Gluconeogenesis from glutamine and lactate in the isolated humanrenal proximal tubule: longitudinal heterogeneity and lack of response to adrenaline

2001 ◽  
Vol 360 (2) ◽  
pp. 371-377 ◽  
Author(s):  
Agnès CONJARD ◽  
Mireille MARTIN ◽  
Jérôme GUITTON ◽  
Gabriel BAVEREL ◽  
Bernard FERRIER
Keyword(s):  
Proximal Tubule ◽  
Human Kidney ◽  
Glucose Production ◽  
Proximal Tubules ◽  
Renal Gluconeogenesis ◽  
Adrenaline Infusion ◽  
Gluconeogenic Substrates ◽  
Stimulation Of

Recent studies in vivo have suggested that, in humans in the postabsorptive state, the kidneys contribute a significant fraction of systemic gluconeogenesis, and that the stimulation of renal gluconeogenesis may fully explain the increase in systemic gluconeogenesis during adrenaline infusion. Given the potential importance of human renal gluconeogenesis in various physiological and pathophysiological situations, we have conducted a study in vitro to further characterize this metabolic process and its regulation. For this, successive segments (S1, S2 and S3) of human proximal tubules were dissected and incubated with physiological concentrations of glutamine or lactate, two potential gluconeogenic substrates that are taken up by the human kidney in vivo, and glucose production was measured. The effects of adrenaline, noradrenaline and cAMP, a well established stimulator of gluconeogenesis in animal kidney tubules, were also studied in suspensions of human renal proximal tubules. The results indicate that the three successive segments have about the same capacity to synthesize glucose from glutamine; by contrast, the S2 and S3 segments synthesize more glucose from lactate than the S1 segment. In the S2 and S3 segments, lactate appears to be a better gluconeogenic precursor than glutamine. The addition of cAMP, but not of adrenaline or noradrenaline, led to the stimulation of gluconeogenesis from lactate and glutamine by human proximal tubules. These results indicate that, in the human kidney in vivo, lactate might be the main gluconeogenic precursor, and that the stimulation of renal gluconeogenesis observed in vivo upon adrenaline infusion may result from an indirect action on the renal proximal tubule.

scholarly journals Acid Stimulation of the Citrate Transporter NaDC-1 Requires Pyk2 and ERK1/2 Signaling Pathways

2018 ◽  
Vol 29 (6) ◽  
pp. 1720-1730 ◽  
Author(s):  
Miriam Zacchia ◽  
Xuefei Tian ◽  
Enrica Zona ◽  
Robert J. Alpern ◽  
Patricia A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Signaling Pathways ◽  
Cultured Cells ◽  
Wild Type ◽  
Experimental Conditions ◽  
Opossum Kidney ◽  
Citrate Transporter ◽  
Stimulation Of

Background Urine citrate is reabsorbed exclusively along the renal proximal tubule via the apical Na+-dicarboxylate cotransporter NaDC-1. We previously showed that an acid load in vivo and media acidification in vitro increase NaDC-1 activity through endothelin-1 (ET-1)/endothelin B (ETB) signaling. Here, we further examined the signaling pathway mediating acid-induced NaDC-1 activity.Methods We transiently transfected cultured opossum kidney cells, a model of the proximal tubule, with NaDC-1 and ETB and measured [14C]-citrate uptake after media acidification under various experimental conditions, including inactivation of Pyk2 and c-Src, which were previously shown to be activated by media acidification. Wild-type (Pyk2+/+) and Pyk2-null (Pyk2−/−) mice were exposed to NH4Cl loading and euthanized after various end points, at which time we harvested the kidneys for immunoblotting and brush border membrane NaDC-1 activity studies.Results Inhibition of Pyk2 or c-Src prevented acid stimulation but not ET-1 stimulation of NaDC-1 in vitro. Consistent with these results, NH4Cl loading stimulated NaDC-1 activity in kidneys of wild-type but not Pyk2−/− mice. In cultured cells and in mice, ERK1/2 was rapidly phosphorylated by acid loading, even after Pyk2 knockdown, and it was required for acid but not ET-1/ETB stimulation of NaDC-1 in vitro. Media acidification also induced the phosphorylation of Raf1 and p90RSK, components of the ERK1/2 pathway, and inhibition of these proteins blocked acid stimulation of NaDC-1 activity.Conclusions Acid stimulation of NaDC-1 activity involves Pyk2/c-Src and Raf1-ERK1/2-p90RSK signaling pathways, but these pathways are not downstream of ET-1/ETB in this process.

In vivo proximal tubular fluid-to-plasma chloride concentration gradient in the rabbit

1982 ◽  
Vol 242 (6) ◽  
pp. F575-F579
Author(s):  
R. C. Vari ◽  
C. E. Ott
Keyword(s):  
Proximal Tubule ◽  
Concentration Gradient ◽  
Concentration Ratio ◽  
Mammalian Species ◽  
Chloride Concentration ◽  
Proximal Tubules ◽  
Plasma Chloride ◽  
Tubule Fluid

It has been reported that the concentration of chloride in the proximal tubule is greater than that in plasma in several mammalian species. Much of the theory concerning fluid and electrolyte reabsorption in the proximal tubule is based on data taken from in vitro isolated proximal tubules of the rabbit nephron. This study measured in vivo the rabbit proximal tubule fluid-to-ultrafiltrate chloride concentration ratio [(TF/UF)Cl] and its relationship to proximal tubule length as estimated by the tubule fluid-to-plasma inulin concentration ratio [(TF/P)In]. From six rabbits, 19 random proximal tubules were micropunctured and analyzed for inulin and chloride concentrations, the latter being measured by microelectrometric titration. Plasma ultrafiltrate was determined by correcting plasma chloride concentration for protein concentration. The average single nephron filtration rate was 20.2 +/- 0.8 nl/min. The (TF/UF)Cl ratio was 1.10 +/- 0.03, which was significantly different from unity. Furthermore, regression analysis yielded no significant correlation between the (TF/UF(Cl and (TF/P)In ratio. This study demonstrates that a tubule lumen-to-plasma chloride concentration gradient exists in the in vivo proximal tubule of the rabbit that is apparently established early and is not correlated with proximal tubule length.

scholarly journals An evaluation of the antioxidant protein α1-microglobulin as a renal tubular cytoprotectant

2016 ◽  
Vol 311 (3) ◽  
pp. F640-F651 ◽  
Author(s):  
Richard A. Zager ◽  
Ali C. M. Johnson ◽  
Kirsten Frostad
Keyword(s):  
Proximal Tubule ◽  
Proximal Tubules ◽  
Monocyte Chemoattractant Protein 1 ◽  
Monocyte Chemoattractant ◽  
Neutrophil Gelatinase Associated Lipocalin ◽  
Monocyte Chemoattractant Protein ◽  
Antioxidant Protein ◽  
Neutrophil Gelatinase

α1-Microglobulin (A1M) is a low-molecular-weight heme-binding antioxidant protein that is readily filtered by the glomerulus and reabsorbed by proximal tubules. Given these properties, recombinant A1M (rA1M) has been proposed as a renal antioxidant and therapeutic agent. However, little direct evidence to support this hypothesis exists. Hence, we have sought “proof of concept” in this regard. Cultured proximal tubule (HK-2) cells or isolated mouse proximal tubule segments were challenged with a variety of prooxidant insults: 1) hemin, 2) myoglobin; 3) “catalytic” iron, 4) H2O2/Fenton reagents, 5) a Ca2+ ionophore, 6) antimycin A, or 7) hypoxia (with or without rA1M treatment). HK-2 injury was gauged by the percent lactate dehydrogenase release and 4,5-(dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide uptake. In vivo protection was sought in rA1M-treated mice subjected to 1) graded myohemoglobinura (2, 4, 8, or 9 ml/kg glycerol injection), 2) purified myoglobinemia/uria, or 3) endotoxemia. In vivo injury was assessed by blood urea nitrogen, creatinine, and the expression of redox-sensitive genes (heme oxygenase-1, neutrophil gelatinase-associated lipocalin, and monocyte chemoattractant protein-1 mRNAs). Although rA1M totally blocked in vitro hemin toxicity, equimolar albumin (another heme binder) or 10% serum induced equal protection. rA1M failed to mitigate any nonhemin forms of either in vitro or in vivo injury. A1M appeared to be rapidly degraded within proximal tubules (by Western blot analysis). Surprisingly, rA1M exerted select injury-promoting effects (increased in vitro catalytic iron/antimycin toxicities and increased in vivo monocyte chemoattractant protein-1/neutrophil gelatinase-associated lipocalin mRNA expression after glycerol or endotoxin injection). We conclude that rA1M has questionable utility as a renal antioxidant/cytoprotective agent, particularly in the presence of larger amounts of competitive free heme (e.g., albumin) binders.

Angiotensin II stimulates ammoniagenesis in canine renal proximal tubule segments

1991 ◽  
Vol 260 (1) ◽  
pp. F19-F26 ◽  
Author(s):  
M. C. Chobanian ◽  
C. M. Julin
Keyword(s):  
Angiotensin Ii ◽  
Proximal Tubule ◽  
Glucose Production ◽  
Renal Proximal Tubule ◽  
Ang Ii ◽  
Ammonia Production ◽  
Proximal Tubule Segments ◽  
Dependent Pathway ◽  
Stimulation Of

To determine whether angiotensin II (ANG II) affects ammoniagenesis in renal proximal tubule, ammonia production was measured in suspensions of canine renal proximal tubule segments (PCT) incubated with L-glutamine and varying concentrations of ANG II. Ammonia production from PCT was significantly increased by 15.5 +/- 1.1% in the presence of ANG II (10(-6) M) at 2 h. Similarly, glucose production significantly increased by 10.0 +/- 0.9%. Half-maximal stimulation occurred at approximately 10(-9) M ANG II. Stimulation of ammonia production by ANG II was blocked in the presence of the ANG II antagonist, [Sar1-Ile8]ANG II (10(-6) M). Enhancement of ammonia production in PCT by ANG II occurred in acidotic and neutral media but not in alkalotic medium. When extracellular [Na+] = intracellular [Na+] ANG II significantly increased ammonia production in PCT. Absence of extracellular Ca2+ or addition of trifluoperazine or N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7) (Ca2(+)-calmodulin-dependent pathway inhibitors) blocked the action of ANG II to enhance ammonia production. We conclude that ANG II stimulates ammonia and glucose production in canine renal PCT via a receptor-mediated signal. The action of ANG II on ammoniagenesis may be mediated by a calcium-calmodulin-dependent pathway. Stimulation of ammoniagenesis in vitro under normal and acidotic conditions may reflect a role in vivo for ANG II in the regulation of renal acid-base metabolism.

Degradation and transport of AVP by proximal tubule

1987 ◽  
Vol 253 (6) ◽  
pp. F1120-F1128
Author(s):  
F. A. Carone ◽  
E. I. Christensen ◽  
G. Flouret
Keyword(s):  
Proximal Tubule ◽  
High Performance ◽  
Reduced Glutathione ◽  
Proximal Tubules ◽  
Electron Microscope Autoradiography ◽  
Lysosomal Fraction ◽  
Renal Brush Border Membranes ◽  
Renal Brush Border

High-performance liquid chromatography (HPLC) analysis revealed that [3,4,5-3H-Phe3,Arg8]vasopressin ([3H]AVP) was not degraded by isolated renal brush-border membranes or by a cortical lysosomal fraction in vitro; however, in the presence of 1 mM reduced glutathione, [3H]AVP was degraded by both preparations. Renal cortical homogenates in vitro and luminal peptidases of proximal tubule in vivo degraded [3H]AVP and in both instances yielded phenylalanine, hexapeptide AVP 1-6, heptapeptide AVP 1-7, octapeptide AVP 1-8, and two uncharacterized products X and Y. These data suggest that filtered AVP is reduced in the proximal tubule by a reduced glutathione-dependent transhydrogenase and subsequently cleaved to [3H]Phe by tubular aminopeptidases. Following microinfusion of [3H]AVP into proximal tubules, 15.7% of the label was absorbed. Five and fifteen minutes after infusion of [3H]AVP, sequestration of total label in proximal tubules was 4.5 and 2.1%, respectively, and quantitative electron microscope autoradiography revealed accumulation of grains over apical endocytic vacuoles and lysosomes consistent with endocytic uptake and rapid lysosomal degradation of AVP and/or a large metabolite. Thus, enzymatic cleavage of AVP by luminal and lysosomal peptidases in proximal tubules could involve disulfide bond, C-terminal, and N-terminal loci.

Growth hormone directly stimulates gluconeogenesis in canine renal proximal tubule

1989 ◽  
Vol 257 (5) ◽  
pp. E751-E756
Author(s):  
S. A. Rogers ◽  
I. E. Karl ◽  
M. R. Hammerman
Keyword(s):  
Growth Hormone ◽  
Proximal Tubule ◽  
Glucose Production ◽  
Renal Proximal Tubule ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Igf I ◽  
Proximal Tubular

To characterize the action of growth hormone (GH) on gluconeogenesis in renal proximal tubule, glucose production was measured in suspensions of canine renal proximal tubular segments incubated with 1 mM L-alanine, 10 mM lactate, 1 mM succinate, and various concentrations (10(-11) to 10(-6) M) of recombinant bovine GH (bGH). Production of glucose increased as a function of time for 120 min. Bovine growth hormone (10(-6) M) increased glucose production at 120 min by 55 +/- 16%. Significant enhancement of glucose production occurred in suspensions of segments incubated with as little as 10(-10) M bGH. Half-maximal stimulation occurred at between 10(-9) and 10(-8) M. To ascertain whether these actions of bGH are mediated directly, we determined the effects of insulin-like growth factors (IGFs) I and II on glucose production. Addition of IGF-I to segments enhanced glucose production in a concentration-dependent manner. However, incubation with bGH did not induce measurable IGF-I production in the segments. In contrast to the action of IGF-I, IGF-II did not affect glucose production. We conclude that bGH acts directly on cells within proximal tubular segments to enhance gluconeogenesis. Stimulation of gluconeogenesis in vitro could reflect a counterregulatory action of GH exerted on renal proximal tubule in vivo.

Thrombopoietin-Induced Stimulation of Megakaryocyte- Enriched Bone Marrow Cultures

1979 ◽  
Author(s):  
K. L. Kellar ◽  
B. L. Evatt ◽  
C. R. McGrath ◽  
R. B. Ramsey
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Bone Marrow Cells ◽  
Rabbit Plasma ◽  
Bone Marrow Cultures ◽  
Plasma Fractions ◽  
Marrow Cultures ◽  
Stimulation Of

Liquid cultures of bone marrow cells enriched for megakaryocytes were assayed for incorporation of 3H-thymidine (3H-TdR) into acid-precipitable cell digests to determine the effect of thrombopoietin on DNA synthesis. As previously described, thrombopoietin was prepared by ammonium sulfate fractionation of pooled plasma obtained from thrombocytopenic rabbits. A control fraction was prepared from normal rabbit plasma. The thrombopoietic activity of these fractions was determined in vivo with normal rabbits as assay animals and the rate of incorporation of 75Se-selenomethionine into newly formed platelets as an index of thrombopoietic activity of the infused material. Guinea pig megakaryocytes were purified using bovine serum albumin gradients. Bone marrow cultures containing 1.5-3.0x104 cells and 31%-71% megakaryocytes were incubated 18 h in modified Dulbecco’s MEM containing 10% of the concentrated plasma fractions from either thrombocytopenic or normal rabbits. In other control cultures, 0.9% NaCl was substituted for the plasma fractions. 3H-TdR incorporation was measured after cells were incubated for 3 h with 1 μCi/ml. The protein fraction containing thrombopoietin-stimulating activity caused a 25%-31% increase in 3H-TdR incorporation over that in cultures which were incubated with the similar fraction from normal plasma and a 29% increase over the activity in control cultures to which 0.9% NaCl had been added. These data suggest that thrombopoietin stimulates DNA synthesis in megakaryocytes and that this tecnique may be useful in assaying thrombopoietin in vitro.

Proximal tubule dysfunction in cystine-loaded tubules: effect of phosphate and metabolic substrates

1996 ◽  
Vol 271 (3) ◽  
pp. F717-F722
Author(s):  
G. Bajaj ◽  
M. Baum
Keyword(s):  
Oxygen Consumption ◽  
Proximal Tubule ◽  
Tricarboxylic Acid Cycle ◽  
Dimethyl Ester ◽  
Proximal Tubules ◽  
Intracellular Atp ◽  
Metabolic Substrates ◽  
Rate Of Oxygen Consumption ◽  
Intracellular Phosphate

Intracellular cystine loading by use of cystine dimethyl ester (CDME) results in a generalized inhibition in proximal tubule transport due, in part, to a decrease in intracellular ATP. The present study examined the importance of phosphate and metabolic substrates in the proximal tubule dysfunction produced by cystine loading. Proximal tubule intracellular phosphorus was 1.8 +/- 0.1 in control tubules and 1.1 +/- 0.1 nmol/mg protein in proximal tubules incubated in vitro with CDME P < 0.001). Infusion of sodium phosphate in rabbits and subsequent incubation of proximal tubules with a high-phosphate medium attenuated the decrease in proximal tubule respiration and prevented the decrease in intracellular ATP with cystine loading. Tricarboxylic acid cycle intermediates have been shown to preserve oxidative metabolism in phosphate-depleted proximal tubules. In proximal tubules incubated with either 1 mM valerate or butyrate, there was a 42 and 34% reduction (both P < 0.05) in the rate of oxygen consumption with cystine loading. However, tubules incubated with 1 mM succinate or citrate had only a 13 and 14% P = NS) reduction in the rate of oxygen consumption, respectively. These data are consistent with a limitation of intracellular phosphate in the pathogenesis of the proximal tubule dysfunction with cystine loading.

scholarly journals Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis

Blood ◽  
1979 ◽  
Vol 54 (1) ◽  
pp. 146-158 ◽  
Author(s):  
KS Zuckerman ◽  
PJ Quesenberry ◽  
J Levin ◽  
R Sullivan
Keyword(s):  
Significant Inhibition ◽  
Erythropoietic Activity ◽  
Limulus Amebocyte Lysate ◽  
Endogenous Erythropoietin ◽  
Significant Change ◽  
In Vitro Effects ◽  
Stimulation Of

Abstract Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU- e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.

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