Histological predictors of renal outcome in lupus nephritis: the importance of tubulointerstitial lesions and scoring of glomerular lesions.

Introduction Lupus nephritis (LN) affects nearly 60% of patients with systemic lupus erythematosus and up to 30% of them will progress to end-stage renal disease (ESRD), despite receiving aggressive immunosuppressive therapy. The prognostic value of ISN/RPS classification is controversial. Therefore, we aimed to identify clinical and pathological predictors of outcome in LN patients independent of this classification. Material and methods Thirty-seven patients with LN who underwent percutaneous kidney biopsy between 1997 and 2016 were included in this study. Twenty clinical and twenty histological variables were tested for their association with a composite end-point of doubling of serum creatinine, ESRD and death. Univariate and multivariate Cox proportional hazard regression analysis were performed to identify independent predictors of outcome. Results During a median follow-up period of 48 months (IQR: 17.5-120 months), 21.6% of patients reached the composite end-point. The overall survival rate of our cohort was 89% at one year, 86% at five years, 74% at 10 years and 64% at 20 years. Patients with Class IV LN showed the worst prognosis with 44% survival at 10 years, while those who additionally showed crescents and global sclerosis on kidney biopsy had an even lower survival of 21% and 0% at 10 years, respectively. After multivariate adjustment, we identified estimated glomerular filtration rate at baseline (HR, 0.91 per ml/min /1.73 m2; 95% CI, 0.84 to 0.99), 24-hour proteinuria at baseline (HR, 2.04 per g/d; 95% CI, 1.19 to 3.5), crescents (HR, 1.068 per %; 95% CI, 1.003 to 1.091), global sclerosis (HR, 1.036 per %; 95% CI, 0.984 to 1.091), presence of adhesions (HR, 9.2; 95% CI, 1.38 to 61.2) and tubulitis (HR, 13.1; 95% CI; 1.3 to 131) as independent predictors of outcome in our cohort of LN. Conclusions Our study identified glomerular (crescents, global sclerosis, adhesions) and tubulointerstitial (tubulitis) lesions, in addition to clinical variables (renal function, 24-hour proteinuria), as important predictors of renal outcome, independent of the ISN/RPS classification. We suggest that the ISN/RPS classification could be improved by a quantitative assessment of glomeruli with active and chronic lesions and by a greater emphasis given to tubulointerstitial lesions.

Immune-complex deposits in pANCA glomerulonephritis: a pediatric case report.

A 15-year-old boy is admitted to the hospital for clinical signs that suggest a pulmonary-renal syndrome (fever, cough, hemopthoic expectoration, oliguria, gross hematuria). A crescentic pANCA positive glomerulonephritis was configured. However, dense subendothelial deposits were identified in electronic microscopy and immunofluorescence staining showed granular deposits of IgG and C3, kappa and lambda in the capillary loops. Although the treatment was strictly followed, after three years and three months of good clinical state, he manifested signs of kidney failure being transplanted. His case represents a specific pattern of rapidly progressive glomerulonephritis leading to kidney failure and emphasizes the importance of clinical attendance especially in a case of two associated glomerulopathies.

The association of polymorphonuclears with humps in acute postinfectious glomerulonephritis.

It is currently considered that hump dense deposits developed during an acute poststreptococcal glomerulonephritis become finally dissolute by three hypothetical mechanisms: loosing their electron density, internalization and processing by podocytes and by incorporation in the glomerular basal lamina (GBM). Analyzing ultrastructurally the association of polymorphonuclear leukocytes and hump deposits, we emphasized features endorsing the hypothesis that the immune complexes of dense deposits are discharged in the circulation under the leukocytes activity. The active polymorphonuclear cells are melting the GBM in the area of contact by complement activation and by the NAPlr bound plasmin. The reversed flow of immune complexes from humps towards the blood circulation leaves fading, wrinkled shaped humps, before total dissolution.

Nanostructural features of diabetic podocytopathy.

The earliest glomerular lesion during the diabetic nephropathy is considered by many authors to be the so-called podocytopathy. Microalbuminuria is an early clinical marker of diabetic nephropathy that results from damages of the glomerular filtration barrier at the level of the highly differentiated podocytes. Thus, the diabetic podocytopathy includes cellular hypertrophy, foot process effacement, detachment from glomerular basement membrane (GBM), and apoptosis. The present paper is reviewing all these features and some additional ultrastructural transformations concerning the podocytes involved in the diabetic kidney disease.

Renal subcapsular tertiary lymphoid aggregates in chronic kidney diseases.

In the last decade, it has been accepted the formation of tertiary lymphoid organs in the renal parenchyma during inflammatory conditions. These organized cellular aggregates contain B- and T-lymphocytes, dendritic cells, surrounded by neo-lymphatic vessels. They have been described in renal allografts, acute and chronic interstitial nephritis, IgA and membranous nephropathies. The functional characteristics of these lymphoid nodules remained still under consideration. After investigating the renal biopsies of 268 patients with primary and secondary nephropathies, we have selected 20 cases showing lymphoid-like cellular aggregates located just beneath the renal capsule and having close contacts with this kidney envelope. All of these cases also showed an associated medium sized lymphatic vessel. The ultrastructure of these nodules proved to contain more or less the same cellular composition: lymphocytes, dendritic cells, seldom plasma cells and macrophages. We consider these particular subcapsular lymphoid-like nodules to be tertiary lymphatic structures in close association with the perirenal lymphatics, and the first to develop in any type of inflammatory and autoimmune renal condition.

Myofibroblast involvement in tubular basement membrane remodeling in type II diabetic nephropathy.

Diabetic nephropathy is always accompanied by tubulointerstitial damage. The mechanisms and the cells involved are not entirely clarified. The damaged tubules may regenerate or undergo necrosis or apoptosis. The purpose of this work was to investigate the structural transformations of both interstitial cells and extracellular matrix of the kidney stromal area in patients with type II diabetes mellitus associated with diabetic nephropathy. Tubulointerstitial fibrosis is characterized by loss of renal tubules and interstitial capillaries and the accumulation of extracellular matrix proteins. Tubular basement membranes were found to be a target of the remodeling process of the stromal area. Thickening, splitting and duplication were the main lesions of these membranes. Much attention has been focused on the importance of myofibroblasts in the progression of renal fibrosis. The results represent strong arguments for a direct involvement of myofibroblasts in the process of renal interstitial remodeling, tubular basement membrane thickening, and stromal fibrosis in the late stages of diabetic nephropathy.

Histological and ultrastructural analysis of the seminiferous tubule wall in ageing testis.

The authors evaluated possible morphological changes of basement membrane (BM) and lamina propria (LP) of seminiferous tubule wall (ST) related to ageing. Surgical samples of testicular tissue from 28 cases with orchiectomy for prostate adenocarcinoma were processed for light microscopy and transmission electron microscopy (TEM) examination. Seven age groups (AgGr) between 50 and 80 years were designed. Tissue samples were immunomarked for collagen IV and smooth muscle actin. Images were acquired and measured with a specialized software. Thirty ST were randomly selected, with ×40-objective, for each case. Five random determinations for each ST and each parameter were performed. Mean values/tubule, case and AgGr were calculated for each parameter. Regression line (RL), slope and significance test for slope were determined for each parameter correlation with ageing. BM mean value was around 0.5 µm, with narrow limits of ranging in AgGr but more extended individual limits. RL showed discrete decreasing trend with ageing but without an obvious statistical correlation. LP mean value was around 6 µm, also with narrow limits of ranging in AgGr and more extended individual limits. RL decreased discretely with ageing but without an obvious statistical correlation. TEM showed more prominent BM material and more collagen fibers and less fibroblasts in LP of older AgGr and higher fibroblasts density in LP of younger AgGr. Our results showed that BM thickness is apparently decreasing with ageing whereas LP presents extremely variable degenerative changes, with a "mosaic", focal distribution and no tendency to advance with ageing.

Telocytes in human isolated atrial amyloidosis: ultrastructural remodelling.

The human heart can be frequently affected by an organ-limited amyloidosis called isolated atrial amyloidosis (IAA). IAA is a frequent histopathological finding in patients with long-standing atrial fibrillation (AF). The aim of this paper was to investigate the ultrastructure of cardiomyocytes and telocytes in patients with AF and IAA. Human atrial biopsies were obtained from 37 patients undergoing cardiac surgery, 23 having AF (62%). Small fragments were harvested from the left and right atrial appendages and from the atrial sleeves of pulmonary veins and processed for electron microscopy (EM). Additional fragments were paraffin embedded for Congo-red staining. The EM examination certified that 17 patients had IAA and 82% of them had AF. EM showed that amyloid deposits, composed of characteristic 10-nm-thick filaments were strictly extra-cellular. Although, under light microscope some amyloid deposits seemed to be located within the cardiomyocyte cytoplasm, EM showed that these deposits are actually located in interstitial recesses. Moreover, EM revealed that telopodes, the long and slender processes of telocytes, usually surround the amyloid deposits limiting their spreading into the interstitium. Our results come to endorse the presumptive association of AF and IAA, and show the exclusive, extracellular localization of amyloid fibrils. The particular connection of telopodes with amyloid deposits suggests their involvement in isolated atrial amyloidosis and AF pathogenesis.

The value of kidney biopsy in diabetes mellitus.

The purpose of this work is to emphasize the value of kidney biopsy in patients with diabetes mellitus and clinical renal impairment. Diabetes is the leading cause of end stage renal disease because diabetic nephropathy develops in 30 to 40% of patients. Multiple genetic predisposing conditions are involved in the development or not of a diabetic nephropathy, therefore supporting the existence of several factors in the pathogenesis of this disease. These predisposing conditions may also favor different other types of glomerulonephritis which can occur independently or in parallel with a diabetic nephropathy. All these renal diseases have different treatments, and therefore they must be correctly identified and managed accordingly. The processing of the kidney biopsy samples requires a very careful and highly qualified management to differentiate precisely the nature of each condition. In addition to the mesangial classical lesions, recent biopsy studies provided evidence that podocytes are injured very early in the diabetic nephropathy. On the other hand, transgenic mice models provide a unique opportunity to investigate the natural course of the disease. The paper underlines the main laboratory techniques required for this activity, and the main structural arguments to perform a satisfactory differential diagnosis.

Interstitial Cajal-like cells (ICLC) in myocardial sleeves of human pulmonary veins.

We present here evidence for the existence of a new type of interstitial cell in human myocardial sleeves of pulmonary veins: interstitial Cajal-like cell (ICLC). This cell fulfils the criteria for positive diagnosis of ICLC, including CD 117/c-kit positivity. Transmission electron microscopy revealed typical ICLC with 2 or 3 very long processes (several tens of mm) suddenly emerging from the cellular body. Also, these processes appear moniliform but extremely thin (0.1-0.4 mm) under the resolving power of the usual microscopy. Cell processes establish close spatial relationships between each other, as well as with capillaries and nerve endings. ICLC appear located among the myocardial cells and particularly at the border between the myocardial sleeve and pulmonary vein wall.

Myocardial interstitial Cajal-like cells (ICLC) and their nanostructural relationships with intercalated discs: shed vesicles as intermediates.

Intercalated discs (ID) are complex junctional units that connect cardiac myocytes mechanically and electrochemically. However, there is limited information concerning the cardiomyocyte interaction with interstitial non-muscle cells. Our previous studies showed that myocardial interstitial Cajal-like cells (ICLC) are located in between cardiomyocytes, blood capillaries and nerve fibres. Typically, ICLC have several very long, moniliform, cytoplasmic processes which establish closed contacts with nerve fibres, as well as each other. We report here ultrastructural evidence concerning the relationships of ICLC processes with ID. The ICLC cytoplasmic prolongations (tens micrometers length) preferentially pass by or stop nearby the ID. Transmission electron microscopy emphasized three distinct connecting features between the tips of ICLC extensions and myocytes at the 'mouth' of ID: free or budding shed vesicles, exocytotic multi-vesicular bodies and direct contacts. In the last case, electron-dense repetitive nanostructures ('pillars') (35-40 nm high and 100-150 nm wide, similar to adhesion molecules) fasten the ICLC to the myocytes. All these features suggest a juxtacrine and/or paracrine intercellular mutual modulation of ICLC and cardiomyocytes in the microenvironment of ID, possibly monitoring the cardiac functions, particularly the electrical activity.

Caveolae in smooth muscles: nanocontacts.

Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask-shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21 +/- 3 nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape-like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20-30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4-6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile compartment. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium - 2.40%; bladder - 3.66%, stomach - 2.61%, aorta - 1.43%). We also observed a higher number of caveolae per length unit of cellular membrane in most visceral SMCs compared to vascular SMCs (myometrium - 1.06/microm, bladder - 0.74/microm, aorta - 0.57/microm, stomach - 0.48/microm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Threedimensional reconstructions (15micro(3)) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), other with mitochondria (10%) and 3% apparently have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae and SR, some of them with nanostructural links between each other or with mitochondria: direct contacts (space <2 nm or none) and molecular links, so called 'feet' (about 12 nm electron dense structures between organellar membranes). Direct contacts possibly allow molecular translocation between the two membranes. Electron-dense 'feet'-like structures suggest a molecular link between these organelles responsible for intracellular Ca(2+) homeostasis (excitation-contraction coupling or pharmaco-mechanical coupling). Close appositions (approximately 15 nm) have also been observed between caveolae and perinuclear SR cisternae, suggesting that caveolae might be directly implicated in excitation-transcription coupling.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC).

We have previously described interstitial Cajal-like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non-conventional light microscopy (Epon-embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent approximately 32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of microm), very thin (0.1-0.5 microm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and MitoTracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil-like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/ 1 microm cell membrane length, or 2-4% of the relative cytoplasmic volume). Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5-10% and 1-2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.

Interstitial Cajal-like cells (ICLC) in atrial myocardium: ultrastructural and immunohistochemical characterization.

We have previously reported (Hinescu & Popescu, 2005) the existence of interstitial Cajal-like cells (ICLC), by transmission electron microscopy, in human atrial myocardium. In the present study, ICLC were identified with non-conventional light microscopy (NCLM) on semi-thin sections stained with toluidine blue and immunohistochemistry (IHC) for CD117/c-kit, CD34, vimentin and other additional antigens for differential diagnosis. Quantitatively, on semi-thin sections, ICLC represent about 1-1.5% of the atrial myocardial volume (vs. approximately 45% working myocytes, approximately 2% endothelial cells, 3-4% for other interstitial cells, and the remaining percentage: extracellular matrix). Roughly, there is one ICLC for 8-10 working atrial myocytes in the intercellular space, beneath the epicardium, with a characteristic (pyriform, spindle or triangular) shape. These ICLC usually have 2-3 definitory processes, emerging from cell body, which usually embrace atrial myocytes (260 nm average distance plasmalemma/sarcolemma) or establish close contact with nerve fibers or capillaries (approximately 420 nm average distance to endothelial cells). Cell prolongations are characteristic: very thin (mean thickness = 0.15+/-0.1 microm), very long for a non-nervous cell (several tens of microm) and moniliform (uneven caliber). Stromal synapses between ICLC and other interstitial cells (macrophages) were found (e.g. in a multicontact type synapse, the average synaptic cleft was approximately 65 nm). Naturally, the usual cell organelles (mitochondria, smooth and rough endoplasmic reticulum, intermediate filaments) are relatively well developed. Caveolae were also visible on cell prolongations. No thick filaments were detected. IHC showed that ICLC were slightly and inconsistently positive for CD117/c-kit, variously co-expressed CD34 and EGF receptor, but appeared strongly positive for vimentin, along their prolongations. Some ICLC seemed positive for a-smooth muscle actin and tau protein, but were negative for nestin, desmin, CD13 and S-100. In conclusion, we provide further evidence of the existence of ICLC in human atrial myocardium, supporting the possible ICLC role in pacemaking, secretion (juxta- and/or paracrine), intercellular signaling (neurons and myocytes). For pathology, ICLC might as well be 'players' in arrhythmogenesis and atrial remodeling.

The predictive value of peritubular capillaries C3d deposition in IgA glomerulonephritis.

The IgA glomerulonephritis (IgAGN) is one of the most common primary glomerulonephritis and has a variable and difficult to predict evolution toward the end-stage nephrosclerosis. The deposition of C3d complement component in peritubular capillaries (PTCs) indicates a variant type of acute rejection while C3d deposition in primary glomerulonephritis (GN) is poorly documented. The aim of this study is to examine C3d expression in peritubular capillaries (PTCs) as a predictive marker and its correlation with the severity of renal injury in IgA glomerulonephritis. Polyclonal FITC conjugated rabbit anti-human C3c and C3d antibodies were used for direct immunofluorescent evaluation of the C3c and C3d deposits in 24 kidney biopsies with IgA glomerulonephritis. The study revealed that the C3d deposits in peritubular capillaries were associated with known predictors for rapid progression of IgAGN: glomerular sclerosis (63.6%), atrophic tubules (90.9%) and interstitial sclerosis (81.8%). The intensity of the C3c glomerular immunofluorescent deposits was related with active lesions. Thus, the predictive value of C3d deposition on PTCs in IgAGN is worth to be taken into consideration as an unfavorable outcome of the disease and request further long run investigations.

Ultrastructural defects of the glomerular basement membranes associated with primary glomerular nephropathies.

It is generally accepted that a glomerular basement membrane (GBM) thinner than 200 nm should be considered below normal. When this abnormality has a global and diffuse distribution, the associated clinical condition is a benign familial hematuria related to mutations of the COL4A4/COL4A3 genes, or an Alport syndrome. More often the GBM defects display a focal and segmental pattern, too small to express a thin glomerular basement membrane disease. The aim of this study is to emphasize statistical data concerning the pathogenic link between the renal glomerular diseases and the preexisting thin and very thin GBM. A series of 487 renal biopsies from adult patients has been thoroughly investigated both for nephropathologic diagnosis and the GBM ultrastructure. It has been statistically concluded that there is a close coexistence of primary glomerulonephritis and thin glomerular basement membranes with the role of a predisposing condition for immune complex deposition.

Neoductular progenitor cells regenerate hepatocytes in severely damaged liver: a comparative ultrastructural study.

In severely injured liver, stem cells give rise to progeny that tend to replace lost hepatocytes. Neoductular reaction appears as an inherent stage of liver reconstruction following severe damage caused by different pathological mechanisms. Few ultrastructural types of progenitor cells have been described, and some molecular phenotypes of progenitor stages have been characterized, but the details of the differentiation process are largely unknown. We prepared for light and electron microscopy examination human liver from biopsies of patients with chronic active hepatitis, and rat liver with allyl alcohol-induced periportal necrosis. We found that progenitor neoductular cells acquire the hepatocytic polarity pattern during a multi-step process apparently involving cell migration and dissolution of neoductular basement membrane. An intermediate stage with "mixed" ductular and hepatocytic polarity was described.

Microscopic and ultrastructural aspects in retinoblastoma.

A number of 12 retinoblastoma cases were studied. We had in view a number of histopathological aspects (tumor type, neighbouring structures invasion) which are considered to be decisive in appreciating the vital prognosis. We mention that undifferential retinoblastoma of histopathological type was met in 8 cases and the neighbouring structures invasion was present in 7 of them. We also studied the ultrastructura of the tumor cells belonging to the malignant tumor.