Identification of lesions indicating rejection in kidney transplant biopsies: tubulitis is severely under-detected by conventional microscopy.

BACKGROUND: In the current international Banff classification of kidney transplant rejection, tubulitis and intimal arteritis are regarded as the key histological features of acute rejection. Grade 1 tubulitis can sometimes be seen in biopsies that do not represent acute rejection; but in the case of intimal arteritis, just one lymphocyte can justify anti-rejection treatment. Our aim was to audit reliability and accuracy of recognizing tubulitis and intimal arteritis using the approach recommended by the Banff classification and correlate any discrepancies with subsequent graft function. METHODS: This is a retrospective review of all kidney transplant biopsies reported as negative for rejection from 1 January 2009 to 31 December 2009 to assess the presence or absence of occult tubulitis and arteritis. Lymphocytes were immunostained with CD3, using Periodic Acid Schiff as a counterstain. Sections were reviewed to detect missed intimal arteritis and tubulitis. Discrepancies between the report and the immunostain results were analysed by biopsy type and broken down by the reporting pathologist. The graft function of any patient with missed lesions was checked to test for adverse impact on the patient. RESULTS: 'Missed' tubulitis was found in 68% of biopsies, but only two such cases subsequently developed biopsy-proven acute rejection. Only one case of missed intimal arteritis was found (1%) and the subsequent clinical course suggested that this was probably early rejection. There was no significant difference between the reporting pathologists. CONCLUSIONS: We conclude that tubulitis is missed very frequently, but the Banff classification seems to be 'calibrated' to allow for this and it does not seriously affect the identification of clinically significant acute rejection. Immunostaining is therefore not indicated in routine practice because (by Banff criteria) it would result in over-diagnosis of rejection. Intimal arteritis can indicate acute rejection even if extremely mild.

Matrix metalloproteinase-8 and -9 are increased at the site of abdominal aortic aneurysm rupture.

BACKGROUND: Abdominal aortic aneurysm (AAA) expansion is characterized by extracellular matrix degradation and widespread inflammation. In contrast, the processes that characterize AAA rupture are not well understood. The aim of this study was to investigate the proteolytic and cellular activity of ruptured AAA, focusing on matrix metalloproteinases (MMPs) and their inhibitors (TIMPs). METHODS AND RESULTS: Anterior aneurysm wall biopsies were taken from 55 nonruptured and 21 ruptured AAAs. A further biopsy from the site of rupture was taken from 12 of the ruptured AAAs. MMP-1, -2, -3, -8, -9, and -13, as well as TIMP-1 and -2, were quantified in each biopsy with ELISA. A comparison of anterior aneurysm biopsies showed no difference in MMP or TIMP concentrations between nonruptured and ruptured AAA. In a comparison of ruptured AAA biopsies, MMP-8 and -9 levels were significantly elevated in the 12 rupture site biopsies compared with their 12 paired anterior wall biopsies, whereas other MMPs and TIMPs showed no difference (MMP-8, P<0.001; MMP-9, P=0.01). MMP-8 and -9 expression was mediated by native mesenchymal cells and was independent of the inflammatory infiltrate. CONCLUSIONS: A localized increase in MMP-8 and -9, mediated by native mesenchymal cells, presents a potential pathway for collagen breakdown and AAA rupture.

Interobserver reproducibility and application of the ISN/RPS classification of lupus nephritis-a UK-wide study.

We sought to assess the interobserver variation of the new International Society of Nephrology/Renal Pathology Society (ISN/RPS) classification of lupus nephritis when compared with the previous World Health Organization classification, when used by pathologists throughout the UK. We also sought differences in how the 2 classifications were applied to a single set of biopsies. Twenty unselected renal biopsies showing lupus nephritis were circulated to pathologists in the UK National Renal Pathology External Quality Assessment Scheme, before the ISN/RPS scheme was published, with a request to apply the World Health Organization classification. The same slides were recirculated approximately 1 year later with a request to apply the ISN/RPS classification. A significant improvement in interobserver reproducibility was demonstrated by the new classification (kappa 0.53 vs. 0.44, P = 0.002). The reproducibility of the assessment of disease activity and chronicity remains suboptimal (kappa = 0.33). The new classification tends to produce more diagnoses of Class IV lupus nephritis, with fewer diagnoses of Classes III and V. The improvement in interobserver reproducibility indicates that an important aim of the new classification has been achieved. Further work is needed to determine whether the increase in diagnosis of Class IV nephritis represents an improvement in biopsy interpretation or a divergence from the previous classification, as the latter could undermine attempts to relate results from the new system to treatment strategies based on clinical trials which used the old.

Apoptosis and caspase-3 in long-term renal ischemia/reperfusion injury in rats and divergent effects of immunosuppressants.

BACKGROUND: Caspase-3 plays a key role in apoptosis, but the involvement of apoptosis and caspase-3 in mediating long-term ischemia/reperfusion (I/R) and immunosuppressive injury are not fully defined. The present study was undertaken to investigate apoptosis and caspase-3 in a renal I/R injury rat model with or without immunosuppression. METHODS: The right renal pedicle was clamped for 45 minutes and left nephrectomy was induced. Cyclosporin A (CsA), tacrolimus (Tac), rapamycin (Rap), or mycophenolate mofetil (MMF) were administered daily. Animals were killed at 16 weeks, and the levels of apoptosis (with in situ end-labeling fragmented DNA), caspase-3 protein (with immunohistochemistry, Western blotting, and activity assay), and messenger RNA (mRNA; with quantitative reverse-transcriptase polymerase chain reaction) were evaluated. RESULTS: Kidneys with I/R injury showed increased apoptosis in tubular and interstitial areas compared with control kidneys. Tacrolimus, Rap, and MMF significantly reduced apoptosis, but CsA did not. Distribution of full-length caspase-3 widened in I/R-injured kidneys from normal distal tubules and collecting ducts to dilated proximal tubules and expanded interstitium, whereas active caspase-3 was mainly scattered in damaged tubules and interstitium. Active caspase-3 staining and 24-kDa active caspase-3 protein was enhanced in I/R-injured and CsA-treated kidneys, but decreased by Tac, Rap, and MMF. These results were also consistent with changes in caspase-3 activity. Although caspase-3 mRNA levels were significantly increased in uninephrectomy and I/R-injured kidneys, they were not significantly affected by the immunosuppressants. In addition, all changes detected were positively correlated with renal structure and function. CONCLUSION: Apoptosis and caspase-3 are not only involved in the long-term renal I/R injury, but also mediate the divergent effects of immunosuppression in this model.

Macrophage-induced rat mesangial cell expression of the 24p3-like protein alpha-2-microglobulin-related protein.

During screening of a murine macrophage cDNA repertoire for factors potentially able to modulate glomerular cell responses to injury, we identified a gene coding for the murine protein 24p3 lipocalin. Immunostaining of normal rat kidney sections showed positive 24p3-like staining in distal tubules/collecting ducts and small muscular arteries. Although most glomeruli were negative, some did exhibit small numbers of positively stained cells. Cultured rat glomeruli and glomerular mesangial cells secreted the 24p3-like protein in response to macrophage-conditioned medium (MPCM) and the cytokine IL-1beta. MPCM derived from TGFbeta-pretreated macrophages enhanced mesangial cell 24p3 secretion. In contrast, addition of anti-IL-1beta neutralising antibody to MPCM or IL-1beta resulted in suppression of 24p3 secretion. Co-culture of mesangial cells with varying numbers of non-LPS-treated macrophages resulted in dose-dependent secretion of 24p3 into culture supernatants. Archival sections from polyvinyl alcohol-treated and cholesterol-fed rats showed positive glomerular staining for 24p3 in and around glomerular foam cells. Nucleotide sequencing of rat mesangial cell-derived 24p3 cDNA revealed it to be identical to rat alpha-2-microglobulin-related protein (alpha2microGRP), the rat homologue of murine 24p3. These data provide the first description of rat alpha2microGRP in the context of mesangial cell pathophysiology.

Microvessel density at presentation predicts subsequent muscle invasion in superficial bladder cancer.

PURPOSE: The purpose of this study was to determine whether angiogenesis, as measured by microvessel density (MVD), at presentation is related to subsequent progression of superficial bladder cancer (SBC). EXPERIMENTAL DESIGN: Archived primary bladder tumors from 180 patients were stained with a monoclonal antibody against cluster determinant 34 to label vessels. Image analysis was used to count MVD in 30 randomly selected areas in each case. RESULTS: Of the 170 patients evaluated, 37 progressed to muscle invasive disease. A strong association was found between the intensity of angiogenesis and clinical stage, pT1 tumors having a higher MVD than pTa disease. The median MVD was significantly higher at presentation in those patients that subsequently developed progressive SBC than in those that did not progress (P < 0.0001). pT1 (P = 0.001), grade 3 disease (P = 0.002), and MVD (P = 0.008) were found to predict subsequent disease progression on univariable analysis. Both MVD (P = 0.007) and pT1 disease (P = 0.044) remained significant predictive factors for subsequent disease progression on multivariable analysis. CONCLUSION: MVD in SBC at presentation is significantly higher in those cases that subsequently progress to muscle invasive disease.

International variation in histologic grading is large, and persistent feedback does not improve reproducibility.

Histologic grading systems are used to guide diagnosis, therapy, and audit on an international basis. The reproducibility of grading systems is usually tested within small groups of pathologists who have previously worked or trained together. This may underestimate the international variation of scoring systems. We therefore evaluated the reproducibility of an established system, the Banff classification of renal allograft pathology, throughout Europe. We also sought to improve reproducibility by providing individual feedback after each of 14 small groups of cases. Kappa values for all features studied were lower than any previously published, confirming that international variation is greater than interobserver variation as previously assessed. A prolonged attempt to improve reproducibility, using numeric or graphical feedback, failed to produce any detectable improvement. We then asked participants to grade selected photographs, to eliminate variation induced by pathologists viewing different areas of the slide. This produced improved kappa values only for some features. Improvement was influenced by the nature of the grade definitions. Definitions based on "area affected" by a process were not improved. The results indicate the danger of basing decisions on grading systems that may be applied very differently in different institutions.

Protocol biopsy of the stable renal transplant: a multicenter study of methods and complication rates.

BACKGROUND: Clinical trials in renal transplantation must use surrogate markers of long-term graft survival if conclusions are to be drawn at acceptable speed and cost. Morphologic changes in transplant biopsies provide the earliest available evidence of damage, and "protocol" biopsies from stable grafts can be used to reduce the number of patients needed in clinical trials. This approach has been inhibited by concerns over safety, but the risk of biopsy of a stable kidney, with no active inflammation or acute functional impairment, has never been formally estimated. METHODS: In accordance with a predefined set of questions, a retrospective audit of a sequential series of protocol biopsies was performed in four major transplant centers. RESULTS: A total of 2,127 biopsy events were assessed for major complications, and 1,486 were assessed for minor ones. There were no deaths. One graft was lost, under circumstances indicating that the loss should have been prevented. Three episodes of hemorrhage required direct intervention. Three further patients required transfusion. There were two episodes of peritonitis, but one was arguably an unrelated event. All serious complications presented within 4 hr of biopsy. CONCLUSIONS: The incidence of clinically significant complications after protocol biopsy of a stable renal transplant is low. Direct benefits to the patients concerned (irrespective of the benefit that may accrue in clinical trials) were not formally assessed but seem likely to outweigh the risk of the procedure. We believe that it is ethically justifiable to ask renal transplant recipients to undergo protocol biopsies in clinical trials and routine care.

Biomarkers assessing warm ischemic injury using an isolated porcine kidney hemoreperfusion model.

Prolonged warm ischemia (WI) occurring in marginal kidney donors together with reperfusion injury determines allograft survival, in which apoptosis and inflammation play crucial roles. There is no single valid biomarker, so far, to assess the degree of kidney donor injury. To define new biomarkers for detecting initial donor ischemic injury, caspase-3, caspase-7, apoptosis, inflammation, HSP70 and renal histological changes were examined in porcine kidneys subjected to 7- 15- 25- or 40-min WI, two-hour cold storage and six-hour hemoreperfusion. Caspase-3 activity was gradually increased by prolonged reperfusion, with a decrease trend against WI time. This result was verified by raised 17 kDa active caspase-3 in postreperfusion kidneys, with elevated 12 kDa active caspase-3 and lowered precursor at seven-minute WI. Active caspase-7 was also doubled by reperfusion with decreased precursor at seven-minute WI, but declined against prolonged WI. Apoptotic cells in tubular and interstitial areas were greatly increased by reperfusion at seven-minute WI, but decreased against prolonged WI. In addition, myeloperoxidase (MPO)+ cells were dramatically increased by reperfusion and presented as a bell-shape against WI time, while HSP70 was significantly increased at 7-min WI, but decreased at 40-min WI after reperfusion. In postreperfusion kidneys, tubular dilation and cell shedding were observed at 7- and 15-min WI, while tubular vacuolation and cell debris were found in tubular lumens at longer WI times. At 40-min WI, early nuclear pyknosis, tubular epithelia detachment and peri-tubular capillary dilation were detected. Furthermore, caspase-3, caspase-7, apoptosis, but not MPO+ cells or HSP70, were correlated with renal function. In conclusion, caspase-3, caspase-7 and apoptosis appear to be better biomarkers than MPO+ cells or HSP70 for assessing warm ischemic injury in donor kidneys. Hemoreperfusion activates caspase-3 and caspase-7, promotes apoptosis of damaged cells in kidneys only with limited WI, which might be beneficial to renal structural re-modeling and functional recovery.

Glomerular changes in microscopic haematuria, studied by quantitative immunoelectron microscopy and in situ zymography.

BACKGROUND: Haematuria of glomerular origin, even if mild, implies the development of defects in the glomerular basement membrane (GBM). In diseases where there is no infiltration of leukocytes into the glomerulus-such as thin basement membrane disease (TBMD) and histologically mild cases of IgA nephropathy (IgAN)-the mechanism by which such defects form is unclear. METHODS: Frozen renal tissue from 18 cases of TBMD, 18 of mild IgAN and 18 cases with no detectable abnormality were studied: (i) by quantitative in situ zymography, to estimate the activity of glomerular collagenases; and (ii) by quantitative immunoelectron microscopy to estimate the amount of major basement membrane proteins per unit length and per unit area of glomerular basement membrane. RESULTS: Cases of IgAN showed considerably more glomerular collagenase activity than normal (P=0.001). Thin basement membrane disease showed no difference in collagenase activity. A count of LCA-positive cells in glomeruli confirmed that the IgAN cases did not show glomerular leukocyte infiltration. Conversely, cases of IgAN showed no difference in GBM composition from normal, nor was any difference in GBM thickness detected in this group. However, cases of TBMD showed considerably less laminin (P=0.0008), fibronectin (P=0.002) and type VI collagen (P=0.0005) per unit length of basement membrane. Collagen IV showed a smaller reduction per unit length (P=0.01), but unlike the other protein studies it appeared to be present in higher concentration per unit area (P=0.03), suggesting that it is more 'compact' in TBMD disease. CONCLUSIONS: Two distinct mechanisms of haematuria seem to be involved in these two conditions. In IgAN there is increased activity of enzymes that can degrade GBM, probably reflecting mesangial cell activation. In TBMD an abnormal composition of the thinned GBM is confirmed. When considered with published reports of genetic abnormalities in TBMD, these results raise the possibility of an abnormal interaction between collagen IV and laminin.

Fatty acids exacerbate tubulointerstitial injury in protein-overload proteinuria.

The role of the albumin-carried fatty acids in the induction of tubulointerstitial injury was studied in protein-overload proteinuria. Rats were injected with fatty acid-carrying BSA [FA(+)BSA], fatty acid-depleted BSA [FA(-)BSA], or saline. Macrophage infiltration was measured by immunohistochemical staining, apoptotic cells were detected by in situ end labeling, and proliferating cells were identified by in situ hybridization for histone mRNA. Macrophage infiltration was significantly greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. The infiltrate was largely restricted to the outer cortex. Apoptosis was greater in the FA(+)BSA group than in the FA(-)BSA and saline groups. Compared with the saline group, apoptosis was significantly increased in the FA(+)BSA group but not in the FA(-)BSA group. Cortical cells proliferated significantly more in the FA(+)BSA and FA(-)BSA groups than in the saline group. FA(+)BSA is therefore a more potent inducer of macrophage infiltration and cell death than FA(-)BSA. The fatty acids carried on albumin may be the chief instigators of tubulointerstitial injury in protein-overload proteinuria.

A computer image analysis system for microvessel density measurement in solid tumours.

Microvessel density (MVD) is a widely used surrogate measure of angiogenesis in pathological specimens and tumour models. Measurement of MVD can be achieved by several methods. Automation of counting methods aims to increase the speed, reliability and reproducibility of these techniques. The image analysis system described here enables MVD measurement to be carried out with minimal expense in any reasonably equipped pathology department or laboratory. It is demonstrated that the system translates easily between tumour types which are suitably stained with minimal calibration. The aim of this paper is to offer this technique to a wider field of researchers in angiogenesis.

Sequential protocol biopsies from renal transplant recipients show an increasing expression of active TGF beta.

Chronic allograft nephropathy (CAN) is a major cause of graft loss after renal transplantation. Implicated in the pathogenesis of this complication is overproduction of the cytokine transforming growth factor beta (TGF beta). In this study we measured changes in CAN's expression in stable patients early after transplantation, and studied links with established risk factors for CAN, such as delayed graft function, acute rejection, and cyclosporine exposure. We took biopsies from 40 renal allografts at time of transplantation (pre-perfusion), and then, using ultrasound guidance, at 1 week and 6 months after transplantation. An immunofluorescence technique was used to stain sections for active TGF beta. These were then assessed by semi-quantitative scanning laser confocal microscopy. There was very little variation in active TGF-beta expression among patients in their pre-perfusion biopsies. Expression had increased by 1 week and then very significantly by 6 months ( P<0.0001). Patients who suffered delayed graft function had increased TGF-beta expression at both time points. There was no difference regarding donor type, acute rejection, and immunosuppressive drug (cyclosporine or tacrolimus). There was no correlation between the amount of TGF-beta expression at any time-point and isotope glomerular filtration rate (GFR) at 12 months. This study demonstrated that in a group of stable renal allograft recipients, TGF-beta expression in the kidney increased after transplantation. As the study used protocol biopsies, this increase is unlikely to be due to acute events, and probably represents a genuine increase.