Corrections in counting paired nuclei labelled with bromodeoxyuridine in tissue sections.

Bromodeoxyuridine is finding increasing use as an alternative to, or in conjunction with, tritiated thymidine for labelling nuclei in DNA synthesis. Precise identification of labelled nuclei is possible, even when there is considerable overlap between neighbouring nuclei. In the sparsely labelled renal cortex of the normal male mouse, 'flash labelling' with bromodeoxyuridine shows single labelled nuclei at 1 h. At 24, 48 and 72 h after injection of bromodeoxyuridine, some labelled cells are seen to lie adjacent and such labelled pairs are presumed to be the result of cell division. Single labelled nuclei at 24, 48 and 72 h might indicate arrest in DNA synthesis or a prolonged G2 period, but it is important to recognize that a correction must be made for paired labelled nuclei in which one member is out of the plane of section. The factors involved in making such a correction are discussed and a correction table calculated. In the normal male mouse renal cortex, we show that nearly all cells labelled at 1 h had divided by 72 h.

Renal allograft rejection--in situ demonstration of cytotoxic intratubular cells.

A nonisotopic in situ hybridization method to detect perforin mRNA was developed in cytospin preparations of IL-2-stimulated normal human lymphocytes and applied to formalin-fixed acutely rejected renal transplant material. Individual cells expressing perforin mRNA were localized in severely damaged tubular areas, and a number of these cells appeared to be located inside the tubular basement membrane in close association with tubular epithelial cells. Immunoperoxidase staining in acetone-fixed cryostat sections of acutely rejected kidney confirmed that a considerable proportion of infiltrating cells was CD8+; many of these were in an intratubular location. In addition, perforin protein was identified in individual cells in similar locations to perforin mRNA-positive cells. Again, some intratubular cells were identified. Our findings illustrate that these cells can be fully activated with definite cytotoxic potential. Previously we have demonstrated that T lymphocytes proliferate within the tubular compartment during tubulitis, a characteristic condition in acute renal allograft rejection, and that there is associated tubular epithelial cell proliferation. In this study we think that we have further clarified the consequences of invasion of tubules by lymphoid cells. Our in situ hybridization method in rapid and convenient and may be applied to archival material.

Renal allograft rejection: beta-chemokine involvement in the development of tubulitis.

BACKGROUND: Tubulitis is a defining feature of renal allograft rejection. Graft dysfunction may result from damage inflicted on tubular epithelial cells by intratubular cytotoxic T lymphocytes. Graft cells are known to produce chemokines during acute rejection, but it is not known whether changes in expression of specific chemokines can influence the composition of the intratubular lymphocyte population. We examined expression of individual chemokines in biopsy sections showing different pathological rejection grades. METHODS: Sections from Banff-graded transplant biopsies were examined for the presence of beta-chemokines (MCP-1, MIP-1alpha, MIP-1beta, and RANTES) by immunofluorescence and semiquantitative confocal laser scanning microscopy. RESULTS: Beta-chemokines were expressed predominantly at the basolateral surface of tubular epithelial cells. Expression of MCP-1 and MIP-1beta was significantly higher in sections showing grade 2 rather than grade 1 acute rejection. RANTES and MIP-1alpha showed no significant variation in level of expression between rejection grades. CONCLUSIONS: Beta-chemokines are expressed by tubular epithelial cells during acute rejection. Consistent expression of RANTES and MIP-1alpha suggests a general role in recruiting T lymphocytes. However, MCP-1 and MIP-1beta may play a more subtle role in recruitment of specific T-cell subsets, such as Th1 cells, during acute cellular rejection.

The proliferative response of the coagulating gland of the castrated mouse under continuous androgen stimulation: an experimental and computer simulation model.

The proliferative response of the coagulating gland of the castrated male mouse has been examined during continuous treatment with testosterone propionate. Fourteen days after castration, s.c. daily injections of testosterone propionate were begun. Mitotic (Im) and labelling (IL) index values were obtained at 3 h intervals for up to 100 h after the initial injection. These showed a biphasic response, in which IL reached a maximum at 30 and 70 h, and Im at approximately 45 and 75 h. Fraction-labelled mitoses (FLM) curves were begun 24, 48, and 72 h after the first androgen injection. In each curve the first wave of labelled mitoses rose to 100% and showed a square form indicating little spread in the durations of the G2 and S phases. Values of 7.5, 1.3 and 0.7 h were obtained for the durations of DNA synthesis (ts), the post-synthetic period (tG2) and of mitosis (tm) respectively. In none of the FLM curves was it possible to demonstrate a second wave of labelled mitoses and direct measurement of the cell cycle time (Tc) was not obtained. Continuous tritiated thymidine labelling indices revealed that after a latent period of 25 h, DNA synthesis began and labelling rose rapidly to 80% by 45 h and then more slowly to 95% by 97 h. Cell population changes during androgen stimulation estimated from measurements of total glandular DNA indicated that the number of cells present in the glands remained constant during the first 30 h after stimulation and thereafter increased to approximately 2-3 times the original value. The data are compared with a mathematical model which assumes that the cell population of castrated mice when stimulated passes from a GO compartment through successive waves of DNA synthesis and mitosis. After each cell division the cells may leave or remain in the proliferative cycle. This model has been subjected to computer simulation using the cell cycle parameters obtained in the kinetic experiments. There was good agreement between the stimulation and experimental results in the Im and IL curves, continuous labelling, and total cell number experiments. The simulation of FLM curves was less successful. Although the first wave of labelled mitoses was clearly seen the model predicts a distinct second wave of labelled mitoses. It is concluded that this does not appear because of variation in the duration of G1.

Remnant kidney pathology after five-sixth nephrectomy in rat. II. Electron microscopy study.

In Wistar male rats, hypertension was induced by 5/6 nephrectomy (5/6N). Electron microscopy revealed an increase in the number of epithelial cells on the Bowman's capsule during the early stages (4 weeks). After 8 and 12 weeks, frequent adhesion was observed between the glomerular tuft and Bowman's capsule. The abnormal podocytes showed nuclear irregularities and distortions. Characteristic foot process fusion formed cytoplasmic plates. There was a considerable increase in mesangial matrix and cells. No immune deposits or breaks in the glomerular basement membrane were observed. In the endothelial cells, the fenestration disappeared in sclerosed glomeruli. Some capillary loops were obliterated by fibrin, macrophages and foam cells. These findings combined with our previous light microscopy and immunofluorescent observations suggest a non-immunogenic glomerular sclerosis.

Remnant kidney pathology after five-sixth nephrectomy in rat. I. A biochemical and morphological study.

In Wistar male rats, hypertension was induced by 5/6 nephrectomy (5/6N). Body weight, blood pressure measurements, morphological and biochemical changes were followed (at four weekly intervals) for 12 weeks after 5/6N. Renal function was assessed by daily total urinary protein (TUP), plasma creatinine concentration [(Cr)p] and creatinine clearance rate. Plasma renin concentration (PRC), aldosterone concentration and erythrocyte content of sodium [Na]E and potassium [K]E were also investigated. Significant increases in systolic blood pressure (SBP), TUP, [(Cr)p] and [Na]E occurred after 4, 8, and 12 weeks of 5/6N. Progressive glomerulosclerosis (GSC), tubular atrophy and interstitial fibrosis were observed. Positive correlations were found between GSC and SBP and TUP. Positive correlations were also found between SBP and [Na]E and [(Cr)P]. PRC was not increased and showed no correlation with SBP. It is concluded that 5/6N produced hypertension associated with a series of morphological and biochemical alterations in kidney structure and function. In this model, mechanisms other than the renin-angiotensin system may be involved.

Three-dimensional reconstruction of juxtaglomerular apparatus (JGA) in five-sixth nephrectomized rats.

In Wistar male rats, hypertension was induced by five-sixth nephrectomy (5/6N). Body weight, systolic blood pressure (SBP) and plasma renin concentration (PRC) were followed for 12 weeks after 5/6N. Three-dimensional reconstruction and morphometry of the JGA were carried out using a computer program "GLOM". Immunohistochemistry and electron microscopy of the JGA were also investigated. A statistically significant increase in SBP was shown after 5/6N. However, PRC showed no increase and was not correlated with SBP. Renin-containing cells were demonstrated in the afferent and efferent arterioles and the interlobular arteries. Electron microscopy revealed granules of various shapes, sizes and electron densities within the JG cell. The frequency of granulated cells in the efferent arteriole was less than that in the afferent arteriole. The afferent arteriole wall volume of 5/6N rats was significantly increased and positively correlated with SBP. The lack of relationship between PRC and SBP in this model suggests that mechanisms other than the renin-angiotensin system may be involved in the pathogenesis of hypertension.

The juxtaglomerular apparatus of the spontaneously hypertensive rat.

Spontaneously hypertensive rats (SHR) are used to study the pathogenesis of essential hypertension. This study investigates the role of the renin-angiotensin system (RAS) in the pathogenesis of hypertension in SHRs and the morphometry of the JGA by a three-dimensional computer reconstruction program "GLOM" and electron microscopy. Systolic blood pressure (SBP) (tail cuff method) was higher in SHRs compared to controls (P less than 0.001). Plasma renin concentration (PRC) was lower in SHRs than in controls (P less than 0.001). Reconstruction of the JGA revealed granulated JG cells in the afferent and efferent arterioles and in the vascular tree away from the JGA area. Electron microscopy showed granulated JG cells in the afferent and efferent arterioles. The percentage volume of the granulated JG cells in SHR was significantly higher than in controls (P less than 0.01). A relationship was found between the percentage volume of granulated JG cells and the SBP in SHRs (r = 0.933, P less than 0.05). The wall/lumen perimeter ratio was also significantly higher in the SHRs compared to the controls (P less than 0.05). Low PRC in SHRs has been reported by several workers. The apparent hyperactivity of the JGA may indicate failure of renin release or an abnormal synthesis/secretion rate.

Three-dimensional reconstruction of human juxtaglomerular apparatus (JGA).

A three-dimensional and morphological study of the human JGA was undertaken to establish a background for understanding the changes in this vital apparatus during various physiological and pathological conditions. Three-dimensional reconstruction was carried out using a computer program "GLOM". Serial sections of normal human kidneys were used after staining with specific human renin antiserum. Three-dimensional reconstruction revealed renin-positive cells in the afferent and efferent arterioles and interlobular arteries away from the JGA area. A close contact was demonstrated between renin-positive cells and the macula densa. The frequency of positively stained JGAs was significantly higher in the superficial glomeruli compared to the deep glomeruli. The high renin content of the superficial glomeruli suggests higher generation of angiotensin, which may contribute to the regulation of the GFR as proposed by other workers. This preliminary study on normal human JGA is to be extended to hypertensive and renal failure patients.

Routine and rapid enzyme linked immunosorbent assays for circulating anti-glomerular basement membrane antibodies.

Microtitre plate modifications of the original tube enzyme linked immunosorbent assay (ELISA) of Wheeler and Sussman were used for the routine and rapid assays of anti-glomerular basement membrane antibodies in human sera. In a prospective study of 238 sera from 200 patients, the routine assay (about 24 hours) detected circulating antibodies in seven sera from three patients with active anti-glomerular basement membrane disease. The remaining sera, from patients with a variety of other glomerulonephropathies, were negative by the assay. The rapid assays took less than four hours, and in a retrospective study, detected anti-glomerular basement membrane antibodies in a range of 15 positive sera, with a level of discrimination similar to that observed in the routine assay and with no false positive/negative results.

Immunoperoxidase techniques and histology in the diagnosis of rhabdomyolysis related acute renal failure.

A case of rhabdomyolysis associated acute renal failure (RM-ARF) occurring as a result of strenuous exercise is presented. Diagnostic renal biopsy was performed. The histological appearances, combined with immunoperoxidase staining for myoglobin, allowed a positive diagnosis of RM-ARF to be made and excluded the possibility of glomerulonephritis. The patient recovered completely after a stormy clinical course.

Tissue artefacts caused by sponges.

Artefacts which occur during the processing of small biopsy specimens can cause sufficient tissue distortion to impair interpretation and can be a considerable source of nuisance. Triangular artefacts were noted in renal and liver biopsy specimens which were caused by foam sponges in embedding cassettes. Scanning electron microscopic examination of the sponges showed they comprised a mesh of scimitar-shaped rigid spikes which closely match the artefacts seen in the tissues.

In vitro bromodeoxyuridine labelling of renal biopsy specimens: correlation between labelling indices and tubular damage.

AIMS: To examine the correlation between bromodeoxyuridine (BrdU) labelling indices (LI) and tubular damage in renal biopsy specimens; to evaluate the diagnostic and prognostic potential of measuring cell proliferation in a variety of renal lesions. METHODS: In vitro BrdU labelling of renal biopsy specimens was undertaken and labelled cells were detected in routinely fixed, paraffin wax embedded sections by immunohistochemistry. The BrdU LI were calculated as percentages for the three types of tubular cells--proximal and distal convoluted tubules and medulla (LI/PCT, LI/DCT, LI/Med)--and a total tubular BrdU LI (LI/Tub) was also calculated for each biopsy specimen. Histological features indicative of tubular damage were also scored and a total tubular damage score obtained for each biopsy specimen. RESULTS: The one hour labelling process did not affect tissue morphology or impede subsequent diagnosis. Four biopsy specimens were obtained from three renal transplant recipients. Diagnosis of 19 non-transplant biopsy specimens revealed a variety of renal lesions. Total tubular damage scores ranged from 0 to 25 and the LI/Tub ranged from 0 to 3.68% in all 23 biopsy specimens. Analyses of variance showed highly significant correlations between the total tubular damage score and both LI/Tub (p = 0.004) and LI/PCT (p = 0.004); a weaker correlation was found between the total tubular damage score and LI/DCT (p = 0.013). CONCLUSIONS: A correlation was found between tubular damage and BrdU LI. This was most clearly seen in the proximal tubules. However, as the study was limited to a few examples of specific forms of glomerular or interstitial disease, firm conclusions about the value of BrdU labelling in routine diagnosis and prognosis could not be drawn.

Plasma oxalate concentration and secondary oxalosis in patients with chronic renal failure.

To examine the association between hyperoxalaemia and secondary oxalosis, measurement of plasma oxalate concentration was combined with a search for tissue deposition of calcium oxalate crystals in patients with chronic renal disease. Two groups of patients were studied. In the first, samples of the inferior epigastric artery were taken from 35 patients at the time of renal transplantation. In the second, sections taken at necropsy from 23 patients with chronic renal failure in whom plasma oxalate had been measured before death were examined. Though plasma oxalate concentrations ranged between 6 and 116 mumol/l (four to 78 times greater than the upper limit of the reference range), no extrarenal deposits of oxalate were found in either study. Renal deposition of oxalate was associated with a plasma oxalate concentration of greater than 20 mumol/l. This study gives no support to the suggestion that hyperoxalaemia of the degree seen in patients with the type of chronic renal failure that is not due to primary hyperoxaluria confers an appreciable risk of extrarenal oxalosis.

Acute posterior multifocal placoid pigment epitheliopathy and sarcoidosis.

We report on a patient with acute posterior multifocal placoid pigment epitheliopathy and sarcoidosis. A review of the literature suggests that sarcoidosis may occur more commonly in this condition than previously suspected.

Renal biopsy appearances in rheumatoid disease.

The renal biopsies of 30 patients with rheumatoid arthritis and clinical evidence of renal disease were reviewed; only patients in whom the intravenous pyelogram was normal were subjected to biopsy, thus excluding those with papillary necrosis and chronic pyelonephritis. Tissue was studied by light, electron and immunofluorescence microscopy. There were 13 cases of mesangial change, 9 of membranous glomerulonephritis, 4 of tubulointerstitial change, 2 cases of focal segmental glomerulosclerosis, 1 case of amyloid and 1 of diffuse proliferative glomerulonephritis with crescents. All 9 patients with membranous glomerulonephritis but only 6 of 13 with mesangial change had received gold or penicillamine. We found no evidence of "glomerulitis" or of a rheumatoid vasculitis.

Isolated C3 deposition in patients without systemic disease.

Deposition of C3 without immunoglobulins ("isolated C3") was found in 9.8% of 540 renal biopsies performed between 1976 and 1982. Thirty-two of these samples were from patients with systemic diseases (16), well defined forms of glomerulonephritis (9), other renal diseases (4) or renal allografts (4). The remaining 22 patients are described. Five had nephrotic syndrome, three of them with minimal changes on light microscopy and good response to corticosteroids. We were left with a group of 17 patients who presented with gross (4) or microscopic (12) hematuria or asymptomatic proteinuria (1) whose biopsies showed mesangial proliferation or/and hyperplasia and who followed a benign course over the mean 3 years of follow-up, with the exception of one whose renal function is declining. This clinicopathological picture is similar to that described in two previous publications suggesting that "Isolated C3 mesangial proliferative nephritis" is a recognizable sub-group within the spectrum of glomerulonephritis.

Plasma renin activity during the development of paracetamol (acetaminophen) induced acute renal failure in man.

Plasma renin activity has been measured daily in 36 patients suffering from self poisoning with acetaminophen. In 3 developing porto-systemic encephalopathy terminal renal failure developed with high plasma renin activity. In 2 who developed acute renal failure without porto-systemic encephalopathy, plasma renin activity was noted to rise before serum creatinine and to return to initial levels after 3 or 4 days while renal failure persisted. Six other patients with similar hepatic damage showed comparable rises in renin without developing renal failure. Our findings are consistent with but do not establish a pathogenetic role for renin in acetaminophen-induced acute renal failure. It is suggested that other factors may act with renin to bring about renal failure.

Renal histology and immunopathology in distal renal tubular acidosis.

Renal biospy studies are reported from 10 patients with distal renal tubular acidosis (DRTA). On the biopsies from 6 patients who had associated immunological abnormalities immunofluorescent studies for immunoglobulins, complement, and fibrin were performed. Interstitial cellular infiltration and fibrosis were common findings in patients with and without immunological abnormalities, and were usually associated with nephrocalcinosis and/or recurrent urinary infection. No immune deposits were demonstrated in association with the renal tubules. This study shows that DRTA in immunologically abnormal patients is not caused by tubular deposition of antibody or immune complexes. The possibility of cell mediated immune damage is discussed.

Membranous glomerulonephritis in transplant kidneys: recurrent or de novo disease in four patients.

Membranous glomerulonephritis in the graft is a uncommon complication of renal transplantation. In the 4 cases we describe, one patient had recurrence of his original disease, but in the other 3 it arose de novo after allografting for focal segmental glomerulosclerosis, cystinuria or secondary amyloidosis. The lesion was accompanied by mesangial changes of lesser degree and by chronic rejection, and it resulted in accelerated loss of graft function. Transmission of host disease to the graft is more likely to occur in patients whose original membranous lesion progressed to renal failure relatively rapidly, while in de novo cases, the antecedent host disease seems often to have been focal segmental glomerulosclerosis. Allograft membranous glomerulonephritis may be less rare than hitherto supposed.