[3]
ISOLATED PERFUSED AND NONFILTERING KIDNEY
31
[3] I s o l a t e d P e r f u s e d a n d N o n f i l t e r i n g K i d n e y B y PATRICIOSILVA
Among the many advantages of the isolated perfused kidney is that because of the nature of the preparation glomerular filtration can be prevented, thus allowing the investigator to study renal functions independently of glomerular filtration and the associated cellular work related to tubular reabsorption of salt and other organic and inorganic compounds. The nonfiltering isolated kidney preparation can be used to determine whether the site of uptake into renal tubular cells is basolateral, independent of glomerular filtration, or lumenal, dependent on glomerular filtration. To my knowledge, the first reported use of the preparation was that of Johnson and Maack t where they used a nonfiltering isolated perfused rat kidney to study the metabolism by the kidney of growth hormone. Since then, it has been used by many other investigators to examine peptide or protein catabolism, uptake of toxic substances, renin secretion, or to determine physiological parameters such as basal oxygen consumption.t-5 There is an additional advantage in the use of the nonfiltering mode of the isolated kidney preparation perfused with an artificial solution over that of the filtering mode, and that is that the nonfiltering preparation never develops the morphological lesions in the thick ascending limb of the loop of Henle6 associated with perfusion regularly found in the filtering one.7, a
Several different methods have been used to prevent glomerular filtration: ureteral ligation, reduction in the pressure of perfusion, increased oncotic pressure of the perfusate, or a combination of these. Ureteral ligation prevents the excretion of urine and, because of the increased back pressure along the urinary tract, it reduces giomerular filtration; however, it does not prevent it completely. While electrolyte and water reabsorption continue, predominantly in the proximal tubule, glomerular filtration continues as well, albeit at a much reduced rate. Another way of reducing i V. Johnson and T. Maack, Am. J. Physiol. 233, F185 (1977). 2 S.T. Kau and T. Maack, Am. J. Physiol. 233, F445 (1977). 3 K. Miura, R. S. Goldstein, D. A. Pasino, and J. B. Hook, Toxicology44, 147 (1987). 4 A. J. Cohen, K. Spokes, R. S. Brown, J. S. Stoff, and P. Silva, Orc. Res. 50, 400 (1982). 5 R. D. Swartz, P. Silva, R. HaUac, and F. H. Epstein, Curr. Probl. Clin. Biochem. 8, 123
(1978). 6 M. Brezis,S. Rosen,P. Silva,and F. H. Epstein,Kidney Int. 25, 65 (1984). 7D. Alcorn,K. R. Emslie,B. D. Ross, G. B. Ryan, and J. D. Tange,Kidney Int. 19, 638 (1981). s H. J. Schurekand W. Kriz, Lab. Invest. 53, 145 (1985). METHODS 1N ENZYMOLOGY, VOL. 191
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formreserved.
32
KIDNEY
[3]
glomerular filtration is to reduce the pressure of perfusion to a level where the hydrostatic pressure in the glomerular capillary is lower than the algebraic sum of the oncotic pressure of the perfusate and the proximal tubular pressure. The problem associated with reducing the pressure of perfusion is that the flow of perfusate, and hence the delivery of nutrients and oxygen to the renal tubular cells, may become rate limiting. A much better approach is to increase the oncotic pressure of the perfusate. This can be readily accomplished by perfusing the kidney with a concentration of bovine serum albumin calculated to provide an oncotic pressure that is high enough to counterbalance the filtration pressure at the glomerulus. Because the flow of perfusate through the isolated kidney perfused with an artificial solution is generally very high, of the order of 20-22 ml/min/g, there is little or no decline in the pressure of filtration along the glomerular capillary (in the filtering kidney this translates into a very low filtration fraction, of the order of 2 to 3%, and an absence of filtration pressure equilibrium; in other words, there is filtration all along the glomerular capillary). There is also no change in the oncotic pressure. Therefore, the balance between filtration pressure and oncotic pressure remains constant along the length of the glomerular capillary. The glomerular capillary pressure in the isolated perfused rat kidney is comparable to that measured in vivo. 9 T h e proximal tubular pressure has been either measured directly or estimated in different isolated perfused rat kidney preparations and found to be either comparable to that measured in vivo or slightly higher.9-11 Thus, the filtration pressure in the glomerular capillary of the isolated perfused kidney depends on the oncotic pressure of the perfusate. The technique used is the same as that of the filtering perfused kidney preparation. A number of techniques have been developed over the years, but for the purposes of a nonfiltering kidney, perfusion of rat kidneys with bovine serum albumin in Krebs-Henseleit solution is the most convenient. The preparation that we use is that developed by Nishiitsutsuji-Uwo et al. ~2 and first reported in 1967. The rat is anesthetized with pentobarbital, 60 mg/kg intraperitoneally. Heparin (1000 U) is injected into the femoral vein. The peritoneal cavity is then opened and the right ureter dissected free and catheterized with a polyethylene PE- 10 catheter. The ureter is then cut below the site of catheterization. The superior mesenteric artery, the right renal artery, and the right adrenal artery (which usually comes offthe right renal artery about 3 mm from the origin of the renal artery) are 9 H. M. Brink, W. M. Moons, and J. F. Slegers, Pfluegers Arch. 397, 48 (1983). to M. Bullivant, IC O. Hicks, and B. H. Smaill, Pfluegers Arch. 389, 251 (1981). " G . De Mello, and T. Maaek, Am, J. Physiol. 231, 1699 (1976). 12j. M. Nishiitsutsuji-Uwo, B. D. Ross, and H. A. K.rebs,Biochem. Z 103, 852 (1967).
[3]
ISOLATED PERFUSED AND NONFILTERING KIDNEY
33
dissected free. Care is taken not to exert any traction on the right renal artery to avoid trauma-induced vasoconstriction. The right adrenal artery is ligated. The superior mesenteric artery is ligated about 1 cm from its aortic origin. Loose ligatures are placed around the origin of the superior mesenteric artery and the renal artery. An incision is made in the mesenteric artery about 5 m m from its origin and the perfusion cannula inserted into the artery. Perfusion is started at this time. The cannula is then threaded across the aorta into the renal artery and both the mesenteric and renal arterial ligatures are tied. This procedure allows perfusion of the kidney without interruption of flow. The renal vein is cut and the kidney removed from the animal and placed in the perfusion cabinet. In this preparation the venous effluent drains over the kidney into the reservoir. If sampling or derivation of the venous effluent is required, the vein can be separately catheterized. The perfusion mediun5 we use consists of a Krebs-Henseleit solution of the following composition (raM): sodium, 145; potassium, 4; calcium, 2.5; magnesium, 1.2; chloride, 103; sulfate, 0.8; phosphate, 1.2; bicarbonate, 25. The pH of the perfusate is maintained at 7.4 by gassing with a mixture of 95% 02 and 5% CO,. With this gas mixture the O2 content of the perfusate is maintained at 400-500 mmHg. The albumin is prepared as a stock solution of 13 g% of bovine serum albumin in Krebs-Henseleit solution. The albumin is dialyzed in a counterttow dialysis system against Krebs-Henseleit solution for a period of 24 hr. One pump runs the perfusate through and another the Krebs- Henseleit solution. The concentration of albumin necessary to produce an oncotic pressure high enough to counterbalance the filtration pressure at the level of the glomeruli can be calculated from the Landis-Pappenheimer equation, where c is the concentration of albumin in grams per deciliter: 2.8c + 0.18(c 2) + 0.012 (c 3) = oncotic pressure The temperature of the system is maintained at 37 ° . The easiest way to attain this is to keep the whole system in a warm, thermostatted cabinet. Another way is to warm the perfusate. This can be done by using a water-jacketed oxygenator in which the water is heated by a circulator. This approach uses much less space. Evaporation of water, particularly during long perfusions, must be compensated by the continuous addition of distilled water at a rate sufficient to maintain the osmolality of the perfusate constant. The glassware used consists of a reservoir that holds the kidney on top and collects the venous effluent on the bottom. It must have a side opening allowing the ureter catheter through so that the urine can be collected. It can also be made out of plastic. We use a bubble oxygenator, consisting of several glass bubbles attached one on top of the other with five openings.
34
KIDNEY
[4]
At the top is the perfusate inlet; at the bottom is the perfusate outlet; a side arm at the bottom allows a small amount, 4 to 5 ml, to remain at the bottom of the oxygenator at all times (this is the overflow that goes back directly to the reservoir); a side arm slightly higher than the previous one is the oxygen/COe inlet; a side arm in the top bubble is the gas outlet. Two pumps are used. It does not matter whether they are of the peristaltic or continuous type. One pump takes the perfusate from the bottom reservoir, through two Millipore (Bedford, MA) filters arranged in parallel, to the top of the oxygenator. The filters used are Millipore type LC l0 g m with a prefilter type AP Cat. #AP20 02200. Both filters are contained in stainless steel holders. The second pump takes the perfusate from the bottom of the oxygenator to the kidney. The arterial line has an in-line Brooks flowmeter with a tantalum float, the only float that is capable of reading the large flows found in this preparation. The arterial line also has a pressure gauge attached to it by a T-type connector. The arterial line has in addition a derivation to the reservoir. A needle valve in it permits adjustment of the pressure/flow through the kidney. We use glass arterial cannulas, but stainless steel needles are also adequate. The glass cannulas are preferable because they allow one to see whether there is air inside as the cannnlation is performed. The problem with glass cannulas is threefold: First, the glass available in the United States is thick walled, therefore the cannulas that are thin enough to insert into the artery easily have high resistance. Second, they break. Third, they must be calibrated to determine their intrinsic resistance, that is, the pressure drop across the cannula. This is necessary to determine the peffusion pressure of the kidney and can be done by establishing a pressureflow relation for each cannula.
[4] M u l t i p l e I n d i c a t o r D i l u t i o n a n d t h e K i d n e y : K i n e t i c s , P e r m e a t i o n , a n d T r a n s p o r t in Vivo By CHARLES J. LUMSDEN and MELVIN SILVERMAN
Introduction In human kidneys almost 200 liters of water and solutes are filtered daily from the blood at the level of the glomeruhis, but only 1 liter is finally voided as concentrated urine. The glomerular ultrafiltrate must therefore METHODS IN ENZYMOLOGY, VOL. 191
Coi~jright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
ISOLATED PERFUSED AND NONFILTERING KIDNEY
31
[3] I s o l a t e d P e r f u s e d a n d N o n f i l t e r i n g K i d n e y B y PATRICIOSILVA
Among the many advantages of the isolated perfused kidney is that because of the nature of the preparation glomerular filtration can be prevented, thus allowing the investigator to study renal functions independently of glomerular filtration and the associated cellular work related to tubular reabsorption of salt and other organic and inorganic compounds. The nonfiltering isolated kidney preparation can be used to determine whether the site of uptake into renal tubular cells is basolateral, independent of glomerular filtration, or lumenal, dependent on glomerular filtration. To my knowledge, the first reported use of the preparation was that of Johnson and Maack t where they used a nonfiltering isolated perfused rat kidney to study the metabolism by the kidney of growth hormone. Since then, it has been used by many other investigators to examine peptide or protein catabolism, uptake of toxic substances, renin secretion, or to determine physiological parameters such as basal oxygen consumption.t-5 There is an additional advantage in the use of the nonfiltering mode of the isolated kidney preparation perfused with an artificial solution over that of the filtering mode, and that is that the nonfiltering preparation never develops the morphological lesions in the thick ascending limb of the loop of Henle6 associated with perfusion regularly found in the filtering one.7, a
Several different methods have been used to prevent glomerular filtration: ureteral ligation, reduction in the pressure of perfusion, increased oncotic pressure of the perfusate, or a combination of these. Ureteral ligation prevents the excretion of urine and, because of the increased back pressure along the urinary tract, it reduces giomerular filtration; however, it does not prevent it completely. While electrolyte and water reabsorption continue, predominantly in the proximal tubule, glomerular filtration continues as well, albeit at a much reduced rate. Another way of reducing i V. Johnson and T. Maack, Am. J. Physiol. 233, F185 (1977). 2 S.T. Kau and T. Maack, Am. J. Physiol. 233, F445 (1977). 3 K. Miura, R. S. Goldstein, D. A. Pasino, and J. B. Hook, Toxicology44, 147 (1987). 4 A. J. Cohen, K. Spokes, R. S. Brown, J. S. Stoff, and P. Silva, Orc. Res. 50, 400 (1982). 5 R. D. Swartz, P. Silva, R. HaUac, and F. H. Epstein, Curr. Probl. Clin. Biochem. 8, 123
(1978). 6 M. Brezis,S. Rosen,P. Silva,and F. H. Epstein,Kidney Int. 25, 65 (1984). 7D. Alcorn,K. R. Emslie,B. D. Ross, G. B. Ryan, and J. D. Tange,Kidney Int. 19, 638 (1981). s H. J. Schurekand W. Kriz, Lab. Invest. 53, 145 (1985). METHODS 1N ENZYMOLOGY, VOL. 191
Copyright© 1990by AcademicPress,Inc. All fightsof reproductionin any formreserved.
32
KIDNEY
[3]
glomerular filtration is to reduce the pressure of perfusion to a level where the hydrostatic pressure in the glomerular capillary is lower than the algebraic sum of the oncotic pressure of the perfusate and the proximal tubular pressure. The problem associated with reducing the pressure of perfusion is that the flow of perfusate, and hence the delivery of nutrients and oxygen to the renal tubular cells, may become rate limiting. A much better approach is to increase the oncotic pressure of the perfusate. This can be readily accomplished by perfusing the kidney with a concentration of bovine serum albumin calculated to provide an oncotic pressure that is high enough to counterbalance the filtration pressure at the glomerulus. Because the flow of perfusate through the isolated kidney perfused with an artificial solution is generally very high, of the order of 20-22 ml/min/g, there is little or no decline in the pressure of filtration along the glomerular capillary (in the filtering kidney this translates into a very low filtration fraction, of the order of 2 to 3%, and an absence of filtration pressure equilibrium; in other words, there is filtration all along the glomerular capillary). There is also no change in the oncotic pressure. Therefore, the balance between filtration pressure and oncotic pressure remains constant along the length of the glomerular capillary. The glomerular capillary pressure in the isolated perfused rat kidney is comparable to that measured in vivo. 9 T h e proximal tubular pressure has been either measured directly or estimated in different isolated perfused rat kidney preparations and found to be either comparable to that measured in vivo or slightly higher.9-11 Thus, the filtration pressure in the glomerular capillary of the isolated perfused kidney depends on the oncotic pressure of the perfusate. The technique used is the same as that of the filtering perfused kidney preparation. A number of techniques have been developed over the years, but for the purposes of a nonfiltering kidney, perfusion of rat kidneys with bovine serum albumin in Krebs-Henseleit solution is the most convenient. The preparation that we use is that developed by Nishiitsutsuji-Uwo et al. ~2 and first reported in 1967. The rat is anesthetized with pentobarbital, 60 mg/kg intraperitoneally. Heparin (1000 U) is injected into the femoral vein. The peritoneal cavity is then opened and the right ureter dissected free and catheterized with a polyethylene PE- 10 catheter. The ureter is then cut below the site of catheterization. The superior mesenteric artery, the right renal artery, and the right adrenal artery (which usually comes offthe right renal artery about 3 mm from the origin of the renal artery) are 9 H. M. Brink, W. M. Moons, and J. F. Slegers, Pfluegers Arch. 397, 48 (1983). to M. Bullivant, IC O. Hicks, and B. H. Smaill, Pfluegers Arch. 389, 251 (1981). " G . De Mello, and T. Maaek, Am, J. Physiol. 231, 1699 (1976). 12j. M. Nishiitsutsuji-Uwo, B. D. Ross, and H. A. K.rebs,Biochem. Z 103, 852 (1967).
[3]
ISOLATED PERFUSED AND NONFILTERING KIDNEY
33
dissected free. Care is taken not to exert any traction on the right renal artery to avoid trauma-induced vasoconstriction. The right adrenal artery is ligated. The superior mesenteric artery is ligated about 1 cm from its aortic origin. Loose ligatures are placed around the origin of the superior mesenteric artery and the renal artery. An incision is made in the mesenteric artery about 5 m m from its origin and the perfusion cannula inserted into the artery. Perfusion is started at this time. The cannula is then threaded across the aorta into the renal artery and both the mesenteric and renal arterial ligatures are tied. This procedure allows perfusion of the kidney without interruption of flow. The renal vein is cut and the kidney removed from the animal and placed in the perfusion cabinet. In this preparation the venous effluent drains over the kidney into the reservoir. If sampling or derivation of the venous effluent is required, the vein can be separately catheterized. The perfusion mediun5 we use consists of a Krebs-Henseleit solution of the following composition (raM): sodium, 145; potassium, 4; calcium, 2.5; magnesium, 1.2; chloride, 103; sulfate, 0.8; phosphate, 1.2; bicarbonate, 25. The pH of the perfusate is maintained at 7.4 by gassing with a mixture of 95% 02 and 5% CO,. With this gas mixture the O2 content of the perfusate is maintained at 400-500 mmHg. The albumin is prepared as a stock solution of 13 g% of bovine serum albumin in Krebs-Henseleit solution. The albumin is dialyzed in a counterttow dialysis system against Krebs-Henseleit solution for a period of 24 hr. One pump runs the perfusate through and another the Krebs- Henseleit solution. The concentration of albumin necessary to produce an oncotic pressure high enough to counterbalance the filtration pressure at the level of the glomeruli can be calculated from the Landis-Pappenheimer equation, where c is the concentration of albumin in grams per deciliter: 2.8c + 0.18(c 2) + 0.012 (c 3) = oncotic pressure The temperature of the system is maintained at 37 ° . The easiest way to attain this is to keep the whole system in a warm, thermostatted cabinet. Another way is to warm the perfusate. This can be done by using a water-jacketed oxygenator in which the water is heated by a circulator. This approach uses much less space. Evaporation of water, particularly during long perfusions, must be compensated by the continuous addition of distilled water at a rate sufficient to maintain the osmolality of the perfusate constant. The glassware used consists of a reservoir that holds the kidney on top and collects the venous effluent on the bottom. It must have a side opening allowing the ureter catheter through so that the urine can be collected. It can also be made out of plastic. We use a bubble oxygenator, consisting of several glass bubbles attached one on top of the other with five openings.
34
KIDNEY
[4]
At the top is the perfusate inlet; at the bottom is the perfusate outlet; a side arm at the bottom allows a small amount, 4 to 5 ml, to remain at the bottom of the oxygenator at all times (this is the overflow that goes back directly to the reservoir); a side arm slightly higher than the previous one is the oxygen/COe inlet; a side arm in the top bubble is the gas outlet. Two pumps are used. It does not matter whether they are of the peristaltic or continuous type. One pump takes the perfusate from the bottom reservoir, through two Millipore (Bedford, MA) filters arranged in parallel, to the top of the oxygenator. The filters used are Millipore type LC l0 g m with a prefilter type AP Cat. #AP20 02200. Both filters are contained in stainless steel holders. The second pump takes the perfusate from the bottom of the oxygenator to the kidney. The arterial line has an in-line Brooks flowmeter with a tantalum float, the only float that is capable of reading the large flows found in this preparation. The arterial line also has a pressure gauge attached to it by a T-type connector. The arterial line has in addition a derivation to the reservoir. A needle valve in it permits adjustment of the pressure/flow through the kidney. We use glass arterial cannulas, but stainless steel needles are also adequate. The glass cannulas are preferable because they allow one to see whether there is air inside as the cannnlation is performed. The problem with glass cannulas is threefold: First, the glass available in the United States is thick walled, therefore the cannulas that are thin enough to insert into the artery easily have high resistance. Second, they break. Third, they must be calibrated to determine their intrinsic resistance, that is, the pressure drop across the cannula. This is necessary to determine the peffusion pressure of the kidney and can be done by establishing a pressureflow relation for each cannula.
[4] M u l t i p l e I n d i c a t o r D i l u t i o n a n d t h e K i d n e y : K i n e t i c s , P e r m e a t i o n , a n d T r a n s p o r t in Vivo By CHARLES J. LUMSDEN and MELVIN SILVERMAN
Introduction In human kidneys almost 200 liters of water and solutes are filtered daily from the blood at the level of the glomeruhis, but only 1 liter is finally voided as concentrated urine. The glomerular ultrafiltrate must therefore METHODS IN ENZYMOLOGY, VOL. 191
Coi~jright© 1990by AcademicPress,Inc. All rightsof reproductionin any formreserved.
