding proteins

This study evaluates the involvement of GTP-dependent regulatory proteins (G-proteins) in the regulation of Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments. Single PCT segments were dissected from rat kidney and permeabilized to allow nucleotides and medium free access to the interior of the cell. A GDP analogue that blocks GTP-dependent activation of the G-protein, GDP beta S (400 microM) significantly inhibited PCT Na+-K+-ATPase activity when Na in the medium (Nam) was greater than or equal to 70 mM. The inhibition was attenuated when Nam was 55 and 35 mM and was no longer significant when Nam was 25 mM. GDP beta S had no inhibitory effect on the activity of purified Na+-K+-ATPase. A nonhydrolyzable GTP analogue, GppNHp (50 microM) significantly increased Na+-K+-ATPase activity when Nam was 25 and 35 mM, but not when Nam was 55-140 mM. Dopamine (DA) and DA1 plus DA2 agonists significantly inhibit Na+-K+-ATPase activity. DA inhibition was competitively abolished by GppNHp. In PCT segments from rats pretreated with pertussis toxin, DA and DA1 plus DA2 agonist inhibition of Na+-K+-ATPase activity was abolished. In PCT segments from rats pretreated with cholera toxin, basal Na+-K+-ATPase activity was increased, but DA significantly inhibited Na+-K+-ATPase activity. Na+-K+-ATPase activity in PCT segments is regulated via a G-protein that stimulates Na+-K+-ATPase activity and a DA-activated pertussis toxin-sensitive G-protein that inhibits Na+-K+-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine causes inhibition of Na+-K+-ATPase activity in rat proximal convoluted tubule segments.

We studied the effect of dopamine (DA) on Na+-K+-ATPase activity in proximal convoluted tubule (PCT) segments dissected from perfused rat kidneys. DA inhibited Na+-K+-ATPase activity in a dose-dependent manner. Inhibition was significant with 10(-7) M DA and maximal with 10(-4) M DA. The inhibition was reversible. Enzyme inhibition occurred in the presence of DA and a DA antagonist, metoclopramide, but not when 10(5) M of the DA1 and DA2 agonists fenoldopam mesylate and LY 171555 were added in the absence of DA. In PCT segments incubated with the DA precursor dopa, Na+-K+-ATPase activity was also inhibited. However, dopa did not inhibit the sodium pump if dopa decarboxylase activity was blocked with benserazide. These findings suggest an intracellular site of action of DA. In tubules incubated in different K concentrations, 10(-5) DA decreased the maximal activity (Vmax) and increased the Km. DA 10(-5) M caused an almost immediate swelling of PCT segments. Swelling did not occur in the presence of both DA and 10(-5) M amiloride. The DA-induced tubular swelling was probably due to inhibition of Na+-K+-ATPase-mediated Na+-transport. We conclude that in rat PCT segments, DA causes a rapid and reversible inhibition of apparent Na+-K+-ATPase activity and an apparent reduction in the affinity for K. The site of action appears to be intracellular.

Microscopic Postmortem Changes in Kidneys of the Domestic Fowl

Eighty-four male white leghorn chickens were killed by CO2 gas to determine the type, rate, and sequence of postmortem microscopic changes in the kidneys of dry and wet intact carcasses. They were held at 29 or 18 C with 50% relative humidity for different times postmortem. Microscopic postmortem changes in the different segments of the nephron underwent a different rate and sequence of cellular changes. Cellular changes occurred earlier at 29 C than at 18 C and earlier in chickens not wetted with detergent solution. The decrease in body temperature of wetted chickens over dry chickens was significant (P less than 0.05). The proximal convoluted tubule (PCT) underwent the earliest postmortem changes, followed by the distal convoluted tubule (DCT), collecting tubule (CT), medullary loop (ML), medullary collecting duct (MCD), and glomerulus. The PCT, DCT, and thin and thick segments of the ML underwent a sequential nuclear change of chromatin margination, progressive shrinkage, pyknosis, karyorrhexis, and karyolysis. Nuclei were pyknotic if cytoplasmic changes were severe. Primary karyorrhexis was the predominant feature of collecting tubules and ducts. As early as one hour after death, some PCT cells of all kidney sections were pyknotic, emphasizing that immediate tissue fixation was necessary for critical evaluation. By 9 and 18 hr postmortem, PCT of dry and wet chickens, respectively, held at 29 C had pyknotic and karyorrhectic nuclei with slight karyolysis and moderate to marked cytoplasmolysis that extended until 36 hr. At this time, DCT were hardly distinguishable because of loss of basophilia. Karyorrhectic nuclei were already evident in collecting tubules and ducts. At 48 hr postmortem, all tubular cells were non-nucleated with homogeneous, acidophilic cytoplasm. Basement membranes no longer stained with periodic acid-Schiff (PAS). Erythrocytes were pyknotic with unstained cytoplasm. Pyknotic glomeruli were first observed at 9 hr postmortem in dry chickens and 12 hr in wet chickens. Histologic appearance of dry chickens at 9 hr and wet chickens at 18 hr when held at 29 C was similar to that of dry chickens at 12 hr and wet chickens at 24 hr when held at 18 C, with minor differences in some tubular changes. At 18 C, pyknotic glomeruli appeared by 6 hr in dry chickens and 24 hr in wet chickens. Widespread bacterial invasion was noted at 72 hr in dry chickens and at 96 hr in wet chickens.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of peritubular protein upon fluid reabsorption in rabbit proximal convoluted tubules perfused in vitro.

This paper describes an investigation of the effects of varying the peritubular protein concentration upon the rate of fluid reabsorption of proximal convoluted tubule segments of the rabbit kidney, isolated and perfused in vitro. Eleven moderately distended tubules, bathed with rabbit serum (60 g l.-1 protein) and perfused with an ultrafiltrate of serum (ca. 0 g l.-1) protein, reabsorbed fluid at a rate of 1.0 +/- 0.07 nl. mm-1 min-1. When the protein of the bathing solution was reduced by replacing the serum with ultrafiltrate there was little change in fluid reabsorption rate. A further eleven moderately distended tubules, bathed with physiological saline containing bovine serum albumin (60 g l.-1) and perfused with saline, reabsorbed fluid at 1.0 +/- 0.06 nl. mm-1 min-1. There was little change in fluid reabsorption when the protein concentration was reduced to either 20 or 0 g l.-1; these tubules responded to 10(-5) M-ouabain in the bath, or a temperature of 25 degrees C, by reducing their absorption rate to 0.13 +/- 0.05 nl. mm-1 min-1. Nine minimally distended tubules reabsorbed fluid at 0.8 nl. mm-1 min-1 when bathed with physiological saline containing bovine serum albumin (60 g l.-1) and perfused with saline. Under these circumstances reabsorption rate fell by 26% when protein was removed from the bath. A study of the pressure-diameter relationship was made for eight tubules typical of those used in this laboratory. The probable range of lumen hydrostatic pressures was discussed for the distended and undistended states. We conclude that the effect of peritubular protein concentration upon fluid reabsorption in proximal convoluted tubule segments perfused in vitro is dependent upon some property of the tubule wall that is changed when distension occurs.

Intrinsic differences in various segments of the proximal convoluted tubule.

Until recently it has not been possible to compare directly the function of superficial and juxtamedullary nephrons. The present studies, using in vitro microperfusion, were designed to examine whether functional differences exist between proximal convoluted tubule segments of superficial and juxtamedullary nephrons. Electrophysiological studies showed that major differences exist between the relative chloride and sodium permeabilities of these segments. In the 1st mm of the superficial proximal convoluted tubule, the permeability to sodium was greater than that to chloride, whereas in the 2nd mm of the superficial proximal convoluted tubule and all later segments, the permeability to chloride was greater than that to sodium. The juxtamedullary proximal convoluted tubule was found to differ from the superficial proximal convoluted tubule in two respects: first, the relative permeabilities to chloride and sodium did not differ in the various segments of the juxtamedullary proximal convoluted tubule; second, the permeability to sodium was greater than to chloride throughout. When perfused with a solution lacking glucose and amino acids, the superficial and juxtamedullary convolutions exhibited the same transepithelial potential change, a reversible decrease to less than -- 1 mV. It thus appears that in both convolutions there exists electrogenic sodium transport coupled to the transport of these organic solutes. This differs from pars recta of both of these nephrons, which have been shown to exhibit electrogenic sodium transport independent of organic solutes. However, when perfused with a solution lacking glucose and amino acids but also containing high chloride and low bicarbonate concentrations, the superficial convolution developed a significantly more positive potential than the juxtamedullary. This difference reflects greater relative chloride permeability in the superficial proximal convolution. These studies show that intrinsic functional differences exist between proximal convoluted tubules obtained from the superficial and juxtamedullary nephron populations.

Proximal tubule potential difference. Dependence on glucose on glucose, HCO 3 , and amino acids.

The effect of various intraluminal substrates on the magnitude of the transepithelial potential difference (PD) across the proximal convoluted tubule (PCT) of the mammalian kidney was investigated in two ways. First, the transepithelial PD was measured before and after the removal of glucose, bicarbonate, and alanine from the lumen. Second, the effects of specific transport inhibitors-ouabain, phloridzin, and acetazolamide-was ascertained when placed either on luminal or blood side. Isolated segments of rabbit PCT were perfused in vitro. Tubules perfused with isosmolar ultrafiltrate (UF) at rates > 10 nl/min had a mean PD of - 5.8+/-0.2 mV (lumen negative). Normal UF was simulated by an artificial perfusion solution. Using the latter, observed PD was - 5.4+/-0.2 mV. A significant reversible decrease in PD was noted when the following constituents were removed singly: glucose (from - 5.7+/-0.4 to - 3.5+/-0.4 mV); alanine (from - 5.8+/-0.4 to - 4.7+/-0.3 mV); and bicarbonate (from - 5.3+/-0.3 to - 3.3+/-0.5 mV). The combined removal of alanine and glucose (replaced with mannitol) reduced the transepithelial PD to - 0.5+/-0.1 mV with removal of glucose and alanine (replaced with mannitol) and decrease of NaHCO(3) to 5.6 meq/liter (replaced with NaCl), as normally occurs in early part of in vivo PCT, resulted in reversible change of PD from - 5.1+/-0.2 to + 3.2+/-0.2 mV. Ouabain (10(-5) M) reversibly decreased the negative control PD from blood side, but had no effect from luminal side. Phloridzin (10(-5) M) reversibly decreased PD from - 6.4+/-0.3 to - 3.7+/-0.4 mV when placed on luminal side but had minimal effect from blood side, - 6.3+/-0.4 to - 5.8+/-0.4 mV. Acetazolamide (Diamox) was without effect from either side. Reversal of bulk flow of water by addition of 31 mosmol/liter raffinose to perfusion ultrafiltrate did not significantly decrease the PD. It is concluded that specific pumps for transport of glucose, amino acids, and bicarbonate exist on the luminal surface. All three constituents are necessary for expression of maximum PD. Removal of these substrates by transport changes PD from - 5.1 mV to + 3.2 mV (lumen positive). This 3.2 mV positive PD is secondary to a chloride diffusion potential and is not effected by ouabain from the blood side.