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Kidney International, Vol. 48 (1995),pp. 690—697

Cellularlocalization of tissue and kallistatin mRNAs in human kidney

LI-MET CHEN, QING SONG, LEE CHAO, and JULIE CHAO

Department of Biochemistty and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA

Cellular localization of tissue kallikrein and kallistatin mRNAs in kidney, tissue kallikrein is localized at the following sites: reab- human kidney. The renal kallikrein-kinin system has been implicated in sorption droplets of the proximal and distal tubules [6], renal the regulation of blood pressure and sodium/water excretion. The activity of renal kallikrein is controlled by a number of factors in vivo. Kallistatin interstitium [7], connecting tubule cells [8] and collecting ducts is a newly identified serine proteinase inhibitor (serpin) which binds to [9]. Also, Vio, Figueroa and Caorsi [10] reported that tissue tissue kallikrein and inhibits its enzymatic activity in vitro. To understand kallikrein is localized in the connecting tubules, but not in the the role of kallistatin in modulating tissue kallikrein's function in vivo, we proximal tubules. Immunoreactive low molecular weight kinino- examined the anatomical relationship between human tissue kallikrein and kallistatin in the kidney by in situ hybridization histochemistry. Tissue gen has been localized in the distal tubules and collecting ducts of kallikrein and kallistatin gene transcripts were identified using digoxige- human kidney [8, 11]. Anatomical co-localization of kallikrein and nm-labeled riboprobes at the cellular level. Antisense and sense ribo- the substrate in the kidney provides the basis for a probes corresponding to the 3' region of the human kallikrein andfunctional role of intrarenal kinins in the regulation of blood kallistatin mRNAs were synthesized by in vitro transcription and used for hybridization. Using an antisense kallikrein riboprobe, sites of kallikrein pressure and electrolyte balance in the kidney. However, little synthesis were localized in the distal tubules, collecting ducts and Henle's information is known regarding the cellular localization of human loops of the kidney. To a lesser degree, juxtaglomerular cells were also tissue kallikrein mRNA in the kidney. stained. Kallistatin mRNA was found at the same sites where kallikrein Tissue kallikrein levels are regulated at both the transcriptional mRNA was localized. The most intense signals of both kallikrein and kallistatin were seen in the distal tubules and collecting ducts. Hybridiza- and post-translational levels [4, 5]. In search of endogenous tion was specific for the target mRNA since sense kallikrein or kallistatin protein factors modulating tissue kallikrein activity and function, riboprobe did not bind to the sections. Immunoreactive human renal we have recently isolated and cloned kallikrein-binding proteins in kallikrein and kallistatin levels were measured in the kidney and urine by humans, rats and mice. The kallikrein-binding proteins, or KBPs, immunoassays using specific antibodies. Co-localization of kallikrein and belong to the serine proteinase inhibitor (serpin) superfamily. The kallistatin mRNA in the kidney suggests a potential role of kallistatin in regulating tissue kallikrein's function. human kallikrein-binding protein gene displays the highest se- quence homology (46.4%) with human al-antichymotrypsin at the nucleotide level [12]. The genes encoding rat and mouse kallikrein-binding proteins also belong to multigene families of Tissue (EC 3.4.21.35) belong to a subgroup of highlyserpins, which are highly homologous to the rodent al-antichy- homologous serine proteinases which liberate kinin peptides frommotrypsin(s) or mouse contrapsin [13]. The physiological function kininogen substrates by limited proteolysis [1—3]. Kinins mediateof kallikrein-binding proteins is not known at the present time. a broad spectrum of biological effects including vasodilation,However, reduced expression of kallikrein-binding protein in inflammation, pain, and smooth muscle contraction and relax-spontaneously hypertensive rats (SHR) suggests that the kal- ation. Localization of tissue kallikrein and/or its mRNA has beenlikrein-binding protein may play an important role in the blood shown in various mammalian tissues including kidney, salivarypressure regulation in SHR [14]. glands, pancreas, brain, pituitary gland, skeletal muscle, heart and The kallikrein-binding protein in humans is designated as vasculature [3, 4]. The pattern of tissue-specific expression iskallistatin or HKBP [151. It is distinct from other serpins with a different among kallikrein gene family members [4, 5]. Theunique reactive site dipeptide Pi-Pi' of Phe-Ser [12]. It rapidly apparent substrate specificity of individual kallikreins suggestsforms an initial reversible complex with tissue kallikrein, which that kallikrein gene family members process various polypeptidebecomes an irreversible SDS-stable complex. Interaction of kal- precursors in addition to kininogen [3, 5] and may be synthesizedlistatin with kallikrein results in the inhibition of kallikrein activity in a coordinated, tissue-specific manner with their particular[15, 16]. The expression of kallistatin has been found in several prohormone substrates. tissues including the kidney, aorta, liver, pancreas, stomach, colon, Previous immunohistochemical studies showed that in humantestis and prostate gland [12], Studies of cellular localization of kallistatin and its anatomical relationship with tissue kallikrein and/or kininogen in human kidney and other tissues will lead to an Received for publication August 9, 1994 understanding of the physiological role of kallistatin. and in revised form April 26, 1995 In the present study, we localized kallikrein and kallistatin Accepted for publication April 27, 1995 mRNAs in human kidney by in situ hybridization histochemistry. © 1995 by the International Society of Nephrology By using their respective antisense riboprobes we found that both

690 Chen et at: Renal kallikrein and kallistatin mRNAs 691

kallikrein and kallistatin are intensively localized in the distalchange. The sections were then incubated with sheep anti- tubules and collecting ducts of human kidney. digoxigenin Fab fragments which were conjugated to alkaline phosphatase (1:500, Nucleic Acid Detection Kit, Boehringer- Methods Mannheim, Germany). The color reaction was performed by Human kidney sections incubating the sections with 4.5 jil of NBT (nitroblue tetrazolium salt), 3.5 jil of X-Phosphate (5-bromo-4-chloro-3-indolyl phos- Normal human kidney paraffin blocks were cut into 5 j.mphate), and 0.24 mg levamisole in 1 ml of 0.1 M Tris-HCI, pH 9.5, sections which were mounted onto gelatin-coated slides. Thecontaining 0.1 M NaC1, and 50 mvt MgCl2. Control sections were tissue was collected within six hours after death. Normal humanhybridized with either labeled sense riboprobes or labeled anti- kidneys were collected from autopsy for immunoassays. sense riboprobes following RNase A treatment [10 jig/mI RNase A in phosphate-buffered saline (PBS), pH 7.2 at 37°C for 30 minI. Preparation of riboprobes A human tissue kallikrein full-length eDNA of 871 base pairs Tissue extraction and urine collection (bp) [17] was generated using reverse-transcription-polymerase Normal human kidney was minced and homogenized in PBS chain reaction (RT-PCR). This amplified eDNA was cloned intousing a polytron. The homogenate was centrifuged at 600 X g for the pSP73 vector containing the SP6 and T7 viral promoters,20 minutes at 4°C. Sodium deoxycholate (Sigma) was added to the which flank the polycloning site. Tissue kallikrein/pSP73 plasmidsupernatant to a final concentration of 0.5% (wt/vol). The solution DNA was digested with ApaI/EcoRI (one in the vector and one inwas incubated at room temperature for 30 minutes followed by the insert) restriction and recircularized by self-ligation.centrifugation at 20,000 x g for 30 minutes at 4°C. The superna- The resulting tissue kallikrein fragment (186 bp) contains 139 bptant was collected and the protein concentration was determined of the coding sequence and 47 bp of non-coding sequence in theas described previously [18] using bovine serum albumin as the 3' region of the tissue kallikrein eDNA. A kallistatin eDNA ofstandard. 1284 bp [12] was cloned similarly by RT-PCR into the pSP73 Human urine was collected over a 24 hour period and then vector. The kallistatin/pSP73 plasmid DNA was digested withcentrifuged at 600 x g for 15 minutes at 4°C. The supernatant was PstI/HindIII (one in the vector and one in the insert) restrictioncollected and stored at —20°C for analysis. enzymes and recircularized by self-ligation. The resulting kallista- tin cDNA fragment contains the last 153 bp of coding sequence in Human urinary kallikrein radioimmunoassay (RIA) the 3' region. The DNA was then linearized by single Purified human urinary kallikrein (HUK) was labeled with 125! digestion and gel purified. using the lodogen (Pierce) method. Radioimmunoassay was In vitro transcription was performed using an RNA Labeling Kitcarried out as previously described [19]. Purified HUK standard (SP6IT7) (Boehringer-Mannheim). One microgram of linear tem-(8 to 1000 pg), serially diluted human kidney extracts and urine, in plate DNA was used in each labeling reaction. The labelinga total volume of 100 p1, were mixed with 200 p1 of rabbit reaction components including transcription buffer, NTP sub-anti-HUK polyclonal antibodies (1:400,000 dilution) and 10,000 strates, SP6 or T7 RNA polymerase were mixed with the linear-cpm of 1251-HUK in 100 p1 of phosphate-buffered saline contain- ized DNA. The mixture was incubated at 37°C for two hours.ing 1% bovine serum albumin. Following incubation, antibody- During the transcription, digoxigenin-labeled uridine-triphos-bound kallikrein was precipitated by the polyethylene glycol phate (DIG-UTP) was incorporated into the transcription prod-method and separated from free kallikrein by centrifugation at ucts and was the target for anti-digoxigenin Fab fragments during5000 rpm, 4°C for 30 minutes. the detection step. The transcribed riboprobes were precipitated by 70% ethanol, dissolved in 100 l diethyl pyrocarbonate-treated Enzyme-linked immunosorbent assay (ELISA) water and stored at —80°C. A 96-well microplate was coated with non-labeled kallistatin IgG (1 jig/ml in PBS, 100 p1/well) at 4°C for overnight. Before In situ hybridization histochemistry samples were added, the plate was blocked with 200 p1 of PBS Human kidney sections were pretreated with xylene to removecontaining 1% of bovine serum albumin at 37°C for one hour and the paraffin and rehydrated through a series of decreasing ethanolwashed three times with the washing solution (PBS containing solutions (100%, 100%, 95%, 70%, 50%). Tissue sections were0.1% of Tween-20). Kallistatin standard (0.4 ng to 25 ng/ml), digested with proteinase K (5 jg/ml in 0.1 M Tris-HCI, pH 8.0,human urine and kidney extracts, which were diluted in freshly containing 50 mtvi EDTA) for 25 minutes at 37°C and incubatedmade dilution buffer (PBS containing 0.05% Tween-20 and 0.5% for 10 minutes in acetic anhydride (0.25% in 0.1 M triethano-gelatin) in a total volume of 100 p1, were added into each well. lamine, pH 7.0). The sections were then dehydrated through aThe sample addition was done in duplicate. The plate was series of increasing ethanol solutions. Hybridization was carriedincubated at 37°C for 1.5 hours and washed three times with the out overnight at 42°C in a buffer containing 0.3 M NaC1, 20 mMwashing solution. Biotin-labeled kallistatin IgG [20] was added Tris-HC1, pH 8.0, 5 mrvi EDTA, 50% formamide, 5 X Denhardt'sinto each well at a concentration of 1 jig/mI in a total volume of (50 >< Denhardt's solution =1%Ficol, 1% polyvinylpyrrolidone, 100 pi. The plate was incubated at 37°C for one hour and washed and 1% bovine serum albumin), 10% dextran sulfate, 100 jig/mI ofthree times with the washing solution. Peroxidase-avidin at a herring sperm DNA and yeast tRNA and antisense riboprobes.concentration of 1 jig/mi, 100 p1/well (diluted in the dilution After hybridization, the kidney sections were washed at 4 X SSCbuffer) was added and incubated at 37°C for 30 minutes. The plate (10 X SSC =3M NaCI, 0.3 M sodium citrate, pH 7.0) at roomwas washed five times with the washing solution and once with temperature for one hour with one change, 2 X SSC for 10PBS. Freshly made substrate solution [10 ml of 0.1 M of citrate minutes with one change, and 0.2 x SSC for one hour with oneacid, pH 4.3, 3 mg of 2,2'-azinobis(3-ethylbenzthiazoline sulfonic 692 Chenet al: Renal kallikrein and kallistatin mRNAs acid), and 10 1.il of 3% H202] was added into each well (100likrein and kallistatin in human kidney suggests that kallistatin islIwelI). After 30 minutes the plate was read with a ELISA readermay play a role in regulating kidney function by modulating at 414 nm for absorbance. kallikrein activity. In human kidney, tissue kallikrein mRNA was localized in- Results tensely in the distal tubules and collecting ducts. This result is Cellular localization of tissue kallikrein mRNA in human kidney consistent with that of detecting immunoreactive tissue kallikrein by immunohistochemistry [8—10]. A small amount of staining was Using digoxigenin-labeled antisense tissue kallikrein riboprobe, tissue kallikrein mRNA was localized primarily in the distalobserved in the Bowman's capsule and the proximal tubules at the urinary pole. The low level staining in these regions may result convoluted tubules (D) and collecting ducts (CD) (Fig. 1A). Kallikrein mRNA was also seen in some juxtaglomerular arte-from either a low level expression of tissue kallikrein or difficulties rioles (JG). To a lesser degree, staining was observed in epithelialof the riboprobes to access their targets. Staining was also seen in cells of glomerular capillaries. Occasionally, the Bowman's cap-some juxtaglomerular arterioles and epithelial cells of glomerular sule and the proximal tubules at the urinary pole were stained. Incapillaries. Localization of tissue kallikrein mRNA in juxtaglo- the medulla (Fig. 1B), collecting ducts (CD), thin segments ofmerular cells and glomeruli suggests that kallikrein may play a Henle's loops (H) and distal straight tubules (DST) were stained.role in the regulation of glomerular filtration rate, blood flow and Control sections of the cortex and medulla were not stained whenrenin generation for mantaining body fluid homeostasis. probed with digoxigenin-labeled sense kallikrein riboprobe (Fig. 3 Previous studies showed that tissue kallikrein mRNA was A, B) or when pretreated with RNase A and then probed withlocalized in the rat renal cortex at the vascular pole of the antisense kallikrein riboprobe (data not shown). glomeruli using a rat tissue kallikrein eDNA probe [21]. Experi- ments with isolated rat glomeruli confirmed that a member of the Cellular localization of kallistatin mRNA in human kidney kallikrein gene family is present in the glomeruli [22]. Positive Figure 2 shows the localization of kallistatin mRNA in the distalsignals observed in the rat glomeruli using tissue kallikrein cDNA convoluted tubules (D), collecting ducts (CD), thin segments ofprobe may be attributed to the cross-hybridization of the probe Henle's loops (H) and distal straight tubules (DST) using anti-with other rat kallikrein gene family members. The riboprobe sense kallistatin riboprobe. The majority of staining occurred inused in detecting human tissue kallikrein mRNA is specific. It has the distal convoluted and straight tubules and collecting ducts.—67% nucleotide sequence homology to the other two kallikrein Similar to the tissue kallikrein mRNA localization, kallistatingene members, hKLK2(hGK1)or hKLK3(prostatespecific mRNA was seen in some juxtaglomerular arterioles (JG). A smallantigen, PSA). The major synthesis site of PSA and hGK1 was amount of staining was observed in epithelial cells of glomerularfound to be only in the prostate gland [23—25]. The signals capillaries, the Bowman's capsule and the proximal tubules at theobtained by binding of antisense tissue kallikrein riboprobe in the urinary pole. Control sections were not stained when probed withkidney by in situ hybridization are not likely attributed to either digoxigenin-labeled sense kallistatin riboprobe (Figs. 3A and 3B)hKLK2orhKLK3 under such stringent hybridizing and washing or pretreated with RNase A and then probed with antisenseconditions used in this study. kallistatin riboprobe (data not shown). Previously we detected kallistatin gene expression in cultured primary human renal proximal tubular cells using RT-PCR fol- Immunoreactive renal tissue kallikrein and kallistatin lowed by Southern blot analysis [12]. In the present study by in situ Immunoreactive kallikrein levels in human urine and kidneyhydridization, kallistatin mRNA was also identified in the proxi- were quantified by RIA using specific antibodies to tissue kal-mal tubules with a low amount of staining compared to that in the likrein [191. Kallistatin levels were quantified by an ELISA usingdistal tubules and collecting ducts. The low signal in the proximal specific antibodies to kallistatin [15]. Figure 4 shows the standardtubules by the antisense riboprobe may be due to the fact that it curve of kallistatin and serial dilutions of human urine and kidneyis less sensitive than PCR in detecting gene expression. Another extracts. Dose-dependent curves of human urine and kidneypossibility is that cultured primary renal tubular cells may contain extracts are parallel with the kallistatin standard curve, indicatingsome distal tubular cells. Identification of the site of synthesis of their immunological identity. The results of tissue kallikrein andkallistatin in the kidney in the vicinity of the tissue kallikrein kallistatin levels in human urine and kidney are summarized inmRNA provides evidence that kallistatin may function in vivo to Table 1. Tissue kallikrein levels are 216.6 15.2 pg/mg proteinmodulate tissue kallikrein's activity or bioavailability. (mean SE, N =3)in the kidney and 267.8 30.7 pg/day (mean Our studies showed that human urine and kidney contain SE, N =11)in urine. Kallistatin levels are 130.79.3 ng/mgimmunoreactive tissue kallikrein and kallistatin. The quantity of protein (mean SE, N =3)in the kidney and 13.62.7 gg/daytissue kallikrein or kallistatin measured in RIA or ELISA repre- (mean SE, N 13) in urine. The results show that renal andsents total immunoreactive kallikrein or kallistatin including free urinary kallikrein and kallistatin can be quantified. and bound forms. The excretion of human tissue kallikrein and kallistatin in urine indicates the renal origin of urinary kallikrein Discussion and kallistatin. This hypothesis is further supported by locating In the present study, we localized tissue kallikrein and kallista-tissue kallikrein and kallistatin mRNA in the distal tubules and tin mRNA in human kidney using digoxigenin-labeled specificcollecting ducts. Table 1 shows the relatively low tissue kallikrein antisense riboprobes by in situ hybridization histochemistry. Thelevels, compared to that of kallistatin, in the kidney. This may be results show that kallistatin mRNA has a similar cellular distribu-attributed to rapid secretion of kallikrein into the urine. The same tion with that of tissue kallikrein in the distal tubules andphenomenon was observed in the synthesis and secretion of other collecting ducts of human kidney. Co-localization of tissue kal-secretory proteins such as rat kallikrein-binding protein, rat Chen et al: Renal kallikrein and kallistatin mRNAs 693

Fig.1. Cellular localization of human tissue kallikrein mRNA in the kidney. The section was hybridized with digoxigenin-labeled antisense tissue kallikrein riboprobe and detected with anti-digoxigenin Fab fragments. Tissue kallikrein mRNA is seen in the distal convoluted tubules (D) and distal straight tubules (DST), collecting ducts (CD), juxtaglomerular arterioles (JO) and thin segments of Henle's loops (H). The majority of the signal is seen in the distal tubules and collecting ducts. G: glomerulus. A. Cortex. B. Medulla (magnification X200). kininogen and al-antitrypsin [141.Thesethree hepatocyte proteins It has been shown that human urinary (or renal) kallikrein have high mRNA levels in the liver but their protein levels in the liver levels are reduced in hypertensive patients [26] and that high are very low since they are rapidly secreted into the circulation. urinary kallikrein activity is associated with a protective effect hypertensive rats [28,29].Inaddition,rattissue kallikreinhas excretion hasalsobeendescribed inanumberofgenetically against hypertension27I.Reduced urinarykallikreinorkininbeenlinkedwithblood pressure regulationbyrestrictionfragment Hcnle's loops(H).Darkerstaining isseeninthedistaltubulesandcollectingducts.0:glomerulus.A. Cortex;B.Medulla(magnificationX200). localized inthedistalconvoluted tubules (D),distalstraight(DST),collectingducts(CD),juxtaglomerular arterioles(JO),thinsegmentsof Fig. 2.Cellularlocalizationofhuman kallistatinmRNAinthekidney.Usingdigoxigenin-labeledantisense kallistatinriboprobe,mRNAis 694

1 #rt'•s - If .4%. , Chen et al:RenalkallikreinandkallistatinmRftL4s hypertensive rats [30,31J.Thesefindingssuggest thatlowrenal length polymorphismsandco-segregation studiesingenetically

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Fig.3. Control sect ion of human kidney. The section was incubated with both digoxigenin-labeled sense kallikrein and kallistatin riboprobes. No signals were observed. Hybridization and immunodectection conditions were the same as described in the text. G: glomerulus. A. Cortex; B. Medulla (magnification, X200). kallikrein levels may contribute to the pathogenesis of hyperten-kallikrein function in vivo. Therefore, alteration of urinary kal- sion. Kallistatin, which binds to human tissue kallikrein andlistatin levels may serve as an index for the renal kallikrein-kinin inhibits its enzyme activity, is a potential regulator of renalsystem in patients with hypertensive diseases. This hypothesis is 696 Chenet al: Renal kallikrein and kallistatin mRNAs

Acknowledgments 2.8 This work was supported by National Institutes of Health grants HL 29397 and HL 44083. Human kidney paraffin blocks and normal human E2.4 kidneys were obtained from the Department of Pathology, Medical 2.0 University of South Carolina. We thank Dr. J0 Ann V. Simson and Dr. 1.6 Carmelann Zintz for their helpful advice and criticism for preparing this 1.2 0 manuscript. 0.8 .00.6 Reprint requests to Julie Chao, Ph.D., Department of Biochemistiy and Molecular Biology, Medical University of South Carolina, Charleston, South (I)0 .00.4 Carolina 29425, USA.

0.2 - Appendix Abbreviations are: RT-PCR, reverse transcription-polymerase chain I I I I I I reaction; RIA, radioimmunoassay; ELISA, enzyme-linked immunosor- 0.04•H 0.08 0.16 0.32 0.64 1.25 2.50 bent assay; HUK, human urinary kallikrein; PBS, phosphate-buffered saline; NBT, nitroblue tetrazolium salt; X-Phosphate, 5-bromo-4-chloro- Kallistatin, ng/welI 3-indolyl phosphate. Fig.4. Enzyme-linked immunosorbent assay of human kallistatin in the kidney and urine. The standard curve of kallistatin which ranges from 0.4 References to 25.0 ng/ml is shown by solid circles and serial dilutions of human kidney extracts and urine are shown by solid triangles and squares, respectively. 1. SCHACJ-ITER M: Kallikreins (kininogenases): A group of serine pro- teases with bioregulatory actions. Pharmacol Rev 31:7—17, 1980 2. CLEMENT5 JA: The glandular kallikrein family of enzymes: Tissue Table 1. Immunoreactive human tissue kallikrein and kallistatin levels specific expression and hormonal regulation. Endocr Rev 10:393—419, 1989 Kidney' Urine 3. BHOOLA KD, FIGUER0A CD, WORTHY K: Bioregulation of kinins: Kallikreins, , and kininases. Pharmacol Rev 44:1—80, 1992 Kallikrein 216.6 15.2 pg/mg 267.8 30.7 sgIday 4. MACDONALD RJ, MARGOLIUS HS, ERDOS EG: Molecular biology of Kallistatin 130.7 9.3 ng/mg 13.62.7jg/day tissue kallikrein. Biochem J 253:313—321, 1988 5. MURRAY SR. Cuo J, Lm F, CHAO L: Kallikrein multigene families Values represent mean SE (N 11 or 13, urine; N =3,kidney). and the regulation of their expression. J Cardiovasc Pharmacol "Specific activity is expressed as immunoreactive kallikrein or kallistatin 15(Suppl 6):S7—S15, 1990 levels per mg protein in the kidney. 6. PINKOs GS, OLE-MolYol 0, AUSTEN KF, SPARGG J: Antigenic sepa- ration of a non-kinin generating TAMe esterase from human urinary kallikrein and immunohistochemical comparison of their localization in the kidney. J Histochem Cytochem 29:38—44, 1981 7. KIMURA K, MORIYA H: Enzyme and immunohistochemical localiza- supported by the finding that rat kallikrein-binding protein tion of kallikrein. II. The human kidney. Histochemistry 80:443—448, mRNA is expressed mostly in the collecting duct of the kidney and 1984 8. FIGUEROA CD, MACIVER AG, MACKENZIE JC, BHOOLA KD: Local- its mRNA levels were significantly decreased in the kidney of isation of immunoreactive kininogen and tissue kallikrein in the spontaneously hypertensive rats [32]. human nephron. Histochemistiy 89:437—442, 1988 Studies have shown that the renal kallikrein-kinin system is 9.SHIMAMOTOK, IIMuRA 0: Physiological role of renal kallikrein-kinin involved in the regulation of sodium/water excretion and arterial system in human. Adv Exp Med Bio 247A:87—96, 1989 10. Vio CP, FIGUEROA CD, CA0R5I I: Anatomical relationship between blood pressure [33]. The kidney plays a major role in the kallikrein-containing tubules and the juxtaglomerular apparatus in the regulation of body sodium and fluid homeostasis. Failure of the human kidney. Am J Hypertens 1:269—271, 1988 renal handling of sodium and water may lead to renal disorders. A11. PROUD D, PERKINS M, PIERCE JV, YATES KN, HIGHET PF, HERRING number of studies have shown that the release of renal tissue PL, MANGKORNKANOKIMARK M, BAHU R, CARONE F, PISANO ii: kallikrein is associated with several factors such as high sodium or Characterization and localization of human renal kininogen. J Biol Chem 256:10634—10639, 1981 potassium intake, antidiuretic hormone (arginine vasopressin) 12. CRAI KX, CHEN, LM, CHAO J, CHAO J: Kallistatin: a novel human effect and neural control [34—361. Intrarenal production of kinins serine proteinase inhibitor. Molecular cloning, tissue distribution, and may increase local renal blood flow, glomerular filtration and expression in Escherichia coli. 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