Transmission of angiosarcomas from a common multiorgan donor to four transplant recipients.

We describe the donor tumor transmission of metastatic angiosarcomas to four transplant recipients through transplantation of deceased-donor organs, i.e. kidneys, lung and liver, from an apparently unaffected common female multiorgan donor. Fluorescent in situ hybridization of angiosarcoma cells confirmed that the tumor was of female donor's origin in male kidney recipients. Recent literature associated increased urokinase-plasminogen-activator-receptor (uPAR) and plasma soluble urokinase-plasminogen-activator-receptor (suPAR) levels with metastatic malignancies. Now we found that, compared to baseline levels, both deceased-donor kidney recipients showed increased uPAR transcripts in mononuclear cells as well as increased plasma suPAR levels after the diagnosis of metastatic angiosarcomas, i.e. 4 months after donor tumor transmission. These results show an association of uPAR/suPAR in donor tumor transmission of metastatic angiosarcomas in humans.

Hyponatremia as a prognostic and predictive factor in metastatic renal cell carcinoma.

BACKGROUND: Low serum sodium has recently been associated with poor survival in localised renal cell carcinoma (RCC). We now show the prognostic effect of serum sodium in patients with metastatic RCC (mRCC). METHODS: Cohort A comprised 120 consecutive patients with mRCC receiving subcutaneous, low-dose interleukin-2 and interferon-alpha. Hyponatremia was assessed in univariate and multivariate analyses. An independent cohort of another 120 patients with mRCC was used for validation (cohort B). RESULTS: In cohort A, estimated 5-year survival was 15% and median survival was 15.1 months. Serum sodium ranged between 126 and 144 mM. Twenty-four patients (20%) had serum sodium levels below normal range (<136 mM). In multivariate analysis, significant independent risk factors for short survival were low serum sodium (P=0.014), high neutrophils (P=0.018), lactate dehydrogenase >1.5 upper normal level (P=0.002), and number of metastatic sites (+3) (P=0.003). In cohort B, serum sodium ranged between 128 and 146 mM. Seventeen patients (14%) had sodium levels below normal range. In multivariate analysis, serum sodium was validated as an independent prognostic factor (P=0.001). A significant association between lack of response and hyponatremia was observed in both cohorts (P=0.003 and P=0.02, respectively). CONCLUSION: Low serum sodium is a new, validated, independent prognostic, and predictive factor in patients with mRCC.

MLPA and cDNA analysis improves COL4A5 mutation detection in X-linked Alport syndrome.

The X-linked form of Alport syndrome (AS) is caused by mutations in the COL4A5 gene encoding the alpha5 chain of type IV collagen. Most COL4A5 mutations are individual, and mutation analysis is complicated by the size of the gene and the number of exons. Larger structural rearrangements account for 10-15% of mutations. We have established a method for mutation analysis of COL4A5 based on reverse transcriptase-polymerase chain reaction analysis of mRNA from cultured skin fibroblasts and multiplex ligation-dependent probe amplification (MLPA) on genomic DNA. One advantage of using skin biopsies for the mRNA analysis is the possibility of immunohistochemical staining for the alpha5(IV) chain on skin sections to support a diagnosis of X-linked AS. A mutation was detected in all five cases included. One patient presenting with AS and diffuse leiomyomatosis was found to have a COL4A5 deletion extending into and comprising COL4A6 exons 1, 1', and 2. We have evaluated the MLPA assay on samples from 67 previously tested AS patients (45 males and 22 females) and 20 controls. We found that the combination of cDNA and MLPA analysis improves the mutation detection rate in COL4A5 and that MLPA should be the first step in genetic testing for X-linked AS.

Temporal profile of calcineurin phosphatase activity during acute allograft rejection in the heterotopic rat heart transplantation model.

BACKGROUND: Regardless of the extensive worldwide use of calcineurin inhibitors, little is known about the behavior of calcineurin phosphatase (CaN) during acute allograft rejection. The aim of this study was to investigate the temporal profile of CaN during acute allograft rejection and reveal if it can be utilized as a pharmacodynamic marker to identify and monitor the rejection process. METHODS: The heterotopic cervical rat heart transplantation model was used (dark Agouti to Lewis). We performed 25 control isogeneic and 46 allogeneic transplantations. Rats were sacrificed at various postoperative time points. CaN activity was measured in isolated peripheral blood and spleen mononuclear cells and in graft heart homogenates. CaN activity was measured as the release of radiolabeled phosphate from a previously phosphorylated 19 amino acid peptide. RESULTS: We have shown that CaN's activity levels are not significantly altered during acute allograft rejection in peripheral blood mononuclear cells and in spleen-isolated mononuclear cells. CaN's intragraft activity decreased with time in both rejectors and controls, and was significantly lower in the allogeneic group. CONCLUSIONS: CaN failed as a pharmacodynamic biomarker of acute allograft rejection in the heterotopic rat heart transplantation model. Further research is required in order to reveal the precise role of CaN during acute allograft rejection.

Kidney volume increases during long-term ciclosporin a treatment.

PURPOSE: Previously, we have for the first time reported enlargement of the pig kidney during long-term ciclosporin (CsA) treatment. In this paper, we summarize our findings of renal enlargement during long-term protocols with various dosages and durations of CsA administration as well as discuss possible pathogenetic mechanisms. MATERIALS AND METHODS: Twenty-two adolescent Gottingen minipigs were allocated into four groups: group A (n = 6) served as controls for 6 months; group B (n = 5) were treated with CsA (10 mg/kg per day) for 6 months, group C (n = 4), with CsA (20 mg/kg per day) for 6 months, and group D (n = 7) with CsA (10 mg/kg per day) orally for 12 months. At regular intervals, renal length and total volume were measured using magnetic resonance imaging; renal biopsies were performed for histological examination. RESULTS: A significant increase in kidney volume occurred in all CsA-treated pigs (groups B, C, and D); whereas the volume remained stable in the control animals (group A). A small but significant rise in kidney length was observed in groups A, B, and C, probably due to the normal growth of the animals. Histological examination was normal after treatment with CsA doses of 10 and 20 mg/kg per day for 6 months but showed definite interstitial fibrosis and glomerulosclerosis after treatment with 10 mg/kg per day CsA for 12 months. CONCLUSION: Long-term CsA treatment produced renal enlargement in pigs before the development of histological changes in the kidney. Thus, renal enlargement may represent an early stage of chronic CsA nephrotoxicity.

Chronic cyclosporine nephrotoxicity: a pig model.

Cyclosporine A (CsA) is one of the keystones in immunosuppressive treatment after solid organ transplantation, despite its major side effects such as nephrotoxicity. The chronic nephrotoxic effects of CsA seen in humans have been difficult to reproduce in small-animal models. The aim of the present study was to examine the chronic nephrotoxicity produced by therapeutic dosages of CsA in a pig model. Among 11 Gottingen minipigs included in the study, three died, yielding data from five animals given CsA (10 mg/kg/d, orally) for 6 months, and three controls. Body weight, blood pressure, glomerular filtration rate (GFR) by plasma clearance of (51)Cr-ethylenediamine-tetraacetic acid, CsA concentration, serum creatinine, and other values were measured every 5 weeks. Our results showed that the whole blood trough CsA levels were lower in pigs than in humans treated with similar CsA doses. Renal biopsies, which were obtained successfully, except one case of macroscopic hematuria, showed no histological changes in the kidney. No significant increase in serum creatinine or blood pressure was observed. Surprisingly, there was a significant increase in GFR during CsA treatment. We conclude that the pig model displays a hyperfiltration that warrants further investigation.

The giraffe kidney tolerates high arterial blood pressure by high renal interstitial pressure and low glomerular filtration rate.

BACKGROUND: The tallest animal on earth, the giraffe (Giraffa camelopardalis) is endowed with a mean arterial blood pressure (MAP) twice that of other mammals. The kidneys reside at heart level and show no sign of hypertension-related damage. We hypothesized that a species-specific evolutionary adaption in the giraffe kidney allows normal for size renal haemodynamics and glomerular filtration rate (GFR) despite a MAP double that of other mammals. METHODS: Fourteen anaesthetized giraffes were instrumented with vascular and bladder catheters to measure glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). Renal interstitial hydrostatic pressure (RIHP) was assessed by inserting a needle into the medullary parenchyma. Doppler ultrasound measurements provided renal artery resistive index (RI). Hormone concentrations as well as biomechanical, structural and histological characteristics of vascular and renal tissues were determined. RESULTS: GFR averaged 342 ± 99 mL min(-1) and ERPF 1252 ± 305 mL min(-1) . RIHP varied between 45 and 140 mmHg. Renal pelvic pressure was 39 ± 2 mmHg and renal venous pressure 32 ± 4 mmHg. A valve-like structure at the junction of the renal and vena cava generated a pressure drop of 12 ± 2 mmHg. RI was 0.27. The renal capsule was durable with a calculated burst pressure of 600 mmHg. Plasma renin and AngII were 2.6 ± 0.5 mIU L(-1) and 9.1 ± 1.5 pg mL(-1) respectively. CONCLUSION: In giraffes, GFR, ERPF and RI appear much lower than expected based on body mass. A strong renal capsule supports a RIHP, which is >10-fold that of other mammals effectively reducing the net filtration pressure and protecting against the high MAP.

Reproducibility of the Banff classification of renal allograft pathology. Inter- and intraobserver variation.

The present study was undertaken to investigate the inter- and intraobserver variation in use of the scoring system for glomerulitis, vasculitis, interstitial inflammation, tubulitis and arteriolar hyalinosis that is an essential part of the recently proposed Banff classification of renal allograft biopsies. Seventy-seven biopsies done less than 90 days after transplantation were included. The scoring was done blindly by five pathologists on biopsies stained with H&E and PAS. The volume fraction of interstitial inflammation was estimated. Spearman rank correlation coefficient and kappa values were used for the evaluation of reproducibility. The results of both inter- and intraobserver variability showed a good correlation and reasonable kappa values for vasculitis, interstitial inflammatory infiltration, and tubulitis. Less-good correlation was found for glomerulitis and arteriolar hyalinosis. The interobserver kappa score for grading of the rejection severity was 0.40 overall but 0.56 when only presence or absence of acute rejection was considered and 0.66 for presence or absence of vasculitis. Weighted kappa values for interobserver vasculitis score and rejection grading were 0.58 and 0.55, respectively. A strong association existed between the volume fraction of interstitial inflammation and the semiquantitative scoring for interstitial inflammation. In conclusion, the good correlations for the key elements in the grading of the allograft biopsies in the present classification system, confirmed the utility of the defined criteria for grading rejection. More precisely defined criteria or simplification of the scoring system are needed for glomerulitis and arteriolar hyalinosis--parameters not used in the diagnosis of rejection.

Endocapillary glomerulitis in the renal allograft.

Endocapillary glomerulitis is characterized by an increase in number of mononuclear cells in the glomerular capillary lumina. This lesion has been described in the early posttransplant period, but its pathogenesis, relation to conventional rejection, and prognostic impact is not well known. Using the definitions, scorings, and gradings of the Banff system for classification and grading of histopathologic changes in the renal allograft, we have analyzed 444 consecutive renal allograft biopsies from the first 90 days posttransplant. Moderate or severe glomerulitis occurred in 13.5% of the biopsies. There was a strong tendency toward clustering of glomerulitis: if one biopsy from a patient had glomerulitis, there was a high probability that it occurred in other biopsies from the same patient. There was some correlation with conventional acute rejection, but 40% of all biopsies with glomerulitis had no rejection and 53% of all biopsies with rejection had no glomerulitis. Graft function at biopsy was nil or decreased in many patients, but this could largely be explained by the independent presence of primary graft dysfunction or conventional rejection, these conditions being a frequent indication for performing a graft biopsy. Moderate or even severe glomerulitis was, however, compatible with a functioning graft. No correlation between glomerulitis and active CMV infection was found. The one-year graft survival of grafts with early posttransplant glomerulitis was 66%. If early conventional acute rejection is taken into consideration, graft survival does not seem to be influenced by the presence of glomerulitis. Early posttransplant endocapillary glomerulitis may be a peculiar pattern of rejection with a pathogenesis different from that of conventional rejection, but the present investigation does not demonstrate any adverse effects on graft function or graft prognosis.

Atubular glomeruli in cisplatin-induced chronic interstitial nephropathy. An experimental stereological investigation.

In two previous studies on chronic renal failure in humans as well as in experimental animals a large number of glomeruli were found not to be connected to proximal tubules (atubular). Using unbiased stereological methods the purpose of the present study was to investigate and quantitate various renal structures, including the presence or absence of atubular glomeruli, in chronic renal failure induced by cisplatin. After administration of cisplatin to rats for ten weeks, the left kidney was perfusion-fixed 3 weeks later. The cisplatin-treated rats were uremic with an average plasma urea of 32.6 mmol/l compared with 10.8 mmol/l in controls. The stereological investigations showed that 31% of the glomeruli in the cisplatin-treated group were atubular, 37% were connected to a normal proximal tubule and 32% to an atrophic tubule. Sclerotic glomeruli were not observed and the total number of glomeruli did not differ between the two groups. The mean glomerular volume was significantly decreased in the cisplatin group, and the mean volume of atubular glomeruli was lower than the mean volume of glomeruli connected to a normal proximal tubule. Significant negative correlation was found between the plasma urea on the one hand and the percentage of glomeruli connected to normal proximal tubules, as well as the length of normal proximal tubules, on the other hand. A significant positive correlation was found between plasma urea and the volume of interstitial fibrotic tissue. The presence of atubular glomeruli and the absence of compensatory hypertrophy of the remaining nephrons might be responsible for the reduction in kidney function in several chronic interstitial renal diseases.

The progression of cisplatin-induced tubulointerstitial nephropathy in rats.

We have previously shown that administration of cisplatin to rats leads to chronic nephropathy with many atubular glomeruli. Using unbiased stereological and histochemical methods our purpose was to investigate the progression of renal structural changes, specifically the events preceding and following the formation of atubular glomeruli in rats after increasing total doses of cisplatin. After administration of cisplatin (2 mg/kg body weight i.p. once weekly) the animals were sacrificed 10 days, three, six, 10 and 20 weeks later. After 4 mg/kg the distal part of most proximal S3 segments appeared necrotic with a negative reaction for enzymes characteristic of brush border and mitochondria. After larger doses, atrophy (and possibly necrosis) was observed in clusters of cortical proximal and distal nephron segments corresponding to an increasing amount of non-functioning nephrons. Hypertrophy/hyperplasia of remaining uninjured nephrons and collecting ducts was observed. The stereological investigations showed an increasing number of atubular glomeruli with open capillaries or glomeruli connected to atrophic tubules with the total dose of cisplatin (30% and 40%, respectively, after 20 mg/kg). The percentage of glomeruli with damaged tubules was significantly higher in the juxtamedullary cortex compared with the superficial cortex. Sclerotic glomeruli were not observed. The findings suggest that a toxic destruction of most S3 segments leads to atrophy or disappearance of the corresponding nephrons with formation of atubular glomeruli, and that the remaining hypertrophic nephrons are connected to the few surviving or regenerated S3 segments.

Glomerular structure in lithium-induced chronic renal failure in rats.

The effects of long-term lithium administration on glomerular structure and intervention with angiotensin converting enzyme inhibitor (ACEI) were studied in rats. Male Wistar rats were fed a lithium-containing diet (Li) or control diet (C) for 16 weeks postnatally. Li-treated rats developed renal failure, hypertension and proteinuria. During the subsequent 24 weeks, subgroups were treated with ACEI. The kidneys were fixed by perfusion, and tissue blocks were serially cut for estimation of glomerular volume and glomerular characteristics by light microscopy. Mesangial and mesangial matrix volume fractions, surface density of capillary walls, basement membrane thickness and foot process width (FPW) were measured by electron microscopy. Glomerular volume was decreased in Li-rats, with increased intra-individual variation. In all Li-rats, some glomeruli (mean 27%) were abnormal, with severe changes in only three rats. Ultrastructural parameters obtained by systematic sampling of three glomeruli in each rat showed no differences among groups. Among Li-treated animals there was a significant correlation between FPW and albumin excretion per unit filtration surface, and between filtration surface per glomerulus and inulin clearance. In conclusion, long-term lithium administration to newborn rats caused marked changes in glomerular volume which were not associated with measurable changes in structural parameters. No effect of ACEI-treatment was detectable.

Interstitial nephritis and glomerulonephritis in visceral leishmaniasis in a dog. A case report.

Renal involvement characterized by interstitial nephritis and glomerulonephritis is reported in a case of visceral leishmaniasis in a dog. Focal interstitial inflammatory infiltrates including lymphocytes and plasma cells together with collections of histiocytes containing Leishmania donovani organisms were found in the renal cortex. The glomeruli showed diffuse mesangial cell proliferation, thickening of the capillary wall and focal, segmental glomerulosclerosis. By electron microscopy, finely granular electron dense deposits were shown in subendothelial, intramembranous as well as subepithelial locations. It is suggested that the interstitial nephritis was caused by the presence of parasitic elements in the renal interstitium, whereas the glomerular involvement may have been due to deposition of antigen-antibody complexes.

The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis.

The new stereological methods for correct and efficient sampling and sizing of cells and other particles are reviewed. There is a hierarchy of methods starting from the simplest where even the microscopic magnification may be unknown to the most complex where typically both section thickness and the magnification must be known. Optical sections in suitably modified microscopes can be used to improve the ease and speed with which even the most demanding of these methods are performed. The methods are illustrated by practical examples of applications to a wide range of histological entities including synapses, neurons and cancer cells, glomerular corpuscles and ovarian follicles.

Some new, simple and efficient stereological methods and their use in pathological research and diagnosis.

Stereology is a set of simple and efficient methods for quantitation of three-dimensional microscopic structures which is specifically tuned to provide reliable data from sections. Within the last few years, a number of new methods has been developed which are of special interest to pathologists. Methods for estimating the volume, surface area and length of any structure are described in this review. The principles on which stereology is based and the necessary sampling procedures are described and illustrated with examples. The necessary equipment, the measurements, and the calculations are invariably simple and easy.

A quantitative immunohistochemical study of the expression of mesangial alpha-smooth muscle actin and the proliferation marker Ki-67 in glomerulonephritis in man.

Using a specific marker 1A4 (DAKO), a quantitative evaluation of alpha-smooth muscle actin (ASMA) in glomeruli has been performed on human renal biopsies from patients suffering from acute, postinfectious, endocapillary glomerulonephritis (GN; 9 biopsies), IgA nephropathy (11 biopsies) and membranoproliferative GN (11 biopsies) and appropriate controls expressing a very weak ASMA reactivity. A significantly increased expression was found in all categories of GN. The glomeruli from IgA nephropathy showed variation of ASMA expression (range 0.1-27.7%) and a pattern of ASMA staining that was mesangial, global and diffuse. This pattern was also seen in cases of IgA nephropathy with focal, segmental, proliferative GN. In all biopsies, the glomerular cell number and proliferation index was determined. All the categories of GN showed significantly increased glomerular cellularity and proliferation index. Among the three types of GN, the glomerular cellularity and proliferation was lowest in IgA nephropathy. The mean number of Ki-67-positive intraglomerular nuclei and the proliferation index were both significantly correlated with the mean number of glomerular cell nuclei. Morphometric estimates demonstrate increased ASMA expression in types of GN with different prognosis. This finding and the lack of correlation with proliferation markers together indicate that the role of ASMA in GN is complex. This method of ASMA estimation may be useful in further studies of its role in disease activity and prognosis.

Loss of heterozygosity at 1p, 8p, 10p, 13q, and 17p in advanced urothelial cancer and lack of relation to chemotherapy response and outcome.

Studies of urothelial tumors have identified structural abnormalities in a number of chromosomes. This study aimed to identify specific genetic changes of patients with advanced urothelial cancers, and relate these changes to increased chemotherapy sensitivity or good prognosis. We screened 56 muscle-invasive bladder cancer tumors for loss of heterozygosity (LOH) at chromosome 1p, 8p, 10p, 13q, and 17p with PCR using 6 microsatellite markers. All patients had recurrent locally advanced or metastatic disease. DNA was extracted after microdissection of the primary tumor and normal tissue from paraffin-embedded specimens. The PCR products were electrophoresed in an ABI Prism 377 DNA sequencer and the alleles from tumor DNA and normal tissue DNA were analyzed using the GeneScan program. The LOH findings were correlated with response to chemotherapy and survival. Allelic loss of specific markers was present in 26-50% of the informative tumors. The most frequent LOH was observed at 17p, supporting the notion that this region may contain genes of importance to urothelial cancer progression. The overall rate of response to chemotherapy was 48%, and ranged from 40% to 56% according to specific LOH changes. The median survival of all patients from start of chemotherapy was 5.8 months and ranged from 5.3 to 7.9 months for patients with specific LOH changes. Response and survival of patients with no lost markers was the same size, compared to patients with one, two, or more lost markers. Specific genetic changes were detected in a significant number of tumors from patients with advanced urothelial cancer. These changes were not predictive of response to chemotherapy or of the duration of survival.

Reproducibility of the WHO classification of glomerulonephritis.

The inter-observer variation of the WHO classification of glomerulonephritis (GN) was studied using Kappa statistics. One hundred renal biopsies were selected with almost equal representation of the following types of GN: minimal change nephropathy, membranous GN, focal proliferative GN, diffuse mesangial proliferative GN, endocapillary GN, membranoproliferative GN, and crescentic GN. Slides stained with silver-methenamine and PAS-hematoxylin were circulated among the members of the panel, who made their diagnoses without knowing those of the other participants and without knowledge of the clinical conditions. There was a very good overall diagnostic agreement of 0.67 with a Kappa value of 0.61, figures which compete very well with other diagnostic systems analysed with Kappa statistics. Analysing the single types of GN, we found that the highest Kappa values were obtained for crescentic GN (0.81), endocapillary GN (0.79) and membranous GN (0.74) and the lowest Kappa values for membranoproliferative GN (0.40) and diffuse mesangial proliferative GN (0.44). Basically, the international classification of GN is founded upon light microscopy. Our results demonstrate that this system works generally well. The diagnostic reproducibility of the types with less satisfactory Kappa values can be expected to be improved by including immunopathology and electron microscopy.

Combined sarcoidosis and disseminated visceral giant cell vasculitis.

A middle-aged man with only slight symptoms of disease of short duration died suddenly. On autopsy he was found to have not only disseminated visceral giant cell vasculitis with involvement of large and medium-sized arteries and veins, but also sarcoid granulomas in many organs, especially in enlarged mediastinal lymph nodes as well as in several vessel walls. Death was caused by myocardial infarction due to granulomatous vasculitis. This combination of diseases has not previously been described, and the question is whether it represents giant cell arteritis and sarcoidosis, separately, or whether it is a manifestation of a broad spectrum of the same disease.