Histopathological Examination of Clustering Microspheres and Podocyte Infolding in the Glomerular Basement Membrane Using Low-vacuum Scanning Electron Microscopy

Abstract

Podocyte infolding (PI) in a thickened glomerular basement membrane (GBM) is a rare pathological finding in electron microscopy (EM). We histopathologically evaluated a 57-year-old Japanese male patient with clustering microspheres and PI in the GBM. EM showed clustering microspheres in irregularly thickened GBM and PI into the GBM. Endothelial cells also invaded the GBM on EM. Low-vacuum scanning EM revealed numerous openings or holes in the GBM. Careful examination by EM is needed to identify further cases of clustering microspheres and PI. It is also important to explore these cases using new approaches such as low-vacuum scanning EM.

Introduction

Podocyte infolding (PI) in a thickened glomerular basement membrane (GBM) is a rare pathological finding in electron microscopy (EM). The concept of PI was proposed by the Japanese Society of Nephrology in 2008 (1). A small number of cases have been reported worldwide, most of which are from Asian countries (1-10). It is important to share information regarding PI to elucidate its pathogenesis and clinical characteristics. We histopathologically evaluated a Japanese man with clustering microspheres and PI in the GBM.

Case Report

A 57-year-old Japanese male patient with hypertension and dyslipidemia was referred to our hospital from a nearby medical clinic because his dipstick urine test result was positive for proteinuria and hematuria. He was admitted to our hospital for a kidney biopsy because of nephrotic proteinuria and microscopic hematuria. On admission, a physical examination revealed the following: height, 161.0 cm; body weight, 78.8 kg; body temperature, 35.9°C; blood pressure, 119/93 mmHg; and heart rate, 60 beats/min with sinus rhythm. A physical examination revealed pitting edema of the lower extremities. His clinical laboratory data were as follows: white blood cell count, 8.0×10³/μL with 60.6% neutrophils; hemoglobin level, 14.9 g/dL; platelet count, 33.7×10⁴/μL; serum creatinine, 0.71 mg/dL; estimated glomerular filtration rate, 88.4 mL/min/1.73 m²; blood urea nitrogen, 13 mg/dL; total protein without M-protein, 5.4 g/dL; albumin, 3.2 g/dL; blood glucose, 103 mg/dL; hemoglobin A1c, 6.4%; C-reactive protein, 0.06 mg/dL; immunoglobulin G, 417 mg/dL; immunoglobulin A, 114 mg/dL; and immunoglobulin M, 143 mg/dL. Serum complement factor 3 and 4 levels and complement hemolytic activity were normal. Tests for antinuclear antibodies, rheumatoid factor, antineutrophil cytoplasmic antibodies, anti-glomerular basement membrane antibodies, hepatitis B virus surface antigen, and hepatitis C virus antibodies were negative. A urinalysis revealed the following: proteinuria, 4.13 g/g creatinine; 10-19 red blood cells/high-power field; N-acetyl-β-D-glucosaminidase, 8.0 IU/L; and β2-microglobulin, 225 μg/L. The selectivity index for proteinuria was 0.19. Percutaneous kidney biopsy was performed. Light microscopy revealed 49 glomeruli, one of which had global sclerosis, another had an adhesive lesion and segmental sclerosis, and some had mild mesangial matrix expansion. Mild cellular infiltration, interstitial fibrosis, and tubular atrophy were observed. Mild arteriosclerosis and absence of arteriolar hyalinosis were observed. Immunofluorescence (IF) showed pseudo-linear and segmental granular staining for IgG along the GBM, and granular staining for IgM in the mesangial area (Fig. 1). Clustering microspheres were found in the irregularly thickened GBM and podocytes were infolded into the GBM by transmission electron microscopy (TEM). No electron-dense deposits were observed in the GBM or mesangium. We also observed the invasion of endothelial cells into the GBM (Fig. 2). TEM revealed a GBM thickness of 820-1,090 nm and foot process effacement of approximately 100%. Kidney biopsy specimens were examined using low-vacuum scanning electron microscopy (LV-SEM). LV-SEM (Hitachi Tabletop Microscope TM4000; Hitachi High-Technologies, Tokyo, Japan) enabled a three-dimensional ultrastructural analysis of kidney biopsy paraffin sections that were stained with periodic acid methenamine silver. LV-SEM is a comparatively inexpensive approach, allowing rapid and wide observation of kidney biopsy specimens without requiring TEM preparation (11). In this three-dimensional approach, the appearance of the subendothelial structure of the thickened GBM was rough, and numerous openings or holes, ranging from 0.12 to 1.64 nm, in the GBM on both the subepithelial and subendothelial sides were observed (Fig. 2). The openings or holes in the GBM were observed both in areas with and without PI. IF staining for type IV collagen α2 and α5 chains, which is useful for the diagnosis of Alport syndrome, revealed normal expression patterns (Supplementary material 1).

Figure 1.

Histological findings of the kidney biopsy specimens. (A, B) Light microscopy shows mild mesangial matrix expansion with an adhesive lesion and segmental sclerosis. (A: PAS staining, original magnification, ×200; B: PAM staining, original magnification, ×200) (C, D) Immunofluorescence shows pseudo-linear and segmental granular staining for IgG along the glomerular basement membrane and granular staining for IgM in the mesangial area. PAS: periodic acid-Schiff, PAM: periodic acid-methenamine silver

Figure 2.

(A) EM shows clustering microspheres in the irregularly thickened GBM (red circle), and podocytic infolding into the GBM (yellow arrow). No electron-dense deposits are present in the GBM or mesangium (A: original magnification ×20,000). (B) The invasion of the endothelial cells into the GBM is observed in EM (B: original magnification ×20,000). (C, D) Low-vacuum scanning EM shows numerous openings or holes, ranging from 0.12 to 1.64 nm, in the GBM on both the subepithelial and subendothelial sides. Podocytes (P, white arrow) and red blood cells (R) are shown (C: PAM staining, original magnification ×5,000; D: PAM staining, original magnification ×5,000). EM: electron microscopy, GBM: glomerular basement membrane, PAM: periodic acid-methenamine silver

He was treated with blood pressure control, and his serum creatinine and albumin levels were 0.81 mg/dL and 3.7 g/dL at one year after the kidney biopsy.

Discussion

We histopathologically examined a Japanese male patient with clustering microspheres and PI without autoimmune disease using LV-SEM. PI has been reported to have unique ultrastructural findings of microspherules, microtubules, and podocytic infolding in thickened GBM (1). According to the literature review of 44 cases of PI by Feng et al., 42 patients (95.5%) had microspheres and 19 patients (43.2%) had microtubules in the GBM on TEM. GBM thickening and dense deposits were found in 41 (93.2%) and 12 (27.3%) patients, respectively (9). In a previous retrospective study that reported a total of 116 patients with PI, microspheres and microtubules were observed in 111 (95.7%) and 71 (61.2%) patients, respectively. In total, 114 patients (98.3%) had GBM thickening (10). In the present case, TEM demonstrated clustering microspheres and podocytic infolding in an irregularly thickened GBM. Foot process effacement and microvillous transformation were observed in most cases of PI (1-10). PI may be a manifestation of podocyte injury.

Reports on PI are limited, and histological findings are rare. Most previous case reports were from Asian countries (1-3,6-8). Recently, however, several cases have been reported in non-Asian countries (4,5). In previously reported cases, the proportion of women was high, and many cases were complicated by autoimmune diseases, such as lupus nephritis or connective tissue disease (1-3,6,9,10). Our case was a middle-aged man who did not have any autoimmune disease and seemed to have different characteristics from the previous cases.

The pathogenesis and clinical characteristics of PI remain unclear. Fujigaki et al. demonstrated that vimentin (a marker for both podocytes and the endothelium) was positive in most intra-GBM microstructures using immunoelectron microscopy, suggesting that microstructures were derived from podocytes or endothelial cells. In this case report, C5b-9 was positive along the entire epithelial side of the GBM and in some microstructures in immunoelectron microscopy (12). The pathogenesis of PI may be related to the activation of the complement pathway. However, several reported patients with PI did not have autoimmune diseases (7,8), as in the present case.

In our case, using LV-SEM, we found numerous openings or holes in the GBM on both the subepithelial and subendothelial sides (Fig. 2). Yoshida et al. described a middle-aged woman with lupus nephritis class V (13). In that case, three kidney biopsies were conducted and evaluated using LV-SEM. In the first and second kidney biopsies, IF revealed coarse granular staining for IgG, C3, and C1q in the capillary walls, and subepithelial deposits were observed on TEM. However, in the third kidney biopsy, negative conversion of IgG and negative C3 and C1q were observed on IF, while electron-dense deposits were unclear on TEM. In contrast, the openings or holes in the GBM gradually became conspicuous toward the glomerular capillary lumina over the course of the three kidney biopsies. It was suspected that the openings or holes in the GBM were formed when the immune complexes injured the GBM and passed through the GBM from the epithelial side toward the glomerular capillary luminal side. Moreover, both podocytes and endothelial cells were reported to invade the GBM in a previously reported case of PI (6). In our case, we also observed the invasion of endothelial cells into the GBM (Fig. 2), suggesting that GBM injuries or abnormalities might be associated with PI development.

In summary, our patient was a middle-aged man without autoimmune disease who was recognized with clustering microspheres and PI in GBM in the EM findings of his kidney biopsy. The kidney biopsy specimens were histopathologically evaluated using LV-SEM. The histopathological examination of the patient's kidney biopsy specimens suggested that both podocyte injuries and GBM injuries or abnormalities were associated with the development of PI. LV-SEM might be useful to evaluate GBM injuries or abnormalities. The detailed profile of PI, such as the necessity of immunosuppressive therapy and clinical outcomes, will become clear by accumulating information about further cases of PI in the future. It could be more meaningful to discover clustering microspheres and PI by careful examination of EM. It is also important to explore these cases using new approaches such as LV-SEM.

This study was approved by the Institutional Review Board of the Tohoku Medical and Pharmaceutical University Hospital (2023-4-019). This study was conducted in accordance with the Declaration of Helsinki of the World Medical Association. Informed consent was obtained from the patient in this study.

Author’s disclosure of potential Conflicts of Interest (COI).

The Division of Integrative Renal Replacement Therapy (T.H. and T.M.) was financially supported by JMS, Terumo, and the Southern TOHOKU Hospital Group (STHG). The other authors declare no conflicts of interest.

Supplementary Material

Supplementary Figure

Results of double immunofluorescence staining for type IV collagen α2(red) and α5(green) chains using frozen biopsy sections. α2(IV) shows positive staining in the mesangial areas of glomeruli and all basement membranes. α5(IV) shows positive staining in the GBM, Bowman’s capsule, and focal tubule basement membrane. Normal expression patterns of α2(IV) and α5(IV) are observed. GBM, glomerular basement membrane.