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Evaluation of Function in Single Segments of Isolated Renal Blood Vessels, Nephrons, and Collecting Ducts

Jared J. Grantham, Larry W. Welling, Richard M. Edwards

10.1002/cphy.cp080109

Source: Supplement 25: Handbook of Physiology, Renal Physiology

Originally published: 1992

Published online: January 2011

Full Article on Wiley Online Library



Abstract

The sections in this article are:

1 Glomerulus and Vessels
1.1 Mammalian
1.2 Nonmammalian
2 Proximal Tubule
2.1 Mammalian
2.2 Nonmammalian
3 Thin Limbs of Henle's Loop
3.1 Mammalian
4 Thick Ascending Limbs of Henle's Loop
4.1 Mammalian
4.2 Nonmammalian
5 Distal Convoluted and Connecting Segments
5.1 Mammalian
5.2 Nonmammalian
6 Collecting Tubules and Ducts
6.1 Mammalian
6.2 Nonmammalian
Figure 1. Figure 1.

Anatomic and functional segments of mammalian nephron and collecting systems that have been microdissected and studied in vitro. Glomeruli and arterioles from superficial or deep positions in cortex are connected to initial portion of proximal tubule. Proximal tubules arc comprised of three distinct segments that in deep nephrons extend into long thin descending limbs of Henle's loop. Superficial nephrons have very short thin descending and ascending segments. All nephrons have medullary and cortical ascending limbs that join short convoluted segments of distal tubule. Deep nephrons have highly complex connecting segments that via arcades unite distal convoluted tubules and cortical collecting duct. Arcades are not present in tubules derived from superficial nephrons. Cortical collecting duct enters outer medulla to form two axial segments in outer stripe. Branched collecting ducts are found in initial and terminal portions of inner medulla.
Figure 2. Figure 2.

Schematic illustration of method to study filtration across single glomerular capillaries in vitro. Glomeruli are held with micro‐pipette and thoroughly rinsed with solution of lower oncotic pressure than the plasma in capillary loops. This causes rapid filtration of fluid into capillaries, and diameter of glomerulus increases as glomerular volume increases. The filtration coefficient (Kf, LpA) is determined from initial slope of increase in glomerular volume versus time. Modification of original technique is shown in which afferent and efferent arterioles are occluded to prevent loss of capillary plasma into bath compartment during filtration.

From Savin (342a
Figure 3. Figure 3.

Data illustrating relationship between filtration coefficient, Kf, and volume of individual rat glomeruli studied in vitro. Wide range of glomerular volume reflects animals from newborn to adulthood, and glomeruli from deep and superficial locations in cortex. ○ 22–36‐day‐old rats; • 69–84‐day‐old rats; × 4–9‐day‐old rats; Δ superficial glomeruli, 120‐day‐old rats; ▴ deep glomeruli, 120‐day‐old rats.

From Savin et al. 344
Figure 4. Figure 4.

Method for studying isolated perfused glomeruli from dog kidney. Concentric pipettes (left) are advanced into afferent arteriole to perfuse capillaries and to record intracapillary hydrostatic pressure. Postglomerular perfusate is collected from efferent areriole with another pipette arrangement (right).

Courtesy of Terrance Fried and Richard Osgood
Figure 5. Figure 5.

Afferent (top) and efferent (bottom) arterioles dissected from rabbit superficial cortex. Each arteriole has been cannulated and lumenal pressure set at 70 mm Hg for afferent arteriole and 20 mm Hg for efferent arteriole. Bars, 20 μm.
Figure 6. Figure 6.

Direct proof of fluid absorption in isolated proximal tubules. Accumulation of fluid droplets on basolateral surface of proximal tubule perfused in oil bath.

Courtesy of Delon Barfuss and James Schafer.) Top: photomicrograph of S2 segment perfused in oil bath. (From Schafer 349.] Bottom: schematic diagram of procedure for collecting absorbate. [From Barfuss and Schafer 14
Figure 7. Figure 7.

Demonstration that rabbit scrum reversibly decreased urate and p‐aminohippurate (PAH) secretion in isolated perfused S2 proximal tubules. Tubules were perfused with a synthetic medium and perfused in the same medium (designated Burg) containing either bovine serum albumin (BSA) or commercial rabbit scrum (RS) proteins from Pel‐Freeze (PF). The nature of the bath proteins had no effect on net fluid absorption. B, bath; L, lumen.

From Tanner et al. 407
Figure 8. Figure 8.

Response of S2 proximal tubules to sudden decrease in medium osmolality. Nonperfused segments were rinsed in medium of 147, 190, or 230 mOsm. Cell volume, adduced from changes in external tubule diameter, rapidly increased, after which cell volume progressively returned toward original baseline. Upon return of tubules to isotonic (295 mOsm) medium, tubule volume decreased below original baseline, indicating that intracellular solutes were lost during volume adjustment in hypotonic medium.

From Dellasega and Grantham 103
Figure 9. Figure 9.

Demonstration of net fluid secretion in nonperfused proximal S2 segment. Tubule at zero time was incubated in normal rabbit serum. Two, 5, and 10 min after adding human uremic serum to bath, tubule lumen appeared and expanded progressively, thereby proving that fluid was secreted by the segment.

Reproduced from Grantham et al. 148: the Journal of Clinical Investigation, 1973, 52: 2441–2450, by copyright permission of the American Society for Clinical Investigation
Figure 10. Figure 10.

Demonstration of net fluid secretion in flounder proximal tubule. Tubule segment was filled with paraffin oil and incubated without perfusion in medium that contained electrolytes but no known organic or inorganic secretagogues. Fluid secretion into tubule lumen was evinced by appearance of fluid that in focal areas broke the oil column.

Courtesy of Klaus W. Beyenbach
Figure 11. Figure 11.

Isolation of juxtaglomerular apparatus. Top: single perfused thick ascending limb from rabbit kidney with attached glomerulus (G). Perfusion was left to right. Arrow, macula densa. × 92.

Courtesy of Kevin Kirk.) Bottom: differential interference‐contrast image of macula densa during perfusion with hypoosmotic solution (70 mOsm). Note dilated intracellular space and pocket of extra‐glomerular mesangial cells (asterisk) (× 2,250
Figure 12. Figure 12.

Cellular heterogeneity in cortical collecting ducts. Left: Hoffman Modulation Contrast images of perfused segment with focal plane of focus on lumenal surface (A) and on midpoint of lateral wall (B). Asterisks indicate intercalated cells. Right: fluorescent images of fluorescein‐labeled peanut lectin bound to lumenal cell surfaces (A) and acridine orange localization of nuclei (B).

From O'Neil and Hayhurst 300
Figure 13. Figure 13.

Transmission electron micrograph cross sections of cortical collecting ducts perfused with hypotonic medium in vitro, (a) control tubule, (b) tubule treated with maximal concentration of vasopressin, demonstrating widely dilated intercellular spaces. L, lumen; BM, basement membrane; N, nucleus; Ic, intercalated cell; V, large vacuole; IS, intercellular space; arrows, cytoplasmic vacuoles in the ordinary living cells.

From Ganote et al. 131


Figure 1.

Anatomic and functional segments of mammalian nephron and collecting systems that have been microdissected and studied in vitro. Glomeruli and arterioles from superficial or deep positions in cortex are connected to initial portion of proximal tubule. Proximal tubules arc comprised of three distinct segments that in deep nephrons extend into long thin descending limbs of Henle's loop. Superficial nephrons have very short thin descending and ascending segments. All nephrons have medullary and cortical ascending limbs that join short convoluted segments of distal tubule. Deep nephrons have highly complex connecting segments that via arcades unite distal convoluted tubules and cortical collecting duct. Arcades are not present in tubules derived from superficial nephrons. Cortical collecting duct enters outer medulla to form two axial segments in outer stripe. Branched collecting ducts are found in initial and terminal portions of inner medulla.



Figure 2.

Schematic illustration of method to study filtration across single glomerular capillaries in vitro. Glomeruli are held with micro‐pipette and thoroughly rinsed with solution of lower oncotic pressure than the plasma in capillary loops. This causes rapid filtration of fluid into capillaries, and diameter of glomerulus increases as glomerular volume increases. The filtration coefficient (Kf, LpA) is determined from initial slope of increase in glomerular volume versus time. Modification of original technique is shown in which afferent and efferent arterioles are occluded to prevent loss of capillary plasma into bath compartment during filtration.

From Savin (342a


Figure 3.

Data illustrating relationship between filtration coefficient, Kf, and volume of individual rat glomeruli studied in vitro. Wide range of glomerular volume reflects animals from newborn to adulthood, and glomeruli from deep and superficial locations in cortex. ○ 22–36‐day‐old rats; • 69–84‐day‐old rats; × 4–9‐day‐old rats; Δ superficial glomeruli, 120‐day‐old rats; ▴ deep glomeruli, 120‐day‐old rats.

From Savin et al. 344


Figure 4.

Method for studying isolated perfused glomeruli from dog kidney. Concentric pipettes (left) are advanced into afferent arteriole to perfuse capillaries and to record intracapillary hydrostatic pressure. Postglomerular perfusate is collected from efferent areriole with another pipette arrangement (right).

Courtesy of Terrance Fried and Richard Osgood


Figure 5.

Afferent (top) and efferent (bottom) arterioles dissected from rabbit superficial cortex. Each arteriole has been cannulated and lumenal pressure set at 70 mm Hg for afferent arteriole and 20 mm Hg for efferent arteriole. Bars, 20 μm.



Figure 6.

Direct proof of fluid absorption in isolated proximal tubules. Accumulation of fluid droplets on basolateral surface of proximal tubule perfused in oil bath.

Courtesy of Delon Barfuss and James Schafer.) Top: photomicrograph of S2 segment perfused in oil bath. (From Schafer 349.] Bottom: schematic diagram of procedure for collecting absorbate. [From Barfuss and Schafer 14


Figure 7.

Demonstration that rabbit scrum reversibly decreased urate and p‐aminohippurate (PAH) secretion in isolated perfused S2 proximal tubules. Tubules were perfused with a synthetic medium and perfused in the same medium (designated Burg) containing either bovine serum albumin (BSA) or commercial rabbit scrum (RS) proteins from Pel‐Freeze (PF). The nature of the bath proteins had no effect on net fluid absorption. B, bath; L, lumen.

From Tanner et al. 407


Figure 8.

Response of S2 proximal tubules to sudden decrease in medium osmolality. Nonperfused segments were rinsed in medium of 147, 190, or 230 mOsm. Cell volume, adduced from changes in external tubule diameter, rapidly increased, after which cell volume progressively returned toward original baseline. Upon return of tubules to isotonic (295 mOsm) medium, tubule volume decreased below original baseline, indicating that intracellular solutes were lost during volume adjustment in hypotonic medium.

From Dellasega and Grantham 103


Figure 9.

Demonstration of net fluid secretion in nonperfused proximal S2 segment. Tubule at zero time was incubated in normal rabbit serum. Two, 5, and 10 min after adding human uremic serum to bath, tubule lumen appeared and expanded progressively, thereby proving that fluid was secreted by the segment.

Reproduced from Grantham et al. 148: the Journal of Clinical Investigation, 1973, 52: 2441–2450, by copyright permission of the American Society for Clinical Investigation


Figure 10.

Demonstration of net fluid secretion in flounder proximal tubule. Tubule segment was filled with paraffin oil and incubated without perfusion in medium that contained electrolytes but no known organic or inorganic secretagogues. Fluid secretion into tubule lumen was evinced by appearance of fluid that in focal areas broke the oil column.

Courtesy of Klaus W. Beyenbach


Figure 11.

Isolation of juxtaglomerular apparatus. Top: single perfused thick ascending limb from rabbit kidney with attached glomerulus (G). Perfusion was left to right. Arrow, macula densa. × 92.

Courtesy of Kevin Kirk.) Bottom: differential interference‐contrast image of macula densa during perfusion with hypoosmotic solution (70 mOsm). Note dilated intracellular space and pocket of extra‐glomerular mesangial cells (asterisk) (× 2,250


Figure 12.

Cellular heterogeneity in cortical collecting ducts. Left: Hoffman Modulation Contrast images of perfused segment with focal plane of focus on lumenal surface (A) and on midpoint of lateral wall (B). Asterisks indicate intercalated cells. Right: fluorescent images of fluorescein‐labeled peanut lectin bound to lumenal cell surfaces (A) and acridine orange localization of nuclei (B).

From O'Neil and Hayhurst 300


Figure 13.

Transmission electron micrograph cross sections of cortical collecting ducts perfused with hypotonic medium in vitro, (a) control tubule, (b) tubule treated with maximal concentration of vasopressin, demonstrating widely dilated intercellular spaces. L, lumen; BM, basement membrane; N, nucleus; Ic, intercalated cell; V, large vacuole; IS, intercellular space; arrows, cytoplasmic vacuoles in the ordinary living cells.

From Ganote et al. 131
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Article Title: Evaluation of Function in Single Segments of Isolated Renal Blood Vessels, Nephrons, and Collecting Ducts
 

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Jared J. Grantham, Larry W. Welling, Richard M. Edwards. Evaluation of Function in Single Segments of Isolated Renal Blood Vessels, Nephrons, and Collecting Ducts. Compr Physiol 2011, Supplement 25: Handbook of Physiology, Renal Physiology: 351-383. First published in print 1992. doi: 10.1002/cphy.cp080109