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M A Knepper

Publications and source records attributed to M A Knepper.

At least 127 records · Page 7Linked to original sources
PubMed1994

Extracellular ATP increases intracellular calcium in rat terminal collecting duct via a nucleotide receptor.

Recent studies in a variety of cell types have revealed several receptor subtypes that bind ATP and trigger increases in intracellular Ca2+ concentration ([Ca2+]i). The present studies were aimed at determining whether similar receptors are present in the rat terminal inner medullary collecting duct (IMCD). [Ca2+]i was measured using fura 2 in tubules dissected from collagenase-treated rat kidneys. ATP (1-100 microM) caused a rapid increase in [Ca2+]i with a prolonged late phase after an initial peak. A similar rise was observed in tubules exposed to UTP or to the poorly hydrolyzable analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S). In contrast, agonists that bind P2x, P2y, P2z, and P2t purinergic receptors did not affect [Ca2+]i. Removal of extracellular Ca2+ inhibited the response to ATP by approximately 50% with obliteration of the late phase. Furthermore, indomethacin attenuated the rise in [Ca2+]i produced by ATP. Adenosine analogues also increased [Ca2]i apparently by binding to distinct adenosine receptors rather than to the ATP receptor. We conclude that there is a nucleotide receptor in the rat terminal IMCD, which, when occupied, mobilizes intracellular Ca2+.

Adenosine↗
PubMed1993

Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.

Vasopressin (antidiuretic hormone) regulates body water balance by controlling water permeability of the renal collecting ducts. The control mechanisms may involve alterations in the number or unit conductance of water channels in the apical plasma membrane of collecting-duct cells. How this occurs is unknown, but indirect evidence exists for the "shuttle" hypothesis, which states that vasopressin causes exocytic insertion of water channel-laden vesicles from the apical cytosol. To test key aspects of the shuttle hypothesis, we have prepared polyclonal antisera against the recently cloned collecting-duct water channel protein and used the antisera in immunolocalization studies (light and electron microscopic levels) in thin and ultrathin cryosections from rat kidney. Labeling was seen exclusively in collecting-duct principal cells and inner medullary collecting-duct cells. Apical membrane labeling was intense. There was heavy labeling of abundant small subapical vesicles and of membrane structures within multivesicular bodies. In addition, labeling of basolateral plasma membranes in inner medullary collecting ducts was present. Depriving rats of water for 24 or 48 hr markedly increased collecting-duct water-channel protein expression determined by immunoblotting and immunolabeling. These results are compatible with at least two complementary modes of water-channel regulation in collecting-duct cells: (i) control of channel distribution between the apical membrane and a reservoir in subapical vesicles (shuttle hypothesis) and (ii) regulation of the absolute level of expression of water-channel protein.

Amino Acid Sequence↗
PubMed1993

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron.

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.

Animals↗
PubMed1993

In vitro perfusion of chinchilla thin limb segments: urea and NaCl permeabilities.

We measured the urea and NaCl permeabilities (Purea and PNaCl, respectively) of the following nephron segments from chinchilla: the upper part of the long-loop descending limb (from outer medulla, LDLu), the middle part of the long-loop descending limb (from outer 30% of the inner medulla, LDLm), the lower part of the long-loop descending limb (from deep inner medulla, LDLl), and the thin ascending limb (from deep inner medulla, ATL). We found that Purea (x10(-5) cm/s) was relatively low in the LDLu (3.3), but that the value was larger in the inner medullary thin descending limb (16.8 for LDLm and 47.6 for LDLl). The ATL had an even higher value (170). Phloretin, 0.25 mM, added to the peritubular bath had no effect on Purea of these segments, suggesting that the rapid transport rate is not due to a phloretin-sensitive facilitated transport pathway like that seen in the inner medullary collecting duct. PNaCl (x10(-5) cm/s) also increased with distance along the length of the thin descending limb (LDLu, 11.7; LDLm, 41.2; LDLl, 98.4; and ATL, 321). Calculations from NaCl dilution potential measurements showed that LDLu was Na+ permselective, whereas LDLl and ATL were Cl- permselective. High solute permeabilities in the inner medullary thin descending limb contradict a major requirement of the passive model of urinary concentration developed previously (J. P. Kokko and F. C. Rector, Jr. Kidney Int. 2: 214-223, 1972; and J. L. Stephenson. Kidney Int. 2: 85-94, 1972).

Animals↗
PubMed1993

Ammonium and bicarbonate transport in isolated perfused rodent long-loop thin descending limbs.

Ammonium accumulates in the renal medullas of antidiuretic mammals. The accumulation process is thought to involve countercurrent multiplication, energy-dependent recycling between the ascending and descending limbs of Henle's loop. To investigate the role of the long-loop thin descending limb (LDL) in countercurrent multiplication of ammonium, we have perfused outer medullary and inner medullary subsegments of the chinchilla LDL (and inner medullary subsegments of rat LDL) in vitro and measured the fluxes of total ammonia and total CO2. No spontaneous fluxes of total ammonia or total CO2 occurred in the absence of imposed concentration gradients. When transepithelial concentration gradients were imposed, passive total ammonia and total CO2 transport were observed in all subsegments, although the permeabilities varied with distance along the descending limb. Passive total ammonia transport occurred through a combination of NH3 and direct NH4+ permeation. The outer medullary segment was the most permeable to NH4+. The deep inner medullary segment was the most permeable to bicarbonate. Addition of carbonic anhydrase to the lumen accelerated passive NH3 entry in the outer medullary LDL, indicating that little or no luminal carbonic anhydrase is endogenously present. The passive secretion of NH4+ and NH3 into the LDL may contribute to the countercurrent multiplication of ammonium in the rodent renal medulla.

Ammonia↗
PubMed1993

Model of ammonium and bicarbonate transport along LDL: implications for alkalinization of luminal fluid.

Luminal fluid exiting the proximal convoluted tubule of a juxtamedullary nephron is alkalinized as it passes through the long-loop thin descending limb of Henle (LDL). Three potential mechanisms of alkalinization are: 1) concentration of bicarbonate by water abstraction, 2) direct bicarbonate entry, and 3) NH3 entry. We have used a mathematical model of the LDL to investigate these mechanisms. With permeabilities of HCO3-, NH3, and NH4+ measured for subsegments of the chinchilla LDL [M. F. Flessner, R. Mejia, and M. A. Knepper. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33):F388-F396, 1993], the osmotic water permeability of each segment [C.-L. Chou and M. A. Knepper. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32):F417-F426, 1992], and appropriate parameters from the literature, we have used the model to calculate hypothetical pH, HCO3- concentration, and NH3 concentration of the luminal fluid as it descends the LDL within an assumed interstitium. After eliminating each mechanism in turn by setting the appropriate permeability to zero, we recalculated the axial profiles. Our results suggest that, although all three mechanisms individually contribute to LDL alkalinization, NH3 entry likely plays the dominant role.

Ammonia↗
PubMed1993

Ammonium and bicarbonate transport in isolated perfused rodent ascending limbs of the loop of Henle.

Ammonium accumulates in the renal medullas of antidiuretic animals, presumably due to countercurrent multiplication, driven by NH4+ absorption from ascending loops of Henle in the outer and inner medulla. Active absorption of NH4+ occurs in the thick ascending limb (TAL). But the passive transport properties of NH3 in the TAL and the transport of ammonium or HCO3- in the ascending thin limb (ATL) have not been studied in rodents. To investigate the potential role of the ascending limb segments in medullary accumulation of ammonium, we perfused isolated subsegments of the chinchilla ATL and of the rat ATL and TAL. After imposing concentration gradients of total ammonia or total CO2 across ATL subsegments, we found very high rates of transfer of both substances, implying that at physiological flow rates the tubule luminal fluid quickly equilibrates with the interstitium. In the medullary TAL, we found a passive NH4+ permeability of 17 x 10(-5) cm/s but a relatively low NH3 permeability of < 0.003 cm/s. The low NH3 permeability prevents backleak of NH3 when NH4+ is actively transported from the lumen. We conclude that the ATL acts as an equilibrating segment and the TAL has special permeability properties that enhance net ammonium absorption and therefore enhance medullary ammonium accumulation.

Ammonia↗
PubMed1993

Vasopressin and oxytocin receptors coupled to Ca2+ mobilization in rat inner medullary collecting duct.

In renal collecting duct epithelial cells, arginine vasopressin (AVP) at greater than nanomolar concentrations has been reported to transiently increase intracellular free calcium ([Ca2+]i) in a manner consistent with activation of the phosphoinositide pathway. To investigate whether any of the known neurohypophysial hormone subtypes are involved, we measured [Ca2+]i in microdissected rat terminal inner medullary collecting duct (IMCD) using fura-2. To allow quantitative comparisons of the response under different conditions, we determined the areas under the response curves (in nM.min) over 1.5 min using numerical integration. AVP, the V1b-receptor agonist [deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin, the V2-receptor agonist 1-desamino-8-D-arginine vasopressin, oxytocin, and the selective oxytocin-receptor agonist [Thr4,Gly7]oxytocin (TG-OXT), each at 10 nM, significantly increased [Ca2+]i (69.52 +/- 10.25, 27.0 +/- 11.7, 24.33 +/- 5.83, 14.75 +/- 2.81, and 14.57 +/- 3.50 nM.min, respectively). In contrast, a V1a-selective agonist ([Phe2,Ile3,Orn8]vasopressin) did not increase [Ca2+]i (0.43 +/- 2.36 nM.min). In desensitization studies, challenge with 10 nM AVP or TG-OXT completely prevented a rise in [Ca2+]i in response to immediate rechallenge with the same agent, but not the other, demonstrating homologous desensitization. The lack of cross-desensitization implies that at least two receptors are present that can trigger a rise in [Ca2+]i in response to neurohypophysial hormones. Antagonists for oxytocin ([des-glycinamide9,d(CH2)5(1),O-Me-Tyr2,Thr4,Orn8]vaso tocin), V2 ([d(CH2)5(1),D-Ile2,Ile4,Arg8]vasopressin), and V1a ([d(CH2)5(1),O-Me-Tyr2,Arg8]vasopressin) receptors partially inhibited the [Ca2+]i response induced by 10 nM AVP (89.5, 81.6, and 51.4% inhibition, respectively). These data are consistent with the view that both an oxytocin receptor and a vasopressin receptor are coupled to a [Ca2+]i mobilization response in rat terminal IMCD. This vasopressin receptor is distinct from both the V1a receptor and the V2 receptor and may be either the V1b receptor or a novel vasopressin receptor subtype.

Animals↗
PubMed1993

Dual actions of vasopressin and oxytocin in regulation of water permeability in terminal collecting duct.

We conducted studies in isolated perfused terminal inner medullary collecting ducts (IMCD) from rats to investigate the roles of oxytocin and vasopressin in the regulation of osmotic water permeability. Vasopressin and oxytocin were found to have both stimulatory effects (at 0.1 nM) and inhibitory effects (at 10 nM) on osmotic water permeability. Measurements of adenosine 3',5'-cyclic monophosphate (cAMP) production demonstrated that both vasopressin and oxytocin increase cAMP production. Both the selective oxytocin-receptor agonist [Thr4,Gly7]oxytocin (10 nM) and the selective V1b agonist [deamino1,D-3-(pyridyl)Ala2,Arg8]vasopressin (10 nM) inhibited vasopressin-stimulated osmotic water permeability. In contrast, the selective V1a vasopressin-receptor agonist [Phe2,Ile3,Orn8]vasopressin (10 nM) had no effect on vasopressin-stimulated osmotic water permeability. These effects on water permeability correlated with the ability of the agents to transiently increase intracellular free calcium. The oxytocin/vasopressin-receptor antagonist [des-glycinamide9,d(CH2)5(1),O-Me-Tyr2,Thr4,Orn8]vasot ocin, which almost completely blocks vasopressin-induced calcium mobilization, also blocked the ability of 10 nM vasopressin to inhibit osmotic water permeability relative to that found with 0.1 nM vasopressin. We conclude the following. 1) Oxytocin, like vasopressin, has dual effects on osmotic water permeability, increasing it at subnanomolar concentrations and inhibiting it at suprananomolar concentrations. 2) Oxytocin, like vasopressin, can increase cAMP production, perhaps accounting for the increase in water permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Vasopressin activates collecting duct urea transporters and water channels by distinct physical processes.

The present studies were performed to investigate the kinetics of regulation of water channels and urea carriers in the rat terminal (IMCD) in response to vasopressin (AVP). The time courses of osmotic water permeability (Pf) and urea permeability (P(urea)) were measured in isolated perfused rat terminal IMCD segments following AVP stimulation and subsequently following AVP washout. Under control conditions, Pf and P(urea) kinetics were similar. Both transport processes exhibited complex patterns of activation with a period of rapid permeability increase followed by a period of slower increase. Both transporters also exhibited complex patterns of reversal following AVP washout, with a rapid permeability decrease (5 min) followed by a slower decrease toward the baseline value. The measurements were repeated in the presence of a lumen > bath osmotic gradient, a condition associated with a decreased rate of apical endocytosis in collecting ducts. The lumen > bath gradient did not alter the kinetics of Pf increase after AVP addition, but completely blocked the decrease in Pf normally seen with washout of AVP. In contrast, the lumen > bath osmotic gradient did not affect the decrease in urea permeability after AVP washout, but blocked the rapid phase of urea permeability increase following AVP addition. Thus imposition of a lumen > bath osmotic gradient resulted in separation of the time courses of P(urea) and Pf changes associated with AVP addition and washout. This finding indicates that the physical processes responsible for AVP-mediated alteration of urea transporter and water channel activity in the apical membrane are distinct.

Animals↗
PubMed1993

Kinetic model of water and urea permeability regulation by vasopressin in collecting duct.

We present a mathematical model describing the kinetics of water-channel and urea-carrier regulation by vasopressin in the apical membrane of collecting duct cells. The rate of change of the number of activated channels or carriers in the apical membrane is modeled as a balance between the rate of activation (or exocytic insertion) and the rate of inactivation (or endocytic retrieval) of transporters. In a three-state version of the model, transporters are assumed to be partitioned into three physical states, i.e., an "activated" state that imparts a permeation pathway to the apical membrane, an "inactivated" state, and a "reserve" state. Both activation and inactivation are represented by first-order kinetic equations describing transition from reserve to activated transporters and from activated to inactivated transporters, respectively. A simplified two-state model is derived from the three-state model, with the assumption that the transformation from inactivated to reserve transporter occurs rapidly relative to the other state transitions. Simulated time courses obtained by solving model equations are compared with experimentally determined time courses to test whether the response to vasopressin in isolated inner medullary collecting duct segments can be explained by direct effects on the rate constants for activation or inactivation. The results indicate that, for both transporters, it must be assumed that vasopressin directly regulates rate constants for both activation (exocytosis) and inactivation (endocytosis) to account for the experimentally determined dynamic responses to vasopressin and its withdrawal. These studies provide a theoretical basis on which to design further experimental studies.

Animals↗
PubMed1993

Vasopressin- and cAMP-induced changes in ultrastructure of isolated perfused inner medullary collecting ducts.

Studies were performed to correlate arginine vasopressin (AVP)-induced changes in epithelial ultrastructure with changes in osmotic water permeability in isolated perfused rat terminal inner medullary collecting ducts (tIMCD). The tubules were perfused in three time periods, i.e., a 40-min basal period, a 40-min period with 0.1 nM AVP in the bath, and a 60-min withdrawal period. In each phase, the osmotic water permeability (Pf) was measured, and the perfused tubules were fixed for electron microscopy. AVP caused a four- to eightfold increase in Pf and induced several ultrastructural changes as follows: increased cell height of IMCD cells, expansion of the intercellular spaces, formation of large vacuoles, and increased coated pit density in the apical plasma membrane [from 0.6 +/- 0.2 (n = 6) to 2.9 +/- 0.3 (n = 7) pits/100 microns membrane length]. During AVP withdrawal, Pf decreased toward the basal value in association with partial reversal of the ultrastructural changes including a decrease in coated pit density to 1.0 +/- 0.2 (n = 4). Stimulation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) (0.1 mM) produced similar changes in Pf. Coated pit density increased to 2.1 +/- 0.4 (n = 4) after cAMP stimulation and after cAMP withdrawal decreased to 1.2 +/- 0.2 (n = 6). In contrast to stimulation with AVP, cAMP stimulation did not result in dilated intercellular spaces or formation of large vacuoles. The only ultrastructural feature that directly correlated with the water permeability was the density of coated pits in the apical membrane. Organelles involved in the endocytic pathway were studied with cationized ferritin or albumin-gold in the luminal perfusate. At the end of 40 min basal perfusion or AVP stimulation, luminal tracer was found almost exclusively in large multivesicular bodies (MVB). Tubules perfused with tracer during AVP withdrawal demonstrated rapid tracer accumulation in small vesicles and small MVB within 3-5 min, a time point corresponding to the rapid phase of Pf decrease. Later (30-60 min) the label was mainly confined to large MVB. Occasionally during AVP stimulation or withdrawal, small coated vesicles and smooth vesicles with coated extensions were noted to contain tracer. The data demonstrate AVP-mediated coated pit formation and cellular changes and show very rapid internalization of apical membrane after AVP withdrawal.

Animals↗
PubMed1993

Structural-functional correlation in chinchilla long loop of Henle thin limbs: a novel papillary subsegment.

The ultrastructural characteristics of thin limb subsegments from chinchilla long loops of Henle were studied in perfusion-fixed kidneys and in isolated perfused tubules. In sections from the perfusion-fixed kidneys, we noted types I, II, III, and IV thin limb epithelia similar to those previously identified in other rodent species. Sections from the deepest 20% of the papillary tip, however, revealed only a single thin limb epithelial type, which had a combination of structural characteristics distinct from previously identified thin limb subtypes. This "papillary type" epithelium had relatively tall cells and a complex cellular organization with extensive interdigitation, numerous shallow tight junctions, and microvilli. In single-tubule studies, thin limb segments dissected from different levels of the outer and inner medulla were perfused in vitro for osmotic water permeability (Pf) measurements and were fixed for ultrastructural examination. Long-loop thin descending limbs (LDL) dissected from the outer medulla (Pf, 2,637 +/- 336 micron/s) had type II epithelium. LDL dissected from the middle of the inner medulla (Pf, 1,570 +/- 76 microns/s) had a type III epithelium. LDL segments dissected from the deepest 20% of the inner medulla had a low but nonzero Pf (68 +/- 9 micron/s) and had the same novel papillary type epithelium seen in sections from fixed kidneys. Thin ascending limbs dissected from inner 50% of the inner medulla had essentially zero Pf (8 +/- 4 micron/s) and had a type IV epithelium. Immunohistochemical localization of CHIP28 water channel protein confirmed the presence of CHIP28 in thin descending limbs throughout the outer 75% of the inner medulla, whereas labeling was essentially absent in the deep inner medulla where the low-PfLDL (novel papillary type epithelium) is located.

Animals↗
PubMed1993

Expression of vasopressin V1a and V2 receptor messenger ribonucleic acid in the liver and kidney of embryonic, developing, and adult rats.

The ontogenic expression of mRNAs encoding the V1a and V2 vasopressin receptors (V1aR and V2R) was visualized in liver and kidney of embryonic, developing, and adult rats using in situ hybridization histochemistry. Transcripts were detected at 16 and 19 days gestational age in kidney, with V1aR mRNA predominating in the developing cortex and V2R in the medulla. V1aR transcripts in 1-day-old kidneys were in vascular elements, in cells of developing medullary collecting ducts, and over mesangial cells of deep glomeruli, consistent with a role for the V1aR in regulating cellular growth. Expression of V1aR mRNA in the adult was found mainly in medullary vascular elements, arcuate and interlobular arteries, short segments of the cortical distal tubule, and transitional epithelium and smooth muscle of the pelvic wall and ureter. V2R mRNA, at 16 and 19 days gestational age, was in cells of developing medullary and cortical collecting ducts and, after birth, in cells of differentiating thick limbs of the loops of Henle, papillary surface epithelium, overlying macula densa, and short distal nephron segments. This distribution is in accord with the known role of V2 receptors in regulating water excretion. In contrast to kidney, liver did not express V2R mRNA and expressed V1aR transcripts only after birth. V1aR mRNA labeling was over cells bordering central veins on day 1 and surrounding central veins by day 5. A gradient was maximal on postnatal day 21, with V1aR mRNA most abundant in hepatocytes surrounding central veins and virtually absent in periportal hepatocytes. By day 60, most hepatocytes expressed V1aR transcripts, and the gradient was reduced. The ontogenic expression and receptor mRNA gradient are consistent with a role for hepatic V1a receptors in glucoregulation. These experiments confirm the presence of both V1a and V2 receptors in kidney and show that vasopressin receptor mRNA expression is developmentally regulated and tissue specific.

Aging↗