CLEARANCE CONCEPTS LN THE KIDNEY
Gina Louise Sirianni
A thesis submitted in confonnity with the requirements for the degree of Master of Science Graduate Department of Pharmacology, University of Toronto
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Gina Louise Sirianni (M.Sc.), 1997 Department of Pharmacology, University of Toronto (Abstract)
The kidney is an important elirninating organ. capable of metabolism and excretion. The fractional excretion value (FE, unbound urinary clearance normalized to glomemlar filtration rate) is typically used to detemine whether a compound is net fdtered (FE = I). reabsorbed (FE < 1) or secreted (FE > 1) by the kidney. It was hypothesized that competing routes of elimination within the kidney wiU influence the clearance eshate of each other. The hypothesis was first tested with cornputer simulations based on a physiological model of the kidney and parameters describing the rend handling of enalapril, an angiotensin converting enzyme inhibitor that is both rnetabolized and excreted unchanged by the kidney. Since the location of endapril metabolism was unknown. simulations were based on two possible models, one which incorporated intracellular metabolism of enalapril, and another which incorporated intraiuminal metabolism of enalapril. Results of the simulation study showed that the excretory clearance estimate was decreased in the presence of luminal or cellular metabolisrn while metabolic clearance was affected differentialiy. depending upon the site of rend metabolism. The renal metabolic clearance was predicted to decrease with excretion for the intracellular model, but would increase for the intraluminal model. Studies were then performed in the nonfiltenng isolated perfused rat kidney to determine the site of rend enalapril metaboiism with data obtained for enalapril and its metabolite. enalaprilat. The observations closely matched the predictions for intracellular metabolism. A final set of studies with enalapril and an esterase inhibitor. paraoxon, was performed in the renal S9 fraction and in the isoiated perfused rat kidney preparation. The results demonstrated that unnary clearance estimates were indeed reduced by the presence of inmenal metabclism. Since the urinary clearance estimate was affected by intrarenal metabolisrn, FE was not an accurate indicator for the net flux of dmg through the kidney. I greatly appreciate the Mie and guidance that my supervisor, Dr. K. Sandy Pang has provided throughout the course of my studies. Through her suggestions, comments. and criticisrns.
Dr. Pang has seengthened rny scientific capabilities and my ability to think cntically. 1 would also like to thank my advisor, Dr. Lado Endrenyi. for his advice and encouragement.
Although the membea of the lab have changed over the past two years. the group has always consisted of intelligent and amicable people. who have been a pleasure to work with. Thank you all for making my stay here an enjoyable and memorable experience. 1 would especially like to thank my "helpers" for kidney surgery, Dr. Wanping Geng. Dr. Margaret Doherty and Mr. Ford Barker. 1 also thank my boyfnend, Armenio Martins, for his encouragement and thoughtfulness. Lastly. but most irnportantly, I thank my mother. father, and brother, Joe for their continuous love. support and encouragement.
Financial support from the University of Toronto Open Fellowship offered by the School of Graduate Studies was appreciated.
3.3 THEORETICAL ...... 26
3.4 METHODS ...... 28
3.5 RESULTS ...... 31
3.5.1 Kidney as the ody elirninating organ ...... 31 3.5.2 Simulations on rend disposition of enalapril ...... 31
3.6 DISCUSSION ...... ,...... 36
3.7 STATEMENT OF SIGNIFICANCE OF CHAPTER 3 ...... 39
CHAPTER 4: INTRACELLULAR AND NOT INTkUUMIbJAL ESTEROLYSIS OF ENALAPRIL IN KIDNEY: ...... -10
4.1 ABSTRACT ...... ,., ...... 41
4.2 INTRODUCTION ...... -42
4.3 MATERIALS AND METHODS ...... 43 Source of materiais ...... 43
Kidney Perfusion ...... 44 Viability of the PK...... 16 Protein Binding ...... 46 Andytical procedures ...... 47 Calculations...... 47 Simulations ...... 49 Statistics ...... 53
4.4 RESULTS ...... 54 4.4.1 Viability of the Perfused Rat Kidney Preparations ...... 54 4.4.2 Protein Binding ...... 55 4.4.3 The Non-Filtering Kidney...... 55 4.4.4 Simulations ...... 57
4.5 DISCUSSION ...... 59 4.6 STATEMENT OF SIGNIFICANCE OF CHAPTER 4 ...... 62
CHAPTER 5: INHIBITION OF ENALAPRIL ESTEROLYSIS BY PARAOXON IN THE ISOLATED PERFUSED RAT KIDNEY INCREASES URINARY CLEARANCE OF ENALAPRIL ...... 63
5.2 INTRODUCTION ...... 65
5.3 MATERIALS AND METHODS ...... 66
5.3.2 Rend S9 Fraction ...... 66 5.3.3 Partition Coefficient for Enaiapril at Various pH ...... 67 5.3.1 Experiments in the Single-Pass Isolated Perfused Rat Kidney (IPK) ...... 68 5.3.4.1 Viability Studies in the IPK with Paraoxon ...... 69 5.3.4.2 Inhibition Experiments with [3~~~nalapnland Paraoxon in the IPK ...... 69 5.3.5 Thin Layer Chromatographic Assay for Enalapril and Enalapnlat...... 71 5.3.6 Statistics ...... 71 5.3.7 Calculations...... 71
5.4 RESULTS ...... 72 5.4.1 S9 Experiments with Paraoxon ...... 72
5.4.2 Partition Coeficient for Enaiapril at Various pH Values ...... 72 5.4.3 PKexperiments ...... 74 5.4.3.1 Viability in the Presence of Paraoxon ...... 74 54-32 Experimenü with [-3 HIEnaiapril and Paraoxon ...... 75
5.5 DISCUSSION ...... 77
5.6 STATEMENT OF SIGNIFICANCE OF CHAPTER 5 ...... 80
CHAPTER 6: DISCUSSION...... 81
6.1 DISCUSSION ...... 82
CHAPTER 7: CONCLUSIONS ...... 86
7.1 CONCLUSIONS ...... 87
CHAPTER 8: REFERENCES ...... 88 vü 8.1 REFERENCES ...... 89 LIST OF TABLES Table Page
3- 1 Physiologicai constants used for simulation of the rend 30 disposition of enalapril in the isolated pemised rat kidney. 3-2 Clearance equations for the kidney as the only eliminating 32 organ, with intracellular and/or intduminal metabolism. 3-3 Parameten predicted for enaiapril disposition in kidney 33 with physiological constants surnmarized in Table 3- 1.
Parameters used for simulations of [3~~enalapnland ['~lenalaprilatdata in the single pas isolated perfused kidney (IPK) and nonfiltering kidney (NFK) preparations. Surnmary of expenmentally observed and simulated data for the IPK and NFK. Cornparison of on enalapril obtained in the IPK before and during infusion of O. 1 pM paraoxon. LIST OF FIGURES
Figure Page 1- 1 Structure of the nepbron. 3 1-2 Rend disposition of enalaprii. 15
3- 1 Physiologicai mode1 for the rend elimination of a subsuate that is 27 both metabolized and excreted by the kidney. Simulation of the effect of rend metabolism on fractionai excretion and the effect of rend secretion on rnetabolic clearance estimates. Left panel: intraceMar metabolism: right panel: intraluminal metabolism. Schematic representation of the single pass nonfiltering isolated perfused rat kidney (NFK) preparation.
Physiological rnodels of the kidney depicting either (A) intracellular or (B)intraluminai metaboiism of enaiapril. Separation of ['~]enalapriland ['H]enalapriln by thin layer chromatography. Simuiated data on the output rates of enalapril or enalaprilat as a fraction of the inflow rate of endapril in the isolated perfused rat kidney or the nonfiltering isolated pemised rat kidney.
Schematic representation of paraoxon infusion into the isolated pefised rat kidney. Schematic representation of the experimental design for isolated perfused rat kidney expenments with paraoxon.
8 Inhibition of [3~]enalaprilatformation from ['~lenalaprilin rend S9 with paraoxon. Partition coefficient of ['~]enala~rilat various pH values. 5% Sodium and glucose reabsorption in the isolated perfused rat kidney at various concentrations of paraoxon. The relationship between urinary clearance of enaiapril and urine flow rate. ABBREVIATIONS AND TERMINOLOGIES
amount of drug in reservoir, at any time t
amount of drug in rend plasma at any time t
amount of dmg in renal tissue, at any time t amount of dnig in urine, at any time t
total amount of drug excreted into unne
angiotensin convening enzyme area under the coficentration-tirne curve area under the concentration-tirne curve for the reservoir
area under the concentration-time curve for urine osmotic pressure exerted by plasma protein bovine serurn dburnin concentration of dmg in artenal plasma
concentration of dnig in renal tissue. at any tirne t
concentration of metabolite in rend tissue. at any time t venous plasma concentration of dnig at midpoinr of urine collection intenral
concentration of hgin venous plasma concentration of metabolite in venous plasma
concentration of hgin rend plasma, at any tirne t
concentration of metabolite in renal plasma, at any time t
concentration of hgin urine, at any time t
concentration of metabolite in urine, at any time t unbound fraction of dmg in plasma efflux clearance of dmg at rend basolateral membrane efflux clearance of metabolite at rend basolateral membrane
influx clearance of dmg at rend basolaterd membrane influx clearance of metabolite at rend basolateral membrane filtration clearance efflux clearance of dmg at renal brush border (apical or luminal)
membrane rend intracelMar metaboiic intrinsic clearance
renal intralurninal me tabolic intrinsic clearance effiux clearance of metabolite at renal bmsh border (apical or luminal)
membrane influx clearance of dmg at rend bmsh border (apical or luminal) membrane CL^ {mi} influx clearance of metabolite at renal brush border (apical or luminal) membrane renal me tabolic clearance total renal clearance unnary clearance
extraction ratio of kidney unbound fraction of metaboiite in plasma unbound fraction of dmg in kidney tissue unbound fraction of metabolite in kidney tissue
fractional excretion glornemlar filtration rate human organic cation transporter 2 isolated pemised rat kidney concentration of dmg at which the rate is half the maximum filtration permeability of the glomerulus Krebs-Henseleit bicarbonate medium nonfiltenng isolated perfused rat kidney Nt-methylnicotinamide organic anion transporter 1 OAT-K 1 organic anion transporter - kidney 1 organic anion transport polypeptide PAH para-aminohippurate hydrostatic pressure of blood at the glomeruIus hydrostatic pressure of fluid in Bowman's capsule total rend plasma flow rate urine flow rate
red blood cells rat organic cation transporter 1 rat organic cation transporter 2 Tm maximum rate of transport TEA tetraethylammonium TLC thin layer chromatography plasma reservoir volume plasma volume of kidney tissue volume of the kidney volume of urine LIST OF PUBLICATIONS
ARTICLES :
a) In press:
G.L. Sinanni and K.S. Pang. Organ clearance concepts: New perspectives on old principles. J.
Phamacokin. Biopham. 254 ( 1997). b) Submitted:
G.L. Sirianni and K.S. Pang. Intracellular and not inualuminal esterolysis of enalapril in kidney: Studies with the single pass perhised nonfiltering rat kidney. Dncg Metnb. Dispos. (Subrnitted).
ABSTRACTS:
G.L. Sinanni and K.S. Pang. Organ clearance concepts: Should we keep on believing in old
principles? Abstract, Visions in Phamacolog~,University of Toronto ( 1996).
G.L. Sirianni and K.S. Pang. Rend clearance concepts: Effect of rend metabolism on secretory
indices. Abstracr, Universities ar Buflalo and Toronto Phannaceutics Spposicirn ( 1996).
G.L. Sirianni and K.S. Pang. Effect of competing excretion and metabolism on clearance estimates for enalapril in kidney. Abstract, American Society for Phannacology and
Experirnental Therapeutics, San Diego ( 1997).
xiv Chapter One: Introduction 1.1 The Kidney The kidney is one of the most important eliminating organs in the body. It is capable of excreting polar compounds from the blood as weli as metabolizing a variety of endogenous and exogenous substrates. Blood flow to the kidneys is high relative to other orgms in the body such as the heart, Liver and brain. Approxknately 20% of the cardiac output in man is delivered to the kidney, an organ which accounts for only about one half of one percent of body weight (Brenner et al.. 1986a). The kidney is a retroperitoneal organ. There are two distinct regions that are visible upon bisection of the organ: an outer region, the cortex. and an inner region. the rnedulla. The medulla of the human kidney is segregated into severai striated conical masses of tissue. cded the renal pyramids. In contrast. the nt kidney, as weli as the kidneys of many other laboratory anirnals. contains only a single renal pyramid (Tisher and Madsen. 1986). The main functional unit of the kidney is the nephron (Braus. 1924), which includes the renal corpuscle (or glornerulus). the proximal tubule, the thin limbs. the distal tubule and the connecting segment (Fig. 1-1). Blood enters the kidney, via the renal mery which branches into smaller arteries until reaching the afferent artenole. leading to the glomerulus. Blood is filtered at the glomenilus. resulting in an ultrafiltrate of plasma entering Bowman's capsule. The ultrafdtrate consists of plasma water and solutes that are capable of passing through the glornerular sieve. The extent of filtration depends upon the balance between the transcapillary hydraulic and the colloid osmotic pressure gradients at the glomerulus. The process can be descnbed by the following equation :
GFR=Kp[{Pb-Pc) -xbj (1-1)
Where GFR is the glomerular filtration rate, Kp is the fütration permeabiiity of the glomerulus. Pb is the hydrostatic pressure of blood at the glomerulus. Pc is the hydrostatic pressure of fluid in
Bowman's capsule. and nb is the osmotic pressure of proteins. Blood which is not filtered leaves the glomerulus via the efferent artenoles and enters the pentubular capillaries. Venous blood collected from the peritubular capillaries is drained into a network of venous vessels which culminate at various junctions within the kidney, and uitimately. at the rend vein. Figure 1-1. Structure of the nephron (adapted from Tisher and Madsen. 1986). Upon leaving Bowrnan's capsule. the ultrafîltrate enters the proximal tubule. which cm be divided into two observable subsections in the human and several subsections in the rat and other laboratory animals. The pars convoluta and the pars recta are the initial convoluted portion and the more distai straight portion of the proximal tubule, respectively. These two distinct regions have ken identified in humans, whereas three or four morphological regions have ken identified in the rat and other marnrnals (Maunsbach, 1966; Woodhall et al., 1978: Tisher et al., 1969). The proximal tubule is divided into three subsections. narnely SI. S2 and S3. In the pars convoluta
(which includes S 1 and part of S2), the epitheliai cells are columnar and possess a brush border membrane which sends microvilli into the lumen of the proximal tubule. The pars recta (which includes the distal portion of S2 and S3 in its entirety) is predorninantly cornposed of cuboidd epithelial cells and also possesses a brush border with elongated microvilli (Tisher and Madsen. 1986). Approximately 80% of fdtered water is reabsorbed in the proximal tubule. with the remainder king reabsorbed in the latter portions of the nephron. Only a small volume of ultrafiltrate leaves the kidney as urine.
Continuing from the proximal tubule is the thin descending limb of the loop of Henle, which eventuaiiy loops upwards to become the thn ascending limb. There are four types of epithelium present in the thin limb. only some of which exhibit microvilli (Kaissling and Kriz. 1979: Barret etal.. 1978). The next segment ef the nephron is the distal tubule. It begins with the thick ascending limb of Henle (pars recta) and continues through the macula densa and finally the distal convoluted tubule (pars convoluta). Of the two types of cells present in the distal tubule, one has microvilli and the other does not (Allen and Tisher, 1976). Nephrons with shorter loops of Henle (those arising from the more superficial nephrons) may incorporate the transition between the thn and thick limbs before the hairpin mm which changes the direction of the loop of Henle from descending to ascending.
The most distal portion of the nephron is the collecting duct. It is divided into subsections based upon location within the kidney (Myers et al.. 1966). These sections include the cortical collecting segment, the outer meduiIary segment and the inner medullary segment. which terminate as the papillary ducts. Two celi types are found in the collecting duct, the principal cells and the intercalated cells. The principal ceils have short, blunt microvilli and a single prominent ciiium 4 whde the intercalated ceils are covered by ridge-like microplicae andor welldeveloped rnicrovilii without cilium (Andrews and Porter. 1974: Bulger et al.. 1974). In the past, the relationship between the peritubular blood supply and the nephron was depicted quite sirnply. Traditionaliy. it was held that the peritubular capiliary arising from a given glomerulus supplied the remainder of that nephron with blood throughout the kidney. However. evidence obtained by use of double injection techniques (Beeuwkes and Bonventre. 1975) provided a more accurate description of the vascular-tubular relationship. With these studies. investigatoa determined that peritubular capillaries arising from a particular nephron wili not necessarily associate with the same nephron throughout the kidney (Briggs and Wright. 1979).
1.2 Renal Clearance
The ability of the kidney to rernove a substrate from the arterial circulation cm be described in ternis of iü clearance. defined as the volume of inflowing fluid that is cleared of substrate per unit time (Gréhant, 1904). Total rend clearance is the sum of the metaboiic and excretory (or urinary) clearances. The urinary clearance is that component which is accounted for by excretion of intact drug into urine. Likewise. the metabolic clearance is that component which is accounted for by drug which is renaüy metabolized. Factors which influence renal clearance are both of renal and extrarend origin.
1.21 Excretion of Drugs by the Kidney The extent to which a compound is excreted by the kidney is dependent upon the processes of filtration. reabsorption and secretion. Filtration and secretion increase the rate of renal excretion, whde reabsorption decreases the rate of rend excretion. Severd factors are known to influence the extent of each of these processes. 1.2.1.1 Filtration
The rate of Ntration of a hgis dependent upon several factors. including molecular size and charge. plasma protein binding, and the glomerular filtration rate (GFR). Compounds with a rnolecular weight less than 5000 can be fiitered at the glomemlus, although the charge of the rnolecule may bit filtration (Chang et al., 1975). It has been shown that negatively charged macromolecules are restricted in their ability to cross the glomerular membrane as compared to sirnilarly sized neutral polymea (Chang et al.. 1975: Rennke & Venkatachalam, 1977). Moreover. positively charged molecules cross the glomerular wall to a greater extent than do neutral molecules of the sarne size (Rennke & Venkatachalam, 1977; Brenner et al.. 1978). For a drug that is freely filtered at the glomemlus. the rate of fdtration wili rise in direct proportion with drue concentration in plasma. plasma protein binding and the GFR:
Rate of Filtration = GFR * fp * CI, ( 1-2) where GFR is the glomerular filtration rate, fp is the unbound fraction of drug in plasma. and CI, is the input plasma concentration of dmg. The plasma concentration is used radier than the whole blood concentration of drug since drug whch is bound to red blood cells is not available for filtration. Likewise. only the fraction of drue in plasma that is not protein bound is capable of undergoing filtration.
The fdtration clearance (CL,,,). or the volume of inflowing fluid cleared of hgper unit time by filtration. cm be obtained by dividing the rate of filtration by the plasma concentration of drug and is given by the product of the unbound fraction of dmg in plasma and the GFR:
CL,,, = fp * GFR
Once again, this equation describes those substances that are freely filtered at the glomemlus. The filtration clearance wiI1 rise in direct proportion with unbound fraction of drug in plasma and with
GFR. Although the plasma concentration of drug is not included in this equation. it wiU influence the filtration clearance indirectly by altering the unbound fraction of drug in plasma. The unbound fraction rnay (or may not) nse with increased drug concentration in plasma (Ekstrand et al.. 1979). For a compound which undergoes glomemlar filtration yet is neither secreted or reabsorbed. the total rend clearance wiii equal the fdtration clearance. If the compound is also completely unbound in plasma (fp = l), its renal clearance would equal the GFR. A substance with these propenies can be utilized as a tool for measuring GFR. Creatinine, an endogenous compound, is ofien used as a marker of GFR, although it is not ideal since it does undergo some tubular secretion (Brenner et al.. 1986b). The unnary clearance of inulin, an exogenous compound. is the most accurate for the estimation of GFR, since it is neither secreted nor reabsorbed, and is not bound to plasma proteins (Levinsky and Levy, 1978).
1.2.1.2 Secretion
In the kidney, secretion refers to movement of drug from blood to urine. Dmg is first transported across the basolateral membrane into the renal epithelium. then crosses the lurninai membrane into the rend tubule. Transporters located on both the basolateral and lurninai membranes determine the extent to which a compound wu be secreted by the kidney. Therefore. the rate of secretion for a dmg will depend upon the intrinsic affinities of the transporters for influx and efflux of the substrate. Accordingly. the rate of secretion cm be described by the following equation:
Tmffp+C1n Rate of Secretion = Km+fp *Ch
Where Tm is the maximum rate of transport, CI, is the concentration of drug in plasma and Km is concentration of dmg at which the rate of transport is equal to half of Tm. This implies that as the concentration of dnig in plasma increases. the rate of secretion will approach a maximum value. As with fütration. only unbound drug cmbe secreted. However, protein binding is readily reversible and as drug enters renal cells. dmg-protein complexes in plasma rapidly dissociate in order to rnaintain equilibrium for the loss of unbound dmg. The dissolution process replaces the unbound dmg that has been transported. In this way. a substance may be aimost completely removed from the peritubular circulation by secretion with a renal extraction ratio approaching unity. Since total renal clearance (CLfOI,~)is a product of the renal extraction ratio [EK = (rate in - rate out) / rate in] and plasma flow rate (QK),a dmg which is completely removed from peritubular circulation (EK s
7 1) has a total renai clearance equal to the plasma fiow rate. Para-amuiohippurate (PAH), is an
exogenous organic acid that is almost completely filtered and secreted by the kidney without king reabsorbed (Brenner, 1986a). Therefore, the renai clearance of PAH provides a measure of renal plasma flow.
The principle mechanisrns for secretion of xenobiotics by the kidney are via the organic anion and organic cation transport systems. The organic anion system is usuaily associated with transport of para-arninohippurate (PM)and inhibition with probenecid (Pritchard and Miller. 1993). Prototypical drugs for the organic cation system are NI-methylnicotinamide (NMN) and
tetraethylammonium (TEA) (Rennick. 198 1). Quinine and quinidine are used as inhibitors of
cationic transport (Toretti et al.. 1962). The two prirnary rules which govem interaction of a
substrate with the organic anion and cation transport systems are summarized as follows: 1 ) within a homologous series of compounds, increased hydrophobicity results in increased affinity for the transporter 2) inhibitory effectiveness increases with increasing ionization. Not all situations conform perfectly with these guidelines. However. deviations can usually be explained by the influence of other factors (UIIrich et al.. 1992).
In addition to the organic anion and cation systems. secretion of drugs by the kidney cm be mediated by P-glycoprotein, which has ken identified on the apical membrane of the proximal tubule (Willingham et aL. 1987). A Na+-dependent nucleoside transport system has also ken identified in the renal brush border membrane (Gutierrez et al., 1992; Brett et (11.. 1993)
1.2.1.2.1 Secretion Across the Basolateral Membrane
Uptake of organic anions across the basolateral membrane against an electrochemical gradient has ken shown to occur via an anion/dicarboxyIate exchanger (Chatsudthipong and
Dantzler, 1992; Shimada et al., 1987: Pritchard. 1990). which exchanges an organic anion for a- ketoglutarate (Shpun et al.. 1995). a-Ketoglutarate is supplied by a Na+/dicarboxylate couansporter, which is dnven by a Na' gradient (out > in) generated by ATP hydrolysis. The transport of anions by this pathway across the basolateral membrane is thus a tertiary active process
(Shrnada et al., 1987; Pritchard, 1988). Recently, a novel cDNA from rat kidney that encodes an 8 anion/dicarboxylate exchanger. OATl. has ken cloned and characterized (Sekine er ai.. 1997). A variety of exogenous and endogenous anionic compounds were found to inhibit the uptake of PAH by oocytes expressing OATl. Several of the anions were also shown to be taken up by oocytes expressing OAT 1 (Selcine et al.. 1997). The uptake of cations across the basolateral membrane is via facilitated diffusion by an electrogenic. canier-mediated system driven by an inside negative transmernbrane potential (Takano et al., 1984: Wright and Wunz. 1987). Recendy, two basolateral membrane cation transporters have ken cloned from rat cDNA libraries, rOCT 1(Grundemann et al.. 1994) and rOCT2 (Okuda et al..
1996). rOCT1 expressed in Xenopus oocytes was shown to transport the classic cation substrates. NMN and TEA as well as other endogenous and exogenous cations (Busch er al.. 1996). Likewise. expression of rOCT2 in Xenopus oocytes stirnulated uptake of TEA but was inhibited in the presence of other organic cations. including quinidine (Okuda et al.. 1996).
1.2.1.2.2 Secretion Across the Luminal Membrane At the luminal membrane, organic anions are effluxed from the ce11 down an electrochemical gradient via an organic anion transport system (Pritchard and Miller. 1993). Recently. two cDNAs encoding organic anion transporters locaiized to the Iuminai membrane have been identified. The fmt. oatp (organic anion-transporting polypeptide). initially denved from nt liwr. has also ken identified in the bmsh border membrane of the rat kidney S3 segment (Jacquemin et al.. 1994:
Bergwerk et al.. 1996). Although this transporter has been found to mediate Na+-independent transport of bromosulphothalein and several anionic steroids. it is not able to transport PAH (Kanai et al.. 1996). OAT-KI, which transports methotrexate. is the second luminal organic anion transporter cloned recently (Saito et al., 1996). lmmunoblot analysis showed OAT-KL to be prirnarily in the brush border membrane (Masuda er al.. 1997). As reported with oatp. OAT-KI does not transport PAH. however. PAH is capable of inhibiting OAT-K1 for the transport of other compounds (Saito et ai., 1997). Currently, it is unknown whether oatp and OAT-K1 mediate the secretion andor reabsorption of organic anions across the apical membrane. Organic cations are secreted across the brush border membrane by a secondary. active organic cation/proton antiporter. which uses potential energy stored as a pH-gradient to drive the 9 uphill movement of cations fiom cell to tubular lumen (Kinsella et al.. 1979). A Na+-H' exchanger on the brush border membrane. generates the inwardiy directed proton gradient (Aronson, 1985).
Human cDNA encoding a cation transporter, hOCT2. was recently cloned and locaiized to the rend bmsh border membrane, by Northem blot analysis (Gorboulev et al.. 1997). Other transporters. locaiked to the renal bmsh border membrane. that mediate secretion. are
P-glycoprotein. a transporter associated with multi-dmg resistance (Ueda et al.. 1992) and a family of nucleoside transporters (Lee et al., 1988; Williams and Jarvis. 199 1).
1.2.1.3 Reabsorption Reabsorption in the kidney refers to the transfer of subsuate from the renal tubule to the peritubular circulation. This process of transfer cm be passive. facilitated or active. The passive reabsorption of a drug rnolecule is dependent on several factors such as lipophilicity. urine flow rate. urine pH and pKa of the drug. Facilitated and active reabsorption are mediated by transport proteins and would thus be subject to the intrinsic properties of the transporter. Arnong a series of structurally sirnilar drugs, lipophiiicity influences drug reabsorption
(Toon & Rowland. 1983: Mayer et al.. 1988). Mayer et al. ( 1988) examined the relationship between iipophilicity and tubular reabsorption for a senes of barbituric acids and found renal clearance to decrease with increasing lipophihcity due to increased reabsorption across the luminal membrane. Urinary pH and pKa of a drug will influence the reabsorptive capacity of the kidney by aitering the ratio of ionized to nonionized àrug (Beckett and Rowland, 1965). Since the nonionized species is much more lipophiiic. a decreased urinary pH would enhance the reabsorption of acidic compounds and retard the reabsorption of basic compounds. while an increased urinary pH would have the opposite effect. UM~flow rate can alter the exient to which a compound is reabsorbed by diluting the concentration gradient of drug in urine (Bloomer, 1966). Since passive diffusion is dnven by the concentration gradient of drug in the renal tubule, diuresis wiii lead to diluted urine and hastened transit time, thus lowering reabsorption and enhancing excretion of a compound. 1.2.1.3.1 Transport Proteins Mediating Reabsorption
Carrier-mediated tubular reabsorption of organic anions (Roch-Rame1 et al., 1994:
Manganel et al.. L985). organic cations (Weber et al.. 199 1: Weber et al.. 1990: McKinney and
Hosford. 1993) and short peptides (Siibemagl et al.. 1987; Hannelore and Herget. 1997) has ken demonstrated in the kidney. In addition, the reabsorption of D-glucose, phosphate. sulfate. amino acids. urea and urate by the kidney is rnediated by transport proteins (Murer and Biber. 1993: Seheand Endou. 1996). Reabsorption of organic anions across the luminal membrane appears to be mediated by a mechanism of anion exchange (Guggino et al.. 1983: Low et al.. 1984). possibly by more than one type of transporter (Jorgensen and Sheikh. 1984). Two recently cloned bmsh border membrane transport proteins, oatp (Jacquemin et al.. 1994) and OAT-Kl (Saito et al.. 1996). described earlier (see section 1.z. 12.7) may be involved in organic anion reabsorption. However. studies need to be forthcorning to demonstrate that these proteins mediate secretion and/or reabsorption across the luminal membrane. Several organic cations have also been shown to undergo countenransport at the renal bmsh border membrane (Rafizadeh et al.. 1986; McKinney and Kunneman, 1985). The reabsorption of short peptides and peptidornirnetics frorn the tubular lumen has been
Weil established (Matthews. 199 1; Miyarnoto et al.. 1985; Silbemagl et al.. 1987). Substrates are CO-transportedwith H'. and therefore utilize a transmembrane. inwardly directed. electrochernical H'-gradient as the driving force for transport across the apical membrane. The H+-gradient required for uanspon is dependent upon activity of the basolateral (Na+-K+)ATPase. since it supplies metabolic energy required for the luminal membrane Na+-H' exchanger which couples the iflux of Na into the celi with the efflux of H' from the ce11 (Ganapathy er ai., 1987). Recently a hgh affuiity proton-coupled transport protein for di- and tripeptides (PepT 2). localized to the apical membrane of renal tubular cells has been cloned (Saito, 1995: Liu, 1995), and has been show to mediate the facilitative reabsorption of a variety of anionic cephalosporins including cefime (Bok 1996: Ganapathy et al., 1997). 1.2.2 Metabolism in the Kidney
Although the kidney has traditionaiiy been considered as an excretory organ. it is also capable of metabolism. In fact, the kidney is enriched with a wide variety of enzymes. including those capable of conjugation, oxidation and hydrolysis. The extent to which metabolism occurs is dependent upon delivery of substrate to the site of the enzyme as weil as its intrinsic metabolic capacity. Metabolism can occur either invacellularly within the renal perinibular epithelium. or intraluminaily by enzymes located on the brush border membrane of the renal tubule. In general. the extent of phase 1 and II drug metabolism in the kidney is lower in cornparison to the Liver. however, the activities of certain conjugation reactions in the kidney are comparable to those observed in the iiver (Hutt and Caldwell, 1990). Conjugation reactions that occur in the kidney include glucuronidation (Howe et al.. 1992), sulfation (Rorniti et al.. 1992). methylation (Weinshilbourn. 1992). acetylation (Pacifici et al.. l988), glutathione conjugation (Pacifici et al.. 1988), and amino acid conjugation (Hun and Caldwell. 1990; Poon and Pang, 1995). Generatly. the levels of these enzymes are less than those found in the liver, with the exception of acyl-CoA synthetase and N-acylüansferase. which are involved in amino acid conjugation. In certain species, the enzymatic activity in the kidney is actudly higher than that in liver (Hutt and Caldwell, 1990). Several oxidative enzymes systems exist in the kidney, including cytochrome P450 (Murray et al., 1988; Hotchkiss et al., 1995; Hamrnond et al.. 1997: Chen et al., 1996: Masubuchi er al..
1996: Wang et al.. 1996) ethoxycoumarin O-deethylase (ECOD)(Pacifici et al.. 1988) and aldehyde oxidase (Moriwaki et al., 1996). Hydrolysis in the kidney is cmied out by enzymes. including members of the esterase and peptidase families. On the renal brush border membrane is a variety of peptidases that effectively cleave peptides from the blood (Hniska et al.. 1975: Johnson and Maack. 1977: Katz and Rubenstein. 1973). These ecto-enzymes include memben of the following families of peptidases: aminopeptidases. gamma-glutamyluanspeptidases, dipeptidyl peptidases Wgler et al-, 19851, endopeptidases (Landry et al., 1993). dipeptidases. dipeptidyl carboxypeptidases, dipeptidyl aminopeptidases (Wada et al., 1992). Lysosornai rend peptidases include dipeptidylpeptidase 1. dipeptidylpeptidase Il. and cathepsin B (Kugler et al., 1985). With the use of specific inhibiton and activaton for diesterases, arylesterases and " C " - esterases, a variety of esterase activity has been identified in rend tissue (Nicot et al.. 1984; Chow and Ecobichon, 1973: Pond, 1995; Pla and Johnson, 1989; Suda, 1986; Dean et al., 1995: Benedito, 1997). Recently a carboxylesterase, temed hydrolase B. was cloned from a rat kidney cDNA
Library, and found to be locaüzed in the proximal tubules (Yan et al.. 1994). This enzyme is identical to hydrolase B found in rat liver, and is inhibited by the oqanophosphate pesticide, paraoxon (Yan et al., 1994).
Additional hydrolases located in the rat kidney include acid phosphatase. p-glucuronidase. cathepsin-D, aryl sulfatase. ~acetylglucosaminidase.acid 5'-nucleotidase. alkaline phosphatase
(Loven er al., 1982; Lrintscheff and Davidoff, 1981). P-lyase (Jones et al.. 1988). and carbonic anhydrase (Wistrand and Knuuttila, 1989).
1.3 Factors Affecting Renal Clearance Total rend clearance is equal to the sum of excretory and metabolic clearances. It follows. therefore. that the factors affecting excretion and metabolism may also influence the total renal clearance estimate. It is important to note, however. that the variable capable of altering one of the processes may not exert the sarne or similar effect on total rend clearance. For example, a drug which does not have an appreciable filtration clearance due to size or charge limitations may be readily secreted by one of the membrane transport systems in the kidney. In this case. the poor filtration clearance would not be reflected in the estimate of total rend clearance. Likewise, pmtein binding wiil decrease total renal clearance only if filtration is a major component of clearance. Hall and Rowland ( 1984) showed that the extent to which protein binding affects renal clearance depends upon the unbound rend clearance (total renal clearance / unbound fraction of dmg). If the unbound rend clearance is low in relation to the organ blood flow. then clearance wiU be low and dependent on plasma binding. However, if clearance is high. elirnination becomes perfusion rate- limited and the clearance estimate wiii be relatively insensitive to protein binding. For compounds that undergo carrier mediated transport in the kidney, the plasma concentration of drug will alter die rend clearance estirnate. At high plasma dmg concentrations. transport mechanisrns could become sanirated. Upon saturation. rend clearance unll be decreased with increasing drug concentration. Conversely. if reabsorption is saturateci, total rend clearance wiU kcrease with increasing drug concentration (van Ginneken and Russel. 1989). In a sirnilx fashion. cornpetition between similar substrates for a common carrier site wili influence renal clearance estirnates by competing with one another for transport (van Ginneken and Russel. 1989).
1.4 Cornpetition Between Excretion and Metabolkm in the Kidney
A widely accepted method of assessing the capacity of the kidney for excretion is the fractionai excretion vaiue (FE. unbound urinary clearance divided by the GFR). An FE value of less than one infers that the compound undergoes net reabsorption. a value greater than one denotes net secretion. and a vaiue equai to one suggests net filtration (Brenner et al., 1986b). Recently, this method of assessing the net handling of a compound by the kidney has ken challenged by Smith and Kugler (1994). who suggest that the presence of intrarenal metabolisrn WU confound interpretation on the secretion of substrate in the kidney, since metabolism wili compete for drug which would othenvise be eliminated unchanged in the urine. The situation is akin to two enzymes which compete for the sarne substrate in an eliminating organ. Moms and Pang ( 1987). explored the effect that two competing enzymes would have on substrate removal in the liver with a simulation study and found that presence of a competing metabolic pathway would alter the amount of substrate seen by an altemate metabolic pathway. In ths way, metabolite formation by one enzyme system is "apparently" influenced by the rnetabolic activity of the other enzyme system. The same would appear to be tnie for excretion and metabolisrn within the kidney. In cases where a drug is both metabolized and excreted unchanged by the kidney, there will be cornpetition between these two routes of elirnination. Consequentiy, the assessrnent of excretory and rnetabolic clearance estimates in the presence of the competing pathway wiiI not be the same in the absence of the alternate eliminatory pathway. Therefore, the traditional means by which the net secretory ability of the kidney is assessed is prone to error. In order to avoid this pitfall, the total renal clearance of the substrate normaiized to the filtration clearance, and not the urinary clearance normalized to the 14 filtration clearance, rnight be a preferable means of determinhg whether a cornpound undergoes net secretion, reabsorption or filtration (Smith and Kugler, 1994).
1.5 Enalapril: An Ideal Dmg for Examining Clearance Concepts in the Kidney Enalapril, an angiotensin converting enzyme (ACE) inhibitor used in the treatment of hypertension and heart failure, is an appropriate substrate for investigation of rend clearance concepts, since it is both excreted unchanged and metabolized by the kidney. Enalapnlat. once formed, is also excreted (Fig. 1-2). It has been shown that ACE inhibiton are capable of reducing blood pressure in laboratory animals (Laffon et al., 1978) as well as in man (Gavras et al.. 1978) and have proven effective in the treatment of hypertension of various origins (Vidt et al.. 1982). The pharmacological activity of enalapril is dependent upon metabolic transformation to its active metabolite, enaiaprilat. Enaiapriiat reduces hypertension by inhibiting the renin-angiotensin- aldosterone system. which regulates artenal blood pressure. Bnefly. ACE inhibition decreases blood pressure. by blocking the conversion of Angiotensin 1. a mild vasoconstrictor. to Angiotensin II, a potent vasoconstrictor and stimulator of aldosterone secretion. Aldosterone markedly increases sodium reabsorption by the kidney. thereby increasing water retention and elevating blood pressure. (Guyton. 1992).
Excretionl Excretion Figure 1-2. Rend disposition of enalapnl. Since enaiapril has been weli studied in the isolated perfused rat kidney (de Lannoy et al..
1989: de Lannoy and Pang. 1993), a set of physiological parameters which describes transmembrane and metabolic clearances of enalapril in the isolated pemised rat kidney exists in the
Literature. Likewise, a set of parameters describing the disposition of enalaprilat in the kidney is also available from multiple indicator dilution studies (Schwab et al.. 1992). With these parameters.
it is possible to build a physiologicai mode1 to descnbe the handling of enalapril by the kidney. This would allow for examination of the effect of one clearance route on the clearance estimate of
the alternate cornpeting route via computer simulation of renal clearances. Enalapril is ideal for use as a mode1 compound in examining clearance concepts within the kidney experimentaily. inhibition of endapril metabolism within the isolated perfused rat kidney (IPK)would provide a means of examining the relationship between metablic and excreto- clearance estimates. If renal metabolism and excretion are competing elimination pathways. the absence of metabolism should result in increased excretoq clearance. Since enalapril is highly
metabolized within the kidney by a carboxylesterase enzyme (de Lannoy et al., 1989). the possibility exists for manipulation of the renal metabolic capacity for endapd by compounds known to inhibit esterase activity.
1.5.1 Renal Clearance of Enalapril in the Isolated Perfused Rat Kidney (IPK) In the IPK. enalapril is. for the most part, hydrolyzed to enalaprilat. Therefore. the renal clearance of enalapril is mainly metabolic. Since excretion of unchanged enalapril into urine is low
(FE value less than one). net reabsorption of enalapril has been suggested to exist (de Lannoy et al.. 1989). However, in Light of the hypothesis that metabolism will result in a deflated FE value, it has been suggested that enalapril may in fact undergo net tubular secretion by the kidney (Smith and Kugler, 1994).
1.5.2 Renal Transport of Enalapril and Enalaprilat Many ACE inhibiton are cleared renally via glomerular filtration and tubular secretion
(Kostis et al., 1987; Mujais et al., 1992; Olson et al.. 1989) and it seems likely that enalapril and enalaprilat couid be transponed by the organic anion transport system. There exists evidence to 16 support this claim. In humans. Noormohamed et al. (1990) demonstrated an increase in peak serum concentration of both enalapril and enalaprilat in the presence of probenecid as compared to the absence of probenecid. This resdted in a substantial increase in the area under the curve for both enalapril and enalaprilat, and a concomitant decrease in the rend excretion of both compounds.
Sirnilarly. an in vivo rat study by Lin et al. (1988) suggested that enalapnlat is uansported via an organic anion transport system since addition of probenecid and PAH decreased the FE value for enalaprilat. Enalaprii was also shown to inhibit contraluminal PAH influx (Ullrich et al.. 1989). suggesting it is transported via the contraiumllial PAH transporter. On the contrary. studies with compounds capable of inhibiting the organic cation transporter system have shown that ACE inhibitors are not likely substrates for this system (Todd and Goa, 1992; Vertes and Haynie. 1993). ui more recent work. utilizing HeLa cells transfected with the organic anion transport protein (oatp)
Pang et al. (1997) suggest that enalapnl is transported by this rend bmsh border anionic transport protein. Since enalapd and enalaprilat, in addition to king organic anions. are dipeptides. they may also be reabsorbed by the di- and tripeptide carrier at the brush border membrane (Silbemagl et al.. 1987: Hannelore and Herget. 1997).
1.5.3 Renal Metabolism of Enalapril
Esterases are divided into 3 categones, the "A", "B". and "CW-esterases. The " A" - esterases. or arylesterases, contain a sulphydryl group at their active cenue and cm be inhibited by mercuric chloride (Augustinsson. 1970). They are known to hydrolyze organophosphate pesticides, such as paraoxon and thus detoxifj them. without king inhibited by them. The " B " - esterases. or aliesterases. include cholinesterase and carboxylesterase. Cholinesterase and carboxylesterase are sensitive to inhibition by organophosphates, whde other aliesterases are unaffected by organophosphate cornpounds (Augustinsson, 196 1; Aldrich. 1953). Cholinesterase. and certain other diesterases, cm also be inhibited by physostigmine (or eserine) (Augustinsson,
196 1). The "C"-esterascs. or acetylesterases. are resistant to organophospate inhibition. yet can be activated by organic mercurials such as phenylmercuric acetate (Aldrich, 1953). Enalapril is a dipeptide, carboxylester compound that is metabolized in the kidney to its dicarboxylic acid metabolite enalaprilat (de Lannoy et al., 1989) (Fig. 1-2). The metabolism of 17 enalapril involves only esterolysis; there is no evidence for hydrolysis of the peptide bond in the kidney (de Lannoy et al.. 1989; de Lannoy and Pang. 1993). Owing to the carboxylester structure of enalapril, rend carboxylesterase(s) is Likely responsible for the hydrolysis of enaiapnl to enalaprilat within the kidney. Although carboxylesterase activity has thus far only ken identified intraceiiularly in the kidney (in the microsomal fraction) the possibility that luminal metabolism of enalapril occurs with ecto-enzymes embedded in the brush border membrane has ken alluded to by Smith and Kugler (1994). The equations used by Kruger and Smith in a simulation study. examining the influence of renal metabolism on FE denote that metabolism occurs subsequent to filtration and secretion. implying that it occurs within the renal tubule.
1.5.4 Paraoxon: A CarboxyIesterase Inhibitor
Paraoxon is the active metabolite of the organophospate pesticide, parathion. and is an esterase inhibitor. Esterases which are subject to the inhibitory capability of paraoxon are those from the "B"-esterase family which includes. cholinesterase and carboxylesterase. Many researchers have utilized paraoxon to irrevenibly inhibit esterase activity (Butterworth et al.. 1993:
Grima et al.. 199 1: Castle. 1988: Brandt er al.. 1980). It has also been used to inhibit the hydrolysis of enalapril in kidney tissue (Grima et al., 199 I ). Detoxification of paraoxon is by hydrolysis to produce diethylphosphoric acid and p- nitrophenol. This reaction is cax-ried out by "Aw-esterases and aliesterases. both of which have ken identified to exist in the liver (Pond et al., 1995). plasma (McCracken et al.. 1993: Pond et al..
1995) and kidney (Pond er ai., 1995; Pla and Johnson. 1989). However, following exposure to paraoxon. detoxification occurs mainly in the liver and plasma in both humans and rats (McCracken et ai., 1993).
1.6 Approaches for Study The influence of competing ehnation pathways on rend clearance estimates can be exarnined with the use of computer simulation studies and experimentation in the isolated pehsed rat kidney (IPK). A physiological mode1 describing the disposition of enalapril in the kidney is available. since trammembrane and metabolic clearance parameters for enalapril exist in the 18 iiterature (de Lannoy et al.. 1989; de Lannoy and Pang, 1993). Equations for total. renal and metaboiic clearances in the kidney cm thus be derived and perturbation of the parameters will reveal the sensitivity of clearance estimates to a particula.parameter. In this way. the effect of reducing the metabolic intrinsic clearance or the excretory clearance on the clearance estimate of the altemate pathway can be ascenained. The IPK is ideal for exarnining the relationship between renal metabolism and excretion since processes occurring within the kidney can be exarnined without influence from other organs. With paraoxon. an inhibitor of enalapril esterolysis (Grima et al..
1991), the effect of renal metabolism on the urinary ciearance estimate for enalapril cm be demonstrated experimentally. Chapter Two: Statement of Purpose of Investigation 2.1 Purpose The purpose of the investigation was to examine the relationship between metabolic and excretory clearances within the kidney. As discussed in the previous chapter. metaboiism and excretion occuning within the kidney will compete for the removal of substrate. Accordingly. metabolic or uinary clearance values eshated in the presence of altemate pathways wili result in inaccuracies. This phenornena is particularly relevant to examination of the net secretory ability of the kidney . When the secretory capacity of the kidney is based on the fiactional excretion value (unbound urinary clearance normalized to GFR). the presence of metabolism in the kidney cculd lead to an erroneous interpretation of net reabsorption. while the compound may actuaUy be net secreted. Examination of the relationship between metabolic and excretory clearances within the kidney was accomplished by theoretical examination and computer simulations based on a physiological model of the kdney. and with experimentation in the isolated perfùsed nt kidney.
Enalapril was chosen as a model subsuate to demonstrate the influence of excretion and metabolism on one another. Since enalapril is both metabolized and excreted unchanged in the kidney. the effect that each eliminatory pathway exerts on clearance estirnates was assessed. Lastly. since enahprit is metabolized via ester hydrolysis. paraoxon, an inhibitor for the hydrolysis of enalapril was used to investigate the influence of rend metabolism on excretory clearance estimates.
2.2 Specific Objectives The first objective was to examine the influence of rnetabolism on estimates of excretory clearance, and of excretion on metabolic clearance estimates for enalapril. A physiological model of the kidney, incorporating parameten which describe the fate of enalapril widiin the kidney. was employed to simulate the effects of metabolism and excretion on clearance estimates. Two sets of parameters: one which incorporated intracelluiar metabolism of enalapril and the other whch incorporated intraluminal metabolism of enalapril, were used to simulate the rend disposition of enalapril. Metabolic intrinsic clearance values as well as secretory and reabsorptive clearance values were varied in order to view their influence on clearance estimates. The second objective was to assess the site of inh-arenal rnetabolism of enaiapril withui the kidney. Data obtained for enalapril in the isolated perfused nonfiltering rat kidney (NFK) were compared to data previously obtained for enalapril in the isolated perfused rat kidney (IPK) to distinguish between intracellular and innaluminal rnetabolism for enalapril (de Lannoy et al.. 1989: de Lannoy and Pang, 1993). The experimental data were correlated to computer simulated data based either on intraceliular or intraluminai metabolisrn. The Fial objective was to address whether inhibition of enaiapril metabolisrn within the kidney would result in an increased excretory clearance for enalapril. Initiaily, paraoxon was assessed for its ability to inhibit the hydrolysis of enaiapril to enalapnlat in the S9 fraction of kidney tissue homogenate. A concentration of paraoxon that was high enough to inhibit enalapd esterolysis yet Iow enough so as not to compromise viability of the kidney. was chosen for use in the isolated perfûsed rat kidney preparation to examine the effect of inhibiting enalapril metabolism on excretory clearance.
2.3 Specific Aims
1. To illustrate. via computer simulation. that metabolism and excretion occumng within the kidney will compete for subsuate removal. thus altering the clearance estimate of each other. il. To determine whether enalapril esterolysis within the kidney occurs intracellularly or intnluminally. by cornpuhg computer simulated data based on the intracellular and intraluminal models of enalapril metabolism with experimental data obtained in the NFK and IPK.
nI. To test the hypothesis that inhibition of intracellular enalapril metabolism by the kidney increases the estimate of excretory clearance in the isolated perfused rat kidney preparation. Chapter 3: Organ Clearance Concepts: New Perspectives on Old Principles
Gina L. Sirianni and K. Sandy Pang
In Press: Journal of Phunnacokinetics and Biophamaceutics
Volume 254 ( 1997)
Gina L. Sirianni performed the simulation work and Dr. K. Sandy Pang developed the equations used for simulation. 3.1 Abstract The removal capacity of an eliminating organ by metabolism andor excretion is often expressed as its clearance. The metabolic and excretory clearances are considered to be munially independent, and the sum of these constitute the whole organ clearance. The influence of metabolism on estimates of the excretory clearance and vice versa was examimi for the kidney with a physiologically-based model. Mass transfer f~st-orderrate equations describing transport and removal were derived. Upon inversion of the matrices origùiating from the coefficients of these equations, the area under the curve (AUC) and clearance (dose/AUC) were obtained. A complex solution was found to exist for the kidney since glornerular fütration. reabsorption and metabolisrn were present. In order to ascertain the effect of excretion on estimates of the metabolic clearance as well as the effect of rnetabolism on estimates of the excretory clearance, intrinsic clearances for excretion or metabolism were set to zero. Clearance values were found to be altered when altemate pathways were present. Whereas excretory clearance estirnates were consistently reduced in the presence of metabolism. metabolic clearance estimates were affected differentially by excretion and varied according to the site of metabolism. Excretion reduced metabolic clearance estimates when metabolism occumd intracellularly. If metabolism occurred intraluminally (e-g. on the rend bmsh border or lurninal membrane), the metabolic clearance estirnate could become higher since the substrate was available to the enzymes following its excretion. As expected, these changes depend on the relative magnitudes of the intrinsic clearances for metabolism and excretion. The above theory was applied to the elimination of enalapril which is both metabolized and excreted by the perfused rat kidney preparation. The data obtained in these studies were consistent with a set of published physiological parametee denoting transfer and intrinsic clearances.
Perturbations on clearance estimates were studied by setting the metabolic/excretory intrinsic clearance to zero, then to some finite value. The fractional excretion (FE or unbound excretory clearance/glomerular fütration rate) was decreased modestly (from 0.64 to 0.44) with intracellular esterolysis. whereas if metabolism had occurred intraluminally, FE would have been significantly decreased (from 1.8 to 0.45). These simulation results show clearly that clearance estimates are affected by the presence of altemate removai pathways, and question the well established principle that metabolic and excretory clearance estirnates are independent of each other.
3.2 Introduction
Clearance concepts, developed initially to descnbe the functionai efficiency of the kidney (Gréhant, 1904) in the rernoval of urea, have since been extended to describe the handling of xenobiotics by the Liver (Lewis. 1948; Wilkinson. 1987) and other eliminating organs. It is univenally accepted that organ clearance is the sum of the metabolic and excretory clearances. parallel cornponents which are devoid of influence on each other. However, the lack of interaction among competing pathways has ken questioned. In iiver. the sirnultaneous presence of high affinjty-high capacity metabolic pathways was shown to reduce substrate metabolism by altemate pathways (Pang et al., 1983: Koster et ai., 1982; Moms and Pang. 1987). In kidney, the presence of metabolism would decrease the extent to which substrates are excreted unchanged (Smith and
Kugler, 1994; Smith et al., 1995; Kluger et al., 1995). Estimation of metabolic and excretory clearances in the presence of altemate eliminatory pathways within elimation organs wiil thus yield inaccuracies. This would particularly affect interpretation of the secretory ability of the kidney, a major excretory organ, since the estimate of fractionai excretion (FE. or unbound urinary clearance nonnalized to glomenilar filtration rate GFR) is often used to infer a net excretory process: FE > 1 for net secretion: FE c 1 for net reabsorption; FE = 1 for net filtration (Brenner et al., 1986b).
In this snidy. a physiological mode1 was developed to study the influence of the metabolic intrinsic clearance on excretory clearance estimates and of the excretory intrinsic clearance on metabolic clearance estirnates, within the kidney. We utiiized the physiological modeling approach since this allows for inclusion of transport barriers (Bishoff and Dedrick. 1968: Lutz et al., 1980: de
Lannoy et al., 1990) that segregate tissue from perfusing blood. The method employs physiological flow rates and volumes and describes dmg rernoval at the designated sites. Identical clearance estimates are obtained between the physiological and cornpartmental approaches, and sirnilarities can be drawn with regard to the intercornpartmentai and elimination rate constants and volume tem(Rowland, 1972: Rowland et al., 1973). The advantage of the physiological approach over cornpartmental modeling lies in the improved understanding of physiological events occurring within the organ. Such physiological models have ken developed for the description of drug and metabolite clearances in the kidney and liver (Hekman and van Ginneken. 1982; de Lannoy and
Pang, 1987: de Lannoy et al., 1993: de Lannoy and Pang, 1993). Enalapril was used for illusiration purposes since the compound is both metabolized and excreted unchanged in the isoiated pemised rat kidney preparation (IPK) (de Lannoy et al.. 1989).
3.3 Theoretical
The present kidney model includes subdivision of the organ into vascular and cellular compartments, and an additional cornpartment that represents the urine (Fig. 3- 1 ). The model considen a dmg which distributes only in plasma; there is no red blood ce11 binding. Drug binding to plasma or kidney proteins. denoted by the unbound fractions. fp or f~,respectively. are constant for ail input conditions. The model incorporates fist-order transport and dimination. In this model. the kidney is the only removal organ for metabolism and excretion. In addition. there are two possible sites for metabolisrn: one is within the ceii and the other, on the lurninal or bmsh border membrane. Although the model incorporates two sites of metabolism. simulations were performed by assuming that either intracellular or intraiumùial metabolism occurs. not both. This was accomplished by setting the altemate metabolic pathway as absent in the simulations. The kidney is subdivided into the vascular, tissue. and tubular lumen (or urine) compartments. Dmg is fxst fiitered by the glomerulus More reaching the peritubular cells via the postglomerular circulation, given by the difference in rend plasma flow rate (QK)and the glomemlar filtration rate
(GFR). The mass balance rate equations that descnbe drug removal from the body (reservoir). and from the rend plasma. tissue, and urine compartments are as follows: For rate of change in reservoir Reservoir
Renal Plasma
Renal Tissue I
CL in,u 1I Urine
Figure 3-1. Physiological mode1 for the rend elimination of a substrate that is both metabolized and excreted by the kidney, the only eliminating organ. The kidney is divided into three compartments: plasma, tissue and urine. The outflow plasma recirculates to the body (reservoir). The shaded areas
(reservoir and urine compartments) represent sampling compartments. Drug entering the kidney is first filtered, and only the postglornerular circulation reaches the rend tubulctr cells. Exchange of drug between renal plasma and tissue is characterized by influx (CL:) and efflux CL:^) clearances across the renal basolateral (b) membrane, and the exchange between urine and tissue is characterized by CL:, and CL> at the luminal (0 membrane. Dmg within tissue is metabolized with a renal metabolic intrinsic clearance, CLinr,K whereas drug within the renal tubule is metabolized with a renal luminal metabolic intrinsic clearance, CLint,u. Setting CLint,u = O provides a model of intracellular metabolism while setting CLinr,K = O provides a model of intraluminal metabolism. Urinary excretion of drug is a function of unne flow (Qu)and the net transfer clearances at both basolateral and luminal membranes. For rate of change in renal plasma
For rate of change in renal tissue
For rate of change in urine
where AR, APK,AK and A,, are the arnounts of substnte in the reservoir. rend plasma. renal tissue. and urine. respectively. at any tirne t; VR, V~KVK, and V, are the correspondhg volumes. CL:, and CL$ are the influx and efflux clearances at the rend basolateral membrane (b), respectively. and CL:^ are the influx and efflux clearances at the lurninal membrane (0. respectively: CLint K is the ceIiular metabolic intrinsic clearance and CL,,,, the intraluminal metabolic intrinsic clearance of the kidney. Q,, is the urine flow rate.
3.4 Methods The coefficients in the mass balance rate equations for the kidney were presented as elements in a four by four square matrices. Inversion of the rnauix was performed with the program ~heoristRon a Macintosh computer (PowerMac 9500). This yielded solutions for the area under the amount-time cuve per unit dose for iv (intravenous) administration of drug: multiplication of this to dose and division by the volume furnished the area under the cwe. AUC. The total amount excreted into urine, A:. was @en by the product of urine flow rate (Q,,) and the area under the urine concentration-time cuve, AUC,. The total organ clearance was estimated by the model-independent relationship, doselAUC~,where AUCR is the area under the cuve in the reservoir. the excretory (or urinary) clearance was given as Ar/AUCR. Metabolic clearance was estirnated as the difference between the total organ clearance and the excretory clearance. The 28 hctional excretion (FE) was estimated as the urinary clearance divided by the fütration clearance. CL, /(fpGFR); the urinary clearance was assessed by the product of the unchanged drug concentration in urine and the urinary flow rate. or the excretion rate (arnount excreted per unit time) divided by the circulating plasma concentration at the midpoint time. In kidney. enzymes were assigneci intracellularly or intralumùiaiiy (dong the renal luminal or bnish border membrane). Thus, wo sets of parametee were used for simulation. Parameters
CL^, = CL> = 25 ml/min/g; CL in,x = 4.77 d/min/g; CL^ = 0.245 ml/rnin/g: CL$ =O ml/min/g) associated with intracellular metabolism were obtained from single pass and recirculating rat kidney perfusion with enalapril (de Lannoy et al.. 1990: de Lannoy and Pang, 1993; de Lannoy et al.. 1989). Upon substitution of these parametes into the appropnate clearance equations for the kidney, it was found that the pararneters slightly under-predicted the total renal and metabolic clearances and FE values in comparison to those observed. The pararneters were subsequently modified to be more consistent with the experimental data. The optirnized panmeters used for simulation (CLhLK= 6 mVdg; cLfn= 0.85 ml/min/g: CL^^ = 1.96 rnl/min/g) are summarized (see Table 3-1). In view of the fact that the luminal membrane is an aitemate metabolism site. the experimental data on enalapril removal in the single pas and recirculating rat kidney preparations were pararnetenzed for luminal rnetabolism. The trammembrane clearances across the basolateral membrane were kept constant (CL!'= CL^^ = 25 mümidg), and a secretory clearance (CL&) of
8.45 mVmin/g, a reabsorption clearance (CL;) of 0.98 rnl/~~m/g,and an intraluminal metabolic intrinsic clearance (CL,,.,) of 1.95 ml/mm/g were found to predict the observed perfusion data equaily weil. The occurrence of both intraceliular and iniraluminai metabolism was. however. not explored. We varied the metabolic intrinsic clearance (increasing from zero) to ascertain its effect on urinary clearance estimates. Anaiogously. we altered the excretory intrinsic clearance (increasing from zero) to view its influence on renal metabolic clearance estimates. Simulations were performed to demonstrate the influence of rneîabolisrn/excretion on the excretay/metabolic clearance estimates, and the changes in the renal metabolic and urinary clearances with changes in
(a) basolateral inflwdefflux clearances, (b) site of rnetabolism (intracellular vs. intraluminal). and (c) reabsorption clearance at the luminal (or brush border) membrane. Table 3-1. input data used for simulation of kidney as the only elirninating organ htraceiidar Model Intraluminal Model Initial or Assigned Initial or Assigned Description Syrnbol Value Value
Cornpartment volumes (dg) Reservoir plasma Rend plasma Rend tissue Renal urine
Flow rate (mVmin/g)
Rend plasma QK 7.1 ld 7.1 ld Urine QU O. 15' O. 15' Glomedar filtration rate GFR 0.6' 0. O' Clearances for enalapril in kidney (mVmin/g)
Rend metabolic intrinsic L ~'~~int.1, 6' Basolateral membrane CL^^ = CL$ 25.F Lurninal influx (reabsorption) CL;,, 0.85' Luminal efflux (secretion) CL,j 1.9€if
Fractions unbound for enaiapril Plasma Kidney
" Obtained experimentally from recirculating IPK (de Lannoy and Pang, 1993). Obtained from Schwab et al. (1992). ' Obtained from Spector ( 1956). d Obtained expenmentally from combined perfusion of the iiver and kidney (de Lannoy and Pang. 10a2\ 'Assigned value. close to values obtained with IPK. ' Obtained by trial and error via simulation. Obtained from fitting of single-pass IPK data (de Lannoy et al.. 1990). Obtained from de Lannoy et al. (1989). Assumed by de Lannoy et al. ( 1990). 3.5 Results 3.5.1 Kidney as the only eüminating organ
The solutions for the AUC of the reservoir and urine compartments were found to be cornplex due to the presence of GFR. luminal reabsorption CL^^ > 0) and metabolism. The solutions for total renal, and urinaq clearances are shown in Table 3-2. Rend metabolic clearance was calculated by subtracting the uinary clearance from the total rend clearance. and fractional excretion was calculated by dividing the unbound urinary clearance by GFR. The areas for the reservoir (AUCR) and urine (AUC,) and the total and urinary clearances were not afTected by tissue binding (f~absent in equations). but were influenced by the trammembrane clearances at both the basolateral and luminal membranes, the rend rnetabolic intrinsic clearances (intracellular or in~aluminal),the urinary and renal flow rates. GFR. and the plasma unbound fraction (Table 3-2). Again, the solutions for the urinary clearance (CL,,,K) and fractional excretion (FE) encompassed the renai metabolic intrinsic clearance tenns. CLinrK and CL,,,,,.
3.5.2 Simulations on renal disposition of enalapril When the parameters in Table 3- 1 were substituted into the general equations shown in Table 3-2 for the kidney, the predicted rend extraction ratio (EK)and the rend metabolic (CLttleL~) and urinary (CL,,K) clearances (also expressed as FE) were remarkably similar to those observed experimentally for enalapril in the IPK (Table 3-3). Values of FE were found to be highly dependent on the eMux and reabsorption clearances at the luminal membrane. and were readily affected by the presence of rend metabolism. Simulation of the total mal, urinary and metabolic clearances was performed for either intracellular or in~aluminalmetabolism, based on the optimized sets of CL:^, CL:,. and CL,,K and
CLinr,,, for intraceliular and intraiuminal metaboiism (Table 3- 1 ). Simulation with the optirnized parameters that descnbed cellular metabolism of enalapril (CL:, = CL$ = 25 ml/min/g) provided the control value for FE (0.64) when renal metabolism was absent (simulation conditions: CLinrK
Table 3-3. Parameten predicted for enalapril disposition in kidney. based on the physiological constants summarized in Table 3- 1.
Observed Data ' Predicted Predicted for for Intracellular Intraluminal Metabolismb N1etabolismb
r - -- -- L Kidney Extraction Ratio: E, 0.25 to 0.29 0.295 O. 295 Total Clearance: CLtOt,~(ml/min/g) 2.1 2.10 2.10 1.95 Me tabolic Clearance: CLmet,K (drnidg) 1.94 to 1.96 1.95 Urinary Clearance: CL,K (mYmin/g) 0.09 to 0.15 O. 145 0.15 3 Fractional Excretion: FE 0.45 to 0.48 0.44 0.45 I I I " Data of de Lannoy and Pang ( 1993) Based on parameten in Table 3- 1
= O; CL$ = 1.96 ml/rnin/g, and CL:,, = 0.85 rnl/min/g). With intracelluiar metabolism in the kidney
(CLin,~= 6 mVmin/g). the FE estimate (0.44) was 31% lower, and decreased funher with increasing CLhL~(Fig. 3-2A). Whereas with metabotism occurring intraluminaily, FE was 1.84 in absence of metabolism (simulation conditions: CLinLu= O: CL:^ = 8.45 mVmin/g and CL!., =
0.98 ml/min/g). and the value was lowered markedly (75%) to 0.45 in the presence of metabolism (CLinLu= 1.95 mVmidg) (Fig. 3-2B). The intraceiiular enzymes act on drug which has entered
rend tubular cells from the postglomemlar circulation. while intralumind enzymes act on drug which is both fütered and secreted. For this reason, changes in FE are greater for intraluminal metabolism. These large changes in FE with luminal metabolism (FE from greater than 1 to less
than 1) wu affect interpretation of the net secretion mechanism. since FE < 1 is normally
construed as net reabsorption whereas an FE value of >l implies net secretion. With increasing reabsorption clearance, the dtimate excretion, monitored as FE was reduced for both instances where metabolism had occurred (Fig. 3-2A and 3-2B). The influence of the luminal emux clearance (CL$) on estimates of the rend metabolic
clearance (ametK)was &O examined. With the relatively high rend metabolic intrinsic clearance
33 for cellular rnetabolism (CLinr,~= 6 rni/min/g). CLmer,~was dfected only rninimdy (from 2 to 1.96 ml/min/g) when CL:^ varied from O and 1-96ml/rnin/g, a 2% decrease (Fig. 3-2C). The sarne insensitivity of the metabolic and total renal clearance to CL:, was also found by de Lannoy er al.
(1989). By contrast, the lurninal efflux clearance was found to significmtly alter the renal intraluminal metabolic clearance estirnate (CLinL,= 1-95 ml/mui/g). The renal metabolic clearance (CL,& increased from 0.2 1 to 1.95 mllmidg when luminal efflux clearance (CL>)varied from
O to 8.45 ml/min/g (Fig. 3-2D). It appears that avid efflux of dmg from ce11 to lumen promotes the rend metabolism by bringing molecules to the intrduminal enzymatic site.
The possibility of other parameten which could describe the enalapril data equaily well was explored by setting the CL$ and CL:' = 5 or 0.5 ml/min/g. We were able to identi@ sets of parameters for both the cellular and lurninal model that yielded close predictions for the experimentally observed data with CL:^ and CL:, = 5 ml/min/g but not 0.5 rnl/min/g. The parameters associated with the models at CL; = CL: = 5 ml/min/g were: for the cellular model. CL^^ = 0.2 rnlhnin/g. CL:, = 0.2 dniinlg. and CLinL~= 46 ml/rnin/g: for the luminal model. CL:. = 50 rnl/rnin/g, CL^ = 0.5 ml/rnin/g, and CL,,,, = 2 rnl/min/g. Simulations performed with these parameten illustrateci the same trends as those observed for the initiai set of parameters shown in Table 3- 1 with CL: = CL:, = 25 rnl/min/g. With the new transmembrane clearances (CL; = CL:,
= 5 ml/rnin/g), the FE estimate was decreased modestiy from 0.58 to 0.44 in the presence of cellular metabolism. However, with intraluminal metabolism. FE was greatly reduced (from 5.26 to 0.34). A comrnon trend evolves: there is reduction in FE with either intraceMar or intraluminal metabolism, and this occurs regardless of the rate of transport at the basolaterai membrane (i.e. 25 or 5 mVmin/g). For the cellular model. the set of parameten associated with CL:,= CL$ = 5 ml/min/g predicted a minimai decrease in the metabolic clearance estimate from 1.96 to 1.95 when dmg efflux across the luminai membrane was present. With the luminal model, CLmeLK was increased frorn 0.25 mVmin/g to 1.94 ml/mui/g when dmg efflux occurs across the luminal membrane. 3.6 Discussion The equations that interrelate the rend clearance to its physiological determinants (Table 3-
2) appear to be new formulations. Although metaboiism and excretion are considered as discrete processes which are independent of each other, the solutions for metabolic clearances include the excretory Uitrinsic clearance and relations for excretory clearances encompass the metabolic intrinsic clearance. When the derived solutions were used for simulations with the assigned parameters. the
results showed that metabolism, occurring either intraceliularly or intraluminally. wiu contribute to an underestimation of the excretot-y clearance and a decreased FE value (Figs. 3-2A & 3-28). On
the other hand. the presence of excretion will underscore the observed metabolic clearance in the
presence of cellular metaboiism (Figs. 3-2C & 3-2D). while uncharactenstically augmenting the metaboiic clearance estimate when metabolism occurs dong the luminal or brush border membrane. The influence of renal excretion on rnetabolic clearance estimates could be surnmarized. The result
is site-specific and the location of metabolism detemiines whether excretion/secretion results in an increase or decrease in the estimate. The magnitude of change in clearance is hrther dependent on the influx/efflux clearances across the basolateral membrane as well as on the relative magnitudes of the metabolic and excretoiy intrinsic clearances. The metabolic and excretory clearances of the kidney are further dependent on the value of the reabsorptive clearance at the luminal membrane
since reabsorption recycles the hgback to the tissue. The situation with enalapril. in ternis of kidney metabolism and excretion is not
straightforward. Net reabsorption of the ACE inhibitor has ken suggested for the perfused rat kidney preparation (de Lannoy et al., 1990; de Lannoy and Pang, 1993), but the excretory clearance
of enalapnl was found to exceed inulin clearance in man (Noormohamed et al., 1990). That intrarenal metabolism would affect analysis of enalapril transport in the kidney has previously ken discussed (Smith and Kugler, 1994: Smith er al., 1995; Kugler et al., 1995). It has ken suggested by Smith and Kluger that FE ought to be replaced by the excretion ratio (ER), or unbound totlil renal clearance normalized by GFR, in order to determine the true secretory abiiity of the kidney for dmgs which are renally metabolized (Smith and Kugler. 1994). A value of the ER (- 7 to 8) was thus calculated for enalapril. and this would suggest net secretion of the dmg. Moreover. the 36 authors had presented a model in which glomenilar fdtration and secretion were followed by metabolkm then in nim. reabsorption (Smith and Kugler. 1994). With the assumption that the discrete processes followed one another in sequential order, the solutions were thus simplified. A linear correlation was found to exist between FEER and the extent of metabolism/reabsorption. These assumptions on sequential processing, however, appear to be closely associated with intraiurninal metabolism because metabolism occurred subsequent to filtration and secretion. the latter of which is construed as the net movement of dmg from blood to lumen. Despite the difference in modeling approach. trends similar to those identified by us could be found: the presence of metabolisrn decreases estimates of the excretory clearance. since the altemate pathway effectively cornpetes for the available dmg (Smith and Kugler, 1994). According to our physiologically based model, processes of transfer. metabolism. secretion and reabsorption occumng within the kidney are dynamic and concurrent. From the solved equations (Table 3-2), we were able to examine the influence of transmembrane clearances at the basolaterd and brush border membranes, and intracellular vs. intraluminal metabolism. With these relationships. we further demonstrated that the FE value (unbound urinary clearance/GFR) in absence of metaboiism (CL,,, = 0) was consistently less than unity when esterolysis occurred within the cell, and the value was indeed decreased in the presence of metabolism. Thus. the change would not have altered the interpretation of net reabsorption of enalapril if esterolysis had occurred within the rend tubula. cells. These trends persisted over a large range of transmembrane clearance (frorn 25 to 5 mllrninhg). If esterolysis of enalapril had occurred intraluminally. FE values, ordinarily exceeding unity in absence of luminal metabolism. could become substantiaily reduced. and metabolism indeed would obscure Our abiiity to identiQ net excretion of enalapnl by the kidney. Clarification on the issue of secretion of enalapril might not have been achieved with use of cornpetitive anionic inhibitors such as p-aminohippurate or probenecid (Kugler et al., 1996) since these may confound transport processes at multiple sites. Recent studies in the non-filtering isolated perfused rat kidney with enalapril provide the disceming data that enalapril esterolysis does occur intracellularly in the perfused rat kidney preparation (Sirianni and Pang, 1997). In the current simulation snidy, we observed different trends, depending upon whether cellular or luminal metabolism of enalapril had occurred. The simulations showed that the presence of renal excretion may increase or decrease the estimate of the renal metabolic clearance, depending upon the site of rend metabolism. In the presence of iniracellular metaboiism, excretion reduced metabolic clearance, and the effect was dependent on the relative ratio of the metabolic intrinsic clearance to the luminal efflux clearance. Ln conmt, excretion tended to increase the estimate of renal metabolic clearance with the presence of luminal metabolism within the eliminating organ. since secretion would bring dmg molecules to interact with the enzymes embedded on the brush border membrane. The pnnciples described for renal intraluminal metabolism could also be extended to the intestine and liver, organs in which enzymes are found lurninally. In the intestine. a drug cm be secreted across the brush border membrane into the intestinal lumen via transport proteins (Saitoh et al., 1996) or P-glycoprotein (Hsing et al.. 1992), and is subject to metabolisrn. The enzymes. y glutarnyltransferase (Inoue et al., 1983; Szewcnik et al., 1980; Meier et al.. 1984) and P glucuronidase (Ho et al., 1986; Ho et al.. 1979) are found within the biliary tree. In the rat and guinea pig liver. it has been demonstrated that )~glutarnyltransferaseis present not only on the epithelial cells of the bile ducc but on the bile canalicular and sinusoidal sides of the hepatocyte
(Lança and Israel, 199 1). ~Glucuronidaseof both bacterial (Maki, 1966) and endogenous (Inoue et al., 1983; Ho et al.. 1979) origins are also present in animal and human bile. In analogy to the decrease in FE observed with renal metabolism, the inü-abiliq hydrolysis of glutathone (Bailaton et ai.. 1986) or glucuronide conjugates (Ho et al., 1986) within the biliary tree would mask the tme rates of excretion.
The principle that metabolism and excretion art devoid of influence on each other could thus be challenged. Since the pathways compete for dmg removal within the organ. they will affect clearance estimates of alternate pathways. The phenomenon is akin to that observed with multiple (parallel) metabolic pathways occuning within an eliminating organ, in that metabolism will regulate the intracellulx substrate concentration and therefore detract from metabolism by altemate pathways
38 (Morris and Pang, 1987). The extents of change, however, strongly depend on the relative values of the inauisic clearances and other prevaiiing feanires of the organ. The definition of organ metabolic or excretory clearance in the past has not Myaddressed the influence exened by competing pathways. and. therefore. the estimates of clearance are probably not as accurate.
3.7 Staternent of Significance of Chapter 3
This chapter clearly demonstrates that excretion and metabolism. when occumng within the same eliminating organ, wdi influence the clearance estirnate of the aiternate eiimination pathway.
Metabolism wili decrease the excretory clearance estimate. Excretion wiU either increase or decrease the metabolic clearance estunate. depending on the location of dmg metabolizing enzymes within the kidney. In the presence of intracellula. metabolism. excretion wili decrease the metabolic clearance estimate. In the presence of intraiuminal metabolism. excretion wili increase the rnetabolic clearance estimate. Chapter 4: Intracellular and Not Intraluminal Esterolysis of Enalapril in
Kidney : Studies With the Single Pass Perfused Nonfiltering Rat Kidney
Gina L. Sirianni and K. Sandy Pang
As Submitted to: Driig Metabolism and Disposition
Gina L. Sirianni performed all experimental work and the simulation study. 4.1 Abstract The esterolysis of enalapril to its dicarboxylate metabolite, enalaprilat. was studied in the isolated pemised, nonfiltering nt kidney preparation (NFK) to ascertain the site of metabolic conversion. Two possible sites of metabolism exist: intraceiiular. by enzymes within the rend peritubular celis, and intraiuminal, by ectoxnzymes embedded on the bmsh border membrane. For the MX. filtration was obliterated with the high oncotic pressure (8% bovine serum albumin in plasma) and ligation of the ureter. thus preventing enalapril from reaching the intraluminal enzyrnatic sites directiy by fdtration. The steady state rend plasma clearance of enalapril in the
NFK was 2.0 mVmin/g, a value similar to that (2. i ddg)obtained previously by de Lannoy et al. (J. Phamacokin. Biophnrm. 21,423,1993) for the isolated perfused kidney. PK. The rate of appearance of the metabolite, enaiaprilat, in venous plasma for the NFK (30 f 3% of the input rate of enalapril) was comparable to that for the IPK (27 + 4%). The identification of the site of enalapril cellular or luminal metabolism was aided by simulations which were based on physiological models and sets of parameters obtained previously on the rend handling of enalapril (de Lannoy et al., J. Phamacokin. Biopham. 18. 56 1. 1990) and enalapnlat (Schwab et al., Am. J. Physiol. 263(RenaI Fliiid Electrolyte Physiol. 32):F858, 1992). These parameten were fùrther optimized by closely matching the simulated data with the experimental observations. The predicted outcornes for total rend clearance were similar for both the "cellular model" and "luminal model": values for the NFK were 59-60% those for the IPK. By contrast, predictions on the venous output rate of enalaprilat (as a percent of the input rate of enalapril) were very different: the b'celluiar model" predicted no change in value between the NFK and the IPK. whereas with luminal metabolism, metabolite appearance was gready magnified for NFK (289% that of the IPK). The experirnentdy observed data correlated better with the presence of intracellular than with intraluminal esterolysis of enalapril. 1.2 Introduction The kidney is capable of both metabolism and excretion, processes that compete for substrate rernoval within the eliminating organ. However, the principles of clearance have been re- exûmined recently, and metabolism and excretion are found to confound the estimates of clearance in the presence of each other (see chapter 3). The postdates are particularly pertinent to the kidney since the urinary (or excretory) clearance, expressed as the fractional excretion. FE (unbound urinary clearance/glomenilar filmtion rate), is often used to infer the secretory potential of the kidney: FE > 1 implies net secretion, FE c 1 implies net reabsorption. and FE = 1 implies net filtration (Brenner, 1986b). It was argued that intrarenal metabolism might mask the me secretory ability of the kidney despite that the FE value was less than unity (Smith and Kugler. 1994). The source of confbsion lies in the traditionai method with which we infer net loss of a compound in the kidney. Rend metabolism will reduce the unchanged drug appearing in the urine. Iowering the FE estimate and affecting the interpretation on net movement of dmg across the kidney.
This clearance principle for the kidney has been exemplified with enalapril. an angiotensin converthg enzyme (ACE) inhibitor. which is metabolized to a single dicarboxylic acid metabolite. enalaprilat; both are excreted unchanged by the kidney (de Lannoy et al., 1989: Tocco et al.. 1982). When viewed theoreticdy with simulations according to a physiologically based mode1 (chapter 3.
Fig. 3-1) the suspicion that metabolism of enaiapnl had reduced the FE value was confirmed (chapter 3, Fig. 3-2A&B). The extent of the decrease, however, was hghly dependent on whether intraceilular or intraluminal metabolism had occurred. With intracellular metabolism, the FE values remained less than unity. regardless of whether rnetabolism occurred or not. With intraluminal metabolism, FE had decreased from a value greater than one (control condition. in the absence of metabolism) to a value less than one in the presence of metabolism. Consideration of the me secretory ability of the kidney towards enalapril therefore necessitates verification of the site of intrarenai metabolism. The discrimination between intracellular and intraluminal metabolism for a dmg is not readily apparent since the solute is transporteci in both directions across the brush border and basolateral membranes. Methods such as microperfusion (Quarnme and Dirks, 1986) or in vitro rend tubule pefision (Burg and Knepper. 1986) wouid be useful in deteminhg the location of metabolism for a compound which 42 is not reabsorbed across the bnish border membrane. Since these techniques involve dkt perfusion of the renal tubule, metabolite appearing in urine would provide evidence of luminal metabolism, whereas the absence of metabolite would suggest inhacellular metabolism. The nonfiltering isolated pemised rat kidney preparation (NFK), used prirnarily for assessrnent of drug transport into renal nibular celis [via reabsorption of filtered component or via the postgiomerular circulation across the basolateral membrane (Giliat et al., 1990; Minami et al., 1992; Suzuki et n1.,1984)], was uWed presently for the snidy of the site of intrarenal metabolism. In this preparation, glornerular fdtration is obliterated by ureter ligation and by the high oncotic pressure exerted by 8% albumin in pefisate. Hence the drug is unable to gain access to the renal tubule directly except through the postglomerular circulation. Since metabolism is a major component of the rend clearance of enalapril. we hypothesized that the site of intrarenal metabolism could be deduced by examining the difference in metabolite data in the nonfiltering, NFK versils the filtering. isolated perfùsed rat kidney or IPK. Due to the lack of glomerular filtration and urine flow in the NFK,it is Iikely that hgand metabolite disposition in the two preparations will differ. Moreover. the expected behaviour of the dmg and metabolite could be predicted for the IPK and NFK by a physiological model that incorporates either cellular or luminal metabolism in the kidney. The correlation between the expected drug and metabolite data and observations provided the basis for model selection. in this study, we studied the handling of tracer concentrations of ['~]enalapril by the NFK such that the denved data were cornpared to those for data on tracer enalapril previously acquired for the IPK (de Lannoy et al.. 1989: de Lannoy and Pang, 1989). Furthemore. the renal handling of the metabolite, enalaprilat, is aiso known (Schwab et al., 1992) such that a set of panmeten was available for simulation (chapter 3. Table 3-1; de Lannoy et al.. 1989: de Lannoy and Pang, 1993; Schwab et al.. 1992; de Lannoy et al., 1990: Spector, 1956).
4.3 Materials and Methods 4.3.1 Source of materials ['~l~nalapril(specific activity 2,654 pCi/mg) and uniabeled enalapril maieate and enalapnlat were supplied by Merck Sharp and Dohme Research Laboratones (West Point. PA). The radiolabeled compounds were purified prior to use, and the radiochernical purity of enalapril 43 was 96% pure as judged by thin layer chromatopphy (TLC. 1-propanol : I M acetic acid : water
( 10: 1: 1 V/V/V with Silica Gel GF plates obtained from Analtech. Newark, DE)
4.3.2 Kidney Perfusion Male Sprague-Dawley rats (Charles River. St. Constant. Quebec. Canada; 385 + 46 g) were fed ad libitum and dowed free access to food and water. The isolated perfised nt kidney preparation was similar to that used by Johnson and Maack (1977). Isolation of the kidney was performed under pentobarbitd anesthesia (50 mg/ kg intraperitoneal injection) according to the technique described by Ross (1972). with rninor modifications. Both IPK and NFK experiments were conducted. For the IPK. mannitol (50 mg/ml) dissolved in heparin (150 U/d) was injected into the pende vein, foilowed by cannulation of the right ureter (PE-50).For the NFK, stimulation of diuresis by mannitol was avoided and the ureter was ligated close to its origin to stop urinary flow. The right adrenal artery was tied near its ongin at the renal artery. A pemisate-filled cannula (18G stainless steel needle) was inserted into the superior mesenteric artery and guided across the aorta into the right renal artery, with immediate perfusion of the kidney. The nght kidney was removed quickly from the rat and placed in a water-jacketed glas holder and maintained at 37°C.
Surgery was usuaily completed within 15 min. The perhsate for the NFK experiments consisted of 88(wlv) bovine serurn alburnin (BSA
Fraction V, Sigma Chemical Co.. St. Louis, MO). but was othewise identical to that used for the IPK; the latter consisted of washed. freshly prepared bovine red blood cells (RBCs) (15% vhr. Ryding-Regency Meat Packen Ltd.. Toronto. Ontario, Canada), 4% (wlv) BSA, 5 rnM glucose and a cornplement of 20 arnino acids, in Krebs-Ringer Bicarbonate solution buffered îo pH 7.4. Travasol 10% (Travenol Laboratories, Deerpark. IL) was used as a convenient source of most of the amino acids (0.86 ml per 10ml perfusate); the remainder of the amino acids (obtained from Sigma
Chemical Co.) was added individualiy. Unlabeled inulin was added to perfusate for assessrnent of the glomerular filtration rate (GFR)in the IPK. Its lack of disappearance from plasma for the NFK reassured that fütration had not occurred. Perfusate plasma was first pumped through an 8 pn filter (Millipore Corp.. Mississauga, Canada) at room temperature. It was then warmed to 37°C and filtered again with a 0.22 pm filter (Millipore Corp.) for the IPK or an 8 pm filter for the NFK in 44 view of the high albumin content. Mer the addition of washed bovine RBCs to the reservoir. the resulting perfùsate was equilibrated with 95% OZ-5% COZ at I Umin (Canox, Mississauga,
Canada) and then pumped through a stainless-steel mesh filter (Fig. 4-1). Perfusion pressure was mcnitored by a sphyngomanometer.
Reservoir (8% BSA) ligated ureter
venous outflow
Figure 4- 1. Schematic represcntation of the single pass nonfiltering isolated perfused rat kidne y (NFK) preparation. Perfusate, consisting of bovine red blood cells, amino acids, dextrose. and bovine semm albumin (BSA), is filtered, pumped. and gassed with a mixture of Ol:COz (955 v/v) pnor to reaching the renal artery. Venous outflow is not recirculated to the reservoir. Perfusion pressure is monitored by a sphyngomanometer. The obliteration of filtration at the giomemlus is accomplished by a high BSA content (8% albumin) which increases the oncotic pressure in the arterial perfusate at the glomerdus, and by ligation of the ureter.
Single-pass and recirculating IPKs were performed for viability studies only (n=4). Single- pass NFK studies with [3~]enalapril(0.014 f 0.0026 PM) (n=4) were performed under constant flow (8 rnllrnin). Al1 kidneys were equilibrated for 20 min at constant pressure (about 75 to 90 mm
Hg), derwhich the flow rate was fixed at 8 mVrnin for an additional 15 or 30 min. Urine sarnples were collected in taro at 5 min intervals. Inflow (reservoir) perfusate samples were taken at the midpoint of urine collection for the recirculating IPKs. In single-pass IPKs or NFKs. three intlow perfusate samples were taken during the entire experiment. Outflow pehsate samples were taken at steady-state, at 3 or 5 min intervals.
4.3.3 Viability of the IPK Viability of the isolated perfused rat kidney (IPK) was assessed by determination of sodium and glucose reabsorption. the pemision pressure, and swelling of the pemised organ (vs. control. the weigh! of the unperfbsed, left kidney). Viability of the NFK was assessed by examination of the pehsion pressure and the % change in kidney weight with perfusion. In addition. the plasma clearance of inulin was estimated for both IPK and NFK.
4.3.4 Protein Binding
The unbound fraction of drug in plasma perfusate containing 88 BSA was determined using equilibrium dialysis at 37°C in a rotating water bath. Prelirninary studies revealed that equilibrium was attained at 8 h. Freshly prepared plasma, identical to that used in the NFK perfusion expenmenü. was spiked with radiolabeled enalapril and used for equilibrium dialysis.
The radioactivity in both plasma and buffer was quantified after 8 h by dual channel liquid scintillation spectrometry (Beckrnan Instruments mode1 6800, Pa10 Alto, CA). The unbound fraction in plasma (fp) was estimated by the ratio of the concentration of radiolabeled endapril in the buffer to that in the plasma. The leakage of protein from the plasma side of the membrane to the buffer side was determined by the Lowry method (Lowry er al.. 1951). Significant volume shifts and protein leakage across the membrane were not observed. The membranes used in the experiments were rinsed with distiiied water, and then irnmersed in scintillation cocktail for B- counting. No radioactivity was detected on the membranes. The binding experiments were repeated with 5% BSA to view binding data for the comparable IPK studies performed by de
Lannoy et al. ( 1989). 4.3.5 Analytical procedures It has been shown that neither enalapril nor its metabolite enalaprilat is significantly bound to red blood celis (de Lannoy er ai., 1989). so chat rneasurements cm be made using plasma concentration and plasma flow rate rather than whole blood concentration and blood perfusate flow rate. The inflow and outflow plasma samples were centrifuged to provide plasma for analysis. TLC was used to separate enalapril and enaiaprilat in inflow and oudlow plasma sarnples. Imtially. we attempted a protein precipitation-drying procedure prior to plating ont0 TLC plates. However. band widening and poor resolution resulted. Thus, plasma (100 pl) was direcdy applied ont0 the ocigin of the TLC plates (Silica gel GF: Analtech Newark. DE). prespotted with unlabeled enalapril and enalaprilat standards. After development of the plates in a system of 1-propanol :1 M acetic acid : water ( 10: 1: 1 v/vhr), the entire TLC plate was scraped into 0.5 cm segments. spanning from 1 cm below the origin up to the solvent front. The R, value (distance compound traveled from originldistance between ongin and solvent front) for authentic enalapril varied from 0.57 and 0.70 and was between 0.3 1 and 0.39 for enaiaprilat. and the radiolabeled compounds were found to closely follow the migration of the unlabeled compounds. Afier the addition of 0.5 ml of water and 5 ml of scintillation cocktail (Ready Safe, Beckman Instruments), the sarnples were kept in the dark for 24 to 48 h before B-counting. In order to correct for recovery from TLC. separate aliquots of plasma (100 pl) were also subjected to B-counting. The arnounts (DPMs) associated with ['~]enalapriland ['~]enalaprilat in the samples were summed. and appropriately corrected for the recovery and volume of the sarnple to provide the concentrations. Sodium and glucose were measured in the plasma and urine sarnples by flarne photometry (IL 943 Rame Photometer, Instrumentation Laboratory, Lexington. MA) and by the oxygen rate method (Glucose Analyzer 2, Beckman Instruments, hc.. Fullerton. CA), respectively. Inulin was assayed by the method of Heyrovsky ( 1956).
4.3.6 CaIculations The rend extraction ratio EK is expressed as the difference between the steady-state input rate (Q&) and output rate [(Q, - Qu) C,] divided by the input rate; C,, and C,, are the steady state infiow and outflow plasma concentrations of dmg. Q, and Quare the plasma and urine flow rates, respectively.
The equation becornes simplified when Qu= O for the NFK.
Total rend clearance of enalapril was calculated as foilows:
The fractional excretion (FE) or the unbound urinary clearance nomalized to GFR, was calculated as foilows:
where Cuand Quare the concentration of dmg in urine and the urine flow rate (the product yields the urinary excretion rate). respectively: GFR is the glomerular filtration rate, and fp is the unbound fraction of enalapril in plasma. The venous rate out of enalaprilat . normalized to the rate of input of enalapril was as follows:
(QK -Qir %plasma rate out of enalaprilat = ) COut{mi1 x 100% QK where C,,{ mi} is the steady state output plasma concentration of enalaprilat. For the NFK. the urine flow rate, Quwas zero. 4.3.7 Simulations
A physiological model of the kidney, which describes either intraceilular or invaluminal metabolism (Fig. 4-2) was considered. In this model, the kidney is subdivided into the vascular, tissue, and tubular lumen (or urine) compaments. The drug is first fütered by the glomerulus be fore reaching the peri tu bdar cells via the postglomedar circulation, given b y the di fference in rend plasma flow rate (a)and the glornerular filtration rate (GFR). Parameters for the transport of enalapril: CL^^ and CL$ for the influx and efflux clearances at the basoiaterai membrane (b). and
CL:, and CL:. for the influx and efflux cleannces at the luminal membrane (0,and corresponding transfer clearances for enalaprilat across the basolateral membrane (CL:, {mi} and ~Lt~.(rni) ) and luminal membrane (CL:, { mi J and cLLf ( mi } ) are described. Esterolysis of enalapril occun intraceliularly with CLh,~,the cellular metabolic intrinsic clearance, or inaaluminally, with CL,,,,. the intraiuminal metabolic intrinsic clearance. heviously solved equations (see Table 3-2) penaining to these kinds of physiological rnodels (Fig. 4-2) were utilized to estimate the total rend (CL,,) and excretory (CL,,) ctearances of enaiapril at steady state. For cellular metabolism
For intraluminal metabolism: Intracellular Metabolism (B) lntraluminal Metabolisrn
Reçervoi r I Reservoir I I I Q, - GFR Renal Renal L D mi Plasma D mi Piasma QK - Qu QK - Qu - jfcL->{mil clK, cqn{mi)
Renal mi Tissue 1
Figure 4-2. Physiological models of the kidney depicting either (A) intracellular or (B) intraluminal
metabolisrn of enalapril. Enalapril is eliminated by the kidney via both excretion and metabolisrn to enalaprilat. which is also excreted. The kidney is subdivided into three compartrnents: plasma, tissue and urine. The outflow plasma can either be recirculated back to the reservoir (the recirculating preparation ), or drained into a collecting vesse1 (single-pass preparation ). Exc hange of drug (D)and metabolite (mi) between renal plasma and tissue is characterized by influx (cL~~,cL~~{mi } ) and efflux (CL$CL:~ {mi})clearances across the basolateral membrane. and the exchange between urine and tissue is chmcterized by influx (CL:,,CL{,(~~})and effiux (~~$~l,f{mi})clearances at the luminal membrane. The arterial plasma and urine flow rates are represented by QK and Q,,. respectively. (A) Dmg within the tissue is metabolized with a renal metabolic intrinsic clearance,
CL,,, K. (B) Dnig within the unne cornpartment or renal tubule is metabolized with a renal metabolic intrinsic clearance, CLint,u. Values for the transfer and metabolic intrinsic clearances of enalapril were modified until the set of parameters closely matched all of the experimental data for the total and unnary clearances of enalapril for the IPK (Eqs. 4-5 and 4-6) when intracellular metabolisrn occurs. The procedure was repeated to obtain another set of parameters which wouid be consistent with the sarne experimental data when intraluminal metabolism occurs (Eqs. 4-7 and 4-8) (chapter 3. section 3.4). Since the
IPK expenments (de Lannoy et al.. 1989, de Lannoy and Pang. 1993) were performed at a higher
molar concentration of enalapril than that for the NFK (- 1 pM vs 0.0 1 pM), due to differences in the specific activities of the enalapril used, we Merascertained in a set of simulations the effect of concentration. Experirnentation in the IPK with various concentrations of enalapril revealed a concentration dependence for metabolic clearance but a lack of change in the excretoty clearance of
enalapril between 1.06 and 12.7 pM (de Lannoy et ni.. 1989). Since metabolic clearance varied
with concentration. simulations were perfonned to obtain an optimized CL,,., for enalapril at 0.0 1
PM used in the NFK. de Lannoy et al. ( 1989) estimated the metabolic intrinsic clearance (CL,,,,)
of 6 ml/min/g for 1.06 pM under the assumption that metabolism occurred intracellulady. The observed data were found equally consistent wiih a metabolic clearance of 1.95 rrii/min/g when intraluminal metabolism occurred (see chapter 3. Table 3-3). Based on rend clearance estimates observed at various concentrations of enalapril in the IPK. simulations were performed according to
Michaelis-Menten kinetics to yield appropriate V-. Y, and CL,,,., [VJ(Y, +[SI)]. For the concentration of 0.01 pM. the rnetabolic intrinsic clearance became 6.25 ml/rnin/g for the cellular mode1 and 3 ml/min/g for the luminal modei. These optimized parameters are sumrnarized in Table
4-1 (chapter 3. Table 3-1; de Lannoy and Pang. 1993; Schwab et al., 1992: de Lannoy et al., 1990: Spector. 1956). For the nonfiltering kidney. the urine flow rate and GFR were set to O and the
unbound fraction of enalapril in plasma equaled that found expenmentally for 8% albumin (fp =
0.32). Table 4-1 Parameters used for simulations of [3wenalapril and [3~]enalapri~atdata in the single pass isolated perfused kidney (PK)and non-filtering kidney (NFK) prepantions. Intmcellular Mode1 lnuaIumina1 Mode1 Initial or Assigned Value Initial or Assigned Valiie Description Symbol LPK / NFK IPK / NFK
Ccirnpartment volumes (mu?) Reservoir plasma v~ Rend plasma v~K Renal tissue v~ Renal urine vu
Fiow rate (mVmidg) Rend plasma QK Urine Qu Glomemlar filtration rate GFR Clearances for endapril in kidney (mYmin/g) rend rnetabolic intrinsic: cellular CLinrk IurninaI CLint,fi basolateral influx/efflux CL:>, = Iurninal influx (reabsorption) CL:, luminal effiux (secretion) CG,
Clearances for enalaprilat in kidney (mVmin/g)
basolateral influx CL:, (mi } basoIatera1 efflux CL$ {mi)
luminal influx (reabsorption) CL:, (mi
luminal efflux (secretion) CL:, (mi]
Fractions unbound for enalapril plasma kidney
Fractions unbound for enalapnlat plasma kidney
" value obtained experimentally by de Lannoy and Pang ( 1993). obtained by Schwab et al. (1992). ' obtained bi Spector et al. (1956). value obtained by parameter optimization procedure (chapter 3. section 3.4). ' optimized value obtained via simulation based on Michaelis-Menten kinetics. ' value obtained by de Lannoy et al. (1990). value for IPK. due to binding to 4% bovine senim albumin. determined by equilibriurn dialysis (de Lannoy et al.. 1989). value for nonfiltering kidney. due to bindinp to 8% bovine serum albumin. determined by equilibrium dialysis. the unbound fraction was not measured. but the value was not important for the efflux of enalaprilat. Simulations were aiso perfomed with the program, scientistR (MicroMath Scientific Sohare. Salt Lake City, Utah). Mass transfer rate equations descnbing the change in concentrations for enalaprii (Eqs. 4-9 to 4- 1 1) and enalapriiat (Eqs. 4- 12 to 4- 14) with tirne for single pass conditions were written, noting that the rate of change for enalapril or enalaprilat in the reservoir is zero:
For enalapril in vascdar plasma in kidney,
For enalapril in kidney tissue.
For enalapril in urine,
For enalaprilat in vascular plasma in kidney.
For enaiaprilat in kidney tissue, For endapriiat in urine,
where C, is the steady state input concentration of enalapril, and C,, C,. and Cu are the concentrations of endapril in the rend plasma, rend tissue, and urine, respectively, and CM{mi }, C,{ mi } . and Cu(mi ) are the corresponding concentrations of enalaprilat : V,,. V,. and Vu are the physiological volumes of the vascuiar plasma space in kidney, the kidney, and urine. respectively, f, and fp{rni) are the unbound fractions of enalapril and enalaprilat in plasma, respectively. and f, and f,{ mi} are the corresponding unbound fractions in kidney. For the cellular model. CL,,., was set as zero; alternately, for the intraiumind model. CL,, was set as zero.
4.3.8 Statistics
Ail data were expressed as mean IS.D. ANOVA was perfomed on the data: a p value of
0.05 was viewed as significant.
4.4 Results
4.4.1 Viability of the Perfused Rat Kidney Preparations The viabiliv of the IPK preparation was established for the flow rate of 8 ml/min. The extents of sodium and glucose reabsorption were 9 135% and 98 +1%. respectively. values which were relatively constant over the 40 min of perfusion under constant flow. These indicators suggest that the viability of the kidney was adequately maintained (de Lannoy et al.. 1989). The arterial pressure was 121 k 34 mm Hg at 8 mYm.in, and the increase in kidney weight aber perfusion
([weight of right kidney - weight of lefi kidney] / weight of lefi kidney) averaged 23 f 108. For the NFK. the mean arterial pressure and increase in kidney weight were 1 13 I34 mm Hg and I 1 f
13%. respectively. The percentage of inulin recovered in outfiow perfixsate was 97 k 4%. and the plasma clearance, normally used as an estimate of GFR, was virtuaiiv zero for the NFK. Apart from the difference in GFR, viability parameters for the IPK and the NFK preparations were not
statisticdly signifiant @ > 0.05).
4.4.2 Protein Binding Equilibrium dialysis yielded an fp value of 0.41 f 0.0 1 (n=4) and 0.32 + 0.0 1 (n=4) for
pefisate plasma containing 5% and 8% (wh) BSA, respectively, at a concentration of 0.39 PM.
Protein leakage from the protein side across the membrane into the buffer side was negligible: the
leakage was 0.42 f 0.07 % for 5% %SA and 0.27 f 0.0 1 for 8% BSA. The volume shifts across
the membrane, and non-specific binding to the membrane were negligible for both the 5 and 8% BSA.
4.4.3 The Non-Filtering Kidney The TLC procedure effectively separated labeled enalaprilat frorn enalapril. albeit there were fluctuations in R, due to the high albumin content in the sampies (Fig. 4-3). The recovery of radioactivity was generally greater than 90%. The inflow sarnples contained only radiolabeled enalapnl, confirming that esterolysis of enalapril had not occurred in the perfusion medium. The enalapnlat found in the plasma venous sarnples was necessarily forrned by the kidney. The steady state extraction ratio for enalapril in the NFKs (n=4) was 0.39 f 0.03. and was slightly hgher than that (0.29) for the IPK @ < .OS), presumably due to the lower rend plasma flow rate for the NFKs
(Table 4-2). Values of the rend clearance (extraction ratio x plasma flow rate) for the NFK ( 1.99 f
0.12 ml/rnin/g) and IPK (2.1 nil/min/g) (de Lannoy and Pang, 1993) were not significantly different
@ > 0.05). The enalapnlat venous output rate. expressed as a percent of input rate of enaiapril. was 30 f 3% for the NFK (Table 4-2); the parameter was not si,@icantly different from that for the IPK (27 + 4%; n= 6) (de Lannoy et al., 1989). Figure 4-3. Profiles of radioactivity recovered on thin layer plates for inflow and outflow plasma sarnples. lnflow and outflow plasma sarnples were assayed for enalapril and enalaprilat by thin layer chromatography (TLC). The total counts were divided by the recovery and volume (0.1 ml) to obtain the concentration (DPWml). The left and nght panels illustrate the radioactive profiles of representative inflow and outflow plasma samples, respectively.
Table 4-2. Summary of experirnentaily observed and simulated data for the IPK and NFK.
Expenmental Observations S imulated DataL'- Cellular Luminal -IPK~ -NFK' -IPK -NFK -IPK -NFK
CL,,, (ml/min/g) 2.1 i 0.4 1.99 + 0.12 2.10 1 .24J 2.10 1.26" predictedo bserved 0.99 0.61 0.995 0.62
CL,, (mYmin/g) 0.15 k 0.07 O** o. 14 0 0.15 0 Enalaprilat plasma rate 27 f 4 30 t3 24 24J 9 26" out (% enalapril rate in) predictedo bserved 0.87 0.79 0.34 0.80
" Parameters in Table 4-1 used for simulation. Data obtained by de Lannoy et al. (1989) and de Lannoy and Pane (1993). Data based on 4 nonfiltering kidney preparations. 'Optimized intrinsic metabolic clearance values used. * ANOVA: p < .O5 vs. PK ** absence of unnary output 4.4.4 Simulations Four cases were considered: cellular vs. luminal metabolism for both the IPK and the NFK.
According to Eqs. 4-5 and 4-7, the optimized parameters (Table 4-1) for the cellular rnetabolism model yielded rend clearances of 2.1 and 1.24 ml/rninfg for the IPK and NFK, respectively, whereas the luminal metabolism model predicted rend clearances of 2.1 and 1.26 ml/min/g for the IPK and NFK, respectively (Table 4-2). Moreover, venous plasma output rate of endapril or enalaprilat, expressed in relation to the input rate of enalapril, were sirnulated according to Eqs. 4-9 and 4-12 and presented in Fig. 44A for the cellular metabolism model and Fig. WBfor the luminal metabolism model. Values of the plasma clearance of enalapril were the rame for the cellular and luminal models for the IPK (2.10 ml/rnin/g), and these were greater than those for the
NFK ( 1.24 and 1.26 ml/rnin/g, respectively). The reduction in total renal clearance in the NFK is due primarily to the lower unbound fraction of drug in the NFK. The enalaprilat plasma output patterns were different for the PK(24 vs. 9% input rate of enalapril) with cellular and intralurninai metabolism. respectively. By contrast, values for the accumulation of the metabolite. enalaprilat, in plasma were similar for the NFK for both the cellular and luminal rnodels (24 vs. 268 of input rate of enalapril). Viewing these altematively. there was no difference in metabolite outfiow levels between the IPK and NFK with cellular metabolism but drastic difference was expected between the IPK and NFK for luminal metabolism. The venous output of enalaprilat for the NFK was anticipated to be almost three-fold (289%) that of the IPK (Table 4-2; Fig. 4-4). The experimental data for the renal clearance of enalapril decreased only by 5% and vimially no difference was observed for enalaprilat plasma output rate for the NFK. in cornparison to data for the IPK. nie predicted/observed output rate of enalapnlat was indeed a sensitive indicator. A better correlation existed for the cellular metabolism model. - Cellular Metabolism - --,+--+--- l+-- +---*-
-' -' NFK - enalaprilat - - IPK - enaîapnl .- .- NFK - enalapril
- Luminal Metabolism - --&--*---&--*---*-- - - - I
--e- NFK - enalaprilat - - IPK - endapril
t
Time (min)
Figure 4-4. Simulated data on the output rates of enalapril and enalaprilat as a fraction of the inflow rate of endapril. (A) Simulations based on the intncellular metabolism of enalapril predicted similar values for the plasma output rates of enalapril and enalaprilat /rate in for the NFK vs. the IPK. (B)
Simulations based on the intr&minal mode1 of enalapril metabolism again predicted similar ratios of enalapt-il rate outhate in for both models. However. a higher enalaprilat rate out was expected for the NFK in relation to the IPK. 4.5 Discussion
Recentiy, it has been proposed that renal metaboiism would mask the tme dmg secretov capacity of the kidney (Smith and Kugler, 1994). It is envisioned that for a compound which undergoes both metabolism and excretion within the kidney. the processes will compete with one another in the removal of the substrate. Undoubtedly, the excretory and metaboiic clearance estimates wiil be affected by the presence of the competing route of elimination (chapter 3: Smith and Kugler, 1994). Since the secretory abdity of the kidney is assessed based on the excretion of unchanged dmg in the urine (i-e. the FE value), the presence of metabolism within the kidney will decrease the arnount of dmg present in the urine, and therefore mask the nue secretory ability of the kidney for that particular compound (chapter 3, Fip. 3-2A&B). Enalapril undergoes esterolysis within the kidney to form its dicarboxylic acid metabolite enalaprilat. The question that arises is whether enalapril is metabolized intracellularly or intralumllidy within the rat kidney. Indeed. previous simulations based on renal physiological models for enalapnl had shown that metabolism within the rend tubule (by ecto-enzymes dong the brush border membrane) could decrease the FE vaiue from above one (net secretion) in the absence of metabolism to a vaiue below one (net reabsorption) when metaboiism occurs (chapter 3.
Fig. 3-2B). This would alter interpretation of the secretory ability of the kidney in the presence of metabolism. If metabolism of enalaprii occurred intracellularly, interpretation on net reabsorptive flux of enalapd would remain unchanged. The decrease in FE value with metabolism was small and the change would not have perturbed FE values enough to exceed one (chapter 3. Fig. 3-2A).
The converse, however, would not hold mie. that is. when metabolism occurred intraluminaily. A large change in FE is expected to be induced by intraluminal metabolism and the interpretation of net reabsorption for enaiapnl would be in error. In order to address the question of intrarenal enalapril metabolism, we perfomed a set of MKexpenments, compared the results to those of the IPK (de Lannoy er al.. 1989: de Lannoy and Pang, 1993), and further examined how these data correlated to the simulations based on physiological rnodels. A necessary step was the establishment of viabiiity of the perfused kidneys (bodi IPK and NFK). Since the number of viability indices is reduced in the NFK due to lack of
59 urine, viability of the IPK preparation: adequate sodium and glucose reabsorption and low weight changes (de Lannoy et al., 1989; de Lannoy and Pang, 1993) was fmt established; comparable weight changes and perfusion pressures were also observed for the NFK. The second step was examination of the eEect of the greater protein binding for enalapril at 8% alburnin for the NFK studies, although that for enalaprilat did not enter into the formulation (Geng and Pang. unpublished equation). The unbound plasma fraction of enalapril was found to be 0.32 for 8% albumin and 0.41 for 5% aibumin with equilibrium didysis at 0.39 W. Simulations based on the
Langmuir isotherm showed that the fp values obtained at these albumin concentrations were consistent with each other and that the unbound hction of enalapril would not change within the concentration range spanning from 0.0001 to 10 pM. Therefore, the unbound fraction of enalapnl obtained for 8% BSA couid be utilized for simulation of NFK data at 0.01 pM enalapril. The value of 0.41 for 5% BSA was lower than that (0.55) observed by de Lannoy et al. (1989). who examined the binding of enalapril at 5% BSA and found a lack of concenrration dependence (0.2 1
- 21.2 PM). The difference might have ken due to the batch of alburnin used. Thus. the respective fp values were used for simulation (i.e. fp = 0.55 for IPK: fp = 0.32 for MK).
A notable observation was the lower plasma flow rate for the NFK preparations in relation to those for the IPKs (Table 4-2). The unbound rend metabolic clearance for the IPK (3.53 ml/min/g) previously obsewed by de Lannoy and Pang (1993) was also lower than that for the
NFK (4.9 1 ml/rnin/g). The difference could not be attributed to the difference in flow rates. However, a higher metabolic clearance value was anticipated for the NFK due to the absence of excretion (cbapter 3, Fig. 3-20, a new paradigm to be reckoned with in clearance concepts. It could merbe surmised that the performance of the kidney preparations was irnproved at the lower flow rate (8 vs. 10 mi/rnin), as also shown by the improved viabiiity of the present IPKs over previous ones (de Lannoy et al.. 1989).
The simulations showed that the E, and total renal clearance of enalapril were poor indices for the discrimination of cellular versus luminal metabolism (Table 4-2). A similar decrease in the total renal clearance of enalaprii was predicted in the NFK with both models. However. the 60 models were discriminated based on the enalaprilat rate out in plasma nomalùed to the input rate of enalapd (Fig. 44; Table 4-2); the parameter was independent of the unbound fraction of enalaprilat. The predicted/observed ratios were between 0.79 and 0.89, with the exception of 0.34 for the IPK, based on the luminal model. Enalaprilat, if formed lumindy in the fdtenng kidney. is expected to be canied down the renal tubule and excreted rapidly into urine radier than king reabsorbed. The ratio is an aberrant value arnong the entire data set. The poor correlation suggests that enalapril deiivered to the kidney is unlikely to be rnetabolized by enzymes on the luminal membrane. The observation appears to be consistent with what is known on enzyme distribution within the kidney. Only peptidases are known to be e~chedon the lumùial membrane (Carone et al., 1982; Kugler er al.. 1985). Yet the presence of carboxylesterases on the luminal membrane has not, to our knowledge, been reported. The interpretation on the net reabsorption of enalapnl by the kidney is justified for the intracellular metabolism of enalapril (de Lannoy et al., 1989).
The above treamient was based on the assumption that transport of enalapril across the basolaterai and brush border membranes was not aitered between the IPK and NFK. On the contrary, Kim et al. ( 1992) had studied the renal handling of the rnodel anion. para-arninohppurate. PAH, in both the iPK and NFK and showed a decrease in luminal efflux of PAH in the NFK to 1/3 the value observed in the IPK. If the situation had prevailed for the present NFK studies. decreases would have resulted for the NFK. The decrease would only affect the output rate of enalaprilat for the luminal model (enalaprilat output rate would be halved) and not the cellular model. Since there was no decrease observed for enalaprilat outflow. it might be surmised that reduction in secretory clearance of enalapril had not occurred for the NFK. Ln addition. the recently cloned organic anion transport polypeptide (oarp) (Jacquernin et al., 1994) was found to be localized dong the brush border membrane of the proximal tubule of the rat kidney (Bergwerk et al., 1996). The transporter. expressed in Hela cells, was unable to mediate the uptake of PAH (Kullack-Ublick et al.. 1994:
Kana1 et al., 1996) whereas it was capable of taking up enalapril (Pang et al., 1997). suggesting that enalapril and PAH might not share the same transporter at the rend bmsh border membrane. Accordingly, the decrease in secretory clearance observed for PAH in the NFK does not necessarily extend to enalapril. In summary, single pass studies were perfomed with tracer ['~lenala~nlwith the nonfiltering kidney, which provided data (extraction ratio, renal and metabolic clearances. and enalaprilat output rate) sirnilar to those obtained previously for the IPK (de Lannoy et ai.. 1989). The "luminal metabolism model" which predicted an increase of the output rate of enalaprilat for the NFK vs. the IPK, did not agree weil with the observations. The composite observations for the
IPK and NFK correlated better with the expected changes with the "cellular rnetabolism model". The data suggest that the metabolism of enalapril is intracellular rather than intralurninal. The previous assumption on net reabsorption of enalapril by the kidney (de Lannoy et ai.. 1989) appears justified, namely, the metabolism of enalapril has not altered the interpretation on the mechanism of excretion of enalapril (chapter 3). Fume studies. with renal membrane vesicles will allow for the direct assessrnent of the metabolic capability of bmsh border membrane enzymes for enalapril.
4.6 Staternent of Signifieance of Chapter 4 This chapter provided evidence to identiw the site of enalapril ester01ysis within the kidney as intraceilular. Since enalapnl is metaboiized intracellularly, the simulations presented in chapter 3
(Fig. 3-2A&C), that were based on the inmcellular model of enalapril handling by the kidney are applicable to enalapril. Chapter 5: Inhibition of Enalapril Esterolysis by Paraoxon in the Isolated Perfused Rat Kidney Increases Urinary Clearance of Enalapril 5.1 Abstract The cornpetition between elimination pathways in the kidney was examined with [3H]enalapnlin renal S9 tissue and the isolated perfused rat kidney preparation (IPK). Studies in rat rend S9 fraction demonstrated that paraoxon, a carboxylesterase inhibitor, effectively inhibited
['Hlenalapril esterolysis to ['~]enalaprilatat concentrations of 0.1,0.5, 1 and 10 PM paraoxon (76 f 7%, 93 f 5%. 96 + 5% and 93 t 6%. inhibition as compared to control. respectively). The inhibition at O. 1 pM paraoxon was significantly less (p < 0.05) than those observed at the three higher concentrations. Subsequent IPK viability studies performed in the presence of 0.1. 0.5 and
1 pM paraoxon reveaied that the inhibitor did reduce viability of the kidney. However. loss of viability was lessened with decreasing concentration of paraoxon. Therefore. O. 1 pM paraoxon was chosen as a suitable concentration to inhibit enalapril metabolism in the IPK. Studies conducted with [3~]enalapnland 0.1 ph4 paraoxon in the IPK consisted of a constant pressure (90- 100 mm
Hg for 20 min) equilibration period foilowed by a constant flow (8 dmin for 30 min) study period. During constant flow KHB was infused (O. 18 1 ml/min) into the arterial inlet to the kidney for 15 min (control), then the KHB was replaced by 4.5 pM of paraoxon solution for infusion for the next 15 min: ths would yield a concentration of 0.1 pM paraoxon reachng the kidney.
Viability of the IPK in the presence of 0.1 pM paraoxon. assessed by sodium and glucose reabsorption. was not significantly aitered in cornparison to the control period. In the presence of
0.1 pMparaoxon, the metabolic and total renal clearance of v~]enalaprildecreased significantly (p
< 0.05) when compared to those for the conuol period. The metabolic clearance decreased from
1.83 k 0.52 to 1.48 I0.47 ml/min/g, and the total renal clearance decreased from 1.87 + 0.46 to
1.57 i: 0.4 1 ml/min/g. By contrast, the urinary clearance (from 0.04 I 0.07 mi/min/g to 0.09 I0.09 ml/min/g) and fractional excretion value (FE) (from 0.23 +. 0.18 to 0.52 t 0.25) of ['~lenalapril were significantly (p < 0.05) increased in the presence of 0.1 pM paraoxon. These results illustrate that decreased metabolism induced a rise in the urinary clearance estimate.
5.2 Introduction The simulation snidy presented in chapter 3 established that renal excretoy and metaboiic clearance estimates for enalapril were altered in the presence of competing routes of elimination. Metabolism was shown to decrease the excretory clearance estimate, while excretion would either increase or decrease the me tabolic clearance estimate. depending upon the location of drug metabolizing enzymes. Since enalapril hydrolysis was subsequentiy shown to occur intracellulary. (chapter 4) metabolism and excretion will both decrease the clearance estirnate of each other. The present chapter describes a set of experiments designed to identib the effect of reduced metabolism on the urinary clearance estimate for enalapril in the isolated perfused rat kidney preparation (IPK). These experiments wdl afford hypothesis testing that metabolism and excretion decrease the clearance estirnate of the alternate pathway, and lend support to the results of the simulation study on enalapril (chapter 3). Since the metabolic clearance of enalapril is much greater than the excretory clearance of enalapril in the PK, inhibition of enaiapnl hydrolysis should result in an observable increase in the excretory clearance for enaiapnl.
A compound that inhibits the renal metabolism of enalapril needs to be identified. Since enalapril is a carboxylester precursor, paraoxon, the active metabolite of the organoposphorus insecticide parathion. was viewed as a possible candidate. Paraoxon was previously shown to inhibit enalapril esterolysis in rat kidney tissue (Grima et al., 199 1). However. an in vitro inhibition study with rat renal S9 tissue was fust perfomed to confm this finding. Various concentrations of paraoxon, ranging from 10 ph4 to O. 1 pM, were tested for the ability of paraoxon to inhibit tracer ['~lenala~rilatformation from ['Hlenalapril. With the knowledge that paraoxon is toxic and may potentialiy be detrimental to the IPK, a series of viabiiity studies was performed in order to determine a suitable concentration of paraoxon for use in the perfusion studies with enalapril. Upon establishing a suitable concentration of paraoxon in the PK, inhibition experiments were perfomed with tracer ['~]enalapnl and paraoxon. Results from this last set of studies will illustrate experimentally, that renal metabolism wili compete with excretion for elunination of substrate, rende~ga lower estimate of the excretory clearance.
5.3 Materials and Methods 5.3.1 Chernicals ['~]~nalaprilwas synthesized by Dr. Alfred Chung (University of Gainsville. Honda) from
['~lproline,according to the method described by Bartroli, et ni. ( 1986) and was 97% pure as judged by thin layer chromatography (TLC,1-propanol : 1 M acetic acid : water v/v/v with silica gel GF plates obtained from Analtech, Newark DE). Non-radiolabelled enalapril and enalaprilat were gifts from Merck Sharp and Dohme Research Laboratones (West Point. PA). Paraoxon was obtained from Sigma-Aldrich Canada Ltd. (Oakville, ON).
5.3.2 Renal S9 Fraction
The renal S9 fraction was freshly prepared on the day of the expriment from male
Sprague-Dawley rats (Charles River Canada, St. Constant, Quebec, Canada) weighing 4 19 f 26 ,o.
Kidney surgery was perfonned according to the method of Ross (1972). and has ken described in detail in chapter 4 (section 4.3.2). The kidney was perfused for approxirnately 1 min with ice cold Krebs-Henseleit bicarbonate solution (KHB), removed from the rat, and placed on ice. Following removal of excess tissue. the kidney was minced and homogenized (Ultra Turrax Tî5 homogenizer,
Janke & Kunkel, KA-Labortechnik, West Germany) with 6 ml of ice cold KHB. The hornogenate was cennifuged at 9,000 x g in a Sorvaü RC-SB Refrigerated Superspeed Centrifuge (Dupont hstruments) for 20 min at WC. The top fany layer was skirnmed off while 4 ml of supernatent (S9 fraction) was removed for dilution with 8 ml of ice cold KHB. This would result in - 1-2 mg/rnl of protein in the incubation mixture. Preliminary experirnents established that incubation of ['~lenalaprilwith 1-2 mg/ml of protein in the incubation mixture would yield a linear production of [3~]enalaprilatfor at least 20 min without substantial depletion of substrate, ['~lenala~ril.
An aliquot (1 ml) of S9 was incubated with 0.5 mi of paraoxon solution on ice. The S9 fraction was warmed to 37°C for 5 min in a rotating water bath (New Brunswick Scientific, Edison NJ) before commencement of the experiment. The incubation experiment was initiated upon addition of 0.5 ml of KHB containing a tracer concentration of ['~lenalaprilto the 1.5 ml mixture of S9 and paraoxon. Controis consisted of 1 ml of S9 used for paraoxon inhibition. incubated with 0.5 ml KHB, and 0.5 mi ['~]enalapril. Concentrations (fmal concentration in reaction mixture) of paraoxon tested were 0.1 pM (n=3). 0.5 pM (n=3). 1 ph4 (n=4) and 10 ph4 (n=3). The reaction of the incubation mixture was terminated at 1, 2.5, 5, 7.5, 10 and 15 min, at which time, 200 pl was removed direcdy into 800 pl of acetonitrile for protein precipitation. The sampie was irnmediately mixed and centrifugeci. The supernatent was transferred into a microlige tube for subsequent assay. At the end of 15 min. an additional 500 pl of the incubation mixture was removed for protein determination and for direct counting of radioactivity to provide the initial concentration of
['~]enala~rilin the reaction tube. Mer norrnalizing the concentration of ['~lenalaprilat by the concentration of protein in the mixture. a cornparison was made between the concentrations of
['~lenalapnlatin the presence and absence of paraoxon at 15 min. in order to determine the ability of paraoxon to inhibit the esterolysis of enalapnl.
5.3.3 Partition Coefficient for Enalapril at Various pH The partition coefficient for enalapnl was determined in a system of 1-octanol and KHB.
KHB was buffered to pH values of 6.4 to 8.2. Equal volumes (0.5 ml) of the buffer containing tracer levels of [%]enalapd and 1-octanol were mixed. The solution was then allowed to sit for Z h. and was then cenmfuged to allow for complete separation of the two phases. A 200 pl sample of the top layer (1-octanol)was removed and subject to pcounting; the remainder of the upper layer was suctioned off and 200 pl of the bonom layer (KHB) was also subject to psounting. The partition coefficient was obtained by dividing the concentration of ['~]enalapril in the octanol by that in buffer. 5-34 Experhents in the Single-Pass Isolated Perfused Rat Kidney (IPK) Two sets of studies were performed in the PK. In the fit study, viability of the IPK was assessed in the presence of a range of paraoxon concentrations. In the second snidy. ['H]enalapnl
metabolism was examined in the presence and absence of paraoxon in order to assess the influence
of metabolism on excretion. Kidney surgery and perfusate composition for the isolated perfused rat kidney preparation had been descnbed in detail in chapter 4 (section 4.3.7). After completion of
surgery for the IPK, the kidney was aiiowed to equilibrate for 15 or 20 min at constant pressure
(90-100 mm Hg), then pefision was continued under constant flow rate (8 mL/min) for the remainder of the experiment. The fmt period of constant flow was used as a control period, during which time KHB was infused (Sage Instruments, Division of Orion Research Incorporated) via an indweliing cannuia into the rend arterial. input am (Fig. 5- 1). Foilowing the control period KHB infusion was replaced by paraoxon infusion. Randomization of control and inhibition periods was not possible since paraoxon irreveaibly inhibited esterase activity (Chernnitius et al.. 1992). as confmed in preliminary expenments.
ressure Monitor
lnflow Perfusate
Figure 5-1. Schematic representation of paraoxon infusion to the kidney. A syringe pump delivers paraoxon to the renal anenal input amvia an indwelling cannula. Paraoxon is mixed with inflow pemisate just prior to reaching the kidney. 5.3.4.1 Viability Studies in the IPK with Paraoxon The fust set of studies was performed in order to determine a suitable concentration of paraoxon for use in the inhibition of enalapril hydrolysis. Fig. 5-2A depicts the experimental design of these viability studies. During the post-equilibration constant flow (8 rnl/min) period, the kidney was perfused for 15 min in the absence of paraoxon, and infused with KHB into the pemisate at a rate of 0.18 1 mVmin. For the next 20 min paraoxon was infùsed (0.18 1 rnllrnin). at concentrations of 45 w,22.5 pM and 4.5 pM to result in concentrations of 1 pM (n=2), 0.5 pM (n= 1) and 0.1 ph4 (n=2) reaching the kidney. Urine was collected in toto at 5 min intervals. inflow reservoir pemisate sarnples were taken at early, rniddle and late tirne points. Viability of the kidney. in the absence and presence of paraoxon, was assessed in terms of glomemlar fdtration rate (GFR). urine flow rate. % weight gain, and sodium and glucose reabsorption. Reabsorption was calculated by dividing the difference between the filtered and excreted loads, by the filtered load. [(fiitered load
- excreted load)/ filtered load x 1001. Sodium and glucose were measured in the plasma and urine sarnples by flame photometry (IL 943 Flame Photometer, Instrumentation Laboratory, Lexington. MA) and by the hexokinase method (Glucose Kit, Sigma-Aldrich, Canada), respectively. GFR was estimateci by the urinary clearance of inulin. Inulin was assayed for by the method of Heyrovsky
( 1956).
5.3.4.2 Inhibition Experiments with [3H]Enalapril and Paraoxon in the IPK
For the inhibition experirnents, the single pass IPK was perfbsed with ['~lenala~ril throughout the entire study. KHB was infused into the inlet ami (0.18 1 rnl/min) for the first 15 min of the constant flow perfusion and was substituted by paraoxon (4.5 PM) to furnish a concentration of O. 1 pMat the inlet perfusate to the kidney. Inflow perfusate sarnples were taken at early, middle and late the points during constant flow. Urine sarnples were collected in toto at 3 min intervals. Oudlow pemisate samples were taken at the rnidpoint of urine collection intervals. A schematic representation of the experimental design is presented in Fig. 5-28. Viability in the presence and absence of paraoxon was assessed by values of the GFR. urine flow rate, 8 weight gain, and sodium and glucose reabsorption. Constant FIow (8 rnl/min) Equilibration Period Constant Pressure (90-1 00 mm Hg) Infusion of KRB Infusion of Paraoxon at 0.181 mumin at 0.1 81 ml/min I O-15min 1 15 - 30 min l 30 - 50 min l 1 lnflow Plasma Sampling: early. middle, late time points 1 I Urine Sampling: 5 min intervals I
Constant Flow (8 rnümin) 1 Equilibration Period \ Constant Pressure (90-100 mm Hg) lnfusion of KRB Infusion of Paraoxon at 0.1 81 ml/min at 0.181 mhin O - 20 min l 20 - 35 min I 35 - 50 min I I -- -- 1 lnflow Plasma Sarnpling: early, rniddle, late time points 1 -- I Urine Sampling: 3 min intervals I -- 1 Outflow Plasma Sampling: rnidpoint of urine collection 1
Figure 5-2. Schematic representation of the experimental design for iPK experirnents. (A) For viability studies with paraoxon in the iPK, the kidney was first equilibrated at constant pressure. after which time the flow rate was set constant to 8 mumin. Infusion of KHB dunng the control period is followed by infusion of paraoxon. Inflow plasma was sarnpled at early. middle and late time points.
Urine was collected at 5 min intervals. (B) For the inhibition studies in the iPK. the kidney was first equilibrated at constant pressure in the presence of ['~lenala~riland then perfused at a constant flow rate of 8 mumin. Infusion of KHB for the control period was followed by infusion of paraoxon.
Inflow plasma was sampled at carly, middle and late time points. Urine was collected at 3 min intervals. Outflow plasma was collected at the midpoint of urine collection. 5.39 Thin Layer Chrornatographic Assay for Enalapril and Enalaprilat
['~]~nalapriland [3~enalaprilatwere separated in S9, plasma and urine sarnples by thin layer chrornatograp hy (TLC). Samples containing [3~]enalapriland ['Hlenalaprilat wece spotted ont0 the origin of silica gel GF TLC plates (Analtech Newark, DE) on top of non-radiolabeled enalapril and enalaprilat. After development of the plates in a system of 1-propanol : 1M acetic asid
: water, (10: l:l, vlvh.) visualization of where enalapril and enalaprilat had migrated was possible under ultraviolet light. Bands of TLC maienal containing enalapril and enalaprilat were scraped from the plates, and 0.5 mi of water and 5 ml of scintillation cocktail (Ready Safe, Beckrnan Instruments) were added to the TLC material. Afier the sarnples were mixe& they were kept in the dark for 48 h pnor to scintillation counting. A direct count of radioacûvity in the sarnple was obrained in order to assess the recovery of radioactivity from TLC plates. Concentration (DPM/ml) was calculated by dividing the DPM for endapril or enalaprilat by the product of recovery and volurne plated. For S9 samples. 200 pl of supernatant, obtained following precipitation of the S9 reaction mixture with acetonitrile. was plated directly ont0 the origin of the TLC plate. Then 100 pl of the supernatant was subject to Bcounting. Plasma samples from iPK experirnents were partially precipitated with an equal vo!urne of acetoninile. Afier mixhg and centrifugation. 250 pl of supernatant was applied to the origin of the TLC plate and 50 pl was subjected to direct B-counting. Urine sarnples. obtained from IPK experiments, were plated directly ont0 the TLC plate.
5.3.6 Statistics
Al1 data were expressed as rnean + standard deviation. Statisticai significance was assessed with the paired t-test: a p value of 0.05 was viewed as significant.
5.3.7 Calculations Equations used to calculate the rend extraction ratio (4).total rend clearance (CL,,,,.,), unnary clearance (CL,,). and rend metabolic clearance (CL,,,) were presented in chapter 4 (section 4.3.6). 5.4 Results 5.4.1 S9 Experiments with Paraoxon
The average dilution of kidney tissue (2.18 f0.12 g) after homogenization was 22-fold for the S9 studies, rendering a protein content of 1.45 t 0.35 mglrnl for incubation. In control experiments, the amount of [3~]enalaprilatformed increased Linearly with tirne. with 80.7 t 13.3% of ['~lenalapril remaining in the incubation mixture at the end of 15 min. For the inhibition studies, the amount of ['~]enalaprilatformed in the presence of paraoxon at 15 min. normalized to the protein content of the incubation mixture, was compared to the arnount of ['~]ena.lapnlat formed in controls. The decrease in [3~]enaIaprilatformation was almost complete for paraoxon concentrations of 0.5, 1, and 10 WM (93 f 5%, 96 + 5 and 93 t 6. respectively) (Fig. 5-3). The percent inhibition observed with 0.1 pM paraoxon (76 f 7%) was somewhat lower and significantly less (p < 0.05) dian those afforded by the other higher concenuations of paraoxon.
5.4.2 Partition Coefficient for Enalapril at Various pH Values In the pH range of 6.4 to 8.23, the partition coefficient (ocranol/KHB) of ['~lenalapril (0.0472 + 0.003) was not altered (Fig. 5-4). Previous studies have shown that the pH of urine obtained from the IPK is - 7.4 (de Lannoy et al., 1989). Since the partition coefficient of enalapnl was unchanged within a fairly wide range of pH values encompassing the previously observed pH of urine in the IPK, urinary pH would not Likely alter the extent of enalapril reabsorption in the IPK. 1 O 1 o. 5 0.1 Concentration of Paraoxon (PM)
Figure 5-3. Ic Inhibition of ['H]enaiaprilat formation from ['Hlenalapnl with paraoxon in S9. The
% inhibition of [3H]enaIapriiat formation at 0.1 pM paraoxon is significantly less (p c 0.05) than that observed at 10, 1, and 0.5 pM of paraoxon.
pH of KHB
Figure 5-4. Partition coefficient of ['~lenala~rilat various pH values. The partition coefficient of enalapril was found not to Vary within the range of pH values tested. 5.4.3 IPK experiments 5.4.3.1 Viabiiity in the Presence of Paraoxon
Viability of the IPK was assessed at 1 pM (n=2), 0.5 pM (n=l) and 0.1 pM (n=2) paraoxon, in the absence of ['Hlenalapril. GeneraUy, for aU concentrations of paraoxon tested a decrease in both Na' and glucose reabsorption was observed in the presence of paraoxon (Fig. 5-
5). In conjunction, increases in urine flow rate and GFR were observed. However. the loss of viability Iessened with decreasing concentrations of paraoxon. The increase in weight of the perfùsed right kidney for pemisions conducted with 1. 0.5 and 0. I pM paraoxon was 50 + 12%.
43% and 30 fi 8 of the unperfused Ieft kidney, respectively.
Sodium 0 Glucose
Input Concentration of Paraoxon (PM)
Figure 5-5. 'k Na* and tk Glucose reabsorption in the PK at various concentrations of paraoxon. 5.432 Experiments with [3A]Enalapril and Paraoxon
Results of the S9 (section 5.4.1) and IPK viability study (section 5 -3.3.1) suggested 0.1 yM paraoxon to be a suitable concentration for use in the PK. The concentration inhibited
['~lenalaprilhydrolysis in vitro by 76% and was least deleterious to the viability of the kidney. In these [3~]endaprilIPG, sodium and glucose reabsorption tended to decrease in the presence of
0.1 pM paraoxon as compared to the control period (Table 5- 1 ). However. these differences were not statisticaiiy significant (p > 0.05) and generaiiy conformed to the time-dependent viability changes observed in the IPK (de Lannoy er al., 1989). The increase in GFR was not significant (p
> 0.05). although urine flow rate increased (p c 0.05) during the inhision of 0.1 pM. when compared to the control period. Kidney weight was increased by 28 t 12% in the inhibition studies with O. 1 pMparaoxon. The viability of these PKs was generally maintained in cornparison to that in other IPK preparations (Ross, 1972: Lieberthal et al., 1987; Brezis et al.. 1986). The extraction ratio of ['Hlenalapril in control period (without paraoxon) was 0.37 + 0.09. and was significantly decreased to 0.3 1 f 0.07 (p < 0.05) in the presence of 0.1 piVI paraoxon. This was accompanied by a sigmficant decrease in the total rend clearance of enalapril from 1.87 f 0.46 to 1.57 f 0.41 mUmin/g (p c 0.05) due to a significant decrease in metabolic clearance (from 1.83 + 0.52 to 1.48 f
0.47 mL/rnin/g, p < 0.05). With inhibition of metabolism, the excretory clearance of enalapril was significandy increased from 0.04 + 0.07 to 0.09 + 0.09 (p c 0.05). Likewise the FE value for enalapnl was significantly increased from 0.23 f O. 18 to 0.52 + 0.25 (p < 0.05) in the presence of paraoxon (Table 5- 1).
5.5 Discussion
It was suggested by Smith and Kugler (1994) that metabolism would reduce the FE estimate for a compound which is both metabolized and excreted unchanged by the kidney. Accordingly, the fractional excretion value, the traditional means for assessing the net flux of a dnig through the kidney, is decreased in the presence of renal rnetabolism. The simulation study presented in chapter 3 indeed confmed the notions that renal excretion and metabolism do exert an influence on the clearance estimate of one another and that the FE for enalapnl is decreased in the presence of renal metabolism.
III this chapter, the dynamics of cornpetition between rend metabolism and excretion was illustrated expenmentally with the isolated perfused rat kidney. It was necessary to find an appropriate inhibitor for enalapril esterolysis in the kidney. An obvious choice was panoxon. since it had been used in many studies as an effective inhibitor of carboxylesterase activity (Butterworth et al., 1993: Grima et al., 199 1: Castle. 1988: Brandt et al., 1980). Paraoxon was previously shown to inhibit enalapnl esterolysis in rat kidney tissue (Grima et al., 1991). and its effectiveness was confirmed presently in renal S9 studies with the rat. Paraoxon inhibited enalapnl metabolism by at least 75% among ail four concentrations (0.1 to 10 PM) tested. Since the viability of the IPK tended to be less compromised at the Iowest concentration of paraoxon. 0.1 pM was chosen as the appropriate concentration for use in the iPK inhibition studies with ['~]enalapril. The infusion of paraoxon into the rat kidney indeed resulted in decreased metabolic and total rend clearances and a concomitant increase in the urinary clearance and FE value for enaiapril. thus providing the expenmentai data for hypothesis testing and validation of the results of the simulation snidy presented in chapter 4. Although the FE value was increased, it rernained much less than unity, and the interpretation of net reabsorption for enaiapril thus appeared to be valid. However, metabolism was not completely inhibited and only in the absence of metabolism could the maximum FE value be obtained. Changes in unnary pH throughout perfusion were ruled out as a potential reason for increased urinary clearance in the presence of paraoxon, since the partition coefficient of enalapril was found not to Vary within a wide range of possible urinary pH values (Fig. 5-4). The extent of inhibition observed with 0.1 pM paraoxon in the IPK was less than that observed in the S9 studies. This discrepancy could be attributed to a difference in accessibility of paraoxon to enzymes in S9 as cornpared to that in the intact kidney. In contrast to S9 where enzymes are freely accessible. the kidney possesses bamee that limit entry of paraoxon to the site of metabolism. Altematively, if paraoxon is extensively bound to protein or red blood cells. the concentration of paraoxon entering the renal tissue would be substantiaily decreased. Although the extent of protein and red blood ceil binding of paraoxon is not known. this second hypothesis appears more likely, since paraoxon is quite lipophilic and should not expenence difficulty entering renal epitheLial celis.
An increase in urine flow rate was observed among the IPKs infused with paraoxon. Diuresis could decrease the extent to which a dnig is reabsorbed (Bloomer, 1966). and the increase in urinary clearance could be attributed to reduced reabsorption of enalapril. This. however, is unlikely since a noticeable increase in urinary clearance was observed imrnediately after infusion of paraoxon began. In addition. data obtained by de Lannoy et al. ( 1989) for enalapnl in the IPK was reexamined in order to determine whether urinary clearance was augmented with increased urine flow rate. A rise in urine flow rate is a property associated with the constant flow IPK and it appears that there is sorne dependency of urinary clearance associated with the increased urine flow rate (Fig. 5-6). However, the increase in urinary clearance observed in the presence of paraoxon is greater than that which would be expected in the absence of paraoxon. Fig. 5-6 shows that urinary clearance estimates made in the presence of paraoxon (filled symbols in Fig. 5-6) tend to fa11 above the line drawn through urinary clearance estimates for enalapril in the absence of paraoxon (de
Lannoy et al.. 1989). This suggests that although the urinary clearance of enalapril is somewhat dependent on urine flow rate, the increase observed in the presence of paraoxon exceeds that which would be expected based on urine flow rate alone. Studies in which paraoxon infusion preceded the KHB infusion would have been definitive in clearly demonstrating that the increase in urinary clearance was independent of increased urine flow rate. Unfortunately, paraoxon is an irrevenible esterase inhibitor (Chernnitius et al.. 1992) and so studies of this nature were not possible. IPKrg(KR8) I PKW9@amoxon) lPK#l 1(KRB) 1PK#ll (paraoxon) iPK#l2(KRB) IPK#l2@araoxon) iPK#l3(KRB) IPK#13(paraoxon) IPK#i4(KRB) IPKX t 4(paraoxon) de tannoy-earty Limes de iannay-iater times
Urine Flow Rate (ml/min/g)
Figure 5-6. The relationship between unn~clearance of enalapril and urine flow rate. The urinary clearance of enalapril in the IPK was plotted against urine flow rate (data of de Lannoy er al.. 1989).
A line was drawn through the urinary clearance estimates to provide an estimate of the cxpectrd increase in urinary clearance with urine flow rate. Unnary clearance rstimates for paraoxon experiments in the IPK were plotted against urine flow rate (empty symbols: control period: filled symbols: paraoxon inhision). Increased urinary clearance of enalapril in the presence of paraoxon is generally greater than that observed in IPK experiments without paraoxon (de Lannoy er al.. 1989).
The accepted method of assessing the net flux of drug through the iudney is by means of the FE value, which is calculated by nonnalizing the unbound urinary clearance to GFR. However. it has ken clearly demonstrated, that for a compound which is both metabolized and excreted unchanged by the kidney. the presence of metabolisrn wiil detract from the arnount of drug that is excreted into the urine. This in turn will Iead to a lower FE value, and perhaps to an erroneous interpretation of the rend secretory capacity for that compound. The concept illustrated here with endapril cm be extended to other dmgs which are both metabolized and excreted unchanged by the kidne y.
5.6 Staternent of Siwcance of Chapter 5
The ability of paraoxon, an esterase inhibitor, to inhibit enalapril esterolysis was verified in renal S9. A concentration of O. 1 pMparaoxon was foound suitable for use in the isolated perfused rat kidney for inhibition of renal esterolysis of enalapril. The decreased metabolic clearance and increased urinary clearance and fractionai excretion value provided the experimenta1 proof that rend metabolism reduces the urinary clearance estimate for enalapnl. Chapter 6: Discussion 6.1 Discussion In the preceduig chapters, the relationships between renal metabolic and excretory clearances in the kidney were exarnined. Through use of both simulations. based on a physiological model of the kidney (chapter 3), and experimentation with the isolated pexfused rat kidney, it was shown that metabolism and excretion will compete with each other for the removal of enalapril in the rat kidney. The clearance estimate of the altemate route of elllnination will be altered. Since the site of intrarenai enalapril hydrolysis was deemed to be intiacellular by the set of nonfiltering kidney studies (chapter 4). metabolism and excretion will both decrease the clearance estimate of the alternate, competing pathway. Although it had been suggested previousiy that urinary clearance estimates made in the presence of rend metabolism would not be accurate, the phenornenon had not ken adequately explored. Kugler and Smith (1994) performed a set of simulations based on the presence of competing elimination pathways. However. they used a simplistic mode1 which failed to incorporate the dynamic nature of competing pathways in the kidney. It considered processes of reabsorption, secretion and metabolism occurring in a sequential manner rather than in unison. In addition. their model tended towards intraIumind metabolism, and the possibility of intraceilular metabolism was not considered. However, they extended simulation results to describe the effects of rend metabolism on the renal clearance of enalapril, a compound that is likely metabolized by a carboxylesterase. enzymes which have yet to be identified intraluminally. In light of the shortcornings associated with this simulation study, our present simulations based on a physiologicai model of the renal disposition of enaiapril appear to be superior for predicting and exarnining events on renal removal. The physiologicai model of the kidney utilized for simulation in chapter 3 described the dynamic nature of the processes occurring widiin the kidney. Accordingly, a more accurate assessrnent of competition between rend metabolism and excretion couId be made. In addition the simulation study presented in chapter 3 incorporated two sets of parameters, one descnbing intracellular metabolism of enalapnl and the other descnbing intralurninal rnetabolism of enalapnl. so that a greater insight into the relationship between metabolism and excretion could be gained. The parameters used for simulation were tested for their ability to accurately predict the 82 experimentdy observed clearance estimates of enalapd obtained in the isolated pefised rat kidney
(de Lannoy et al., 1993). The theoretical examination substantiated the fact that metabolism and excretion affect the estimates of each other in the kidney. However. the trends differed between intraluminal and intracellular metabolism. For both models. the excretory clearance decreased in the presence of metabolism. However. divergent trends existed for the influence of excretion on metabolism. The intracellular rnodel predicted a decrease in metabolic clearance in the presence of excretion, while the intraluminal rnodel predicted an increase in the rnetabolic clearance with increased excretion. Differences between the effect of excretion on metabolism with the two models can be attributed to the fact that excretion cornpetes for substrate with intracellular enzymes. whereas excretion into the lumen delivers dmg to the site of metabolism when enzymes are located within the renal tubule. Another difference between the two models was in the extent to which the fractionai excretion value (FE) was increased in the absence of metabolism. For the luminal rnodel. FE increased from a value of less than one, to a value grearer than one. This wodd obscure the interpretation on the mechanism of rend excretion of enalapnl in the kidney from net reabsorption to net secretion. when assessrnent was based solely on the urinary excretion clearance of unchanged drug. With cellular rnetabolism, however. the FE value would be increased but not to a value pater than unity. ln this case net reabsorption of enalapnl by the kidney would be implied both in the presence and absence of metabolism. Experimental results obtained in the isolated perfused rat kidney supported the findings of the simulation study. With inhibition of enaiapril esterolysis by paraoxon. a carboxylesterase inhibitor, the renal metabolic clearance of enalapril decreased significantly, leading to an increase in the excretory clearance. The FE value for enalaprii was also increased with metabolic inhibition. but remained less than unity. However, metabolism was not completely inhibited. Only with complete inhibition of metabolism could the maximal urinary clearance be attained. IPK viability snidies, performed in the presence of various concentrations of paraoxon, demonstrated that with increasing concentrations of paraoxon. kidney viability was compromised. Since cornplete inhibition of esterolysis would have required a higher concentration of paraoxon than that used in the study, it could not have been achieved without reducing viability of the PK. Akhough enalapril was used as a model dmg, the concepts uncovered through simulation and experimentation cm be extended to other dnigs that are both metabolited and excreted unchanged by the kidney. The situation may be more complex for a dmg metabolized by multiple enzymes in the kidney since competition would occur not only between metabolism and excretion. but also among metaboiic pathways. Moms and Pang (1987) demonstrated this competition between enzymes by means of a computer simulation study based on a physiological model of the Liver. They showed that enzymes present within the Liver, that compete for the sarne substrate. wiU alter the extent to which a given metabolite is fomed. In addition to examining clearance concepts in the kidney, the location of intrarenal enalapril esterolysis was identified. Since the simulation results presented in chapter 3 highlighted a difference between intracellular and intraiurninal metabolism, it was necessary to elucidate the site of enalapril metabolism in order to understand the set of accompanying changes in the renal disposition of enalapril. The approaches used included simulation and experimentation in the nonfdtenng isolated perfused rat kidney (NFK), a preparation used to identifj the uptake site of substrate into renal celis (Gillat et al., 1990; Suzuki et al., 1984; Nakane et al., 1978). Absence of glomeruiar filtration in this preparation will disallow access of dmg into the peritubular cell via the basolateral membrane. This perturbation of the isolated perfùsed rat kidney has provided a means of distinguishing between intracellular and intrduminal metabolism in the kidney. Based on simulations of enalapril and enalaprilat data in the NFK as compared to the IPK. it was deterrnined that metabolite data could be used to distinguish whether intracellular or intraluminal metabolism occurs for enalapril in the kidney. With intracellular metabolism. a small increase in the rate out of enalaprilat, norrnalized to the rate in of enaiapril, was predicted. With intraiurninal metabotism a large increase (-300%) was predicted for the enalaprilat rate out. When data for the NFK were compared to those previously obtained from IPK experiments with enalapril (de Lannoy et al..
1989. 1993), the lack of change in enalaprilat rate out suggested the location of enalapril metabolism was intraceilular. The results of this study were supported by the fact that esterase activity has yet to be identified intralurninaily. However, confmation that metabolism of enalapril is not mediated by enzymes embedded on the bmsh border membrane could be obtained in studies involving bmsh border membrane vesicles (Okajima et al., 198 1; Brunette et al.. 1984). 84 In summary, the presented studies clearly demonstrate that the estimates for metabolic or excretory clearances of enalapril are influenced by the presence of one another, implying that the fractional excretion value is in some cases not an ideal indicator for assessing the net flux of drug through the kidney. For a compound which is both metabolized and excreted unchanged by the kidney, the magnitude of change wiU depend on the extent of rend metaboiism. These findings challenge traditionai clearance concepts that do not dow for cornpetition dynamics between eliminaùon pathways in an eliminating organ. Chapter 7: Conclusions 7.1 Conclusions Simulations based on a physiologicd mode1 of the kidney and a set a parameters descnbing the renal handling of enalapril demonstrated that renal metabolism and excretion will alter the clearance estimate of the aiternate, competing pathway. With the assurnption of intraceliular enalapril hydrolysis, metabolism and excretion both decreased the clearance estimate of the other route of elimination. Conversely, simulations based on intraIumina1 metabolism of enaiaprii predicted decreased urinary clearance in the presence of metabolism and increased metabolic clearance in the presence of excretion. Snidies in the nonfiltering isolated perfused nt kidney identified the site of intrarenal enalapril hydrolysis as intracellular. Therefore. both renal metabolism and excretion of enalapril wiil decrease the clearance estirnate of the competing pathway. Inhibition of enalapril esterolysis in the isolated perfused rat kidney with paraoxon exemplified the conclusions drawn from the simulation study since a decrease in metabolism resulted in an increased urinary clearance estimate and fractional excretion value. In conclusion. clearance estimates for the kidney are intricately dependent upon the presence of altemate elimination pathways. nierefore. an interpretation on the net flux of dmg throuph the kidney. based on the urinary clearance estimate is prone to error when made in the presence of renal me tabolism. Chapter 8: References 8.1 References
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