gene expression of prothrombin in the human kidney and its potential relevance to kidney stone...

British Journal of Urology (1998), 81, 666–672

Gene expression of prothrombin in the human kidney and itspotential relevance to kidney stone diseaseA.M.F. S TAPLETON, T.L. TIMME* and R.L. RYALL†Division of Surgery, Repatriation General Hospital, Adelaide, Australia, *Scott Department of Urology, Baylor College of Medicine,Houston, Texas, USA and †Flinders Medical Centre, Adelaide, Australia

Objective To determine whether urinary prothrombin Results Six kidney specimens showed clear evidence ofprothrombin gene expression; expression in the kidneyfragment 1 (UPTF1), which shows considerable promise

as a critical determinant of calcium oxalate (CaOx) was less than that in the liver.Conclusion This is the first demonstration of prothrom-stone formation, is manufactured by the human kidney.

Materials and methods Ribonucleic acid was isolated bin gene expression within the human kidney, afinding that not only has implications for CaOx stonefrom eight kidneys, two spleens and one liver. Using

reverse transcriptase-polymerase chain reaction, disease but also potentially for blood coagulation.Keywords Kidney stones, macromolecules, prothrombin,mRNA corresponding to the UPTF1 portion of pro-

thrombin was analysed by agarose-gel electrophoresis urinary inhibitors, calcium oxalateand Southern blotting.

it was found to be the principal protein occluded withinIntroductionCaOx crystals generated in undiluted urine in vitro [10]. Itis present in CaOx and calcium phosphate stones [11],Calcium oxalate (CaOx) renal lithiasis is a common

disease that aCects almost one in every 10 men in and has significant inhibitory activity when tested inundiluted human urine [8]. Its true identity as UPTF1, aWestern countries during their lifetime. Although there

are well described technologies available for the urinary form of serum fragment 1 (F1) was revealed byN-terminal amino-acid sequence analysis [12] and detailedtreatment of a calculus once clinically evident, our

understanding of the mechanisms that influence stone biochemical and immunological experiments [13].Prothrombin F1 is found in the blood in small quantitiesformation remain comparatively poor. Reasons for this

discrepancy are many and varied, and include the fact and is liberated by the degradation of prothrombin in thecoagulation cascade [14]. It contains at its N-terminus 10that urine, the medium in which stones develop, is an

ever-changing collection of salts, cells, proteins and other carboxyglutamic acid (Gla) residues that confer upon it astriking ability to attract and bind calcium ions in solutionmaterials. Typically, human urine is occasionally super-

saturated with CaOx in every person, prompting the [15]. Almost a decade ago, the addition of F1 to a seeded,inorganic solution was noted to inhibit the rate of growthquestion of why all people do not succumb to stone

disease. The answer is partly because urine contains of hydroxyapatite crystals [16]. Moreover, the results ofthat study suggested that the Gla portion of F1 wasseveral inhibitors of crystal growth and aggregation that

accompany the passage of these salts from the body. responsible for the inhibitory eCects, as another fragmentof prothrombin, prethrombin 1, which lacks Gla, hadOne popular theory regarding the genesis of kidney

stones centres around the failure of such inhibitors [1,2] markedly reduced inhibitory activity.Fragment-1 also has a regulatory role in the pro-to prevent large clumps of crystals from forming. There

is indirect evidence that urinary macromolecules in duction of prothrombin by hepatocytes [17]; F1 is presentin urine, as is F1+2, another prothrombin fragment,general, and proteins in particular, play a significant

role in the prevention of stone disease [3–7]. although the latter has been found in concentrations of# one-tenth those of F1 [18]. The SDS-PAGE gel charac-One protein, urinary prothrombin fragment 1 (UPTF1),

has shown considerable promise as an important inhibitor teristics of UPTF1 diCer from those of F1 [13,19], thusjustifying the use of the name UPTF1 to distinguishof CaOx crystallization [8,9] and is the focus of the current

study. Initially described as crystal matrix protein (CMP), these two fragments. Prothrombin is produced byhepatocytes and has a plasma concentration of0.07–0.1 mg/mL; UPTF1 may therefore be a product ofAccepted for publication 8 January 1998

666 © 1998 British Journal of Urology

PROTHROMBIN AND STONE FORMATION 667

the renal passage of F1. However, it is also possible that antisense primer was 5∞ GCT TGC GAC CTT GAC CATCTT 3∞.UPTF1 could be manufactured in the kidney and released

into the luminal fluid of the collecting system, under the An aliquot of 5 mg of RNA from each sample was firsttreated with RNAse-free DNAse (Pharmacia, Piscataway,control of a site-specific mechanism. UPTF1 has been

detected immunohistochemically in luminal cells of the NJ, USA) in a total volume of 20 mL according to themanufacturer’s instructions, to prevent the potentialdistal convoluted tubule and the thick ascending limb of

the loop of Henle in a proportion of nephrons [20], and contamination from genomic DNA in the amplificationreaction. Next, 16 mL of this product was used as ais therefore well placed to act as a crystallization inhibitor

in concentrated urine downstream of these cells. Notably, template to create cDNA using MMTV reverse tran-scriptase (RTase), while the remaining 4 mL was used inthe expression of UPTF1 is reportedly increased signifi-

cantly in individuals with a history of renal lithiasis, a control reaction without RTase. The final concen-trations of reagents in the RTase-positive reaction (80 mL)although few patients were studied [20]. Therefore, it

was the aim of the present study to determine whether were: 2 mL RNAse inhibitor (Perkin Elmer Cetus,Norwalk, CT, USA), 25 mmol/L pd(N)6 primersUPTF1 is specifically manufactured within the renal

parenchyma. (Pharmacia), 50 mmol/L of each of the four deoxyribonu-cleotide triphosphates, 25 mmol/L MgCl

2, 200 units

RTase and 1×PCR II buCer (Perkin-Elmer). The samplesMaterials and methodswere heated at 22°C for 10 min, 42°C for 15 min, and99°C for 5 min, then cooled to 4°C. Two microlitres of

TissuesRTase-positive product (#0.1 mg cDNA) or 2 mL ofRTase-negative product, were added to a solution withMacroscopically normal human tissue was harvested

fresh, away from the site of known pathology, from a final concentration of 1×PCR buCer (Perkin-Elmer),0.5 mmol/L of both primers from one set for either UPTF1surgical specimens in accordance with guidelines from

the Baylor ABliated Hospitals’ Institutional Review or HPRT, 200 mmol/L for each of the four deoxynucleo-tide triphosphates, and 0.625 Units of Taq polymeraseBoard, snap-frozen in liquid nitrogen and stored at –80°C

before use. There were kidney specimens from eight (Perkin-Elmer) in a final volume of 27 mL. Amplificationwas performed in a model 9600 thermal cycler (Perkinindividuals, spleen from two, pancreas from one and

liver from two. The renal tissue consisted primarily of Elmer), with each cycle consisting of a denaturation stepof 30 s at 94°C, a primer-annealing step of 30 s at 55°Ccortex with some associated medulla, although the rela-

tive ratios in each specimen were not well defined. Tissue and a primer extension-step of 60 s at 72°C for a totalof 30 cycles, followed by a 3 min extension at 72°CRNA was isolated by homogenizing the tissue in guanidi-

nium isothiocyanate, followed by ultracentrifugation before holding at 20°C.The final products (typically 10 mL) were loadedthrough a caesium chloride gradient. RNA was further

purified by phenol-chloroform extraction and ethanol directly on a 3% NuSeive5SeaKem (251) gel (FMCBioProducts, Rockland, ME, USA) containing 5 mg/mLprecipitation. The resultant RNA was quantified using

spectrophotometry at a wavelength of 260 nm. ethidium bromide and electrophoresed at 90 V in40 mmol/L Tris-acetate, 1 mmol/L EDTA, pH 7.8. Analiquot of 250 ng of HaeIII-digested W.5X 174 viral DNA

PCR amplificationwas used to assess molecular weight. The gels werephotographed on a UV transilluminator with PolaroidPCR primer sets were designed such that the products

matched no sequence, as available in GenBank (version type 57 film (Kodak, Rochester, NY, USA) with a Wratten#22 A filter.86), other than that a 145 bp fragment of the amino

terminus of prothrombin (UPTF1) mRNA or a 245 bpfragment of hypoxanthine phosphoribosyl transferase

Southern blotting(HPRT) mRNA. HPRT was chosen as a control to indicatewhether tissue mRNA was present and adequate for Southern-blot analysis of the PCR products of the RTase-

positive and -negative reactions was performed as pre-reverse-transcription (RT) PCR. The PCR primers weresynthesized by Nucleic Acids Core Laboratory, viously described [21] with minor modifications. Briefly,

after photographing the gel containing the PCR products,Department of Molecular and Human Genetics, BaylorCollege of Medicine, Houston, Texas, USA. The sense the gel was denatured in 0.5 mol/L NaOH, 1.5 mol/L

NaCl for 30 min, then after a brief wash in water,strand primer for UPTF1 was 5∞ TTG CTG CAT GTC TGGAAG GTA 3∞ and the antisense primer was 5∞ GGA TGG washed with 1.5 mol/L Tris-HCl, pH 7.8, in 1.5 mol/L

NaCl for an additional 30 min. The DNA was transferredGTA GTG GAG TTG ATT 3∞. The sense primer for HPRTwas 5∞ TTC TTT GCT GAC CTG CTG GAT 3∞ and the overnight to a nylon membrane (BioTrans+, ICN) by

© 1998 British Journal of Urology 81, 666–672

668 A.M.F. STAPLETON, T. L. TIMME a nd R. L. RYALL

capillary action with 20×SSPE. The filter was baked at mRNA in all samples and the reduction of liver substraterequired because of overloading on prior experiments.80°C for 2 h, then hybridized in 7.5% SDS, 0.5 mol/L

Na2HPO

4(pH 7.2), 1 mmol/L EDTA, 4×Denhardt’s solu- High levels of prothrombin gene expression were also

detected in liver samples from other individuals (datation, and 50 mg/mL sonicated salmon sperm DNA at65°C for 2 h. The primers for UPTF1 used in the PCR not shown).reaction were end-labelled with c-32P-ATP using polynu-cleotide kinase and 10 pmoles of oligonucleotide. The Discussionoligonucleotide was purified with a nucleotide-removalkit (Qiagen, Chatsworth, CA, USA) and added directly to Protein gel electrophoretic analyses of organic material

from CaOx crystals generated from human urine in vitrothe blot in the same hybridization solution, where it wasincubated at 65°C for 16 h, washed twice for 20 min reveals UPTF1 as a major constituent [10]. Matrix

extracted from human uroliths containing calcium aseach with 40 mmol/L Na2HPO

4(pH 7.2) and 1% SDS,

then autoradiographed at −80°C for several hours. the predominant mineral cation also contains UPTF1[11]. The protein potently inhibits CaOx crystal growthand, more importantly, aggregation [8], arguably theResultskey event in stone pathogenesis; it has been immunoloca-lized to the distal tubule within a subset of nephrons inThose control samples to which RTase was not added,

and which were run in parallel with the test samples, the human kidney [20]. Evidence that this protein maybe important in stone formation is strengthened by theshowed no appropriately sized band on both agarose-gel

electrophoresis nor any signal upon Southern blotting. finding of increased levels in kidneys from stone formerscompared with those from normal individuals [20], andThe PCR-negative control sample, in which substrate

was substituted with sterile water, remained undetect- the observation that the protein’s amino acid composi-tion diCers between patients with recurrent stoneable in all agarose gels (Fig. 1, lane 14). The 267 bp

fragment corresponding to the mRNA of HPRT (a positive formation and healthy subjects [22]. Being aware thatthe vast majority of the plasma pool of prothrombincontrol) was detectable with mild variation in intensity

in all 12 tissue samples treated with RTase, indicating originates in the liver, we intended to determine whetherthe prothrombin F1 found in urine is simply a conse-that amplifiable mRNA was present in all of these

samples. quence of renal processing of plasma prothrombin or isactively synthesized by the kidney, given that the sub-Following the RTase-positive PCR-based assay, the

expected 145 bp fragment corresponding to the cellular machinery for production of this Gla-containingprotein is present in the kidney [23].N-terminal region of the mRNA of prothrombin was

clearly detectable within the liver sample (lane 13, The present results indicate that prothrombin geneexpression is present within the human kidney. TheFig. 1a). Within the same gel there were faintly positive

bands at 145 bp corresponding to three of the eight agarose gels are shown primarily to indicate that ampli-fiable RNA is present in the samples, seen clearly usingrenal samples examined (lanes 3, 4, 6, Fig. 1a). In the

remaining lanes, which represent samples of spleen (two) the HPRT oligonucleotides. That there are faint bandsin some of the kidney samples using the UPTF1 oligo-and pancreas (one) there were no clear identifying bands

visible. In the adjacent RTase-negative agarose gel there nucleotides is very encouraging, as the sensitivity of thisdirect assay is often inadequate when low copy numberswere DNA smears in several of the lanes that were not

representative of amplified fragments. Importantly in this of the appropriate mRNA are present. There were nodiscrete bands visible in the RTase-negative lanes. Thegel, there was no band visible in lane 13 which contained

the liver sample. Southern blot analyses complement the agarose gels welland remove suspicion of doubt as to the presence ofSouthern-blot analysis of the RTase-positive PCR-

amplified products of prothrombin showed that six of prothrombin mRNA in six of eight kidneys, one spleenand in liver.eight kidney specimens had clear evidence of substrate

(lanes 2–7, Fig. 1b), as did one of two specimens from It is not clear why two of eight kidney extracts werenegative for prothrombin mRNA; they clearly hadspleen (lane 11). The specimen from normal pancreas

was negative (lane 12) and the positive tissue control evidence of amplifiable HPRT mRNA. It is possible thatthese cases represent false-negative results. The precisefrom liver was strongly positive, as anticipated (lane 13).

Although it was not intended to use this technique to amount of renal cortex as opposed to medulla in eachspecimen was not measured, and as the medulla containsquantify the relative amounts of substrate between

samples, it is apparent that prothrombin gene expression less detectable protein [20], the amount of specific mRNAmay have been below the threshold of detection usingfrom liver was significantly higher than that observed

in kidney, given the similar amount of detectable HPRT this methodology for these two cases. Also, the PCR



Fig. 1. In each panel, Lane 1 represents WX174 DNA digested with Hae III, here used asa fragment size marker. Lanes 2–13represent human tissue samples of RNAderived from kidney (lanes 2–9), spleen(lanes 10–11), pancreas (lane 12), and liver(lane 13). Lane 14 represents the PCRnegative control in which water was addedin place of tissue RNA.a, PCR-amplified products identified withethidium bromide in 3% agarose gels ofhuman tissue RNA, treated with (RTase+ve) and without (RTase –ve) reversetranscriptase using primer sets for a 145 bpfragment of the amino region ofprothrombin (and therefore also of F1) anda 267 bp fragment of hypoxanthinephosphoribosyl transferase (HPRT). In theRTase –ve gels, there are no definite bandsto indicate an amplified product; however,in some there is an indeterminate smear.The 145 bp fragment corresponding toprothrombin mRNA was clearly detectablein lane 13 (liver), and less obviously in lanes3, 4 and 6 (kidney). The fragmentcorresponding to mRNA of HPRT wasdetectable in all 12 tissue samples treatedwith RTase, indicating that amplifiablemRNA was present in each specimen tested.b, Southern blot analysis of the RTase+vePCR-amplified products representative ofprothrombin mRNA displayed in a using a32P-labelled probe specific for the N-terminalregion of prothrombin. Six of eight kidneysamples were positive (lanes 2–7), as wasone (lane 11) of two samples of spleen. Thesample from normal pancreas was negative(lane 12). As anticipated, the sample fromnormal liver was strongly positive (lane 13).The RTas −ve PCR products showed noreactivity to the specific probe.

a

b

control fragment (HPRT) is of a diCerent size to the one In feline kidney fibroblast cells there is evidence of amembrane-bound protease that activates prothrombinunder investigation and therefore may not have been

the ideal positive control for amplifiable material within to release thrombin [25], which itself has many diversefunctions, including the alteration of vascular tone,the samples. Pancreatic tissue was used as a likely

negative control, although the prothrombin gene was wound healing and inflammatory reactions [26,27].Such a specific prothrombin activator was also detectedexpressed in this organ, albeit in only one individual.

Although no function has been postulated for prothrom- in multicystic dysplastic kidney cells (dog kidney), Verocells (African green monkey kidney) and RK13 cellsbin gene expression in the pancreas, such expression

within organs other than the liver has been reported (rabbit kidney) [25]. Suzuki et al. [28] have recentlypresented evidence of prothrombin gene expression (F1,previously.

It is possible that renal prothrombin gene expression F2 and thrombin) in the human and the rat kidney.Hence, there is prior evidence that prothrombin can bemay not be limited to its N-terminal portion and that

the complete prothrombin molecule is produced, and activated outside the blood coagulation pathway, andthat prothrombin probably plays a physiological role inpossibly released, into the plasma pool, or indeed the

urinary tract. Using Northern blot analysis, Jamison and vivo, not restricted to the haemostatic process, and whichmay be controlled by site-specific mechanisms.Friezner Degen [24] showed that in rats the prothrombin

gene is expressed in the kidney, although only prenatally. Prothrombin is clearly expressed in the brain [29],



highly selective phenomenon. Clin Chem 1991; 37:where the presence of thrombin has been associated1589–94with inhibition of neurite outgrowth and retraction of

11 Stapleton AMF, Dawson CJ, Grover PK et al. Furtherextended neurites, and is thought to be critical to neuralevidence linking urolithiasis and blood coagulation: urinarydiCerentiation [30,31]. Thrombin may have a site-prothrombin fragment 1 is present in stone matrix. Kidneyspecific function in vivo within the kidney and theInt 1996; 49: 880–8

fragments of prothrombin in urine may be degradation12 Stapleton AMF, Simpson RJ, Ryall RL. Crystal matrix

products produced for purposes other than control of protein is related to human prothrombin. Biochem Biophysurolithiasis within the kidney. If this is so, then equimolar Res Commun 1993; 195: 1199–203amounts of F1 and F1+2 in the urine might be antici- 13 Stapleton A, Ryall R. Blood coagulation proteins andpated; however, that the urinary concentration of F1 is urolithiasis are linked: crystal matrix protein is the F1

activation peptide of human prothrombin. Br J Urol 1995;10 times that of F1+2 mitigates against this possibility.75: 712–9The present findings support the hypothesis that the

14 Davie EW, Fujikawa K, Kisiel W. The coagulation cascade:kidney manufactures the F1 N-terminal region of pro-initiation, maintenance, and regulation. Biochem 1991;thrombin. This protein fragment has many of the features30: 10363–70expected of a macromolecule considered to play a signifi-

15 Burnier JP, Borowski M, Furie BC, Furie B. Gamma-cant role in stone formation and further studies arecarboxyglutamic acid. Mol Cell Biochem 1981; 39: 191–9

clearly warranted to clarify the nature of its role.16 Romberg RW, Werness PG, Riggs BL, Mann KG. Inhibition

of hydroxyapatite crystal growth by bone-specific and othercalcium-binding proteins. Biochemistry 1986; 25: 1176–80Acknowledgements

17 Kisiel W, Hanahan DJ. The action of factor Xa, thrombinThe authors gratefully acknowledge the support from Dr and trypsin on human factor II. Biochim Biophys Acta

1973; 329: 221–32Timothy C. Thompson, PhD, Houston, Texas, USA. Also,18 Bezeaud A, Guillin M-C. Quantitation of prothrombinwe appreciate the provision of human tissues from the

activation products in human urine. Br J Haematol 1984;Tissue Bank, Department of Pathology, Baylor College of58: 597–606Medicine, Houston, Texas, USA.

19 Stapleton AMF. Crystal Matrix Protein and Urolithiasis:Getting Blood Out of a Stone [PhD thesis]. Adelaide: FlindersUniversity of South Australia, 1995

References 20 Stapleton AMF, Seymour AE, Brennan JS, Doyle IR,1 Fleisch H. Inhibitors and promoters of stone formation. Marshall VR, Ryall RL. The immunohistochemical distri-

Kidney Int 1978; 13: 361–71 bution and quantification of crystal matrix protein. Kidney2 Coe FL, Nakagawa Y, Parks JH. Inhibitors within the Int 1993; 44: 817–24

nephron. Am J Kidney Dis 1991; 17: 407–13 21 Thompson TC, Park SH, Timme TL et al. Loss of p533 Leal JJ, Finlayson B. Adsorption of naturally occurring function leads to metastasis in ras+myc-initiated mouse

polymers onto calcium oxalate crystal surfaces. Invest Urol prostate cancer. Oncogene 1995; 10: 869–791977; 14: 278–83 22 Doyle IR. The Role of Urinary Proteins in Calcium Oxalate

4 Blomen LJMJ, Bijvoet OLM. Physicochemical consideration Urolithiasis [PhD thesis]. Adelaide: Flinders University ofin relation to urinary stone formation. World J Urol 1983; South Australia, 19921: 11–25 23 Hauschka P, Friedman P, Traverso H, Gallop P. Vitamin

5 Addadi L, Berkovitch-Yellin Z, Domb N, Gati E, Lahav M, K-dependent c-carboxyglutamic acid formation by kidneyLeiserowitz L. Resolution of conglomerates by stereoselective microsomes in vitro. Biochem Biophys Res Commun 1976;habit modifications. Nature 1982; 296: 21–6 71: 1207–13

6 Nancollas GH, Gardner GL. Kinetics of crystal growth of 24 Jamison CS, Degen SJF. Prenatal and postnatal expressioncalcium oxalate monohydrate. J Crystal Growth 1974; of mRNA coding for rat prothrombin. Biochim Biophys Acta21: 267–76 1991; 1088: 208–16

7 Ryall RL, Stapleton AMF. Urinary macromolecules in 25 Sekiya F, Usui H, Inoue K, Fukudome K, Morita T.calcium oxalate stone and crystal matrix: good, bad, or Activation of prothrombin by a novel membrane-associatedindiCerent. In: Khan SR, ed. Calcium oxalate in biological protease. An alternative pathway for thrombin generationsystems. Boca Raton: CRC Press, 1995: 265–90 independent of the coagulation cascade. J Biol Chem 1994;

8 Ryall R, Grover P, Stapleton A et al. The urinary F1 269: 32441–5activation peptide of human prothrombin is a potent 26 Tapparelli C, Metternich R, Cook NS. Structure and functioninhibitor of calcium oxalate crystallization in undiluted of thrombin receptors. Trends Pharmacol Sci 1993; 14:urine in vitro. Clin Sci 1995; 89: 533–41 426–8

9 Stapleton AMF, Ryall RL. Crystal matrix protein — getting 27 Friezner Degen SJ. The prothrombin gene and its liver-blood out of a stone. Miner Electrol Metab 1994; 20: 399–409 specific expression. Semin Thromb Haem 1992; 18: 230–42

10 Doyle IR, Ryall RL, Marshall VR. Inclusion of proteins into 28 Suzuki K, Suga K, Miyazawa K, Tsugawa R. Where is theorigin of crystal matrix protein? — expression of prothrom-calcium oxalate crystals precipitated from human urine: a



bin mRNA in human and rat kidney. Br J Urol (Supp 2) factors, enzyme inhibitors, bone proteins and plasma1997; 80: 322 proteins for its protection. If there is such a range of

29 Dihanich M, Kaser M, Reinhard E, Cunningham D, Monard ‘potent’ inhibitors available in urine, why do stones formD. Prothrombin mRNA is expressed by cells of the nervous at all and why do they continue to grow in theirsystem. Neuron 1991; 6: 575–81 presence? Surely, patients who form stones cannot be

30 Jalink K, Moolenaar WH. Thrombin receptor activationdeficient in all of these or, alternatively, excrete them in

causes rapid neural cell rounding and neurite retractionforms that are less potent than those excreted byindependent of classic second messages. J Cell Biol 1992;non-stone-formers?118: 411–9

Unquestionably, urine does have some ability to31 Gurwitz D, Cunningham DD. Thrombin modulates andmodify the rate of crystal growth and/or agglomerationreverses neuroblastoma neurite outgrowth. Proc Natl Acad

Sci USA 1988; 85: 3440–4 of CaOx crystals. However, it seems more likely that thismay be due to the combined eCorts of a fortuitouscollection of excretory products (which share thecommon property of being highly negatively chargedAuthorsand having a strong aBnity for calcium ions whether inA.M.F. Stapleton, FRACS, PhD, Consultant Urologist.solution or on the surface of crystals of calcium salts)T.L. Timme, PhD, Assistant Professor.rather than to a single ‘Compound X’. Indeed, most ofR.L. Ryall, PhD, Professor of Surgery.the molecules listed above involve calcium for theirCorrespondence: Dr A.M.F. Stapleton, Repatriation General

Hospital, Daw Park, SA 5041, Australia. activity in the body. Perhaps they are only present asadventitious inclusions in stones, attracted secondarilyby the calcium ions on the crystal surface or in theEditorial commenthydration layer surrounding the crystals? Moreover, asmany of the macromolecular inhibitors occur in bloodIn this paper, the authors have clearly identified gene

expression for prothrombin in the human kidney. This or in renal tubular cells, is it more likely that theirpresence in calcium stones is attributable either tocomplements their previous observation that a fragment

of prothrombin is not only capable of inhibiting the periods of microhaematuria or to cellular damage causedby the crystals or stones themselves, and that theircrystallization of CaOx in vitro but is also present in the

matrix of calcium-containing kidney stones. Their con- absence from stones not containing calcium is becausesuch stones lack the calcium necessary to attract andclusion from these findings is that this fragment may

play a role in controlling an individual’s ability to form attach them?There are also other problems with the inhibitor-CaOx-containing stones in his/her urinary tract.

Thus, another macromolecule is added to the ever control theory. For example, how do inhibitors accountfor the observations that: (i) the incidence of stones hasincreasing list of candidates for the role of the elusive

‘Compound X’ that, it is claimed, will eventually explain generally increased in most countries since 1945 andthe peaks and troughs in stone incidence noted duringwhy some people form stones while others do not. Apart

from the low molecular weight inhibitors of crystalliz- that time precisely reflect periods of economic boom andrecession respectively; (ii) there is a diCerence in theation such as citrate, pyrophosphate, ADP, ATP, phos-

phocitrate and at least two phosphopeptides, there is prevalence of stone disease between countries that paral-lels the level of aAuence within each country; (iii)now a dazzling array of macromolecules including vari-

ous glycosaminoglycans, Tamm-Horsfall mucoprotein military and naval personnel form more stones whenthey are moved from a temperate to a hot, sunny(now referred to as ‘uromodulin’), nephrocalcin, various

plasma proteins, uropontin (or osteopontin), a- environment; (iv) there is a seasonal variation in stoneincidence within a given population; (v) men form more1-microglobulin, b-2-microglobulin, urinary prothrom-

bin fragment 1, inter-a-inhibitor and numerous other calcium-containing stones than women and adults formmore stones than children; (vi) fluctuations in dietaryexotic entities. From every part of the human anatomy

inhibitors apparently rain down on the kidney to protect habits appear to influence the risk of stones in thepopulation.it from forming calcium-containing stones (but curiously,

none of the other types of stone!). Further inhibitors are Whereas all of these observations have been shownto be accounted for by fluctuations in the supersaturationclaimed to be added in the bladder.

One might be forgiven for asking first, why the human of urine with one or both calcium-containing salts, itwould be surprising if they were also mirrored by inversebody should apparently have developed so many protec-

tive systems against a disorder that rarely causes death changes in blood-clotting factors, bone proteins, enzymeinhibitors, plasma proteins, etc. This raises the questionand second, why the kidney should rely on such appar-

ently unrelated biochemical entities as blood-clotting of whether the proposed protective inhibitors are



generally inadequate to deal with increases in the pre- citrate, Tamm Horsfall mucoprotein and, possibly,nephrocalcin as credible candidates for ‘Compound X’.vailing supersaturation and level of crystalluria in urine

and, if so, how can they then be considered to ‘control’ This paper may add one other to the list.stone-formation?

If I am ever to be persuaded that one particularinhibitor is specifically designed to control stone forma- W.G. Robertson

Institute of Urology and Nephrologytion, it would seem reasonable that the kidney would bethe most likely organ involved in its production. This UCL London

UKwould limit the current list considerably, leaving only


gene expression of prothrombin in the human kidney and its potential relevance to kidney stone...

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