Periodic Acid Methenamine Silver Staining Research Articles

A Novel Deep Learning Approach for Analyzing Glomerular Basement Membrane Lesions in a Mouse Model of X-Linked Alport Syndrome

Alport syndrome is a rare kidney disease typically more severe in males due to its X-linked inheritance. However, female patients with heterozygous X-linked Alport syndrome (XLAS) can develop renal failure over time, necessitating accurate pathological assessment for effective therapy. A key pathological finding in female XLAS patients is the mosaic pattern of partial loss of α5 chains of type IV collagen. This study, using a mouse model of XLAS with a nonsense mutation (R471*) in the Col4a5 gene, analogous to human XLAS, aimed to examine the consistency of this pattern with the glomerular basement membrane (GBM) structure. A modified periodic acid-methenamine silver (PAMS) staining method was developed for clearer GBM visualization. The integrated images from COL4α5-stained fluorescence, PAMS, and low-vacuum scanning electron microscopy (LVSEM) into a single-slide section and applied supervised deep learning to predict GBM lesions. Results showed significant individual variability in urinary protein levels and histological lesions. Pathological parameters, including crescent formation, focal segmental glomerulosclerosis, and the COL4α5/α2 ratio, correlated with clinical parameters like urinary protein and plasma creatinine levels. Integrated LVSEM analysis revealed dense GBM regions corresponded to areas where COL4α5 was preserved, while coarse GBM (basket-weave lesions) occurred in COL4α5-deficient regions. These advanced techniques can enhance biopsy-based diagnosis of Alport syndrome and aid in developing AI diagnostic tools for diseases involving basement membrane lesions.

Morphological and etiological analyses of C3 and non-C3 glomerulonephritis in primary membranoproliferative glomerulonephritis using periodic acid-methenamine silver stain electron microscopy: a retrospective multicentered study.

This study elucidated the etiology of C3 glomerulonephritis (C3GN) and non-C3GN with primary membranoproliferative glomerulonephritis (MPGN) using transmission electron microscopy (TEM) and periodic acid-methenamine silver stain (PAM-EM). Thirty-one primary MPGN cases were analyzed by TEM and PAM-EM to distinguish among MPGN I, MPGN II, MPGN III Burkholder subtype (MPGN IIIB), and Anders and Strife subtype (MPGN IIIA/S). Each case was also classified into C3GN or non-C3GN according to the standard C3GN definition using immunostaining. Four cases of MPGN II met C3 glomerulopathy; whereas, four cases of MPGN IIIB did not meet C3 glomerulopathy. Seven of 11 cases (64%) of MPGN I without GBM disruption and 7 of 12 cases (58%) of MPGN IIIA/S with GBM disruption met the non-C3GN criteria with significant immunoglobulins' deposition. Regardless of the C3GN or non-C3GN diagnosis, the deposits in primary MPGN I and MPGN IIIA/S exhibited ill-defined, amorphous, and foggy characteristics similar to those found in postinfectious GN but were different from immune complex (IC) deposits seen in MPGN IIIB. Not only C3GN but also non-C3GN was due to mechanisms other than IC deposition as found in postinfectious GN. Consequently, GBM disruption of MPGN IIIA/S was not due to IC deposition.

#6227 SPONTANEOUS REMISSION OF MEMBRANOUS VARIANT OF PGNMID WITH MONOCLONAL IGG2κ

Abstract Background and Aims The clinical significance of the morphological patterns of glomerular injury and of each IgG subclass in proliferative glomerulonephritis (GN) with monoclonal immunoglobulin deposits (PGNMID) is not well understood. Literature suggests that PGNMID with certain histological features such as membranous or mesangio-proliferative features or non-IgG3 subclass staining may be associated with a more favourable renal prognosis. We present a patient with membranous variant of PGNMID with monoclonal IgG2κ who had spontaneous remission without recurrence over a 2-year follow-up. Method Not applicable Results A 62-year-old Malay gentleman presented with new onset nephrotic range proteinuria, on a background of type 2 diabetes mellitus with diabetic duration of 5 years, hypertension and hyperlipidemia. He was mildly hypertensive (blood pressure 148/87) and non-edematous at presentation. Investigations revealed preserved kidney function [serum creatinine (sCr) 66µmol/L], bland urinalysis, and 24-hour urinary total protein (UTP) of 3.74g/day. Serum albumin (sAlb) was 35g/L. Serum complements were not low. Autoimmune markers inclusive of anti-double stranded DNA antibodies, anti-neutrophil cytoplasmic antibodies, and anti-phospholipase A2 receptor (PLA2R) antibodies were negative. Viral serologies were also unremarkable. No monoclonal band was detected on serum electrophoresis, while serum immunofixation was not performed. Kidneys were normal sized. Renal biopsy performed demonstrated 28 glomeruli, of which only 1 was globally sclerosed. Glomeruli capillary walls were diffusely thickened by vacuolations. Capillary loops were mostly single contoured although occasional focal double contouring was seen. Masson trichome stain revealed fuchsinophilic subepithelial immune deposits while periodic acid methenamine silver stain showed argyrophilic basement membrane spikes. Variable mesangial expansion and hypercellularity was observed, as well as mesangiolysis. Focal leukocyte margination was present, but significant endocapillary proliferation was not seen. There were no Kimmelstiel-Wilson lesions. Tubular atrophy with interstitial fibrosis was overall mild (10% of parenchymal area) and there were no tubulointerstitial infiltrates. Immunofluoresence showed granular staining of IgG (1-2+), C3 (2-3+) along glomerular capillary walls, as well as presence of kappa light chain restriction (κ 1-2+; λ negative in glomeruli). Immunostaining for PLA2R was negative. IgG subclass analysis performed in view of kappa restriction revealed isolated deposition of IgG2. Electron microscopy was not available. Overall findings were most consistent with membranous variant of PGNMID with monoclonal IgG2κ. Age-appropriate and symptom-directed malignancy screening did not reveal significant abnormalities. Given that the patient was minimally symptomatic, a trial of conservative therapy was initiated with optimized non-immunosuppressive anti-proteinuric therapy using renin-angiotensin-aldosterone and sodium-glucose co-transporter-2 inhibitors. Gradual spontaneous remission was observed without recurrence over 2 years of follow-up (sCr 77µmol/L; sAlb 40g/L; 24-hour UTP 0.22g/day). Conclusion Our case adds to the literature that a membranous variant of PGNMID with monoclonal IgG2κ may be associated with better renal outcomes. Despite the lack of proliferative changes, it is possible that membranous variants may share similar underlying disease mechanisms as the other proliferative variants. Large international registries to allow the correlation of morphological features and IgG subclasses with clinical outcomes are required to confirm our observation.

Membranous nephropathy associated with multicentric Castleman\u2019s disease that was successfully treated with tocilizumab: a case report and review of the literature

BackgroundMulticentric Castleman’s disease is a life-threatening disorder involving a systemic inflammatory response and multiple organ failure caused by the overproduction of interleukin-6. Although renal complications of Castleman’s disease include AA amyloidosis, thrombotic microangiopathy, and membranoproliferative glomerulonephritis, membranous nephropathy is relatively rare. We experienced a case of secondary membranous nephropathy associated with Castleman’s disease.Case presentationThe patient was a 43-year-old Japanese man who had shown a high zinc sulfate value in turbidity test, polyclonal hypergammaglobulinemia, anemia, and proteinuria. A physical examination revealed diffuse lymphadenopathy, an enlarged spleen and papulae of the body trunk. A skin biopsy of a papule on the patient’s back showed plasma cells in the perivascular area and he was diagnosed with multicentric Castleman’s disease, plasma cell variant. Kidney biopsy showed the appearance of bubbling in the glomerular basement membranes in Periodic acid methenamine silver stain and electron microscopy revealed electron dense deposits within and outside the glomerular basement membranes. Since immunofluorescence study showed predominant granular deposition of IgG1 and IgG2, he was diagnosed with secondary membranous nephropathy associated with Castleman’s disease. He was initially treated with prednisolone alone, however his biochemical abnormalities did not improve. After intravenous tocilizumab (700 mg every 2 weeks) was started, his C-reactive protein elevation, anemia, and polyclonal gammopathy improved. Furthermore, his urinary protein level declined from 1.58 g/gCr to 0.13 g/gCr. The prednisolone dose was gradually tapered, then discontinued. He has been stable without a recurrence of proteinuria for more than 6 months.ConclusionsTocilizumab might be a treatment option for secondary membranous nephropathy associated with Castleman’s disease.

Case Report: Polyvinylpyrrolidone deposition disease from repeated injection of opioid substitution drugs: report of a case with a fatal outcome

Background: Intravenous injection of oral opioid substitution drugs (OSD) is widespread among injecting drug users. Several OSDs contain the polymer polyvinylpyrrolidone (PVP) as an excipient. Parenterally administered PVP of high molecular weight may accumulate in tissues and organs. This phenomenon was first described in the 1950s, when PVP was utilised in medication for parenteral use. We report a case of an opioid-addicted patient with extensive PVP–deposition caused by repeated injections of OSDs. Case presentation: A 30-year-old male drug addicted patient in opioid substitution therapy (OST) was repeatedly referred to his local hospital in a poor general condition. Work-up revealed severe normocytic anaemia, renal insufficiency, pancreas insufficiency and pathological fractures. Biopsies from fractured bones, bone marrow and gastric mucosa showed extensive infiltrates of histiocytes with intracytoplasmic vacuoles. Vacuole content stained slightly bluish in hematoxylin and eosin stain, red in Congo red stain and black in periodic acid methenamine silver stain. The morphological appearance and staining properties were in accordance with the diagnosis of PVP deposition. The patient had been injecting both buprenorphine tablets and a specific methadone syrup for several years. The methadone syrup contained large amounts of high molecular weight PVP, making it the most likely cause of the deposition. His health quickly deteriorated and he died, impaired by multi-organ failure and cachexia, five years after the first diagnosis of PVP-deposition. The autopsy revealed extensive PVP-deposition in all sampled organs and tissues. Conclusions: Histological investigation and the correct identification of PVP in the biopsies led to the discovery of a severe adverse effect from long-standing misuse of a drug. The disseminated PVP deposition likely contributed to multi-organ dysfunction and cachexia with a fatal outcome. The deposited PVP likely originated from repeated injections of a certain methadone syrup.

Elevated blood pressure in high-fat diet-exposed low birthweight rat offspring is most likely caused by elevated glucocorticoid levelsdue to abnormal pituitary negative feedback.

Being delivered as a low birthweight (LBW) infant is a risk factor for elevated blood pressure and future problems with cardiovascular and cerebellar diseases. Although premature babies are reported to have low numbers of nephrons, some unclear questions remain about the mechanisms underlying elevated blood pressure in full-term LBW infants. We previously reported that glucocorticoids increased miR-449a expression, and increased miR-449a expression suppressed Crhr1 expression and caused negative glucocorticoid feedback. Therefore, we conducted this study to clarify the involvement of pituitary miR-449a in the increase in blood pressure caused by higher glucocorticoids in LBW rats. We generated a fetal low-carbohydrate and calorie-restricted model rat (60% of standard chow), and some individuals showed postnatal growth failure caused by growth hormone receptor expression. Using this model, we examined how a high-fat diet (lard-based 45kcal% fat)-induced mismatch between prenatal and postnatal environments could elevate blood pressure after growth. Although LBW rats fed standard chow had slightly higher blood pressure than control rats, their blood pressure was significantly higher than controls when exposed to a high-fat diet. Observation of glomeruli subjected to periodic acid methenamine silver (PAM) staining showed no difference in number or size. Aortic and cardiac angiotensin II receptor expression was altered with compensatory responses. Blood aldosterone levels were not different between control and LBW rats, but blood corticosterone levels were significantly higher in the latter with high-fat diet exposure. Administration of metyrapone, a steroid synthesis inhibitor, reduced blood pressure to levels comparable to controls. We showed that high-fat diet exposure causes impairment of the pituitary glucocorticoid negative feedback via miR-449a. These results clarify that LBW rats have increased blood pressure due to high glucocorticoid levels when they are exposed to a high-fat diet. These findings suggest a new therapeutic target for hypertension of LBW individuals.

Observation of Corneal Wound Healing and Angiogenesis Using Low-Vacuum Scanning Electron Microscopy.

PurposeWound healing processes in a rat corneal alkali burn model were observed using low-vacuum scanning electron microscopy (LV-SEM), a new observation method that can use paraffin sections for light microscopic immunostaining.MethodsInjured cornea was observed under immunohistochemistry, LV-SEM, and transmission electron microscopy. In LV-SEM, periodic acid-methenamine silver staining was used to observe collagen and platinum blue staining was used to observe vascular endothelial cells. Analyses of the messenger RNA expression involved in neovascularization processes after wound creation were also performed.ResultsLV-SEM depicted progression of corneal wound healing in a stereoscopic fashion. In neovascularization processes after wound creation, LV-SEM with osmification clearly demonstrated detachment of pericytes from the vascular endothelial cells, in association with up-regulation of angiopoietin-2 messenger RNA expression.ConclusionsLV-SEM enables high magnification observation of paraffin sections used for immunohistochemistry. LV-SEM provides easy, detailed observations and offers a promising new observational modality in the field of ophthalmology.Translational RelevanceHigh magnification analysis was easily available using LV-SEM with conventional paraffin sections for light microscopy.

Concurrent Kidney Glomerular and Interstitial Lesions Associated with Kimura’s Disease

Background: Kimura disease (KD) is a chronic benign inflammatory disorder that is usually manifested as a deep, subcutaneous mass with or without regional lymphadenopathy in the head and neck region. Various types of glomerulonephritis are associated with KD, including minimal change disease (MCD), membranous glomerulopathy (MN), and immunoglobulin (Ig)A-nephropathy. Kidney interstitial lesion associated with KD is rarely reported. The aim of this study was to expand the spectrum of kidney lesions associated with KD. Methods: From 2007 to 2016, 12 cases of KD with kidney lesions were retrospectively reviewed. Pathological examinations included hematoxylin and eosin staining, periodic acid-schiff staining, periodic acid-methenamine silver staining, and Masson staining, immunofluorescence, and electron microscope analyses. Results: Anatomic sites of subcutaneous involvement included head and neck area, arm, and groin. Most cases had elevated IgE level and peripheral eosinophilia. Nephrotic syndrome was the most common kidney manifestation. Pathological results showed 3 types of glomerulonephritis, including 9 cases of MCD, 2 cases of IgA nephropathy, and 1 case of MN. Of note, 4 MCD cases showed concurrent glomerular and interstitial lesions associated with KD, including 2 distinct patterns: (1) a diffusely eosinophilic and lymphatic infiltration similar to KD lesion developed elsewhere and (2) exclusively eosinophilic infiltration in the interstitium. Eight out of 10 patients were responsive to steroid treatment and had complete remission of proteinuria and recovery of kidney function. Conclusion: Our report suggested that, in addition to glomerulonephritis, interstitial lesions may also be associated with KD. Pathologist should pay special attention to differentiated diagnosis when such pathological changes are identified in patients with subcutaneous mass or cervical lymphadenopathy with concurrent kidney manifestation.

SUN-017 LOW-VACUUM SCANNING ELECTRON MICROSCOPY ANALYSIS FOR HUMAN BIOPSY SAMPLES

One of the major goal of biological microscopy is to elucidate the structural evidence relating functional activity or pathophysiological condition. High resolution imaging by electron microscopy can provide detailed tissue architectures, but the specific and skillful technics are required and it takes long time to the sample preparation. Low-vacuum scanning electron microscopy (LV-SEM) developed recently is a simple and fast technic to observe higher resolution of histological paraffin sections prepared for conventional light microscopy. Periodic acid methenamine silver (PAM) stain is a routine staining for basement membrane in renal biopsy, and contains silver, a heavy metal, which enhances the backscattered electron signal. To evaluate the benefit of LV-SEM, we observed PAM stained glomerular basement membrane (GBM) in human renal biopsy samples. The study was performed under the declaration of Helsinki principles. The study protocol was approved by the Institutional Review Board of the Tohoku Medical and Pharmaceutical University. All participants provided written informed consent after a full explanation of the purpose of the study and the potential risk involved. The human kidneys were obtained from patients with membranous nephropathy, IgA nephropathy, Alport syndrome, or thin basement membrane disease by renal biopsy and embedded in paraffin. Four-μm sections were deparaffinized with xylene, hydrated with ethanol, and stained with periodic acid methenamine silver (PAM). Miniscope TM4000 (Hitachi High-Technologies) was used to observe the GBM by detecting backscattered electron signal. After evacuation of the specimen chamber a few minutes, the sections were observed under the electron beam accelerating voltage of 10 kV for higher magnification (x2,500 to x20,000) or 15 kV for lower magnification (x40 to x2,000) with 30 Pa. The observations at higher magnification were easily performed under LV-SEM by zooming up from x40 to x20,000. The PAM-positive GBMs were bright under LV-SEM. The GBMs showed dark electron dense deposits in membranous nephropathy and IgA nephropathy, and characteristic coarse meshwork appearances in Alport syndrome. The GBMs in thin basement membrane disease seemed relatively thinner than the others. Direct observation of conventional renal biopsy paraffin sections by LV-SEM is a simple and beneficial method for the rapid and detailed analysis of the diseases. It will likely be useful for the observation of GBM morphological change.

An IgA1-lambda-type monoclonal immunoglobulin deposition disease associated with membranous features in a patient with IgG4-related kidney disease: a case report

BackgroundIgG4-related disease (IgG4-RD) is a newly recognized fibroinflammatory condition. The kidney is one of the organs commonly affected by IgG4-RD. Tubulointerstitial nephritis (TIN) is the main feature, and membranous nephropathy (MN) has also been described frequently. In MN, polyclonal immunoglobulins and complements are deposited in granular form along the glomerular basement membranes (GBMs). Unusual cases of monoclonal immunoglobulin deposition disease (MIDD) associated with membranous features have been reported. MIDD is morphologically similar to MN but contains immunoglobulins considered to be derived from single B-cell clone.Case presentationWe describe a 65-year-old man who was referred to our hospital because of hyperproteinaemia, eosinophilia, anaemia, and proteinuria. A renal biopsy demonstrated infiltration of plasma cells and eosinophils in the interstitium, and the ratio of IgG4-positive plasma cells to IgG-positive plasma cells was 55%. The patient was diagnosed as having IgG4-related TIN. Periodic acid methenamine silver staining under light microscopy revealed a bubbling appearance and spike formation in the GBM. On immunofluorescence, the expression of IgG and complements was negative; however, IgA was positively expressed in a granular pattern along the GBM. An IgA subclass analysis revealed a significant deposition of IgA1-lambda (IgA1-λ). Electron microscopy revealed irregular and small non-organized and non-Randall-type granular electron-dense deposits in the GBM that were shaped like snow leopard spots.ConclusionsAfter corticosteroid therapy was initiated, the patient’s eosinophilia remarkably improved and his serum creatinine, IgG, and IgG4 levels decreased to within the normal ranges. However, massive proteinuria persisted. To our knowledge, this is the first reported case of IgG4-related TIN associated with IgA1-λ-type MIDD with membranous features.

A case of membranoproliferative glomerulonephritis associated with metastatic colon cancer

A case of membranoproliferative glomerulonephritis associated with metastatic colon cancer

A large-sized bubbling appearance of the glomerular basement membrane in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis

We report an unusual pathological finding, a large-sized bubbling appearance of the glomerular basement membrane (GBM), in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis. The first renal biopsy specimen from 10 years ago, when systemic lupus erythematosus was diagnosed, demonstrated mild mesangial proliferation and subepithelial deposits (WHO classification: III + V). Light microscopy of the current biopsy using periodic acid methenamine silver (PAMS) stain demonstrated a large-sized bubbling appearance of the GBM; however, very weak immunoglobulin and complement deposition was observed in immunofluorescence studies. Routine electron microscopy demonstrated partial subendothelial expansion with electron-lucent materials, but no electron-dense deposits or amyloid fibrils. Electron microscopy with PAMS stain revealed electron-lucent endothelial scalloping, including some cellular components and microspheres in the GBM; however, it is not clear if these materials are derived from endothelial cells. One possibility is that these unique findings represent a recovery phase of lupus membranous nephritis; another is that these findings correspond to a new disease entity.

Differential diagnosis of trichosporonosis using conventional histopathological stains and electron microscopy

Although Trichosporon is a causative pathogen of white piedra and summer-type hypersensitivity pneumonitis, fatal disseminated trichosporonosis cases have recently been increasing. However, Trichosporon is often confused with other fungi, especially Candida, in pathological specimens. The aim was to determine the utility of histopathological stains and electron microscopy for diagnosing trichosporonosis. Autopsy cases of trichosporonosis, candidiasis, aspergillosis and cryptococcosis were investigated using histopathological stains and electron microscopy. Using Grocott's method, Trichosporon was weakly detected compared with other fungi. In contrast, diluted periodic acid methenamine silver (PAM) stain clearly enhanced the intensity of staining of Trichosporon compared with Candida. Furthermore, Alcian blue and colloidal iron stains predominantly detected Trichosporon. Electron microscopy after staining with diluted PAM demonstrated that Trichosporon has a variety of hyphal sizes and laminar deposition of rough silver granules, whereas Candida has uniform pseudohyphae and fine granules. The average diameter and population area of the granules were significantly higher in Trichosporon compared with Candida (P<0.01). Meanwhile, the laminar structure was preserved in the cell walls of Trichosporon without silver stains, whereas a low-density structure was observed in Candida. Histopathological staining patterns and electron microscopic findings can facilitate the diagnosis of trichosporonosis.

A Pathological Study of Bamboo Nodule of the Vocal Fold

We examined pathologically a bamboo nodule of the vocal fold by means of immunohistochemical studies and scanning electron microscopic examination. A 38-year-old female showed a high index of antinuclear antibodies without any systemic symptoms but had complained of progressive voice disorder for 9 months. She had used her voice excessively in her occupation and for singing. Hematoxylin and eosin staining showed submucosal edema with lymphocyte and neutrocyte infiltrations and hyaline degeneration. Periodic acid methenamine silver staining showed hypertrophy of the basal lamina of the blood vessels. Immunohistochemical study showed IgG-positive cells in the blood vessel walls. Scanning electron microscopic study demonstrated immune complexes deposited as fine granules of high electrodense materials in the hypertrophic walls of the micro-blood vessels. After surgical resection of the bamboo nodules and advising her to avoid using her voice excessively, her voice improved gradually and the levels of IgG, immune complexes, and antinuclear antibody decreased for 1 year. These findings suggest that the bamboo nodules were not induced by an organ-specific reaction to an autoimmune disease, but mechanical damage to the micro-blood vessels induced by phonation injury of the vocal fold might have caused the deposit of high-molecular weight immune complexes in the damaged micro-blood vessels at the midportion of the vocal folds, which induced secondary inflammatory change at the midportion of the vocal fold.

Proposal of podocytic infolding glomerulopathy as a new disease entity: a review of 25 cases from nationwide research in Japan

A rare and peculiar glomerulopathy has begun to be recognized in Japan. The Japanese Society of Nephrology has established a research working group and has collected cases from all over Japan in an attempt to understand the complete spectrum of this glomerulopathy. The diagnostic criterion, which was needed to collect the cases, was proposed as a glomerulopathy showing microspheres or microtubular structures or both associated with podocytic infolding into the glomerular basement membrane (GBM) on electron microscopy. The lesion shows a non-argentaffin hole in the GBM with periodic acid methenamine silver staining and is similar to membranous glomerulonephritis. Twenty-five cases were collected from 17 institutions. Patients were 20-69 years old (19 women, 6 men). Seventeen patients also had collagen diseases such as lupus nephritis and Sjögren's syndrome. All patients had proteinuria. Proteinuria showed a remission in 15 of 23 patients within 12 months, but proteinuria remained higher than 1.0 g/day in five patients despite different types of therapy. Podocytic infolding including microspheres showed either positive or negative staining for immunoglobulins. Cluster formation of microspheres was found in 4 of 17 patients with collagen disease, and in five out eight patients without collagen disease. Electron-dense deposits in the GBM were also found in 6 of 17 patients with collagen disease but were not found in eight patients without collagen disease. Some patients might have a subtype of lupus nephritis, class V, or membranous glomerulonephritis. However, we propose a new disease entity, podocytic infolding glomerulopathy, as a common basis of all 25 patients, because we suspect that microspheres or microtubular structures or both can be derived from podocytic infolding.

Intramembranous microspherical structures in focal segmental glomerulosclerosis

A 45-year-old male had proteinuria for 3 years. For persistent proteinuria over 2 g/day, he underwent renal biopsy. Light microscopy revealed focal segmental glomerulosclerosis together with diffuse capillary wall thickening. Periodic acid methenamine silver (PAM) staining showed a prominent bubbly appearance without spike formation could be found in almost all capillary walls. Electron microscopy revealed many microspheres measuring 50-70 nm in diameter distributed in diffuse and global fashion together with the thickened glomerular basement membrane. A few cytoplasmic processes of the podocytes showed infolding to the GBM. The patient exhibited no symptoms and no physical and serological findings suggesting autoimmune disease, such as systemic lupus erythematosus or Sjögren's syndrome. Therefore, the present case is important, because the peculiar microstructure in the GBM was noted in focal segmental glomerulosclerosis, which has never been reported in the literature.

The Effect of Calcium Channel Blocker Verapamil on Gentamicin Nephrotoxicity in Rats

Aminoglycoside antibiotics are obligated nephrotoxins and inevitably cause renal failure during prolonged use. Experimental models of gentamicin-induced nephrotoxicity have shown histopathological, ultrastructural and functional alteration with blood urea nitrogen and serum creatinine increase leading to acute renal insufficiency (ARI). The aim of our study was to emphasize effects of verapamil, a calcium channel blocker, on gentamicin-induced ARI in rats. Experiments were done on 50 male Wistar rats (250-300 g) divided in three experimental groups. G-group animals (20 rats) were treated daily with gentamicin in dose of 100 mg/kg during 8 days. GV-group animals (20 rats) were treated daily with verapamil in dose of 3 mg/kg and the same dose of gentamicin as in G-group during 8 days. The control group (10 rats) received 1 ml/day saline intraperitoneally. Histological examinations were done using hematoxylin and eosin, periodic acid Schiff and methenamine silver staining methods. Morphometric parameters included measurement of glomerular area, major and minor axis, perimeter, diameter, roundness, and mean optical density. Biochemical analyses were used to determine concentrations of blood urea, serum creatinine, sodium and potassium. In G-group rats' glomerular basement membrane was diffusely and unequally thickened with polymorphonuclear neutrophils infiltration, while coagulation-type necrosis and vacuolization of cytoplasm of proximal tubules epithelial cells were observed. In GV-group rats' glomeruli were slightly enlarged with thickened basement membrane in some segments but without coagulation-type necrosis. Morphometric analyses showed statistically significant differences between the G-group and control group of animals in glomerular size, mean optical density and average roundness (p<0,05). On the other hand, morphometric analyses between GV-group and control group animals did not show statistically significant differences in any of parameters measured. Blood urea and serum creatinine concentration in G-group were significantly elevated in comparison with GV-group (p<0,05) but sodium and potassium levels in G-group were decreased compared to GV-group without statistical significance. Our results show that verapamil modify some of morphological and functional kidney alterations induced by gentamicin.

Histochemical and immunohistochemical study of the glomerular development in human fetuses

Few studies exist that establish the normal morphological patterns of glomerular development, though this is one of the organs that continues to evolve morphologically during the postnatal period up to 4 weeks after birth. In our study one kidney from each autopsy of a total of 86 autopsies was analyzed [15 weeks to 40 weeks of gestational age (GA)]. We examined the variation in the nephrogenic zone thickness, the area and diameter of the glomerular tuft, the area and diameter of the glomerular capsule, and the immunohistochemical markers, anti-CD31 and anti-CD34 antibodies, which accompany the development of the glomerular microvasculature. Periodic acid-methenamine silver (PAMS) stain was used for the morphological and morphometrical analyses, and it was particularly useful in fetuses in which autolysis had occurred. The length of the nephrogenic zone (NZ) decreased with the increase of the GA (P < 0.001) according to the formula: GA = 36.5 - (0.05 x length of NZ). The areas of the Bowman capsule (P < 0.0001), the capillary tuft (P < 0.0001), and the capillary tuft diameter (P = 0.00393) of the intermediary glomeruli increased with the advance of GA, with a positive significant correlation. The same parameters of the juxtamedullary and superficial glomeruli had no correlation with the advance of GA. The cells of the primary structures in the "S" shape of the primitive nephrons were negative for CD31 and CD34. Staining for both antibodies was found, for all GAs, in the endothelial cells of the mature glomeruli tuft and in the renal interstitial vessels. The data obtained in this work contribute to the evaluation of renal maturity in autopsied fetuses and are particularly important in fetuses when autolysis has occurred, to which the parameters used in this study can also be applied. The establishment of normal morphometric and immunohistochemical parameters for the evaluation of renal maturity increases the diagnostic precision of renal pathological alterations in aborted material and perinatal autopsy.

Leishmania (Leishmania) chagasi-infected mice as a model for the study of glomerular lesions in visceral leishmaniasis

Renal involvement in visceral leishmaniasis (VL) is very frequent but the pathogenesis of this nephropathy is poorly understood. In previous studies using dogs with VL we have detected new immunopathological elements in the glomeruli such as T cells and adhesion molecules. Although Leishmania (Leishmania) chagasi-infected dogs and hamsters are considered to be good models for VL, their use is limited for immunopathologic studies. The use of isogenic mouse strains susceptible to L. (L.) chagasi infection was an alternative but, on the other hand, the renal lesions of these animals have not yet been characterized. Thus, our purpose in the present study was to characterize mice infected with L. (L.) chagasi as a suitable model to study VL nephropathy. Kidney samples were obtained from control mice (N = 12) and from BALB/c mice (N = 24) injected intraperitoneally with 20 million L. (L.) chagasi amastigotes 7, 15, and 30 days after injection and processed for histopathological studies and detection of IgG deposits. Glomerular hypercellularity was clearly visible and, upon Mason's trichrome and periodic acid methenamine silver staining, a pattern suggestive of mesangial proliferative glomerulonephritis was observed in mice with VL. Time-dependent IgG deposits were also seen in infected mice. We consider L. (L.) chagasi-infected mice to be a suitable model for studies of the immunopathogenesis of glomerular lesions in VL.

Need Based Structural Changes in the Ductulus Efferentis During Sperm Movements in the Human Epididymis

The dynamic structures of the human ductuli efferentes, which connect between the rete testis and the ductulus epididymidis, are described in the present paper. Maturation of sperm takes place in the tubule and after this process the group of sperm is transferred to the ductus. All ductuli do not show the same histological contents. Each ductulus shows variety of different configuration depending on the functional phase (the state of sperm maturation) of the tubule. The basic pattern of all the tubuli is the same, however, they differ in the thickness, cytoplasmic components of epithelial cells and luminal contents. Larger tubule has big lumen whereas small is almost without a lumen. During the process, spherical and luminal macrophages act as nurse macrophage by using G1 granules obtained from the epithelial cell, and irregular shaped macrophage in the small tubule acts as scavenger macrophage. It has been shown that the epithelial cell of the large tubule show intense excretory activity and the secretory substance is different from other granules and lysosomes by periodic acid methenamine silver staining.