Defined human renal tubular epithelia in culture: growth, characterization, and hormonal response

1985 ◽  
Vol 248 (3) ◽  
pp. F436-F443 ◽  
Author(s):  
P. D. Wilson ◽  
M. A. Dillingham ◽  
R. Breckon ◽  
R. J. Anderson
Keyword(s):  
Primary Cultures ◽  
Thick Ascending Limb ◽  
Marker Enzyme ◽  
Nephron Segments ◽  
Growth Characterization ◽  
Proximal Straight Tubule ◽  
Collecting Tubule ◽  
Culture Growth ◽  
Renal Tubular

Individually microdissected nephron segments of defined epithelial origin from human kidneys were cultured in vitro in the present studies. Nephron segments of proximal convoluted tubule, proximal straight tubule, cortical thick ascending limb of Henle, and cortical collecting tubule were grown in defined media. Each cell type retained differentiated characteristics as assessed by ultrastructural morphology, marker enzyme profiles, and adenylate cyclase response to selected hormones. These studies demonstrate the feasibility of using primary cultures of well-defined segments of the human nephron to study human renal tubular epithelia in vitro.

Sites of antinatriuretic action of insulin along rat nephron

1992 ◽  
Vol 263 (1) ◽  
pp. F175-F179 ◽  
Author(s):  
E. Feraille ◽  
S. Marsy ◽  
L. Cheval ◽  
C. Barlet-Bas ◽  
C. Khadouri ◽  
...  
Keyword(s):  
Initial Rate ◽  
Dose Dependence ◽  
Proximal Convoluted Tubule ◽  
Proximal Tubules ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Collecting Tubule ◽  
Collecting Tubules

This study was aimed at identifying the renal sites of the antinatriuretic action of insulin by evaluating whether this hormone may alter the function of Na-K-ATPase in specific nephron segments. For this purpose, possible actions of insulin on the rate of 86Rb uptake were evaluated in vitro on single segments of proximal convoluted tubule (PCT), thick ascending limb, and collecting tubule microdissected from collagenase-treated kidneys of normal rats. Results indicate that physiological concentrations of insulin inhibited by 44% the initial rate of ouabain-sensitive 86Rb uptake in the medullary and cortical thick ascending limb, whereas it increased it by 40% in proximal tubules and by 60% in both cortical and medullary collecting tubules. The kinetics and dose dependence of insulin actions were different in the thick ascending limb, the PCT, and the collecting tubule, with the latter less sensitive but displaying an earlier response to insulin than the PCT and the thick ascending limb.

Binding of [3H]bradykinin in isolated nephron segments of the rabbit

1984 ◽  
Vol 246 (5) ◽  
pp. F732-F737 ◽  
Author(s):  
K. Tomita ◽  
J. J. Pisano
Keyword(s):  
Prostaglandin Synthesis ◽  
Distal Convoluted Tubule ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Binding Data ◽  
Proximal Straight Tubule ◽  
Collecting Tubule ◽  
Collecting Tubules ◽  
Stimulation Of ◽  
Cortical Collecting Tubule

Binding of intrinsically labeled [3H]bradykinin was studied in isolated nephron segments of the rabbit. Highest binding was observed in the cortical collecting tubule (5.76 +/- 0.34 X 10(-18) mol/mm) and the outer medullary collecting tubule (5.24 +/- 0.25 X 10(-18) mol/mm, means +/- SE, n = 6). Small but significant binding was also seen in the glomerulus, proximal straight tubule, cortical thick ascending limb of Henle's loop, and distal convoluted tubule. Lysyl-bradykinin, methionyl-lysyl-bradykinin, and tyrosine-8-bradykinin (but not des-arginine-9-bradykinin, vasopressin, angiotensins I and II, or prostaglandins) competed with [3H]bradykinin. The site of highest kinin binding (collecting tubule) is downstream from the highest concentration of kallikrein (granular portion of distal convoluted tubule). The binding data indicate that the major sites of kinin action in the kidney are the cortical and medullary collecting tubules. One action of kinins could be the stimulation of prostaglandin synthesis in the collecting tubules, which are known to actively synthesize prostaglandins.

Dependency of Microdissected Nephron Segments upon Oxidative Phosphorylation and Exogenous Substrates: A Relationship between Tubular Anatomical Location in the Kidney and Metabolic Activity

1989 ◽  
Vol 77 (3) ◽  
pp. 287-295 ◽  
Author(s):  
Shozo Torikai
Keyword(s):  
Collecting Duct ◽  
Anatomical Location ◽  
Thick Ascending Limb ◽  
Tubular Cells ◽  
Renal Tubular Cells ◽  
Nephron Segments ◽  
Henle's Loop ◽  
Renal Tubular ◽  
Substrate Deprivation ◽  
Exogenous Substrates

1. In order to examine the possibility of heterogeneity in the dependence of renal tubular cells upon oxidative phosphorylation and exogenous substrates, the effects of antimycin A and substrate deprivation on adenosine 5′-triphosphate (ATP) content were examined in isolated rat nephron segments in vitro at 37°C. 2. Antimycin A (5 μmol/l) caused varying decrements in cell ATP level within 5 min in the following order: proximal tubules > cortical thick ascending limb of Henle's loop (cTAL) > cortical collecting duct (cCD) in the cortex, and thin descending limb of Henle's loop (TDL) > medullary thick ascending limb of Henle's loop (mTAL) > outer medullary collecting duct (omCD) in the inner stripe of the outer medulla. In the thick ascending limb and the collecting duct, the segments located in the cortex were more sensitive than those in the medulla. 3. Substrate deprivation for 30 min markedly decreased the cell ATP content in cortical and medullary proximal tubules and also in medullary TDL, whereas it caused only a slight decrease in cTAL and mTAL with no change in cCD and omCD. 4. Media made hypertonic by the addition of 200 mmol/l NaCl under aerobic conditions, increased the requirement for exogenous substrates in TDL and mTAL, but not in omCD. This stimulation was seen to a lesser extent in media made hypertonic by the addition of mannitol instead of NaCl. 5. Taking into consideration a knowledge of rat kidney architecture and intrarenal gradient of oxygen partial pressure, it is likely that the observed dependency upon both oxygen and exogenous substrates in the renal tubular cells reflects adaptation of such cells to their anatomical location, and to the availability of those substances in situ. Furthermore, extracellular sodium concentration and osmolarity stimulate metabolic requirements to a different extent among the nephron segments.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F114-F120 ◽  
Author(s):  
A. I. Katz ◽  
A. Doucet ◽  
F. Morel
Keyword(s):  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Sensitivity Limit ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Hydrolysis Of ◽  
Total Enzyme

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.

scholarly journals Proximal and Distal Nephron-specific Adaptation to Furosemide

2021 ◽  
Author(s):  
Aram J. Krauson ◽  
Steven Schaffert ◽  
Elisabeth M. Walczak ◽  
Jonathan M. Nizar ◽  
Gwen M. Holdgate ◽  
...  
Keyword(s):  
Cell Number ◽  
Differentially Expressed ◽  
Sodium Reabsorption ◽  
Thick Ascending Limb ◽  
Distal Nephron ◽  
Transcriptional Responses ◽  
Cell Hyperplasia ◽  
Diuretic Resistance ◽  
Nephron Segments ◽  
Renal Tubular

ABSTRACTFurosemide, a widely prescribed diuretic for edema-forming states, inhibits sodium reabsorption in the thick ascending limb of the nephron. Tubular adaptation to diuretics has been observed, but the range of mechanisms along the nephron has not been fully explored. Using morphometry, we show that furosemide induces renal tubular epithelial hyperplasia selectively in distal nephron segments. By comparison, we find progressive cellular hypertrophy in proximal and distal nephron segments. We next utilize single cell RNA sequencing of vehicle- and furosemide-treated mice to define potential mechanisms of diuretic resistance. Consistent with distal tubular cell hyperplasia, we detect a net increase in DCT cell number and Birc5, an anti-apoptotic and pro-growth gene, in a subset of DCT cells, as the most prominently up-regulated gene across the nephron. We also map a gradient of cell-specific transcriptional changes congruent with enhanced distal sodium transport. Furosemide stimulates expression of the mitogen IGF-1. Thus, we developed a mouse model of inducible deletion of renal tubular IGF-1 receptor and show reduced kidney growth and proximal, but not distal, tubular hypertrophy by furosemide. Moreover, genes that promote enhanced bioavailability of IGF-1 including Igfbp1 and Igfbp5 are significantly and differentially expressed in proximal tubular segments and correspond to IGF-1R-dependent hypertrophy. In contrast, downstream PI3-kinase signaling genes including Pdk1, Akt1, Foxo3, FKBP4, Eif2BP4, and Spp1 are significantly and differentially expressed in distal nephron segments and correspond to IGF-1R-independent hypertrophy. These findings highlight novel mechanisms of tubular remodeling and diuretic resistance, provide a repository of transcriptional responses to a common drug, and expand the implications of long-term loop diuretic use for human disease.

Difference in the Na affinity of Na(+)-K(+)-ATPase along the rabbit nephron: modulation by K

1990 ◽  
Vol 259 (2) ◽  
pp. F246-F250 ◽  
Author(s):  
C. Barlet-Bas ◽  
L. Cheval ◽  
C. Khadouri ◽  
S. Marsy ◽  
A. Doucet
Keyword(s):  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Kidney Cells ◽  
Nephron Segments ◽  
Adaptive Property ◽  
Collecting Tubule ◽  
Pump Activity ◽  
K Concentration ◽  
Cortical Collecting Tubule

The sensitivity of Na(+)-K(+)-ATPase to Na was determined in single segments of rabbit nephron isolated by microdissection. In the cortical collecting tubule (CCT), Na(+)-K(+)-ATPase was threefold more sensitive to Na (apparent K0.5 approximately 3 mM) than in proximal convoluted tubule and cortical thick ascending limb (apparent K0.5 approximately 10 mM). Furthermore, increasing K concentration from 5 to greater than 100 mM markedly reduced the affinity of the pump for Na in all three nephron segments. In fact, the main shift in Na affinity occurred when K changed from 100 to 120 mM; in the CCT, increasing K concentration from 100 to 120 mM while maintaining Na concentration at 10 mM reduced Na(+)-K(+)-ATPase activity by greater than 35%. These findings confirm that, in kidney cells as in other cells, intracellular Na limits the rate of Na(+)-K(+)-ATPase. Thus any alteration of intracellular Na concentration modifies the pump activity in a way that contributes to the restoration of intracellular Na homeostasis. This adaptive property is particularly efficient in the collecting tubule in which the apparent K0.5 of the pump for Na is close to normal intracellular Na concentration. Furthermore, changes in intracellular K concentration, which usually accompany those of Na so as to maintain the total cation concentration constant, potentiate the regulatory role of Na through modifications of its affinity for the pump.

Enhanced glomerular filtration and Na+-K+-ATPase with furosemide administration

1987 ◽  
Vol 252 (5) ◽  
pp. F910-F915 ◽  
Author(s):  
P. Scherzer ◽  
H. Wald ◽  
M. M. Popovtzer
Keyword(s):  
Glomerular Filtration ◽  
Collecting Duct ◽  
Thick Ascending Limb ◽  
Cortical Collecting Duct ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Proximal Straight Tubule ◽  
X 24 ◽  
Furosemide Administration

To evaluate the effect of furosemide on kidney function, glomerular filtration rate (GFR), urinary Na excretion (UNaV), Na reabsorption (NAR), and Na+-K+-ATPase in isolated nephron segments were measured in 1) rats treated with furosemide 10 mg X 100 g-1 X 24 h-1 ip for 7 days, and 2) rats receiving an oral Na load for 12 days. In furosemide-treated rats, GFR rose from 0.61 +/- 0.03 (mean +/- SD) to 0.83 +/- 0.06 ml/min (P less than 0.01), UNaV rose from 904 +/- 71 to 1,402 +/- 85 mueq/day (P less than 0.001), and net NAR rose from 87.5 +/- 3.7 to 116.7 +/- 9.0 mueq/min (P less than 0.01). Na+-K+-ATPase remained unchanged in the proximal convoluted tubule (PCT), proximal straight tubule (PST), cortical thick ascending limb of Henle's loop (cTALH), and medullary thick ascending limb of Henle's loop (mTALH), but was increased in the distal convoluted tubule (DCT) and in cortical collecting duct (CCD) from 48.5 +/- 1.2 to 75.3 +/- 0.7 (P less than 0.001) and from 18.6 +/- 0.7 to 27.1 +/- 2.7 (P less than 0.02) X 10(-11) mol X mm-1 X min-1, respectively. In Na-loaded rats GFR rose from 0.61 +/- 0.04 to 0.86 +/- 0.03 ml/min (P less than 0.001), UNaV rose from 1,064 +/- 118 to 18,532 +/- 2,045 mueq/day (P less than 0.001), net NAR from 88.1 +/- 3.0 to 107.8 +/- 3.9 mueq/min and Na-K-ATPase in the mTALH rose from 40.3 +/- 1.4 to 56.2 +/- 2.11 (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of metabolic acidosis and alkalosis on NEM-sensitive ATPase in rat nephron segments

1992 ◽  
Vol 262 (4) ◽  
pp. F583-F590 ◽  
Author(s):  
C. Khadouri ◽  
S. Marsy ◽  
C. Barlet-Bas ◽  
L. Cheval ◽  
A. Doucet
Keyword(s):  
Metabolic Acidosis ◽  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Nephron Segments ◽  
Medullary Thick Ascending Limb ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Nephron Segment ◽  
Electrogenic Proton Pump ◽  
Chloride Treatment

An N-ethylmaleimide (NEM)-sensitive adenosinetriphosphatase (ATPase) displaying the kinetic and pharmacological properties of an electrogenic proton pump has been described in the different segments of rat nephron, where it mediates part of the active tubular proton secretion. This study was therefore designed to evaluate whether changes in urinary acidification observed during metabolic acidosis or alkalosis were associated with alterations of the activity of tubular NEM-sensitive ATPase, and if so, to localize the nephron segments responsible for these changes. Within 1 wk after the onset of ammonium chloride treatment, rats developed a metabolic acidosis, and NEM-sensitive ATPase activity was markedly increased in the medullary thick ascending limb of Henle's loop and outer medullary collecting tubule, and slightly increased in the cortical collecting tubule. Conversely, treatment with sodium bicarbonate induced a metabolic alkalosis that was accompanied by decreased NEM-sensitive ATPase activity in medullary thick ascending limb and outer medullary collecting tubule. NEM-sensitive ATPase activity was not altered in any other nephron segment tested in alkalotic and acidotic rats, i.e., the proximal tubule and the cortical thick ascending limb of Henle's loop. Changes qualitatively similar were observed as soon as 3 h after the onset of NaHCO3 or NH4Cl-loading. In the medullary collecting tubule, alterations of NEM-sensitive ATPase activity are in part due to hyperaldosteronism observed in both acidotic and alkalotic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Gluco- and mineralocorticoids control adenylate cyclase in specific nephron segments

1990 ◽  
Vol 258 (4) ◽  
pp. F812-F820 ◽  
Author(s):  
A. Doucet ◽  
C. Barlet-Bas ◽  
S. Siaume-Perez ◽  
C. Khadouri ◽  
S. Marsy
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mechanism Of Action ◽  
Thick Ascending Limb ◽  
Aluminum Fluoride ◽  
Camp Production ◽  
Nephron Segments ◽  
Glucocorticoid Administration ◽  
Collecting Tubule ◽  
Adrenalectomized Rats

Adrenal insufficiency is associated with an impairment of kidney diluting and concentrating ability, defects that may result from alterations of vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The purpose of this study were 1) to localize the sites of decreased vasopressin-stimulated adenylate cyclase (AC) activity along the nephron of adrenalectomized rats; 2) to determine whether the response of AC to other hormones is altered by adrenalectomy; 3) to evaluate whether changes in AC are due to the deficiency in mineralocorticoids and/or glucocorticoids; and 4) to characterize the mechanism of action of corticosteroids on the AC system. Results indicate that adrenalectomy reduced AC stimulation by vasopressin, glucagon, and calcitonin in the thick ascending limb, whereas only the response to vasopressin decreased in the collecting tubule. Glucocorticoid administration curtailed adrenalectomy-induced alterations of AC in the thick ascending limb, whereas that in the collecting tubule was prevented by mineralocorticoids. Adrenalectomy did not alter forskolin-stimulated AC, whereas it decreased responses to aluminum fluoride and cholera toxin. Finally, alterations of fluoride- and cholera toxin-stimulated AC were prevented by glucocorticoid and mineralocorticoid repletion in the thick ascending limb and collecting tubule, respectively.

Handling of angiotensin II and oxytocin by renal tubular segments perfused in vitro

1977 ◽  
Vol 232 (4) ◽  
pp. F319-F324 ◽  
Author(s):  
D. R. Peterson ◽  
S. Oparil ◽  
G. Flouret ◽  
F. A. Carone
Keyword(s):  
Angiotensin Ii ◽  
Rabbit Kidney ◽  
Experimental Conditions ◽  
Radioactive Label ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Renal Tubular ◽  
Hydrolysis Of ◽  
Cortical Collecting Tubule

In order to study renal tubular handling of two small peptide hormones, [14c]angiotensin II ([14C]AII) and [3H]oxytocin ([3H]OT) were microperfused through rabbit kidney tubule segments in vitro. The reabsorption and tubular sequestration of radioactive label were determined, and the collection fluid was electrophoretically analyzed. The data suggest that [14C]AII is extensively hydrolyzed in the pars recta of the nephron and is rapidly reabsorbed across the tubular epithelium. Under similar experimental conditions, hydrolysis of [3H]OT was not observed in either the proximal straight or cortical collecting tubule segment, and the rate of reabsorption was low. Thus, tubular handling of OT appears to differ from that of AII, probably because of differences in molecular structure.

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