Introduction

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The kidney functions as an organ of drug elimination that can remove drugs from the blood by glomerular filtration and tubular secretion. Renal tubule organic cation secretion involves transport-mediated passage from the peritubular capillaries, across the basolateral membrane, and into the tubule cell, followed by transport into the tubule lumen, across the luminal membrane. In the kidney, numerous endogenous or exogenous organic cations can be secreted by the renal tubules (Rennick, 1981).

An abundance of information pertaining to the renal tubule organic cation transport system comes from studies with tetraethylammonium (TEA) as a prototypical organic cation substrate. Original studies demonstrated that TEA is secreted by the proximal tubules of the nephron (Rennick and Moe, 1960). Most subsequent studies have focused on the proximal tubule as the only component of the nephron that has the capacity for transport of organic cations, with little attention given to distal tubules and their ability to transport TEA, or other organic cations. Transport of TEA across the basolateral membrane of proximal tubules is a saturable, energy-dependent, carrier-mediated process that is driven by the inside negative membrane potential and is independent of pH (Takano et al., 1984; Sokol and McKinney, 1990). Efflux from the tubule cell into the lumen is mediated by a saturable H⁺/organic cation exchanger that uses a proton gradient derived from the Na⁺/H⁺ exchanger also located in the luminal membrane (Takano et al., 1984; Rafizadeh et al., 1987). However, the TEA model alone may not be sufficient to account for the renal tubule secretion pathways of other organic cations.

Amantadine, a clinically used organic cation drug, is eliminated by the kidneys, and renal tubule secretion is important in this process (Bleidner et al., 1965; Aoki et al., 1979). Amantadine has been used extensively in our laboratory as a prototypical substrate for characterizing the mechanisms of renal tubule organic cation transport. The mechanisms controlling amantadine secretion by the kidney appear to be different than for TEA. Amantadine transport and accumulation have been demonstrated in proximal and distal tubules of male and female rats, with transport properties being heterogeneous within tubules, between tubules, and between sexes (Wong et al., 1991-1993; Escobar et al., 1994, 1995; Escobar and Sitar, 1995, 1996). It is believed that these findings are representative of amantadine influx via an energy-dependent saturable component of the basolateral membrane (Escobar and Sitar, 1995). Transport sites for amantadine in proximal and distal tubules can be subdivided into bicarbonate-dependent (high-affinity, high-capacity) sites responsible for most of the amantadine uptake and less efficient bicarbonate-independent (lower affinity, lower capacity) sites (Escobar et al., 1994; Escobar and Sitar, 1995). Although a bicarbonate-dependent basolateral transport component has been reported for the cation N¹-methylnicotinamide (NMN) (Ullrich et al., 1991), a similar transport phenomenon has not been demonstrated for TEA. Membrane potential and activity of the basolateral membrane Na⁺/K⁺-ATPase are not rate limiting for the renal tubule uptake of amantadine (Escobar and Sitar, 1995, 1996). These findings suggest that most amantadine uptake occurs as a nonelectrogenic step at the basolateral membrane as opposed to electrogenic uptake for TEA. Considering the apparent differences in amantadine and TEA renal tubule transport characteristics, we hypothesize that amantadine and TEA are selective for distinct basolateral membrane organic cation transporters in the kidney. Identifying distinct renal organic cation transporters and their substrate specificity by using TEA and amantadine as organic cation probes may allow for the prediction of potential drug interactions in the kidney.

	Materials and Methods

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Renal Tubule Preparation. Experimental procedures involving the use of animals have been approved by the University of Manitoba Protocol Management and Review Committee. Separation of proximal and distal tubules was performed by the Percoll density-gradient centrifugation method (Vinay et al., 1981; Gesek et al., 1987) as modified by Wong et al. (1990) and current modifications to improve the tissue preparation. The modified procedures are as follows: Four male Sprague-Dawley rats (Charles River breeding stock; University of Manitoba, Canada) weighing 250 to 300 g were anesthetized with pentobarbital sodium (50 mg/kg). Kidneys were removed, immediately decapsulated, and then placed in ice-cold Krebs-Henseleit solution (KHS) (pH 7.4). KHS contained 118 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 1.4 mM KH₂PO₄, 25 mM NaHCO₃, 2.5 mM CaCl₂, and 11 mM glucose. Renal cortical sections were then dissected from the medullary tissue approximately 1 mm from the corticomedullary junction and placed in ice-cold KHS buffer (20 ml). Next, the cortical sections were finely minced with a tissue chopper (Mickle Lab. Engineering Co. Ltd., Gomshall, Surrey, UK). Minced tissue was placed in 10 ml of cold KHS and added to a KHS-collagenase solution containing 15 ml of KHS, 1 ml of 10% BSA, and 10 mg of low trypsin collagenase A (0.23 U/mg lysozyme) and oxygenated for 2 min with 95% O₂/5% CO₂.

Amantadine and TEA Transport Studies. Linear rates for the energy-dependent renal tubule uptake of amantadine and TEA were determined in the presence (KHS buffer) and absence (CT buffer) of bicarbonate. For the amantadine transport assays, tubes were prepared (in triplicate) that contained a fixed amount of [³H]amantadine (1 nM) and unlabeled amantadine (final assay concentrations 10-500 µM) in a volume of 150 µl of either KHS or CT buffer. Proximal or distal tubule suspensions (50 µl in the appropriate buffer) were added to each assay tube to begin the transport reaction. After addition of the tubule suspension, the assay tubes were incubated for 30 s in a 25°C water bath with shaking (100 oscillations/min). The reactions were terminated by addition of 2 × 4 ml of ice-cold KHS, followed by rapid filtration under negative pressure, through glass fiber filters (no. 32; Schleicher & Schuell, Inc., Keene, NH). The filters were immediately placed into scintillation vials containing 4 ml of Ready Safe scintillation fluid (Beckman Instruments Inc., Fullerton, CA) and counted in a Beckman model LS5801 scintillation counter.

Inhibition Studies. Inhibition of [¹⁴C]TEA (10 µM) energy-dependent uptake by unlabeled amantadine and NMN (10-1000 µM) and inhibition of [³H]amantadine uptake (10 µM) by TEA and NMN (10-1000 µM) were determined in proximal and distal tubules in KHS and CT buffers (pH 7.4). The same procedures as described for the transport assays were used for the inhibitor studies.

Chemicals. [³H]Amantadine (28 Ci/mmol) was obtained from Amersham International (Buckinghamshire, UK). [¹⁴C]TEA was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Collagenase was obtained from Boehringer Mannheim (Laval, Quebec, Canada). Unlabeled amantadine was obtained from Dupont Canada Inc. (Mississauga, Ontario, Canada). Unlabeled TEA and NMN were obtained from Sigma Chemical Co. All other chemicals were of the highest grade available from commercial suppliers.

Data Analysis. For individual experiments, each data point for the transport and inhibition studies was performed in triplicate. Data are expressed as means ± S.E. of at least four experiments. Transport rates are reported as specific uptake (nonspecific uptake subtracted) of amantadine or TEA by the tubules in nanomoles per milligram of protein per minute. Apparent K_m and V_max values were determined by nonlinear regression fit to the Michaelis-Menten equation with a nonlinear regression program (WinNonlin version 1.1; Pharsight Corp., Palo Alto, CA). IC₅₀ values were determined from the amantadine inhibition profiles by regressive probit analysis of increasing inhibitor concentrations (Cheng and Prusoff, 1973). Dixon (1953) and Cornish-Bowden (1974) analyses were used to determine the nature of inhibition. K_m and V_max data from these experiments were analyzed by a two-way ANOVA model with the factors buffer (bicarbonate versus nonbicarbonate) and tubule (proximal versus distal). Observed transport rates for inhibition data were compared within tubule group with the repeated measures ANOVA model. Multiple comparisons of the significant ANOVA were performed by Tukey's honestly significant difference (HSD) test. Differences between means with p  .05 were considered significant. All statistical analyses were performed with Systat for Windows 6.0.1 (SPSS Inc., Chicago, IL).

	Results

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Amantadine and TEA Transport Studies. Data characterizing the degree of separation of proximal and distal tubules with our methodology have been previously reported in detail by our laboratory (Wong et al., 1991, 1993; Escobar et al., 1994; Escobar and Sitar, 1995, 1996). Visual enumeration of the tubules in either fraction by hemocytometry indicates purity of 80%. Enzyme assays demonstrate that alkaline phosphatase activity is dominant in proximal tubules, whereas hexokinase activity is dominant in distal tubules, which is consistent with their reported distribution in the nephron (Scholer and Edelman, 1979; Vinay et al., 1981; Gesek et al., 1987). TEA uptake was linear for at least 60 s (r² for linear regression ranged from 0.89 to 0.98) in proximal tubules and distal tubules in the presence and absence of bicarbonate, as shown in Fig. 1. Amantadine uptake into proximal and distal tubules was linear for at least 30 s (data not shown), as previously reported by our laboratory (Wong et al., 1990). Both proximal and distal tubule segments accumulated [¹⁴C]TEA and [³H]amantadine in a saturable manner (data not shown). Eadie-Hofstee plots for energy-dependent TEA uptake versus concentration are shown in Fig. 2. The biphasic nature of the plots reveals that TEA uptake into proximal and distal tubules may be characterized by two transport sites, a high-affinity transport site and a lower affinity transport site. TEA concentrations of 10 to 60 µM were used to characterize the high-affinity TEA transport site, and concentrations of 100 to 500 µM were used to characterize the lower affinity transport site. Figure 3 shows K_mTEA1 and V_maxTEA1 (K_m and V_max for TEA uptake at the high-affinity site). K_mTEA1 and V_maxTEA1 were similar in CT and KHS buffers in both proximal and distal tubules. The lack of a difference in kinetic parameters between buffer groups allowed us to combine the data from each buffer group such that we could increase the power (n = 16, compared to 8) of detecting a difference in K_m or V_max between proximal and distal tubules. Comparing proximal and distal tubules when data from both buffer groups were combined indicated that K_mTEA1 was less (higher affinity) in proximal (33.4 ± 4.8 µM) than in distal (49.4 ± 4.8 µM) tubules (p < .05). The difference in V_maxTEA1 in proximal tubules (0.295 ± 0.034 nmol · mg¹ · min¹) compared with distal tubules (0.209 ± 0.034 nmol · mg¹ · min¹) approached significance (p < .08). For the observed difference between mean proximal and distal tubule V_maxTEA1, a power calculation with grouped standard deviation (0.133 nmol · mg¹ · min¹) indicated that at least 37 replicates would be required to detect a significant difference when symbol 97 = 0.05 and symbol 98 = 0.20 and thus was not pursued. For the lower-affinity site, K_mTEA2 and V_maxTEA2 were similar between tubule fragments and were not dependent on the presence of bicarbonate in the medium (Fig. 4). K_m and V_max for the lower affinity TEA uptake sites were 5- to 10-fold and 3- to 4-fold greater, respectively, than for the high-affinity sites.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Representative plots of a single experiment showing TEA (10 µM) uptake versus time into isolated renal cortical proximal (top) and distal (bottom) tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. Total TEA uptake is expressed as nanomoles per milligram of protein.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Eadie-Hofstee plots for rate of TEA uptake into isolated renal cortical proximal (top) and distal (bottom) tubules. v, rate of TEA uptake (nmol · mg1 protein · min1); [s], concentration of TEA (µM). Each data point represents the mean ± S.E. of six to eight separate determinations.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Calculated apparent Km (top) and Vmax (bottom) values for [14C]TEA uptake by high-affinity transport site in proximal and distal tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. TEA concentrations used in each experiment were 10, 20, 35, and 60 µM. Values are means ± S.E. from eight separate determinations. Treatment groups were compared by two-way ANOVA, with tubule and buffer as the grouping variables. *p < .05, p < .08, proximal tubules versus distal tubules when data from KHS and CT groups are combined.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Calculated apparent Km (top) and Vmax (bottom) values for [14C]TEA uptake by lower affinity transport site in proximal and distal tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. TEA concentrations used in each experiment were 100, 200, 300, and 500 µM. Values are means ± S.E. of four to six separate determinations. Treatment groups were compared by two-way ANOVA, with tubule and buffer as the grouping variables. Km and Vmax for the proposed low-affinity site were not dependent on tubule type, buffer, or tubule-buffer interactions. p > .1.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Measured apparent Km (top) and Vmax (bottom) values for [3H]amantadine uptake by isolated renal proximal and distal tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. Amantadine concentrations used in each experiment were 10, 20, 50, 100, 300, and 500 µM. Values are means ± S.E. of four separate determinations. Treatment groups were compared by two-way ANOVA, with tubule and buffer as the grouping variables. ***p < .001 versus KHS within tubule group. p < .001, proximal versus distal tubule within same buffer group.

Inhibition Studies. We first evaluated the ability of TEA to inhibit the energy-dependent renal tubule uptake of amantadine (Fig. 6). TEA concentrations ranging from 10 to 1000 µM were unable to impede the uptake of amantadine into isolated renal proximal and distal tubules in bicarbonate or phosphate buffer at pH 7.4. Conversely, amantadine was able to inhibit TEA uptake into proximal and distal tubules (p < .05) compared with the respective control (Fig. 7). The inhibition profiles were similar in bicarbonate and phosphate buffers.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   TEA inhibition of 10 µM amantadine uptake into isolated renal cortical proximal and distal tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. Values are means ± S.E. of four separate determinations.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Amantadine inhibition of 10 µM TEA uptake into isolated renal cortical proximal (top) and distal (bottom) tubules in the presence (KHS) and absence (CT) of bicarbonate at pH 7.4. Values are means ± S.E. of five to seven separate determinations. *p < .05, **p < .01, ***p < .001, versus control (no amantadine present); repeated measures ANOVA followed by Tukey's HSD test.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   NMN inhibition of amantadine (squares) and TEA (circles) uptake into proximal (top) and distal (bottom) tubules. Assays were performed in KHS (solid symbols) and CT buffers (open symbols) buffers at pH 7.4. Values are means ± S.E. of four separate determinations. *p < .05, **p < .01, ***p < .001, versus respective amantadine or TEA control; repeated measures ANOVA followed by Tukey's HSD test.

	Discussion

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Amantadine uptake into proximal and distal tubules from the rat could be described by a high-affinity-capacity, bicarbonate-dependent transport site and a lower affinity-capacity, bicarbonate-independent transport site and is concordant with previous studies (Escobar et al., 1994; Escobar and Sitar, 1995). The K_mA and V_maxA values reported herein were somewhat higher than those reported by Escobar et al. (1994). However, the same qualitative effects on amantadine transport were maintained, namely, a decrease in V_max and an increase in K_m in the absence of bicarbonate.

The uptake of TEA into isolated proximal and distal tubules was best characterized by a high-affinity, low-capacity component and a lower affinity, higher-capacity component. To the best of our knowledge, this is the first report to describe energy-dependent distal tubule transport of TEA. Unlike amantadine, the uptake of TEA at these two sites was similar in the presence and absence of bicarbonate. Most other studies have described only a single transport site for TEA, with widely varying affinity (Schali et al., 1983; Takano et al., 1984; Wright and Wunz, 1987; McKinney et al., 1990; Ullrich et al., 1991; Takami et al., 1998). The one exception (Grundemann et al., 1997) demonstrated a high-affinity component (20 µM) and a low-affinity component (620 µM) for TEA uptake by the OCT2p transporter transfected into human 293 cells. We believe that the failure of others to detect two transport sites for TEA relates to the selection of TEA concentrations used in attempts to characterize its transport properties. The difference in K_mTEA1 and possibly V_maxTEA1 between proximal and distal tubules suggests that the composition of transporters involved in uptake of TEA is different between the tubule segments, with a higher affinity-capacity component existing in the proximal tubules. The observed differences in the bicarbonate effect on amantadine versus TEA transport further support the division of basolateral membrane organic cation transporters into those that are bicarbonate dependent and those that are independent (Escobar et al., 1994).

The inhibition studies showed that TEA could not inhibit amantadine uptake. The highest concentration of TEA (1000 µM) was greater than the high- and lower-affinity K_m values for TEA uptake in proximal and distal tubules. This saturating concentration of TEA would be expected to inhibit amantadine uptake if the two compounds entered the tubules via identical transporters. Conversely, amantadine can block TEA uptake completely. The fact that amantadine inhibited TEA uptake but TEA did not inhibit amantadine uptake suggests that 1) TEA is not transported and does not interact with the bicarbonate-dependent and bicarbonate-independent amantadine transporters; 2) amantadine interacts with the TEA transporters but is not transported; or 3) TEA transporters also transport amantadine, but the proportion of uptake at this site is so small that any inhibition of amantadine uptake by TEA is masked by the larger bicarbonate-dependent amantadine transport component.

In addition to TEA, NMN has been used to characterize organic cation transport in the kidney (Kinsella et al., 1979; Holohan and Ross, 1980). NMN has been demonstrated to inhibit renal tubule transport of TEA (Montrose-Rafizadeh et al., 1989; Ullrich et al., 1991), but interactions with amantadine uptake have not been reported. The fact that NMN does not inhibit amantadine uptake in proximal tubules at concentrations that inhibit TEA uptake strengthens the argument that TEA and amantadine may characterize different transport sites.

The distinctness of the organic cation transport sites for TEA and amantadine are further supported by the difference in K_m for amantadine uptake versus the K_i for amantadine inhibition of TEA transport. Theoretically, the K_m/K_i ratio should be near 1 when TEA transport sites are the same as those previously identified for amantadine and competitive inhibition is assumed. The observed K_m/K_i ratio is substantially greater than 1 for both proximal and distal tubules and is greater in CT than in KHS buffer. Thus, the bicarbonate-dependent and bicarbonate-independent amantadine transport sites may be different from those described by TEA.

Our previous model to explain organic cation transport by renal proximal tubules (Escobar and Sitar, 1995) has been revised to reflect the findings of this study (Fig. 9). Transport site 1 in Fig. 9 represents the high-affinity-capacity, bicarbonate-dependent amantadine transporter responsible for approximately 80% of basolateral amantadine uptake into the tubule cell. Transport site 2 represents a lower affinity-capacity, bicarbonate-independent amantadine transporter responsible for about 20% of amantadine uptake. Our data show that TEA is not a substrate for transport site 1 or 2. Basolateral uptake of TEA may be best characterized by two additional bicarbonate-independent transport sitesa high-affinity, low-capacity site (site 3) and a lower affinity, higher-capacity site (site 4). Sites 3 and 4 may also represent higher-affinity, lower capacity transport sites for amantadine, as identified by amantadine inhibition of TEA uptake. In proximal tubules, NMN appears to interact with the TEA transport sites but not the amantadine transport sites. On the luminal membrane, exit of TEA is mediated by an H⁺/organic cation exchanger (transport site 5) that uses the H⁺ gradient (out  in) created by the Na⁺/H⁺ exchanger (Takano et al., 1984; Rafizadeh et al., 1987). Transport of certain organic cations (not including TEA) across the luminal membrane may also be mediated by the ATP-dependent P-glycoprotein (transport site 7) (Dutt et al., 1994). Further studies are necessary to evaluate whether the mechanisms of luminal transport of amantadine are similar to those of TEA and whether amantadine is a substrate for P-glycoprotein. Our data suggest that similar organic cation transport mechanisms for amantadine and TEA exist in the distal tubule. However, for both amantadine and TEA, existing in vivo evidence is insufficient to determine the relative contribution of the proximal and distal tubules to total renal secretion of these compounds.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Revised model for organic cation transport by rat renal proximal tubules when amantadine and TEA are used as prototypical substrates to characterize this system. Site 1, high-affinity-capacity, bicarbonate-dependent amantadine transporter. Site 2, low-affinity-capacity, bicarbonate-independent amantadine transporter. Site 3, high-affinity, low-capacity TEA transporter (inhibited by amantadine). Site 4, low-affinity, high-capacity TEA transporter (possibly inhibited by amantadine). Site 5, luminal membrane Na+/H+ exchanger. Site 6, H+/organic cation exchanger. Site 7, P-glycoprotein. A+, amantadine; OC+, organic cation; (+), activation; (), inhibition.

rOCT1, rOCT1a, rOCT2, and rOCT3 are members of the organic cation transporter family that are expressed in the rat kidney and have been demonstrated to transport TEA (Grundemann et al., 1994; Okuda et al., 1996; Zhang et al., 1997; Kekuda et al., 1998). Of the cloned rat organic cation transporters, rOCT1 is thought to be localized in basolateral membranes of S1 rat proximal tubule segments (Koepsell, 1998). rOCT2 is located in the basolateral membranes of S2 and S3 rat proximal tubule segments and possibly distal tubules (Koepsell, 1998). Our data probably reflect TEA uptake by at least two of these transporters. For the proposed high-affinity TEA transport site, our K_mTEA1 values (33 µM for proximal tubules and 49 µM for distal tubules) closely correspond to those estimated for rOCT1a (42 µM) expressed in Xenopus oocytes (Zhang et al., 1997) and rOCT1 (38 µM) and rOCT2 (42 µM) expressed in MDCK dog kidney distal tubule cell lines (Urakami et al., 1998). For the proposed lower affinity TEA transport site, our K_mTEA2 values (246-331 µM) were intermediate between those reported for TEA uptake by rOCT1 (K_m = 95 µM) and rOCT2 (K_m = 500 µM) (Grundemann et al., 1994; Koepsell, 1998). The estimated K_m for TEA uptake by rOCT3 was 2.5 mM by tracer uptake studies (Kekuda et al., 1998). Thus, rOCT3 should not be a major contributor to TEA uptake in our preparation. Our K_mTEA1 and K_mTEA2 values are similar to those reported for rOCT1, rOCT1a and rOCT2 and suggest that basolateral TEA uptake into isolated renal tubules may be mediated by some combination of uptake by these transporters. The fact that expression of rOCT1 and rOCT2 in Xenopus oocytes (Grundemann et al., 1994; Koepsell, 1998) and MDCK cells (Urakami et al., 1998) gives different estimates of K_m suggests the cell expression system used may influence kinetic determinations. It is not known which expression system compares best to that of normal renal tubule cells. Therefore, our ability to identify the two TEA transporters detected in this study is limited. Because TEA does not inhibit amantadine uptake, rOCT1, rOCT1a, and rOCT2 may be excluded as the bicarbonate-dependent amantadine transporters. This hypothesis remains to be tested by evaluating the ability of specific inhibitors of rOCT1 and rOCT2 to block amantadine and TEA uptake into the renal tubule preparations.

NKT, NLT, and RST are kidney-expressed proteins that are highly homologous to the OCT family and have been speculated to transport organic cations (Simonson et al., 1994; Mori et al., 1997; Lopez-Nieto et al., 1997). These proteins may contribute to amantadine transport in the kidney.

In summary, it has been proposed that the basolateral uptake of type 1 (small, more hydrophilic) organic cations in the proximal tubule is mediated by a single, multispecific, saturable component of the membrane, namely, OCT1 (Koepsell, 1998). Use of different prototypical type 1 organic cation substrates (amantadine versus TEA) gives a vastly different depiction of organic cation transport in the kidney. This discrepancy suggests that multiple transporters with dissimilar controlling mechanisms may mediate the renal tubule basolateral transport of type 1 organic cations in proximal and distal tubules. Considering the observed differences in amantadine and TEA renal tubule transport, we conclude that amantadine and TEA identify distinct organic cation transport sites in the kidney. Identification of substrate specificity for the different renal organic cation transporters may enable the prediction of potential drug interactions in the kidney.