The Kidney as Excretory Organ

Most drugs are eliminated in urine ei-ther chemically unchanged or as metab-olites. The kidney permits eliminationbecause the vascular wall structure inthe region of the glomerular capillaries allows unimpeded passage of bloodsolutes having molecular weights (MW)5000. Filtration diminishes progres-sively as MW increases from 5000 to70000 and ceases at MW 70000. Withfew exceptions, therapeutically useddrugs and their metabolites have muchsmaller molecular weights and can,therefore, undergo glomerular filtra-tion, i.e., pass from blood into primaryurine. Separating the capillary endothe-lium from the tubular epithelium, thebasal membrane consists of chargedglycoproteins and acts as a filtrationbarrier for high-molecular-weight sub-stances. The relative density of this bar-rier depends on the electrical charge ofmolecules that attempt to permeate it.Apart from glomerular filtration, drugs present in blood may passinto urine by active secretion. Certaincations and anions are secreted by theepithelium of the proximal tubules intothe tubular fluid via special, energy-consuming transport systems. Thesetransport systems have a limited capac-ity. When several substrates are presentsimultaneously, competition for thecarrier may occur.During passage down the renal tu-bule, urinary volume shrinks more than100-fold; accordingly, there is a corre-sponding concentration of filtered drugor drug metabolites. The resultingconcentration gradient between urineand interstitial fluid is preserved in thecase of drugs incapable of permeatingthe tubular epithelium. However, withlipophilic drugs the concentration gra-dient will favor reabsorption of the fil-tered molecules. In this case, reabsorp-tion is not based on an active processbut results instead from passive diffu-sion. Accordingly, for protonated sub-stances, the extent of reabsorption isdependent upon urinary pH or the degree of dissociation. The degree of disso-ciation varies as a function of the uri-nary pH and the pKa, which representsthe pH value at which half of the sub-stance exists in protonated (or unproto-nated) form. This relationship is graphi-cally illustrated with the example ofa protonated amine having a pKa of 7.0.In this case, at urinary pH 7.0, 50 % of theamine will be present in the protonated,hydrophilic,membrane-impermeantform (blue dots), whereas the other half,representing the uncharged amine(orange dots), can leave the tubular lu-men in accordance with the resultingconcentration gradient. If the pKa of anamine is higher (pKa = 7.5) or lower (pKa= 6.5), a correspondingly smaller orlarger proportion of the amine will bepresent in the uncharged, reabsorbableform. Lowering or raising urinary pH byhalf a pH unit would result in analogouschanges for an amine having a pKa of7.0.The same considerations hold foracidic molecules, with the importantdifference that alkalinization of theurine (increased pH) will promote thedeprotonization of -COOH groups andthus impede reabsorption. Intentionalalteration in urinary pH can be used inintoxications with proton-acceptor sub-stances in order to hasten elimination ofthe toxin (alkalinizationphenobarbi-tal; acidification amphetamine).

Enterohepatic Cycle

After an orally ingested drug has beenabsorbed from the gut, it is transportedvia the portal blood to the liver, where itcan be conjugated to glucuronic or sul-furic acid (shown in B for salicylic acidand deacetylated bisacodyl, respective-ly) or to other organic acids. At the pH ofbody fluids, these acids are predomi-nantly ionized; the negative charge con-fers high polarity upon the conjugateddrug molecule and, hence, low mem-brane penetrability. The conjugatedproducts may pass from hepatocyte intobiliary fluid and from there back intothe intestine. O-glucuronides can becleaved by bacterial inthe colon, enabling the liberated drugmolecule to be reabsorbed. The entero-hepatic cycle acts to trap drugs in thebody. However, conjugated productsenter not only the bile but also theblood. Glucuronides with a molecularweight (MW) > 300 preferentially passinto the blood, while those with MW >300 enter the bile to a larger extent.Glucuronides circulating in the bloodundergo glomerular filtration in the kid-ney and are excreted in urine becausetheir decreased lipophilicity preventstubular reabsorption.Drugs that are subject to enterohe-patic cycling are, therefore, excretedslowly. Pertinent examples include digi-toxin and acidic nonsteroidal anti-in-flammatory agents.Conjugations (B)The most important of phase II conjuga-tion reactions is glucuronidation. Thisreaction does not proceed spontaneous-ly, but requires the activated form ofglucuronic acid, namely glucuronic aciduridine diphosphate. Microsomal glucu-ronyl transferases link the activatedglucuronic acid with an acceptor mole-cule. When the latter is a phenol or alco-hol, an ether glucuronide will beformed. In the case of carboxyl-bearingmolecules, an ester glucuronide is theresult. All of these are O-glucuronides.

Amines may form N-glucuronides that,unlike O-glucuronides, are resistant tobacterial Soluble cytoplasmic sulfotrans-ferases conjugate activated sulfate (3’-phosphoadenine-5’-phosphosulfate)with alcohols and phenols. The conju-gates are acids, as in the case of glucuro-nides. In this respect, they differ fromconjugates formed by acetyltransfe-rases from activated acetate (acetyl-coenzyme A) and an alcohol or a phenol.Acyltransferases are involved in theconjugation of the amino acids glycineor glutamine with carboxylic acids. Inthese cases, an amide bond is formedbetween the carboxyl groups of the ac-ceptor and the amino group of the do-nor molecule (e.g., formation of salicyl-uric acid from salicylic acid and glycine).The acidic group of glycine or glutamineremains free.

Biotransformation of Drugs

Many drugs undergo chemical modifi-cation in the body (biotransformation).Most frequently, this process entails aloss of biological activity and an in-crease in hydrophilicity (water solubil-ity), thereby promoting elimination viathe renal route. Since rapid drugelimination improves accuracy in titrat-ing the therapeutic concentration, drugsare often designed with built-in weaklinks. Ester bonds are such links, beingsubject to hydrolysis by the ubiquitousesterases. Hydrolytic cleavages, alongwith oxidations, reductions, alkylations,and dealkylations, constitute Phase I re-actions of drug metabolism. These reac-tions subsume all metabolic processesapt to alter drug molecules chemicallyand take place chiefly in the liver. InPhase II (synthetic) reactions, conju-gation products of either the drug itselfor its Phase I metabolites are formed, forinstance, with glucuronic or sulfuric ac-id.The special case of the endogenoustransmitter acetylcholine illustrateswell the high velocity of ester hydroly-sis. Acetylcholine is broken down at itssites of release and action by acetylchol-inesterase so rapidly as tonegate its therapeutic use. Hydrolysis ofother esters catalyzed by various este-rases is slower, though relatively fast incomparison with other biotransforma-tions. The local anesthetic, procaine, is acase in point; it exerts its action at thesite of application while being largelydevoid of undesirable effects at other lo-cations because it is inactivated by hy-drolysis during absorption from its siteof application.Ester hydrolysis does not invariablylead to inactive metabolites, as exempli-fied by acetylsalicylic acid. The cleavageproduct, salicylic acid, retains phar-macological activity. In certain cases,drugs are administered in the form ofesters in order to facilitate absorption(enalaprilenalaprilate; testosteroneundecanoatetestosterone) or to re-duce irritation of the gastrointestinal mucosa (erythromycin succinateerythromycin). In these cases, the esteritself is not active, but the cleavageproduct is. Thus, an inactive precursoror prodrug is applied, formation of theactive molecule occurring only after hy-drolysis in the blood.Some drugs possessing amidebonds, such as prilocaine, and of course,peptides, can be hydrolyzed by pepti-dases and inactivated in this manner.Peptidases are also of pharmacologicalinterest because they are responsiblefor the formation of highly reactivecleavage products andpotent mediators frombiologically inactive peptides.Peptidases exhibit some substrateselectivity and can be selectively inhib-ited, as exemplified by the formation ofangiotensin II, whose actions inter aliainclude vasoconstriction. Angiotensin IIis formed from angiotensin I by cleavageof the C-terminal dipeptide histidylleu-cine. Hydrolysis is catalyzed by “angio-tensin-converting enzyme”. Pep-tide analogues such as captopril block this enzyme. Angiotensin II is de-graded by angiotensinase A, which clipsoff the N-terminal asparagine residue.The product, angiotensin III, lacks vaso-constrictor activity.

The Liver as an Excretory Organ

As the chief organ of drug biotransfor-mation, the liver is richly supplied withblood, of which 1100 mL is receivedeach minute from the intestinesthrough the portal vein and 350 mLthrough the hepatic artery, comprisingnearly 1/3 of cardiac output. The bloodcontent of hepatic vessels and sinusoidsamounts to 500 mL. Due to the widen-ing of the portal lumen, intrahepaticblood flow decelerates. Moreover,the endothelial lining of hepatic sinu-soids contains pores largeenough to permit rapid exit of plasmaproteins. Thus, blood and hepatic paren-chyma are able to maintain intimatecontact and intensive exchange of sub-stances, which is further facilitated bymicrovilli covering the hepatocyte sur-faces abutting Disse’s spaces.The hepatocyte secretes biliaryfluid into the bile canaliculi (darkgreen), tubular intercellular clefts thatare sealed off from the blood spaces bytight junctions. Secretory activity in thehepatocytes results in movement offluid towards the canalicular space.The hepatocyte has an abundance of en-zymes carrying out metabolic functions.These are localized in part in mitochon-dria, in part on the membranes of therough (rER) or smooth (sER) endoplas-mic reticulum.Enzymes of the sER play a most im-portant role in drug biotransformation.At this site, molecular oxygen is used inoxidative reactions. Because these en-zymes can catalyze either hydroxylationor oxidative cleavage of -N-C- or -O-C-bonds, they are referred to as “mixed-function” oxidases or hydroxylases. Theessential component of this enzymesystem is cytochrome P450, which in itsoxidized state binds drug substrates (R-H). The FeIII-P450-RH binary complex isfirst reduced by NADPH, then forms theternarycomplex,O2-FeII-P450-RH,which accepts a second electron and fi-nally disintegrates into FeIII-P450, oneequivalent of H2O, and hydroxylateddrug (R-OH).

Compared with hydrophilic drugsnot undergoing transport, lipophilicdrugs are more rapidly taken up fromthe blood into hepatocytes and morereadily gain access to mixed-functionoxidases embedded in sER membranes.For instance, a drug having lipophilicityby virtue of an aromatic substituent(phenyl ring) can be hydroxylatedand, thus, become more hydrophilic. Besides oxi-dases, sER also contains reductases andglucuronyl transferases. The latter con-jugate glucuronic acid with hydroxyl,carboxyl, amine, and amide groups; hence, also phenolic products ofphase I metabolism (Phase II conjuga-tion). Phase I and Phase II metabolitescan be transported back into the blood— probably via a gradient-dependentcarrier — or actively secreted into bile.Prolonged exposure to certain sub-strates, such as phenobarbital, carbama-zepine, rifampicin results in a prolifera-tion of sER membranes.This enzyme induction, a load-depen-dent hypertrophy, affects equally all en-zymes localized on sER membranes. En-zyme induction leads to acceleratedbiotransformation, not only of the in-ducing agent but also of other drugs (aform of drug interaction). With contin-ued exposure, induction develops in afew days, resulting in an increase in re-action velocity, maximally 2–3 fold, thatdisappears after removal of the induc-ing agent.

External Barriers of the Body

Prior to its uptake into the blood (i.e.,during absorption), a drug has to over-come barriers that demarcate the bodyfrom its surroundings, i.e., separate theinternal milieu from the external mi-lieu. These boundaries are formed bythe skin and mucous membranes.When absorption takes place in thegut (enteral absorption), the intestinalepithelium is the barrier. This single-layered epithelium is made up of ente-rocytes and mucus-producing gobletcells. On their luminal side, these cellsare joined together by zonulae occlu-dentes (indicated by black dots in the in-set, bottom left).


A zonula occludens ortight junction is a region in which thephospholipid membranes of two cellsestablish close contact and becomejoined via integral membrane proteins(semicircular inset, left center). The re-gion of fusion surrounds each cell like aring, so that neighboring cells are weld-ed together in a continuous belt. In thismanner, an unbroken phospholipidlayer is formed (yellow area in the sche-matic drawing, bottom left) and acts asa continuous barrier between the twospaces separated by the cell layer – inthe case of the gut, the intestinal lumen(dark blue) and the interstitial space(light blue).

The efficiency with whichsuch a barrier restricts exchange of sub-stances can be increased by arrangingthese occluding junctions in multiplearrays, as for instance in the endotheli-um of cerebral blood vessels. The con-necting proteins (connexins) further-more serve to restrict mixing of otherfunctional membrane proteins (ionpumps, ion channels) that occupy spe-cific areas of the cell membrane.This phospholipid bilayer repre-sents the intestinal mucosa-blood bar-rier that a drug must cross during its en-teral absorption. Eligible drugs are thosewhose physicochemical properties al-low permeation through the lipophilicmembrane interior (yellow) or that aresubject to a special carrier transportmechanism.

Absorption of such drugs proceeds rapidly, because the absorbingsurface is greatly enlarged due to theformation of the epithelial brush border(submicroscopic foldings of the plasma-lemma). The absorbability of a drug ischaracterized by the absorption quo-tient, that is, the amount absorbed di-vided by the amount in the gut availablefor absorption.In the respiratory tract, cilia-bear-ing epithelial cells are also joined on theluminal side by zonulae occludentes, sothat the bronchial space and the inter-stitium are separated by a continuousphospholipid barrier.With sublingual or buccal applica-tion, a drug encounters the non-kerati-nized, multilayered squamous epitheli-um of the oral mucosa.

Here, the cellsestablish punctate contacts with eachother in the form of desmosomes (notshown); however, these do not seal theintercellular clefts. Instead, the cellshave the property of sequestering phos-pholipid-containing membrane frag-ments that assemble into layers withinthe extracellular space (semicircular in-set, center right). In this manner, a con-tinuous phospholipid barrier arises alsoinside squamous epithelia, although atan extracellular location, unlike that ofintestinal epithelia. A similar barrierprinciple operates in the multilayeredkeratinized squamous epithelium of theouter skin. The presence of a continu-ous phospholipid layer means thatsquamous epithelia will permit passageof lipophilic drugs only, i.e., agents ca-pable of diffusing through phospholipidmembranes, with the epithelial thick-ness determining the extent and speedof absorption. In addition, cutaneous ab-sorption is impeded by the keratinlayer, the stratum corneum, which isvery unevenly developed in various are-as of the skin.

SDS-PAGE and Western bolt analysis

Tachyzoites from three genotypic strains（RH，ME49-PLK and VEG）were lysed using M-PER Mammalian Protein Extraction Reagent(Pierce,Rockford,IL)containing 10 µg/ml each of antipain,leupeptin,chymostatin,pepstatin A,phenylmethylsulfonylfluoride and phenanthroline (Sigma,Dt.Louis MO).Aliquots equivalent to 15×106 tachyzoiteswere subjected to 12% SDS-PAGE under reducing conditions and transferred to nitrocellulose.Following transfer,theblots were blocked with 10% horse serum and then incubated overnight with anti-TgPCNA1 OR 2 mouse polyclonal sera(1:2500).Antigen detection was accomplished by incubating blots with alkaline phosphatase-conjugated anti-mouse IgG（Promega，Madison WIS）diluted 1:7,500,followed by development in nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate.

ResultsCloning and sequence comparison of the PCNA genes in T.gondii

Analysis of EST database entries along with conventional cDNA strategies identified two unique PCNA cDNAs in T.gondii,designated TgPCNA1 and TgPCNA2.The 951bp open reading frame of TgPCNA1 predicts a protein of 316 amino acids with a predicted molecular weight of 34,476 daltons.The TgPCNA2 clone contains a smaller open reading frame of 864 bp encoding a protein of 261 amino acids and a predicted molecular weight of 32,263 daltons.Consistent with all known eukaryotis PCNAs,the TgPCNA sequences contain basic helix-loop-helix DNA binding motifs(Fig.2-1)that demonstrate >50% similarity to the equivalent region in human PCNA[8].Other conserved motifs include the poltmerase-δ and p21 putative binding sites (D-SHV-,Fig.2-1)as well as the C-terminal sequences (-F/YLAP,Fig.2-1)that appear essential for proper folding. PfPCNA1 contains 10 and PfPCNA2 contains 9 additional C-terminal amino acdis while T.gondii PCNA1 and 2 contain C-terminal insertions of 53 and 28 amino acids.respectively.

Medical reports

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