Minimal change disease (MCD) and primary (idiopathic) focal segmental glomerulosclerosis (1FSGS), referred to collectively as “primary podocytopathies”, are major causes of nephrotic syndrome in children and adults, and are thought to be due to direct podocyte damage visible only at the electron microscopic level. Lupus podocytopathy (LP) is a newly recognized entity that involves severe podocyte injury in the setting of systemic lupus erythematosus, in the complete absence of peripheral capillary wall immune deposits. All of these pathologic diagnoses hinge on the ultrastructural finding of severe podocyte injury and foot process effacement. In addition to these ultrastructural changes, we have observed the presence of fine granular anti-IgG antibody immunoreactivity on podocyte cell bodies in kidney biopsies of patients with MCD, LP, and some patients with the tip lesion variant and NOS variants of 1FSGS. To validate this finding, we compared antibody staining from primary podocytopathy biopsies with those in biopsies from patients with other disease states, including lesions associated with severe podocyte injury in the absence of immune deposits: secondary (adaptive) focal segmental glomerulosclerosis, thin basement membrane disease, diabetic nephropathy, and renal amyloidosis. We found that a fine granular pattern of anti-IgG immunoreactivity on podocyte cell bodies is a specific morphologic feature of the primary podocytopathies, including virtually all cases of MCD that we encountered, some instances of tip lesion variant and NOS variant of 1FSGS, and one cases of LP. The antigen targeted by the anti-IgG immunostaining in these biopsies exhibited one of several oligoclonal IgG heavy chain subtype plus light chain profiles. Ultra-high resolution microscopy revealed fine linear anti-IgG staining along filtration slit diaphragms, suggesting that IgG deposition may potentially be targeting a filtration slit-associated antigen such as podocin. Our findings suggest the possibility of a direct antibody-mediated mechanism of podocyte injury in the primary podocytopathies, one that potentially targets podocyte-specific protein structures, and which may provide a specific and more rapid diagnostic marker for this group of diseases. The findings also suggest an etiologic relationship between MCD and some instances of 1FSGS.

Nephrotic syndrome (NS) is a set of symptoms defined by heavy proteinuria and associated with a host of kidney diseases that cause injury to the glomerulus, the filtration apparatus of the kidney. Primary podocytopathies (Px), a group of diseases including minimal change disease (MCD), primary focal segmental sclerosis (pFSGS), and lupus podocytopathy (LP), are the principal cause of idiopathic NS in both children and adults. The hallmark feature of Px is the ultrastructural finding of podocyte foot process effacement (FPE), so current differential diagnosis of Px relies on technically exhaustive electron microscopy (EM) analysis. During routine immunofluorescence (IF) microscopy of many Px cases, we have observed punctate IgG (P-IgG) immunoreactivity in the glomerulus. P-IgG may represent a disease-specific reactivity that could not only provide clues for understanding Px etiology but could also serve as a diagnostic tool. However, we have found that P-IgG may be misinterpreted as protein reabsorption granules (PRGs), a morphological feature seen in proteinuric conditions. We sought to definitively characterize the key characteristics of PRGs and P-IgG in order to differentiate these features. To accomplish this, we reviewed prior IF immunostaining of MCD biopsies to evaluate anti-human IgG and anti-human albumin staining. We conducted additional IF staining on archived MCD biopsies using antibodies against IgG and against the markers of PRGs: albumin, megalin, and cubilin. We found that the P-IgG demonstrates a diffuse, global distribution pattern that is specific to glomerular epithelium and is fine and scattered. Conversely, the PRGs are coarse, clustered, and frequently demonstrate a focal, segmental pattern in the glomeruli and tubules. Co-staining with albumin and megalin revealed that the P-IgG and the PRGs do not colocalize in the tissue. While the cubilin antibody positively stained the tubular epithelium, it did not stain glomeruli. Our results showed that punctate IgG and protein reabsorption granules are morphologically and constitutionally distinct and do not colocalize with each other, indicating that P-IgG is highly likely to represent a distinct process from epithelial protein reabsorption.

Immunolocalization of proteins within the cell is a significant and powerful tool that improves understanding of cellular functions and processes, such as molecule secretion during immune responses. Immunogold electron microscopy (IEM) is an immunohistochemistry technique that uses gold-conjugated antibodies and electron microscopy (EM) to identify and localize antigens at the ultrastructural level. Here, we are trying to develop and optimize an IEM staining protocol that targets glomerular proteins of interest in Minimal Change Disease (MCD), and eliminates background staining, and preserves tissue morphology. Using this optimized protocol, we hope to learn more about the relationship between IgG and Nephrin in MCD. Kidney biopsies diagnosed with MCD, Membranous Nephropathy (MN), and Thin Basement Membrane Disease (TBMD) and previously embedded in paraffin blocks were retrieved from the tissue archive of the Renal Pathology Laboratory at Boston Medical Center. MN and TBMD were selected as positive controls for IgG and Nephrin staining protocols, respectively. Co-staining of IgG and Nephrin was performed after the protocols for each target were optimized. During protocol development, it was observed that section quality is significantly affected by the angle and sharpness of the knife, and the thickness of the section. Moreover, section quality highly impacted gold particle localization. Ultimately, co-staining of IgG and Nephrin was successful in MCD cases. However, further improvements are needed to optimize IgG and Nephrin staining, and in turn, our understanding of MCD.

Nephrotic syndrome is characterized by proteinuria, hypoproteinemia, edemas and hyperlipidemia. It occurs in primary (e.g. focal segmental glomerulosclerosis, FSGS or minimal change disease, MCD) and in secondary glomerulopathies (e.g. kidney amyloidosis). In primary forms, great attention is paid to the potential genetic background of the disease and due to new molecular genetic methods genes, whose mutations cause different nephropathies (e.g. ACTN4 or INF2) were identified. The aims of presented doctoral thesis were following. Firstly, to continue the mutational analysis of ACTN4 that was described in the author's diploma thesis in other glomerulopathies. Secondly, to implement the mutational analysis of INF2 and subsequently analyse this gene in patients with FSGS/MCD and in patients from special group characterized by positive family history for end stage renal disease (ESRD) in combination with advanced chronic kidney disease (CKD) or already developed ESRD at the time of diagnosis. Thirdly, mutational analysis of NPHS2 and TRPC6 (methods implemented in laboratory earlier) in selected patients from the special group. Finally, expression analyses of genes important for podocyte function or connected with human immune system. This part also verifies the applicability of NPHS2/SYNPO expression...