Vascular Hyalinosis Research Articles

#6323 RENAL CONTRAST-ENHANCED ULTRASOUND AND GLOMERULONEPHRITIS: A GOOD MARKER OF DISEASE ACTIVITY

Abstract Background Contrast-enhanced ultrasound (CEUS) could provide a lot of information of renal blood flow. Renal blood flow is an excellent marker of inflammatory and fibrotic processes in the kidney such as glomerulonephritis. Therefore, in patients with these diseases, renal CEUS can be both a prognostic marker and an index of response to therapy. Currently in the literature there are preliminary studies showing the role of CEUS in terms of histology and outcome in the evaluation of patients with glomerulonephritis. In this pilot study we used perfusion CEUS parameters (WiR and WoR; WiAUC and WoAUC) as possible markers of renal microvascular damage. We compared the perfusion parameters with the clinical and histological findings of our patients. Method Eighteen patients with clinical and laboratory signs of glomerulonephritis were enrolled. All patients underwent ultrasound-assisted renal biopsy and B-mode ultrasound examination, color Doppler ultrasound and contrast-enhanced ultrasound. Time Intensity (TI) curves were obtained from renal CEUS. TI curves and several quantitative perfusion parameters were defined using VueBox® Quantitation Software. Results From April to October 2022, 18 patients (12 men and 6 women) with clinical and laboratory suspicion of glomerular disease were enrolled. Median age was 48.05 years, all patients underwent renal biopsy and contrast-enhanced ultrasound at basal condition. Table 1 shows patients characteristics. CKD patients were stratified into two groups according to their eGFR: group I (eGFR < 60 ml/min/1.73 m2, n. = 4) and group II (eGFR ≥ 60 ml/min/1.73 m2, n. = 3). Moreover, we divided patients into two groups based on the presence of nephrotic-range proteinuria (≥ 3.5 g/24h). At this time, seven patient data are suitable for analysis. The small sample size at present does not allow for statistical significance. However, in our study, the persistence of contrast agent signal during the wash-out phase was differed markedly in correlation with the degree of CKD. In particular, we observed a slower wash-in phase in the presence of vascular hyalinosis (Fig. 1). Conclusion Our experience seems to confirm that CEUS parameters can be excellent indicators both in terms of patient disease outcomes and histological data. CEUS can effectively and quantitatively demonstrate renal microvascular perfusion in patients with glomerulonephritis. Future goal is that CEUS could also be a prognostic and follow-up marker after renal biopsy in patients with glomerulonephritis.

ACTIVITY OF ANGIOTENSIN-CONVERSING ENZYME-2 IN ACUTE PULMONARY INFLAMMATION

Relevance. Angiotensin converting enzyme-2 (ACE2), which is the gateway to coronavirus, is also an important component of the tissue renin-angiotensin system with a number of anti-inflammatory effects. It is known that ACE2 is expressed in the lungs of patients with coronavirus pneumonia, but it is not clear how this depends on the stages of development and the severity of inflammation.
 Objective: to establish the effect of acute inflammation on pulmonary expression of angiotensin-converting enzyme-2.
 Material and methods. In Wistar rats (n=20), in compliance with bioethical standards, a sterile nylon thread 2.5 cm long and 0.2 mm thick to a depth of 2.5 cm was introduced into the trachea. The animals were observed and removed from the experiment at 7, 14, 21 and 28 days, microscopic and immunohistochemical (monoclonal antibodies against ACE2; clone 4G5.1; EMD Millipore Corporation; Temecula, CA US) studies were performed.
 Results. The microscopic picture of the lungs indicated the development of acute bronchopulmonary inflammation during the first week, the formation of peribronchial and alveolar abscesses in the second week with the onset of resolution of bronchopneumonia with the organization of abscesses in the third week and the development of diffuse fibrosis of the parenchyma and vascular hyalinosis in the fourth week of observation. The exudative phase of acute inflammation was accompanied by inhibition of ACE2 activity in bronchial epithelial cells, type II alveolocytes and vascular endothelium. With the transition of inflammation to the stage of proliferation and fibrosis, ACE2 activity was restored.
 Conclusion. The detected phase change in ACE2 activity can cause a wavy recurrent course of coronavirus infection, since an increase in the amount of ACE2 protein during attenuation of acute inflammation contributes to an increase in target cell infection.

MO073HISTOLOGICAL PREDICTORS OF PROTEINURIA AND RENAL OUTCOMES IN PRIMARY MEMBRANOUS NEPHROPATHY: IS INTERSTITIAL FIBROSIS THE MAIN CHARACTER?

Abstract Background and Aims Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in older white adults, with an incidence of 12 cases per millions of people per year. Primary MN (PMN, 75%-80% of MNs) is an organ-specific autoimmune disease caused by antibodies anti-PLA2R and anti-THSD7A. Regardless of treatment one third of patients progresses to end-stage renal disease and two third develop non-progressive chronic kidney disease. Renal biopsy is the gold standard for MN diagnosis. Several clinical and biochemical markers have been associated with the risk of progressive loss of kidney function while contrasting results have been obtained by the few studies which have examined the prognostic value of histologic findings. In this study the clinical outcome of patients with PMN has been considered based on the prognostic value of histological findings. Method Forty-nine patients with PMN of our Nephrology Unit at Padova University Hospital from 2003 to 2018 were considered. 16 patients were excluded from the study due to missing data. Age, comorbidities, proteinuria (g/day) and renal function (eGFR, CKD-EPI) were collected. eGFR decline and decrease of proteinuria were used as clinical outcomes. The follow-up was considered from renal biopsy to the last visit (in absence of GFR decline or decrease in proteinuria). Histological grading (0-3) was assigned to parameters (glomerulosclerosis (GS), tubular atrophy (TA), interstitial fibrosis (IF), vascular hyalinosis (VH)) and were evaluated separately or in combination (as GSTIV score). Morphometric analysis was used to quantify IF and expressed in percentage as the mean of area covered by pixel. Statistical analysis was performed using Fisher’s exact test and Mann-Whitney U-test where appropriate. Cox regression analyses (univariate and multivariate) were performed to identify variables associated with both renal outcomes and p<0.05 was considered as significant. ROC curves were used to determine interstitial fibrosis cut-off values predictive for both outcomes. Area under the curve (AUC) between 0.8 and 1.0 was considered as significant. Diagnostic accuracy was assessed by Specificity (Sp), Sensibility (Se), positive (PPV) and negative (NPV) predictive values and positive (LR+) and negative (LR-) likelihood ratios. Results Patients with no decrease of proteinuria had a greater degree of IF vs those with a full response (p=0.006). Univariate Cox analyses identified age ≥65 years (HR 4.92), pre-existing CKD (HR 12.98) and IF (HR3.05) as significant predictors of renal function decline in all patients. Multivariate Cox analysis confirmed these variables (age ≥65 years HR 3.05, CKD HR 6.35, IF HR 3.03). In patients without CKD only IF was significantly associated with eGFR decline in both Cox univariate and multivariate analysis (HR 4.34 and 5.05 respectively). ROC analysis showed that IF threshold of 17.80% identified patients with eGFR reduction (AUC 0.65, Se 0.50, Sp 0.79, PPV 0.75, NPV 0.45, LR+ 2.38, LR-0.63) and IF threshold of 18.04% the lack of proteinuria reduction (AUC 0.78, Se 0.70, Sp 0.83, PPV 0.67, NPV 0.80, LR+ 4.12, LR- 0.36). Conclusion Our study shows that IF could be used as a histologic predictor of renal and proteinuria outcomes. Biopsy report should therefore also include quantitative IF data that could be helpful for the choice of a more appropriate therapeutic approach.

Vasa Vasorum Lumen Narrowing in Brain Vascular Hyalinosis in Systemic Hypertension Patients Who Died of Ischemic Stroke.

Ischemic stroke is a major cause of death among patients with systemic hypertension. The narrowing of the lumen of the brain vasculature contributes to the increased incidence of stroke. While hyalinosis represents the major pathological lesions contributing to vascular lumen narrowing and stroke, the pathogenic mechanism of brain vascular hyalinosis has not been well characterized. Thus, the present study examined the postmortem brain vasculature of human patients who died of ischemic stroke due to systemic hypertension. Hematoxylin and eosin staining and immunohistochemistry showed the occurrence of brain vascular hyalinosis with infiltrated plasma proteins along with the narrowing of the vasa vasorum and oxidative stress. Transmission electron microscopy revealed endothelial cell bulge protrusion into the vasa vasorum lumen and the occurrence of endocytosis in the vasa vasorum endothelium. The treatment of cultured microvascular endothelial cells with adrenaline also promoted the formation of the bulge as well as endocytic vesicles. The siRNA knockdown of sortin nexin-9 (a mediator of clathrin-mediated endocytosis) inhibited adrenaline-induced endothelial cell bulge formation. Adrenaline promoted protein-protein interactions between sortin nexin-9 and neural Wiskott–Aldrich syndrome protein (a regulator of actin polymerization). Spontaneously hypertensive stroke-prone rats also exhibited lesions indicative of brain vascular hyalinosis, the endothelial cell protrusion into the lumen of the vasa vasorum, and endocytosis in vasa vasorum endothelial cells. We propose that endocytosis-dependent endothelial cell bulge protrusion narrows the vasa vasorum, resulting in ischemic oxidative damage to cerebral vessels, the formation of hyalinosis, the occurrence of ischemic stroke, and death in systemic hypertension patients.

Morphological Characteristics of Intestinal Vessels of Animals with an Experimental Model of Diabetes Mellitus Type 2 Complicated by Microangiopathy

Diabetes mellitus and its complications are considered far from unresolved problems of modern medicine.An important aspect in solving fundamental problems is experimental research aimed at assessing the stateof change of pathogenetic important key positions in the development of such a formidable complicationof diabetes as angiopathy. On the other hand, surgeons’ great interest in bariatric and metabolic surgery,as method of surgical treatment of type 2 diabetes mellitus, requires in-depth studies to assess the stateof angiogenic changes in the organs of the gastrointestinal tract, namely in the intestine. The articledescribes the morphological changes in the vascular capillary bed in the dynamics of the development ofa new experimental model of diabetes mellitus, namely the chemical method of diabetic angiopathy. Thedevelopment of an experimental model of diabetic angiopathy showed the staged nature of the changes inthe form of the initial signs of angiopathy, with perivascular infiltration and edema in the form of congestiveplethora in the flesh until the development of vascular hyalinosis, which indicates the irreversibility of theprocesses of pathological transformation. Thus, the results of morphological studies in the dynamics ofmodeling of diabetic angiopathy indicate the development of diabetes mellitus with its complication in theform of microangiopathy.

P0126RENAL TRANSPLANT PATIENTS UNDER CALCINEURIN INHIBITOR THERAPY RAPIDLY ACQUIRE AN ABERRANT LYSOSOMAL LESION IN PROXIMAL TUBULAR CELLS

Abstract Background and Aims Calcineurin inhibitor therapy has changed the field of (renal) transplantation by considerably prolonging graft survival. Yet, all immunosuppressive calcineurin inhibitors are nephrotoxic that eventually contribute to complete scarring of the renal allograft. In renal biopsy analysis many histopathological features have been considered indicative of CNI nephrotoxicity, i.e. striped fibrosis, vascular hyalinosis, isometric tubular vacuolization, glomerulosclerosis, cellular infiltration and tubular atrophy, however, all are rather aspecific and can be secondary to many other causes. During the course of evaluating the specificity of a recently discovered proximal epithelial lysosomal lesion (i.e. multiple enlarged (>1,2µm) dysmorphic lysosomes containing dispersed electron dense non-membrane bound aggregates) in patients with Chronic Interstitial Nephropathy in Agricultural Communities (CINAC), we observed it to be present in renal transplant patients treated with cyclosporine or tacrolimus. Here, we test the hypothesis whether this lysosomal lesion is acquired during CNI therapy. Method A retrospective transmission electron microscopic analysis was performed to evaluate the presence of the typical lysosomal lesion on the following biopsies from renal transplant patients: 20 deceased donor implantation biopsies; 5 living donor implantation biopsies. For another 10 additional deceased donor renal allograft recipients, we evaluated implantation as well as protocol biopsies taken after 6 and 12 months of CNI treatment that started immediately after transplantation. Also included were 24 indication biopsies of CNI treated renal transplants. Results Of the total set of implantation biopsies (n=35), 2 (6%) were positive for the aberrant lysosomal phenotype on EM, whereas in the protocol and indication biopsies prevalence of the lesion was considerably higher ranging between 56% (protocol) and 80% (indication) of cases. Conclusion CNI therapy is associated with the fairly rapid appearance of a particular proximal tubular lysosomal phenotype observable on EM, that was not (or rarely) present at implantation. Whether this lesion is related to CNI toxicity and indicative for the outcome for the graft and/or patient survival after renal transplantation has to be investigated in a prospective trial.

Histological Characterizations of the Brain Vascular Hyalinosis in Patients with Systemic Hypertension‐Induced Ischemic Stroke

BackgroundIschemic stroke is a major cause of death among patients with systemic hypertension. The narrowing of the brain vascular lumen increases the incidence of stroke and the formation of hyalinosis lesion is an irreversible process that occurs in the late stage of the disease and contributes to the vascular narrowing and stroke. Understanding pathologic mechanisms of brain vascular hyalinosis, thus, should contribute to the development of new therapeutic agents that inhibits and/or reverse the formation of hyalinosis, thereby reducing the incidence of ischemic stroke. Despite the clinical importance of this lesion, properties of brain vascular hyalinosis have not been well investigated. Thus, the present study performed detailed histological examinations of the hyalinosis lesions of postmortem brain vascular tissues of human patients who died of ischemic stroke due to systemic hypertension.Methods & ResultsHematoxylin and eosin staining, as well as periodic acid‐Schiff staining, showed the presence of vascular hyalinosis in patient brain tissues. In these lesions, the protein organization of the extracellular matrix was changed, the number and size of smooth muscle cells were altered, and plasma proteins, such as apolipoprotein E and fibrin, infiltrated into the vessel wall. Signs of oxidative stress and lipid peroxidation were also found. These changes are pronounced at the loci of the infiltration of the vessel walls by ApoE. We found increased number of vasa vasorum in adventitia of the small arteries. The formation and deposition of hyaline occur both from the side of the lumen of the artery and from the side of the vase vasorum. Transmission electron microscopy showed that with the development of hyalinosis, the number of smooth muscle cells that undergo atrophy. The deposition of proteins in the arterial wall is also associated with the degree of damage to endothelial cells in the lumen of the vessel and in the vasa vasorum.ConclusionsThese results demonstrate that, at the beginning of the development of hyalinosis, smooth muscle cells and vasa vasorum are increased. This process seem to involve oxidative stress, which damages endothelial cells both in the lumen of the arteries and in the vasorum vase and contributes to the infiltration of blood plasma proteins into the vascular wall. The resulting hyalinosis decreases the lumen of the arteries and increases the incidence of stroke.Support or Funding InformationSupported by NIH

Endothelial Cell Bulge Occludes the Vasa Vasorum in the Cerebellar Vascular Hyalinosis Lesions

BackgroundThe remodeling of the cerebral vascular wall contributes to the occurrence of ischemic stroke in systemic hypertension patients, representing a leading cause of death in the U.S. and worldwide. Cerebral vascular remodeling in systemic hypertension is characterized by progressive degradation of structural components of each layer of the vessel wall with the subsequent transformation to hyaline material leading to the thickening vascular wall and lumen narrowing. The vasa vasorum as a network of small blood vessels supplying the blood to the vascular wall tissues contributes to the damage vascular structure and results in the formation of hyalinosis. The occlusion of vasa vasorum contributes to development of cerebral vascular hyalinization in systemic hypertension. However, mechanisms of occlusion of vasa vasorum that contribute to the brain vascular remodeling events have not been well defined. Lack of such knowledge interferes with the development of therapeutic strategies to reduce the occurrence of ischemic stroke.Methods & ResultsLight microscopy and transmission electron microscopy (TEM) of hyalinized cerebral vessels from human patients who suffered from systemic hypertension and died by ischemic stroke visualized the occlusion of the vasa vasorum due to the formation of the “bulge” structure that protrude from the endothelial cells. Treatment of microvascular endothelial cell with adrenaline (a major biogenic amine that are increased in the blood due to disbalance of neurohumoral homeostasis in systemic hypertension) resulted in a cytoskeletal rearrangement that contributes to the formation of the bulge‐like structure in the vasa vasorum of the brain vessels with hyalinosis lesion. The adrenaline activated clathrin‐mediated endocytosis of endothelial cells that in turn elicited SNX9/N‐WASP‐dependent disruption of cytoskeleton. siRNA knockdown of SNX9 inhibited endocytosis and prevented the adrenaline‐induced bulge formation in human microvascular endothelial cells. TEM visualized the adrenaline‐induced an increase endocytosis of the endothelial cells. Immunofluorescence staining showed the actin filament structural reorganization and TEM confirmed myofilament damage within endothelial cells. Adrenaline treatment induced post‐translational modifications of N‐WASp as a regulator of actin polymerization.ConclusionsThis is the first demonstration that adrenaline contributes to the occlusion of vasa vasorum in the hyalinized cerebral vascular wall that will allow to the development new strategies that can be used to prevent ischemic stroke.Support or Funding InformationSupported by NIH

Abstract 40: Time Course of Cerebral Small Vessel Disease Lesions and Sensory Motor Changes in Spontaneously Hypertensive Stroke Prone Rats

Introduction: Animal models of human cerebral small vessel disease (CSVD) are important for the study of the disease underlying mechanisms and testing therapeutic interventions. Spontaneusly Hypertensive Rats - Stroke Prone (SHRSP) are used as an animal model of human CSVD. However, there is a lack of data regarding the time course of cognitive and motor impairment. Methods: Male age-matched SHRSP and Wistar Kyoto rats (WKY) were studied. Sensorimotor testing (open field test) and tail-cuff systolic blood pressure (SBP) measurements (Visitech Systems, Inc.) were performed weekly starting from 6 until 24 weeks of age. Brain MRI at 7 and 24 weeks was acquired using a 9.4T MRI system. Brain histology was completed at the same time points. Statistical analysis was performed using a linear mixed model with repeated measurements. P< 0.05 was considered significant. Results: 20 SHRSP and 20 WKY male rats were studied. 10 per group were euthanized following brain MRI at 7 weeks and the rest were followed until 24 weeks. SHRSP weighed on average 30 grams less than WKY throughout the study (P=0.0003). At 7 weeks SBP was not different (WKY 106.6±5.4 vs SHRSP 120.8±5.4, P=0.06). SHRSP started to develop hypertension at 9-12 weeks and maintained hypertension until 24 weeks (average group difference across time P<0.0001; SBP at 21 weeks WKY 134.8±5.4 vs SHRSP 168.9±5.4, P<0.0001). Sensorimotor testing showed higher total distance travelled (TDT) at 7 weeks in SHRSP that trended down with ageing. Both groups became similar at 21 weeks (TDT: at 7 weeks WKY 3.78±1.3 vs SHRSP 7.8±1.3, P =0.037; at 21 weeks: WKY 4±1.3 vs SHRSP 4.4±1.3, P=0.83; average group difference across time P=0.014). Brain MRI was normal at 7 weeks, but small white matter hyperintensities were seen at 24 weeks. Brain Histology showed normal histology on hematoxylin & eosin staining at 7 weeks in both groups, while at 24 weeks SHRSP showed CSVD histopathological changes including microbleed formation, homeostasis and vascular hyalinosis. Conclusions: SHRSP develops hypertension, sensorimotor deficits and CSVD pathology as they age suggesting their utility as human CSVD model. Intervention time points should be selected carefully in future therapeutic drug interventions.

Histological grading in primary membranous nephropathy is essential for clinical management and predicts outcome of patients.

AimsDiagnosis of primary membranous nephropathy (PMN) is mainly based on immunofluorescence/immunohistochemistry findings. However, assessment of specific features on optical microscopy can help to estimate the severity of the disease, guide treatment and predict the response. The aim of this study was to identify, classify and grade the precise histological findings in PMN to predict renal function outcome and guide treatment.Methods and resultsHistological parameters, including focal segmental sclerosis (FSGS), tubular atrophy (TA), interstitial fibrosis (IF) and vascular hyalinosis (VH), were re‐evaluated in 752 patients with PMN. Their predictive value was estimated separately, and also in a combination score (FSTIV) graded from 0 to 4. Finally, the impact of histology was assessed in the response to immunosuppressive treatment. Mean age of patients was 53.3 (15–85) years and most presented with nephrotic syndrome. FSGS was present in 32% and VH in 51% of the patients, while TA and IF were graded as stage ≥1 in 52% and 51.4%, respectively. The follow‐up period was 122.3 (112–376) months. FSGS, TA and IF and VH were associated with impaired renal function at diagnosis (P = 0.02, P < 0.0001, P = 0.001 and P = 0.02, respectively) and at the end of follow‐up (P = 0.004, P < 0.0001, P < 0.0001 and P = 0.04, respectively). In multiple regression and binary logistic analysis, the presence of FSGS and degree of TA were the most significant parameters predicting renal function outcome, defined either by eGFR (end), FSGS (r = 0.6, P < 0.0001) and TA (r = 0.6, P < 0.0001), or by the endpoint of >50% eGFR reduction, FSGS (P = 0.001) and TA (P = 0.02). Also, patients presented with FSGS, IF, VH and/or with FSTIV > 1 could benefit from immunosuppression, regardless of clinical presentation.ConclusionsThe presence and degree of four histological indices, FSGS, VH, TA and IF, assessed separately or in combination, and FSTIV score not only predict renal function outcome after long‐term follow‐up, but can also help in the choice of appropriate treatment. Decisions concerning immunosuppressive treatment can be guided by pathology regardless of clinical findings.

A comparative study of the atypical CT findings and pathological features of bladder leiomyoma

Objective To investigate the relationship between the atypical CT findings and pathological features of bladder leiomyoma. Methods A retrospective analysis of the CT findings and pathological features was conducted in 9 cases of atypical bladder leiomyoma. All the 9 cases were females aged 27 to 53 years (average 38 years). All patients had plain and enhanced spiral CT scans and were treated surgically. Results The atypical CT findings of bladder leiomyomas showed as similar malignant tumor signs (n=3), solid mass with obvious inhomogeneous enhancement (n=5) and solid mass with no enhancement (n=1). All cases had lesions appearing as solid lobulated cystic mass in different degrees, whose solid part showed intermediate density on CT, with heterogeneous enhancement after contrast agent injection. Tumor cell and interstitial cell swelling to varying degrees, vascular hyalinosis, myxoedema, hyalinosis and cystic degeneration were found in these lesions. CT findings showed the consistency with similar malignant tumor signs and pathological showed bigger tumor volume, obvious interstitial edema, red degeneration, cystic degeneration and significantly increasing enlargement circuitous blood vessels. While the CT findings of the obvious inhomogeneous enhancement solid mass and pathological showed moderate tumor size, relatively obvious interstitial edema and cystic degeneration, significantly increasing enlargement circuitous blood vessels were consistent. The consistency was found among mild enhancement solid tumors and pathological showed mild interstitial edema, mild degeneration and no enlargement circuitous blood vessels. Conclusion Through the contrast analysis, atypical CT performance and pathological features might have some certain correlation. Key words: Urinary bladder neoplasms; Leiomyoma; Diagnosis; Tomography, X-ray computed; Pathological features

Clinical and morphological characteristics with markers of reparation in neuropathic diabetic foot ulcers.

Aim. To compare the clinical and morphological characteristics of chronic diabetic foot ulcers and the markers of repair.Materials and Methods.We included 26 patients with neuropathic diabetic foot syndrome who had signs of severe peripheral neuropathy. Biopsies were performed from the margin and central part of the lesion and were fixed in a 10% formalin solution before being placed on paraffin slides and stained with hematoxylin and eosin. We assessed the percentages of necrotic, granulation and fibrotic tissues and the severity of vascular hyalinosis. Immunohistochemistry was performed with initial antibodies to Ki-67 (a marker of proliferation), smooth muscle actin (a marker of myofibroblast synthesis) and cytokeratin (a marker of epithelisation). For analysis, the samples were divided into three groups by the length of time the ulcer had been present: group 1 (≤90 days; 9 samples), group 2 (91–365 days; 10 samples) and group 3 (365 days; 9 samples).Results. The patients of group 3 were older than those of groups 1 and 2 (53.7±2.7 vs 51.7±5.9 vs 59.9±5.6 years; p=0.04). There were no differences in the duration of diabetes, glycated haemoglobin or severity of neuropathy. The percentage of necrotic tissue was higher in group 1 (33.7%±21.7% vs 11.0%±3.9% vs 12.8%±6.1%; p=0.02) and the percentage of fibrotic tissue was highest in group 3 (21.1%±21.0% vs 35.5%±19.8% vs 54.4%±23.9%; p=0.001). However, the amount of granulation tissue was not different between the groups (45.2%±21.1% vs 53.5%±21.1% vs 32.8%±26.3%; p=0.4). There was also no difference in the severity of vascular hyalinosis between the groups (p=0.9). Expression of Ki-67 was higher in groups 1 and 2, implying a greater capacity to regenerate. The expression of smooth muscle actin and cytokeratin was higher in groups 1 and 2 but without statistical significance.Conclusion. The morphological characteristics and regenerative capacities of neuropathic diabetic foot ulcers differ with the duration the ulcer has been present. Patients with ulcers for less than 1 year were characterised by higher cell proliferation but lower fibrosis. Neuropathic diabetic foot ulcers that are unable to heal over a year are characterised by incomplete regeneration and higher levels of fibrosis. Thus, different treatment approaches are needed depending on how long an ulcer has been present.

Diffuse Glomerular Nodular Lesions in Diabetic Pigs Carrying a Dominant-Negative Mutant Hepatocyte Nuclear Factor 1-Alpha, an Inheritant Diabetic Gene in Humans

Glomerular nodular lesions, known as Kimmelstiel-Wilson nodules, are a pathological hallmark of progressive human diabetic nephropathy. We have induced severe diabetes in pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha (HNF1α) P291fsinsC, a maturity-onset diabetes of the young type-3 (MODY3) gene in humans. In this model, glomerular pathology revealed that formation of diffuse glomerular nodules commenced as young as 1 month of age and increased in size and incidence until the age of 10 months, the end of the study period. Immunohistochemistry showed that the nodules consisted of various collagen types (I, III, IV, V and VI) with advanced glycation end-product (AGE) and N ε-carboxymethyl-lysine (CML) deposition, similar to those in human diabetic nodules, except for collagen type I. Transforming growth factor-beta (TGF-β) was also expressed exclusively in the nodules. The ultrastructure of the nodules comprised predominant interstitial-type collagen deposition arising from the mesangial matrices. Curiously, these nodules were found predominantly in the deep cortex. However, diabetic pigs failed to show any of the features characteristic of human diabetic nephropathy; e.g., proteinuria, glomerular basement membrane thickening, exudative lesions, mesangiolysis, tubular atrophy, interstitial fibrosis, and vascular hyalinosis. The pigs showed only Armanni-Ebstein lesions, a characteristic tubular manifestation in human diabetes. RT-PCR analysis showed that glomeruli in wild-type pigs did not express endogenous HNF1α and HNF1β, indicating that mutant HNF1α did not directly contribute to glomerular nodular formation in diabetic pigs. In conclusion, pigs harboring the dominant-negative mutant human MODY3 gene showed reproducible and distinct glomerular nodules, possibly due to AGE- and CML-based collagen accumulation. Although the pathology differed in several respects from that of human glomerular nodular lesions, the somewhat acute and constitutive formation of nodules in this mammalian model might provide information facilitating identification of the principal mechanism underlying diabetic nodular sclerosis.

Collapsing glomerulopathy superimposed on diabetic nephropathy: insights into etiology of an under-recognized, severe pattern of glomerular injury

Collapsing glomerulopathy (CG) represents severe podocyte injury with massive proteinuria, rapid progression and relative resistance to therapy. It is associated with multiple etiologies, including obliterative arteriopathy in transplants. However, its association with diabetic nephropathy (DN) has not been reported. Renal biopsies performed in diabetic patients for either increasing proteinuria or deteriorating renal function, or both, were retrospectively reviewed. The clinicopathologic features and immunohistochemical staining of podocytes were analyzed. Of 534 patients with DN, 26 human immunodeficiency virus (HIV)-negative patients were found to have CG superimposed on DN (5% DN cases). At the time of biopsy, their mean serum creatinine was 3.8 mg/dL and proteinuria was 9.8 g/24 h. Renal biopsy showed CG in 2-30% (mean 16% of glomeruli), with segmental (2%) and global (33%) glomerulosclerosis. DN classification was Class IV-12, III-8, IIb-4 and IIa-2. Vascular sclerosis was moderate (44%) and severe (56%). Extensive arteriolar hyalinosis with >50% luminal stenosis was seen in 85% of cases. Markers of podocyte differentiation were lost, consistent with other types of CG. Cytokeratin was focally positive in 70% and VEGF overexpressed in 43%. Follow-up on 17 patients: 13 developed end-stage renal disease (ESRD) in 7 months from the time of biopsy. The development to ESRD in these patients was more rapid than diabetic controls without CG (P=0.005). The remaining four, 5-24 months follow-up, had an increase in creatinine with stable proteinuria. CG contributes to an increased level or new onset of proteinuria in DN which may be intractable. CG in DN with advanced vascular hyalinosis is presumably due to ischemic podocyte injury and is of prognostic significance.

The 2,6‐Disubstituted Naphthalene Derivative FDDNP Labeling Reliably Predicts Congo Red Birefringence of Protein Deposits in Brain Sections of Selected Human Neurodegenerative Diseases

Deposition of conformationally altered proteins prominently characterizes pathogenesis and pathomorphology of a number of neurodegenerative disorders. 2-(1-{6-[(2-[F-18]fluoroethyl) (methyl)amino]-2-naphthyl} ethylidene) malononitrile ([F-18]FDDNP), a hydrophobic, viscosity-sensitive, solvent-sensitive, fluorescent imaging probe has been used with positron emission tomography to visualize brain pathology in the living brain of Alzheimer disease (AD) patients. Its non-radiofluorinated analog FDDNP was shown to label senile plaques and neurofibrillary tangles (NFTs) in brain tissue sections. This work aimed at evaluating FDDNP labeling of various protein deposits in fixed, paraffin-embedded brain tissue sections of selected neurodegenerative disorders: AD, cerebral amyloid angiopathy (CAA), transmissible spongiform encephalopathies, progressive supranuclear palsy (PSP), Pick disease (PiD), Parkinson disease, dementia with Lewy bodies, multiple system atrophy (MSA). Cerebral hypertensive vascular hyalinosis (HVH) was used as negative control. Significant agreement between amyloid histochemical properties and FDDNP labeling of the deposits was established. FDDNP labeling showed high positive predictive value for birefringence in senile plaques and NFTs in AD, prion plaques and amyloid deposits in CAA. No FDDNP labeled structures were observed in HVH, PSP, PiD or MSA tissue sections. Our findings may be of significant value for the detection of neuropathological aggregates with [F-18]FDDNP in some of these disorders in the living brain of human subjects.

Cyclosporine reduction causes decreasing of angiotensin II and transforming growth factor-beta expression in chronic allograft nephropathy

Summary Background High cyclosporine (CsA) levels might lead to nephrotoxicity due to increasing angiotensin II (AII) and transforming growth factor-beta (TGF-β) production. We hypothesized that a chronic reduction in CsA levels would decrease local renal AII and TGF-β expression and result in improvement of renal function and renal pathological changes in renal transplant recipients. Methods We determined the AII and TGF-β expression by immunohistochemical staining (IHCS) in sequential human renal biopsy specimens in patients with chronic allograft nephropathies (time between biopsies: 15.9 ± 0.8 months) after they had their CsA levels reduced by 50%. Pathological evaluation included percentage expression (%) of interstitial fibrosis and tubular atrophy (FIB), vascular sclerosis (VS), transplant glomerulopathy (TG), and vascular hyalinosis (VH). Serum creatinine (CR, mg/dl) and BUN (mg/dl) levels were also investigated. Results Renal pathological score significantly improved with chronic reduction in CsA blood levels (FIB: from 52 to 26%, p p p p p p Conclusion These data indicate that chronic reduction in CsA diminishes production of renal tissue AII and AT1 receptor expression, and results in decreased fibrosis and improvement of renal function in patients with chronic allograft nephropathy.

Neuropathological alterations in drug abusers : The involvement of neurons, glial, and vascular systems.

Because the effects of drug abuse on the cellular elements of the human brain have not been studied systematically, an investigation was performed using histology, immunohistochemistry, and morphometry. The main cortical and subcortical brain areas of 50 polydrug deaths were analyzed as compared with controls.In the brains of drug abusers, a significant neuronal loss was present. Interestingly, the number of glial fibrillary acidic protein (GFAP)-positive astrocytes was reduced. the numerical density of perivascular and parenchymal microglia was increased in the white matter and in most subcortical regions. In the white matter there were widespread β-amyloid precursor protein deposits. Furthermore, there was a prominent vascular hyalinosis, endothelial cell proliferation, and a loss of immunoreactivity for collagen type IV within the vascular basal lamina.The neuronal loss seems to be the result of a direct impairment of nerve cells and, indirectly, to a damage of astrocytes, axons, and the microvasculature. The reduction of GFAP-positive astrocytes is also indicative of a drug-induced damage. The axonal injury suggests a toxic-metabolic drug effect, whereas the concomitant activation of microglia is indicative of a long-standing progressive process. The noninflammatory vasculopathy can be considered as the morphological substrate of a disturbed blood-brain barrier. Our findings demonstrate that drugs of abuse initiate a cascade of interacting toxic, vascular, and hypoxic factors that finally result in widespread disturbances within the complex network of central nervous system cell-cell interactions.

Histologic Change of Peritoneal Membrane in Relation to Adequacy of Dialysis in Continuous Ambulatory Peritoneal Dialysis Patients

Long-term exposure of peritoneal membrane to bioincompatible dialysis solutions leads to structural changes and loss of ultrafiltration capability. We studied the possible relationship between histologic change and the transport characteristics of peritoneal membrane and adequacy of dialysis in continuous ambulatory peritoneal dialysis (CAPD) patients. The study included 18 CAPD patients (11 men, 7 women) who underwent a peritoneal biopsy either at initiation of treatment (group A, n = 9) or after a mean of 4 years on CAPD (group B, n = 9). The morphologic changes in the mesothelial cells and the vascular compartment and the thickness of the submesothelial collagenous zone were estimated and compared with observations from 6 patients with normal renal function who underwent biopsy of the parietal peritoneum during abdominal surgery. The relationship of the observed changes in CAPD patients to results from a peritoneal equilibration test (PET) and to adequacy of dialysis [total weekly creatinine clearance (CCr) and Kt/V urea] were also investigated. The main histologic changes in both groups of patients were loss of mesothelial cells and decrease in the normal mesothelial surface, thickening of the submesothelial collagenous zone, and presence of vascular hyalinosis. The thickness of the submesothelial collagenous zone in both groups of patients was significantly greater than that found in controls (410 mum and 580 mum vs 50 mum, p < 0.05). Although no significant difference was found between morphologic change in the peritoneal membrane of uremic patients starting on CAPD and those who had been on peritoneal dialysis (PD) for a mean period of 4 years, a trend was observed toward more severe lesions in the latter patients. The PET, CCr, and Kt/V urea were not significantly different in the two groups of patients. Those parameters also showed no significant changes when examined at initiation of CAPD and after a mean of 4 years of PD in the same patients (group B). No significant correlations were observed between the histologic changes and the PET, CCr, or Kt/V in both groups of patients. Significant structural changes are observed in the peritoneal membrane of uremic patients, and those changes become worse with CAPD treatment. Structural changes are not followed by functional changes during the first 4 years on CAPD.

DECREASING EXPRESSION OF TRANSFORMING GROWTH FACTOR-BETA IN HUMAN RENAL BIOPSIES WITH IMPROVEMENT OF RENAL PATHOLOGICAL SCORE AND RENAL FUNCTION FOLLOWING CHRONIC CYCLOSPORINE REDUCTION

920 Transforming growth factor-beta (TGF-β) is produced by a variety of cells including several renal cell types. TGF-β is a growth-regulating protein that has been shown to enhance collagen production in cultured cells. High cyclosporine-A (CsA) levels might lead to nephrotoxicity with increasing TGF-β and collagen synthesis in renal transplant recipients. We hypothesized that local renal TGF-β expression may increase with high circulating CsA levels. Therefore, the current study was designed to determine the TGF-β1 isoform expression in human renal biopsy tissue with high and low CsA levels by immunohistochemical staining (IHCS). Kidney biopsies from nine transplant recipients with chronic allograft nephropathy were obtained before (CsA level: 321±106 ng/ml) and after (CsA level: 141±40 ng/ml) reduction in CsA dosage (time between biopsies: 15.8±0.8 months). TGF-β1 IHCS score (S) (0-4) and positive staining area (%) were significantly decreased in patients with reduction in CsA levels. TGF-β1 was localized in the glomeruli and tubular cells, and was minimally evident in control biopsy specimens (IHCS score 0.2-0.4). Pathological evaluation included percentage expression (%) of interstitial fibrosis and tubular atrophy (FIB), vascular sclerosis (VS), transplant glomerulopathy (TG), and vascular hyalinosis (VH). Serum creatinine (CR, mg/dl) and BUN (mg/dl) levels were also investigated. Mean±SE, *p<.05 vs High CsA group. (Table)TableThese data indicate that chronic reduction in CsA may diminish production of renal TGF-β expression, and results in decreased renal fibrosis and improvement of renal function in patients with chronic allograft nephropathy.

Histopathological findings in renal allografts at time of transplantation and correlation with onset of graft function.

Histopathological changes quantified using the chronic allograft damage index (CADI) have been shown to predict subsequent graft outcome and developing chronic rejection. The aim of the study reported here was to investigate the extent to which cadaveric renal allografts exhibit histopathological changes at time of transplantation, focusing on changes covered by the CADI. We also analysed whether any histopathological change predicts delayed graft function. One hundred and twenty-eight cadaveric kidney allografts with adequate protocol biopsy were studied. Tubular epithelial anisometric vacuolization was the commonest change, found in 62% of grafts and scored moderate or severe in 28% of these cases. Other prevalent changes were interstitial fibrosis, vascular hyalinosis, glomerular sclerosis and tubular basement-membrane thickening in 40%, 37%, 28% and 22% of biopsies, respectively. Intensities were, however, scored as mild in over 95% of specimens. The mean CADI for all grafts was 0.74. A significant difference in CADIs was seen between grafts from donors under and over 40 years of age. Grafts with early, delayed and no function had a similar incidence of histopathological changes. Histopathological changes in renal allografts were mostly uncommon and mild at time of transplantation, but some grafts exhibited changes which were quantified using the CADI. Though histopathological changes quantified with the CADI are predictive of subsequent graft function, they did not affect onset of graft function.