J Clin Pathol 1984;37:738-742

Immunocytochemical localisation of renin innephroblastomaGBM LINDOP, S FLEMING, AAM GIBSON*

From the University of Glasgow, Department of Pathology, Western Infirmary, and the *Department ofPathology, Royal Hospital for Sick Children, Glasgow

SUMMARY An antibody to pure human renin and an immunoperoxidase technique were used tostain immunoreactive renin in nephroblastomas. In an unselected series of surgically removednephroblastomas immunoreactive renin was found in 10 of 19 tumours (53%). Tissue obtained atnecropsy from 12 cases of disseminated nephroblastoma was also studied, and immunoreactiverenin in metastatic tumours was found in three cases. Patients' blood pressures and serumpotassium concentrations showed no. evidence of excess renin activity. It is therefore suggestedthat if nephroblastomas contain renin as commonly as this study suggests, then any hormonesecreted may be biologically inactive.

Some malignant tumours secrete hormones, andthere is evidence that nephroblastomas may occa-sionally secrete renin.'-3 Biochemical study of therenin secreted by one of these cases showed that thehormone was mainly biologically inactive.4 Thisraises the possibility that nephroblastomas may se-crete renin more commonly than is realised.We have used an antibody to pure human renin

and an immunoperoxidase technique to localiserenin in the kidney5 and its tumours.6 This studyreports the application of this technique to showimmunoreactive renin in a series of nephro-blastomas.

Material and methodsWe studied 19 surgically excised primary nephro-blastomas. These represent an unselected seriesfrom the files of the Pathology Department, RoyalHospital for Sick Children, Glasgow. They werefixed in neutral buffered formalin, postfixed in cor-rosive formol saline, and, after dehydration, embed-ded in paraffin wax. From each case two blocks wereselected to include not only tumour but also adja-cent kidney, where the renin containing cells of thejuxtaglomerular apparatus provided a known posi-tive staining reaction. Sections (5,m) were stainedwith haematoxylin and eosin, Wilson's stain,Bowie's stain, and periodic acid Schiff after diastase.Consecutive sections were retained forimmunocytochemistry.

Accepted for publication 4 Apnil 1984

The peroxidase-antiperoxidase (PAP) techniquemodified as described previously7 was used for theidentification of human renin in paraffin embeddedtissue. The antiserum was raised in rabbits usinghuman renin purified from a juxtaglomerular celltumour as the antigen. The purity of the renin andthe specificity of the antibody have beendocumented,' ` and appropriate controls were usedas previously described.7

Since we were able to localise renin in the juxta-glomerular apparatus of kidney obtained at nec-ropsy, we also studied postmortem tissue from 12children with disseminated nephroblastoma. Weexamined paraffin embedded sections of metastaticnephroblastoma in lung, liver, and lymph nodes.

All sections were examined independently by twoobservers (GBML and SF), and only those tumoursclassified as positive by both were included. The his-tological features were assessed by an experiencedpaediatric pathologist (AAMG) without knowledgeof the immunocytochemical findings.

Results

HISTOLOGYThe series of nephroblastomas included a full rangeof differentiation: well and poorly differentiatedmesenchyme and well and poorly differentiatedepithelial structures. A comparison of the natureand degree of differentiation of the tumours showedno relation between the morphological assessment

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Immunocytochemical localisation of renin in nephroblastoma

of differentiation and the presence or absence ofcells containing immunoreactive renin.

IMMUNOCYTOCHEMISTRYThe renin containing cells in the juxtaglomerularapparatus of the kidney adjacent to the tumours

OAStza AW f st

Fig. 1 Glomerulus with its afferent arteriole in the kidneyadjacent to a nephroblastoma which containsimmunoreactive renin. There is hyperplasia of thejuxtaglomerular renin secreting cells. Renin PAP.Interference contrast microscopy. Original magnificationxl60.

4'7

immunoreactive rein. One is a spnl cel wit loncytopasmi pocese (aros whic cotiv:auamagnfictio x 630.

-wt r

renwin. PAP.s Orntiginal

magnification x630.

Fig. 3 Renin positive cells are in the better differentiatedmesenchyme ofthe tumour. They are more rounded withround nuclei and are clearly perivascular Renin PAP.Original magnification x396.

A srt v2__-

g. 4 b s jr..> , M-

r'.;w.; ...#4 '^s'A >

Fig. 4 Dilated blood vessel at the junction oftheundifferentiated blastema and mesenchyme ofanephroblastoma with tumour cells round the blood vessel.The cells containing immunoreractive renin arecytologically indistinguishable from adjacent tumour cells.Renin PAP. Original magnification x396.

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Lindop, Fleming, Gibson

endothelium (Figs. 3, 4 and 5). The renin containingZ cells were most commonly found in the well dif-

ferentiated mesenchyme of the tumours, and oftenthey were on the border between this tissue and theundifferentiated metanephric blastema. Occasion-ally, cells containing immunoreactive renin werefound within areas of undifferentiated blastema,always close to capillary blood vessels within the

,. I .' tumour (Fig. 5).A,Wf We found immunoreactive renin in metastatic

Xinephroblastoma from three of the 12 postmortem

Wgl?tcases studied. The metastatic sites were lung, liver,

tjj; and lymph node. The distribution of the renin posi-tive cells in the metastatic nephroblastoma was iden-tical to that of the primary tumours in the kidney.

Sections stained with aldehyde fuchsin andBowie's stains showed granular cells associated withthe blood vessels in a similar position to the renin

t«? .> containing cells in the adjacent sections stained withthe renin PAP method.

Fig. 5 Poorly differentiated blastemal area ofanephroblastoma which contains scattered renin positivecells. They are all related to small blood vessels (arrows).Renin PAP. Original magnification X396.

always showed a positive staining reaction. Thesecells showed cytoplasmic positivity, which was bothgranular and diffuse. There was often hyperplasia ofcells containing immunoreactive renin (Fig. 1).

Cells containing immunoreactive renin werefound in 10 of 19 primary nephroblastomas. Thesecells were variable in size and morphology. Theywere sometimes spindle cells with oval nuclei andlong cytoplasmic processes (Fig. 2); at other timesthey appeared plump and round with round nuclei(Fig. 3). In some areas they were cytologically indis-tinguishable from adjacent tumour cells (Fig. 4).The intensity of the staining reaction varied from

a pale brown granular positivity to almost black, astaining reaction as intense as the renin containingcells in the adjacent juxtaglomerular apparatus.While the appearance and intensity of staining of therenin positive cells varied, their position was con-stant: they were always related to the blood vesselsof the tumour. These varied from small capillaries(Fig. 5) to large dilated thin walled vessels (Figs. 3and 4). Examination of serial sections showed thatwhen apparently isolated positive cells were seen

(Fig. 2) adjacent sections showed that they were

usually related to an underlying blood vessel. Thecells containing immunoreactive renin were usuallyclose to the blood vessels but external to the

CLINICAL MEASUREMENTSBlood pressure measurements and serum potassiumconcentrations were obtained from the clinical caserecords where available. Where there was more thanone measurement the mean was used. There was nosignificant difference (Student's t test) in the bloodpressure measurements between children whosetumours contained cells positive for immunoreactiverenin (mean 124/79 mmHg) and those whosetumours contained negative cells (mean 110/74mmHg). After nephrectomy no significant differ-ence in blood pressure was recorded in either groupas a whole or in individual cases. Similarly, there wasno difference in serum potassium concentrationbetween the renin positive group (mean 4 1 mmol/l)and the renin negative group (mean 4-1 mmol/l).These measurements were also unaffected bynephrectomy.

Discussion

The in vitro specificity of this renin antiserum is wellcharacterised.9 '° It has been found to stain reninreliably with the PAP technique in several studies ofthe kidney,"-'3 and we have used it to detectimmunoreactive renin in other renal tumours.68The cells containing immunoreactive renin in this

series of nephroblastomas could have grown intoprimary renal tumours from the adjacent renal cor-tex along with the blood vessels, since it is thoughtthat tumours stimulate the growth of blood vesselsfrom the surrounding tissue.'4 But this would notexplain their presence in metastatic nephroblastomain other organs. Moreover, our previous study failedto show immunoreactive renin in metastatic tumours

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Immunocytochemical localisation of renin in nephroblastoma

growing in the renal cortex.8 In some of the caseswhich we have studied there were renin positivecells which were cytologically malignant and indis-tinguishable from adjacent tumour cells. It thereforeseems likely that the renin containing cells innephroblastoma are tumour cells.The renin in these cells could either have been

taken up from the plasma or synthesised by the cellsin the tumour. The renin staining was often granular(like the juxtaglomerular renin secreting cells in theadjacent kidney), and occasional cells containedgranules with the staining reactions of renin storagegranules.The renin secreting cells in the kidney are thought

to be derived from undifferentiated mesodermalcells adjacent to the blood vessels in the meta-nephric renal cortex.'5 Nephroblastoma is a tumourof pluripotent mesodermal cells which can differen-tiate into all structures of the normal kidney. It istherefore not surprising that it can differentiate intorenin secreting cells and that these renin secretingcells would be associated with the blood vessels,mimicking the known development pattern for jux-taglomerular cells.'5There are several descriptions of renin secreting

nephroblastomas.'-3 16-18 The evidence is based onhigh concentrations of renin in the preoperativeplasma and in the tumour, and normal plasma con-centrations after nephrectomy. In two cases'6"7ultrastructural examination showed secretorygranules with morphological features consistent withrenin granules within tumour cells. The situation ofthese granular cells within the tumours was notdescribed. Patients with renin secreting tumoursoften have evidence of increased renin activity in theform of hypertension and hypokalaemia.' 15 20 Theseare usually cured by nephrectomy. Analysis of theclinical case records in our series showed nosignificant difference in the blood pressure values orserum potassium concentations between those caseswhose tumours contained immunoreactive renin andthose cases whose tumours did not. Moreover, thesevariables were not affected by nephrectomy. Thusthere was no clinical evidence of excess renin activ-ity. In support of this conclusion is the fact that therewas often hyperplasia of the renin containing juxta-glomerular cells in the adjacent kidney. Previouslydescribed renin secreting nephroblastomas have hadabsent renin activity' and absent granular cells' 1' inthe adjacent kidney, as has been described in otherrenin secreting tumours.6 It is possible either thatthe renin containing cells which we have shown to becommonly present in nephroblastoma did notrelease the hormone in amounts sufficient to raiseplasma angiotensin II concentrations (and hencesuppress physiological renin secretion) or that any

hormone secreted was biologically inactive.Whether or not nephroblastoma commonly secretesrenin will be decided by the biochemical measure-ments of renin concentrations in tumour tissue, ad-jacent kidney, and in pre- and postoperative plasma,which are now underway.We have found a similar distribution of cells con-

taining imm'unoreactive renin in a series of renal cellcarcinomas.8 Interestingly, the renin positive cellswere also usually close to the blood vessels of thetumour, and in one case studied in detail7 the reninsecreted by the tumour was shown to be mainlybiologically inactive and to have a higher molecularweight than active renin.' It may therefore havebeen a prohormone. Similarly, the biochemistry ofthe renin present in the tumour and the plasma fromone case of renin secreting nephroblastoma' hasbeen studied in detail: it was mainly biologicallyinactive, had a higher molecular weight, and wasalso probably a prohormone.4The antibody used in the present study binds to

both active and inactive renin in vitro.' 22 Hence thebiological nature of the immunoreactive renin whichwe have shown to be present in these tumoursremains to be elucidated. Our work suggests thatwhile secretion of biologically active renin bynephroblastoma may be rare, as in other tumours2"secretion of biologically inactive hormone may becommon.

We are grateful to Professor Pierre Corvol and Pro-fessor Joel Menard of Inserm u36 7005 Paris for thegenerous gift of the antibody, which made this workpossible.

References

Mitchell JD, Baxter TJ, Blair-West JR, McCredie DA. Reninlevels in nephroblastoma (Wilms' tumour). Report of a reninsecreting tumour. Arch Dis Childh 1970;45:376-84.

2 Masovari I, Kontor, Kallay K. Renin-secreting Wilms' tumour(Letter). Lancet 1972;i: 1180.

3Ganguly A, Gribble J, Tune B, Kempson RL, Luetscher A.Renin secreting Wilms' tumour with severe hypertension. AnnInt Med 1973;79:835-7.

4Day RP, Luetscher JA. Big renin: a possible prohormone inkidney and plasma of a patient with Wilms' tumour. i ClinEndocrinol Metab 1974;38:923-6.

5 Lindop GBM, Reid R, Downie TT, et al. Blood vessels and bloodpressure. In: Woolf N, ed, The biology and pathology of thevessel wall. Eastbourne: Praeger, 1983.

6 Lindop GBM, Stewart JA, Downie TT. The immunocytochemi-cal demonstration of renin in a juxtaglomerular cell tumour bylight and electron microscopy. Histopathology 1983;7:421-31.

'Lindop GBM, More IAR, Leckie B. An ultrastructural andimmunocytochemical study of a renal carcinoma secretinginactive renin. J Clin Pathol 1983;36:639-45.

Lindop GBM, Fleming S. Renin in renal cell carcinoma-animmunocytochemical study. J Clin Pathol 1984;37:27-31.

9Galen FX, Devaux C, Guyenne TT, Menard J, Corvol P. Multi-

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0 Galen FX, Guyenne T, Devaux C, Auzan C, Corvol P, Menard J.Direct radioimmunoassay of human renin. J Clin EndocrinolMetab 1979;48:1041-3.

Menard J, N Goc J, Bariety J, Guyenne PT, Corvol P. Directradioimmunoassay and immunocytochemical localization ofrenin in human kidneys. J Clin Sci 1979;57: 105s-8s

12 Camilleri JP, Phat VN, Bariety J, Corvol P, Menard J. Use of aspecific antiserum for renin detection in human kidney. J His-tochem Cytochem 1980;28:1343-6.

Nochy D, Barres D, Camilleri JP, Bariety J, Corvol P, Menard J.Abnormalities of renin-containing cells in human glomerularand vascular disease. Kidney Int 1983;23:375-9.

14 Folkman J, Merler E, Abernethy C, Williams G. Isolation of atumour factor responsible for angiogenesis. J Exp Med1971; 133: 275-88.

Phat VN, Camilleri JP, Bariety J, Corvol P, Menard J.Immunohistochemical characterization of renin-containingcells in the human juxtaglomerular apparatus duringembryonal and fetal development. Lab Invest 1981;45:387-90.

lb Luciani JC, Baldet P, Dumas R, Jean R. Etude du systemerenine-angiotensine dans deux cas de tumeur de Wilms avec

Lindop, Fleming, Gibsonhypertension arterielle severe. Arch Franc Pediat1979;36: 240-9.

Sheth KJ, Tang TT, Blaedel ME, Good TA. Polydipsia, polyuriaand hypertension associated with renin-secreting Wilms'tumour. J Pediatr 1978;92:921-4.

Spahr J, Demers LM, Shochat SJ. Renin producing Wilmstumour. J Pediatric Surg 198 1; 16:32-4.

9 Brown JS, Fraser R, Lever AF et al. Hypertension and secondaryhyperaldosteronism associated with a renin secreting renaljuxtaglomerular-cell tumour. Lancet 1973;ii: 1228-31.

211 Schambelen M, Howes EL, Stocbigt JR, Noukes CVA, BigleriEG. Role of renin and aldosterone in hypertension due to arenin secreting tumour. Am J Med 1973;55:86-92.

21 Leckie BJ, McConnell A, Grant J, Morton JJ, Tree M, Brown JJ.An inactive renin in human plasma. Circ Res 1977;40(suppl1):46-51.

22 Guyenne IT, Galen FX, Devaux C, et al. Direct radioimmuno-assay of human renin. Hypertension 1980;2:465-70.

Odell WD, Wolfsen AR. Humoral syndromes associated withcancer. Ann Rev Med 1978;29:379-406.

Requests for reprints to: Dr GBM Lindop, PathologyDepartment, Western Infirmary, Glasgow GIl 6NT, Scot-land.

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