Journal of Clinical Pathology, 1978, 31, 620-625

Tamm-Horsfall protein in the glomerular capsularspaceA. R. McGIVEN, J. S. HUNT, W. A. DAY, AND R. R. BAILEY

From the Department of Pathology, University of Otago, Christchurch Clinical School of Medicine andthe Department of Renal Medicine, Christchurch Hospital, Christchurch, New Zealand

SUMMARY Tamm-Horsfall protein was detected within the capsular space by immunofluorescencein 7 of 72 consecutive patients on whom renal immunopathological studies were performed. Threepatients showed prominent aggregates or crescentic collections affecting 30-50% glomeruli; theremaining four patients showed smaller aggregates between lobules. All patients showed pathologicalevidence of tubulointerstitial disease. It is suggested that Tamm-Horsfall protein in the capsularspace is a sign of intratubular urinary backflow and that Tamm-Horsfall antiserum is a usefuladdition to the reagents used in the immunofluorescence study of renal biopsies.

Tamm-Horsfall protein (THP) is a urinary glyco-protein secreted by the renal tubular cells of theascending loop of Henle and distal convoluted tubule(McKenzie and McQueen, 1969; Schenk et al., 1971;Wallace and Nairn, 1971). It has a molecular weightof 7 x 106 daltons, composed of subunits each ofapproximately 105 daltons (Fletcher et al., 1970) andis a prominent component in tubular casts(McQueen, 1962). We report here the demonstrationof THP in the capsular space in seven patients, whohad had renal immunopathological examinationsduring the past year at the Christchurch Hospital,and conclude that this indicates evidence of renaltubular backflow associated with tubulo-interstitialdamage.

Methods

Specimens from patients 1, 4, 5, and 6 were needlebiopsies. An open biopsy was obtained during theremoval of an adrenal tumour from patient 2, andtransplanted kidneys were obtained from patient 3at necropsy and from patient 7 at operation. Thetissue was examined by conventional histology,immunofluorescence, and electron microscopy.

HISTOLOGYTissue blocks were fixed in formalin and examinedafter staining by haematoxylin and eosin, periodicacid Schiff, methenamine silver, and Massontrichrome methods.

Received for publication 1 December 1977

IMMUNOFLUORESCENCEBlocks were stored at - 70'C and 4,um sections werecut in a cryostat at - 20'C. All fluorescence stainingwas carried out by placing one drop of a 1:5 dilutionof conjugated antisera on frozen sections of renaltissue for 30 minutes, followed by two 5-minutewashes in phosphate buffered saline (pH 7 2). Inaddition to specific antisera for immunoglobulins,albumin, fibrin, and complement components,antiserum against Tamm-Horsfall glycoprotein wasused.THP was isolated from pooled human urine by

precipitation with 0-58 M NaCl. Anti-THP serumwas prepared by injecting New Zealand white rabbitssubcutaneously with 1 mg THP at multiple sites ontwo occasions at intervals of four weeks. Rabbitswere bled from an ear vein one week later. Rabbitantihuman THP was purified by passing a 10 mlsample of immune serum through a 10 x 3-5 cmcolumn of THP linked to cyanogen bromide acti-vated Sepharose 4B (Pharmacia, Sweden) and elutingthe bound immunoglobulin with 0-2 M glycine HCI,pH 2-0. This fraction, which showed a single lineagainst THP on immunodiffusion, was conjugatedto fluorescein isothiocyanate (Nairn, 1976) and un-conjugated dye was removed by chromatography onSephadex G25 using phosphate buffered saline aseluate. The conjugated antiserum was concentratedto 6 ml and stored at - 20'C.

In control studies, a 1:20 dilution of anti-THPserum was absorbed with an equal volume of THP(1 mg/ml) at 370C for one hour and centrifuged, andthe supernatant was used on sections staining for

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THP. Positive staining was abolished in absorbedserum but persisted in sections stained with theequivalent concentration of unabsorbed serum.

ELECTRON MICROSCOPYUltrathin sections prepared from glutaraldehyde-fixed tissue embedded in Spurr's resin were examinedafter staining with lead citrate (McGiven et al.,1977).

Results

Seven of 72 renal biopsies studied for the presence ofTamm-Horsfall protein revealed THP within theglomerular capsular space in addition to the usuallocalisation in the ascending limb of the loop ofHenle, distal tubules, and casts. The quantity ofTHP varied from occasional small droplets betweenglomerular lobules to larger aggregates (Figs 1 and2), peripheral droplets often confluent and adherentto the parietal epithelial cells (Fig. 3), and prominentcrescentic accumulations, which almost filled thecapsular space in the most advanced cases (Fig. 4).The patients' ages ranged from 20 to 49 years, and

the clinical diagnoses and immunopathologicalfindings are summarised in the Table. Three patientsshowing the greatest accumulation of THP in thecapsular space are considered in more detail:

Case 1

A man of 36 years presented with hypertension andrenal failure for investigation. He had recentlysuffered from a prostatic abscess.

HISTOLOGYThe glomeruli showed a mild increase in themesangium, and the capsular space, which wasprominent, frequently contained remnants of pro-teinaceous material (Fig. 5). Some tubules weredilated and there was marked tubular degenerationwith proteinaceous material, inflammatory andepithelial cells, and erythrocytes in the lumen ofseveral tubules. The interstitium showed oedema and

Fig. 1 Patchy aggregates of Tamm-Horsfall protein incapsular space (case 6). x 150

Fig. 2 Tamm-Horsfall proteinstains brightly in tubular cast aswell as in distal convolutedtubules. Prominent aggregates incapsular space and betweenglomerular loops (case 7). x 150.

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A. R. McGiven, J. S. Hunt, W. A. Day, and R. R. Bailey

Fig. 3 Tamm-Horsfall protein adheres to parietalcapsular epithelial lining. Occasional interlobularaggregates (case 7). x 150

Fig. 4 Prominent crescentic Tamm-Horsfall proteinaggregate in capsular space (case 1). x 150

Renal immunopathological information in seven patients with glomerular Tamm-Horsfall protein

Case Sex Age Diagnosis Histology Immunofluorescence Glomlerular THP immunofluorescence

1 M 36 Hypertension and Interstitial C3 in vessels + + + Droplets and crescentic stainingrenal failure nephritis

2 M 27 Adrenal cortical Increased mesangial Glomeruli show Traceadenoma matrix. C3 trace

Tubules dilated.Patchy inflammation

3 M 49 Septicaemia. Mild membranous Glomeruli show Trace and occasional capRenal transplant changes. IgM ++ +(polycystic Interstitial oedema C, + +kidneys) and fibrosis

4 M 20 Nephrotic syndrome Proliferative exudative Glomeruli show + Occasional interlobular aggregatesglomerulonephritis. C, +Marked tubular C4 +degeneration Clq trace

5 M 29 Lupus nephritis Diffuse endocapillary Glomeruli show + Occasional aggregateslupus nephritis. IgG + + + +Tubular degeneration IgA + +

C, +++Clq ++++C4 +

6 F 41 Papillary necrosis Segmental sclerosis. Glomeruli show + + AggregatesInterstitial fibrosis and IgM tracetubular degeneration C, +

Vessels showC, +

7 M 26 Acute graft Acute rejection. Tubular basement + + + Droplets and crescentic stainingrejection Patchy infarction membranes show(Goodpasture's linear C, +syndrome)

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Fig. 5 Glomerulus showingirregular crescent ofprotein-aceous material in capsularspace (case 1). Periodic acidSchiff x 420

increased fibrous tissue together with some focalcollections of chronic inflammatory cells. Theprimary renal lesion appeared to be an interstitialnephritis.

IMMUNOFLUORESCENCEMost glomeruli showed collections of THP in thecapsular space. In some glomeruli, aggregationsappeared to be trapped between lobules of theglomerular tuft, and in others, a prominent crescenticcollection in the capsular space capped theglomerulus (Fig. 4).

Case 6

A woman aged 41 years presented with hypertensionand left ventricular failure and was shown by intra-venous urography to have evidence of papillarynecrosis.

HISTOLOGYGlomeruli showed patchy segmental sclerosis withsome peritubular fibrosis and collections of chronicinflammatory cells around damaged renal tubules.Vessels showed mild thickening.

IMMUNOFLUORESCENCEGlomeruli showed occasional traces of IgM andsome patchy C3, which was also present in smallarteries. Small collections of THP were seen in thecapsular space and between lobules of the glomeruli(Fig. 1).

Case 7

A man aged 26 years suffered from Goodpasture'ssyndrome, for which he had been treated by bilateralnephrectomy and plasmapheresis in 1975. In 1976he received a renal transplant which failed and wasremoved in 1977. A further transplant, which wasacutely rejected and removed after three weeks, is thesubject of this report.

HISTOLOGYThe kidney showed well-defined areas of infarctionof medulla and cortex, and adjacent areas showedevidence of acute rejection. Patchy collections oflymphoid cells were seen around peritubular vesselsand tubules which appeared rather dilated (Fig. 6)and often contained casts. Interlobular arteriesshowed prominent intimal proliferation, almostleading to occlusion. Glomeruli showed a mildincrease in mesangial matrix, and the capsular spaceoften appeared dilated, sometimes with a prominentlayer of lining cells.

IMMUNOFLUORESCENCEImmunofluorescence revealed aggregations of THPin the capsular space in approximately 50% ofglomeruli. This varied from a patchy irregularpattern where THP appeared to be trapped betweenglomerular loops (Fig. 2) to a capsular drop pattern,often becoming confluent, where aggregates adheredto the parietal epithelium lining the capsular space(Fig. 3), and more extensive crescentic aggregates

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A. R. McGiven, J. S. Hunt, W. A. Day. and R. R. Bailey

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resembling those seen in case 1. Tubular castsstained strongly (Fig. 2).

ELECTRON MICROSCOPYMaterial was observed in the capsular space, whichwas more electron dense than plasma seen in thelumen of capillary loops. At high magnification thegranular material tended to be arranged in filamentsand showed a periodicity of about 100 A, resemblingthat seen in preparations of THP isolated fromurinary casts, which show a periodicity of 110 A(Fletcher et al., 1970).

Discussion

Tamm-Horsfall protein has been studied extensivelyin normal renal tubular epithelial cells (McKenzieand McQueen, 1969; Schenk et al., 1971; Wallaceand Nairn, 1971) and is recognised as being a

significant component in tubular casts (McQueen,1962). The present report appears to be the firstdemonstration of this urinary glycoprotein occurringin relation to the renal glomerulus and its capsule.

Normally, THP is demonstrated in the cytoplasmof the cells of the ascending limb of the loop ofHenle and distal convoluted renal tubule, and, in ourexperience, when present in casts is usually seen inthe collecting system, distal to the site of THPsecretion, where the cytoplasm of the tubular cellsembracing the casts does not stain for THP.

Theoretically, THP in the capsular space couldresult from filtration of a plasma component, secre-tion by a glomerular constituent such as mesangialcells, or perhaps from secretion by the parietal

Fig. 6 Interstitial infiltrate ofmononuclear cells around dilatedtubules in kidney showing acuterejection (case 7). Haematoxylinand eosin x 210

epithelial cells lining the capsular space, particularlywhen tubular epithelium has replaced the normalepithelial lining.We cannot entirely exclude the possibility that

THP entered the capsular space from the glomerulus.THP is not detectable in normal serum but it hasbeen suggested that in reflux nephropathy THP mayreach the circulation (Hodson et al., 1975). If it werederived from serum, THP might be expected to havea more widespread glomerular distribution.

Should THP be secreted by mesangial cells orcells lining the capsular space, we would haveexpected to detect its presence in these cells byimmunofluorescence but have failed to do so.A more likely explanation of our findings is the

retrograde passage ofTHP from its site ofproductionin the distal tubule, along the loop of Henle andproximal convoluted tubule into the capsular space.The patients reported above were in a series of 72

patients examined in a period of approximately oneyear. In each case only a proportion of glomeruliwere affected. However, each patient had histo-pathological evidence of tubulo-interstitial damage,suggesting that there was an obstructive element intheir renal condition.

There has been recent interest in the developmentof immunological reactions against THP in renaldisease. Experimental studies in pigs (Hodson et al.,1975) suggested that reflux nephropathy led to theformation of Tamm-Horsfall antibody, and Hansonet al. (1976) proposed that these antibodies might beuseful in distinguishing between upper and lowerrenal tract infections. Fasth et al. (1977) were unableto show a correlation between antibody titres and

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Tamm-Horsfall protein in the glomerular capsular space 625

the presence of vesico-ureteric reflux or kidneyscarring. Cell-mediated immunity against THP hasbeen reported in patients with autoimmune liverdisease associated with renal tubular acidosis(Tsantoulas et al., 1974). Antibodies and cell-mediated immunity against Tamm-Horsfall proteinwere not studied in our cases. Indeed, the detectionof cell-mediated activity against THP by lymphocyteblast cell transformation may prove difficult as wehave found that some preparations of THP act asmitogens on lymphocytes from normal individuals.The addition of Tamm-Horsfall antisera to the

study of renal biopsies can provide useful evidenceof the retrograde tubular passage of urine, whichmay be associated with tubulo-interstitial damageand intrarenal reflux and not be detectable by othermethods of investigation.

We thank Dr K. L. Lynn for advice and the Depart-ment of Medical Illustration. This study wassupported by a grant from the Medical ResearchCouncil ofNew Zealand.

References

Fasth, A., Hanson, L. A., and Asscher, A. W. (1977).Autoantibodies to Tamm-Horsfall protein in detectionof vesicoureteric reflux and kidney scarring. Archivesof Disease in Childhood, 52, 560-562.

Fletcher, A. P., McLaughlin, J. E., Ratcliffe, W. A., andWoods, D. A. (1970). The chemical composition andelectron microscopic appearance of a protein derivedfrom urinary casts. Biochimica and Biophysica Acta,214, 299-308.

Hanson, L. A., Fasth, A., and Jodal, U. (1976). Auto-antibodies to Tamm-Horsfall protein, a tool fordiagnosing the level of urinary-tract infection. Lancet,1, 226-228.

Hodson, J., Maling, T. M. J., McManamon, P. J., andLewis, M. G. (1975). Reflux nephropathy. KidneyInternational, 8, Supplement, 50-58.

McGiven, A. R., Day, W. A., and Hunt, J. S. (1977).Glomerular lesions in argyric NZB/NZW mice. BritishJournal of Experimental Pathology, 58, 57-62.

McKenzie, J. K., and McQueen, E. G. (1969). Immuno-fluorescent localization of Tamm-Horsfall muco-protein in human kidney. Journal of Clinical Pathology,22, 334-339.

McQueen, E. G. (1962). The nature of urinary casts.Journal of Clinical Pathology, 15, 367-373.

Nairn, R. C. (1976). Fluorescent Protein Tracing, 4thedition. Churchill Livingstone, Edinburgh and London.

Schenk, E. A., Schwartz, R. H., and Lewis, R. A. (1971).Tamm-Horsfall mucoprotein. 1. Localization in thekidney. Laboratory Investigation, 25, 92-95.

Tsantoulas, D. C., McFarlane, I. G., Portmann, B.,Eddleston, A. L. W. F., and Williams, R. (1974). Cell-mediated immunity to human Tamm-Horsfall glyco-protein in autoimmune liver disease with renal tubularacidosis. British Medical Journal, 4, 491-494.

Wallace, A. C., and Nairn, R. C. (1971). Tamm-Horsfallprotein in kidneys of human embryos and foreignspecies. Pathology, 3, 303-310.

Requests for reprints to: Professor A. R. McGiven,Department of Pathology, The Christchurch ClinicalSchool, Christchurch Hospital, Christchurch, NewZealand.

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