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IgA nephropathy (IgAN) is one of the most diffuse glomerulonephrites worldwide, and many issues still remain regarding our understanding of its pathogenesis. The disease is diagnosed by renal biopsy examination, but potential pitfalls still persist with regard to discriminating its primary origin and, as a result, determining patient outcome remains challenging. In this pilot study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) was performed on renal biopsies obtained from patients with IgAN (n = 11) and other mesangioproliferative glomerulonephrites (MesPGN, n = 6) in order to enlighten proteomic alterations that may be associated with the progression of IgAN. Differences in the proteomic profiles of IgAN and MesPGN tissue could clearly be detected using this approach and, furthermore, 14 signals (AUC ≥ 0.8) were observed to have an altered intensity among the different CKD stages within the IgAN group. In particular, large increases in the intensity of these signals could be observed at CKD stages II and above. These signals primarily corresponded to proteins involved in either inflammatory and healing pathways and their increased intensity was localized within regions of tissue with large amounts of inflammatory cells or sclerosis. Despite much work in recent years, our molecular understanding of IgAN progression remains incomplete. This pilot study represents a promising starting point in the search for novel protein markers that can assist clinicians in better understanding the pathogenesis of IgAN and highlighting those patients who may progress to end-stage renal disease.

Background The current study evaluates the application of histology and in situ proteomics (MALDI-MSI) in Fabry nephropathy (FN), showing investigative and classification role for this coupled approach. Methods A retrospective series of 14 formalin fixed paraffin embedded (FFPE) renal biopsies with diagnosis of FN and 1 biopsy from a patient bearing a galactosidase-alpha (GLA) genetic variant of unknown significance (GVUS, c.376A>G) have been classified for clinical characteristics. Groups were compared for histological differences (following the ISGFN scoring system). Moreover, renal biopsies from these cases have been analyzed with MALDI-MSI as previously described to find proteomic signatures among different mutations and phenotypes. Results Comparison of clinical features revealed lower mean 24 h proteinuria in females (225 mg/24 h) than in males (1477.5 mg/24 h, p = 0.006). As for clinical characteristics, females significantly differed from males only for lower arterial sclerosis, with a mean value of 0.82 vs. 1.05 (p = 0.001). Proteomic analysis demonstrated specific signatures in different subgroups of FN patients. Moreover, MALDI correctly classified cases with undetermined mutation or GVUS. Conclusions The present study demonstrated the feasible application of MALDI-MSI in the analysis of FN FFPE renal biopsies, allowing the detection of putative signatures for phenotypic distinction and demonstrating genetic classification capabilities.

To the Editor, Many different molecular techniques (polymerase chain reaction (PCR), DNA sequencing, fluorescence in situ hybridization (FISH)) have been introduced in thyroid pathology [1]. Fewer studies evaluated the role of proteomic analysis in the research of new useful targets [2, 3]. Matrixassisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. Therefore, we are investigating the potential role of MALDIIMS in detecting new diagnostic targets in papillary thyroid carcinoma (PTC). We have addressed the issue of molecular stratification of PTC in a small cohort of samples, evaluating both the genomic profile of the main genes of interest (BRAF, N-H-K RAS point mutations, and PAX8/PPARγ or RET/PTC rearrangements) and the proteomic profile shown byMALDIIMS analysis. We have collected ex vivo cytological specimens (see “Technical Note”), in order to analyse a sample perfectly mimicking the in vivo fine-needle aspiration (FNA) biopsy, while respecting the ethical requirements. Unsupervised proteomic analysis of the samples was followed by comparison with histopathology and genetic classification of the patients (Table 1). Data generated by MALDI-IMS were submitted to hierarchical cluster analysis (HCA), in order to evaluate the different proteomic expressions in the cases under study. HCA allowed to cluster proteomic spectra based on their similarity on a dendrogram: spectra showing comparable features were grouped under the same node of the dendrogram; then, it was possible to select nodes and assign them a specific colour (Fig. 1) [4]. Node A groups together three cytological specimens (two collected from patient 1 and one from patient 2), all histologically diagnosed as hyperplastic benign nodules. Node B groups together specimens collected from patients 3, 4 and 5, who were affected by PTC comparable for stage and histotype (Table 1). Node C, instead, groups a microcarcinoma (follicular variant (fv) of PTC), from patient 6, and a nodule histologically classified as uncertain malignant potential (UMP) tumour (patient 7). The last one, originated in a multinodular goitre environment and showed ambiguous morphological features, defined as borderline between a hyperplastic nodule and an “incipient” fv,PTC. The three distinct nodes (A, B, C) generated by HCA analysis confirmed that MALDI-IMS can potentially discriminate between benign and malignant thyroid lesions. Moreover, the homologies between cases 6 and 7 highlight that MALDI-IMS is able to identify a PTC even when the classic diagnostic morphological aspects are still unclear and ambiguous (mild nuclear clearing, rare grooves, no pseudoinclusions). This finding is in agreement Veronica Mainini and Fabio Pagni equally contributed to this paper.

Lipids extracted from detergent-resistant membrane fractions, thought to derive from membrane domains, were analyzed for fatty acid composition. The proportion of palmitic acid in fractions isolated from neurons (cerebellar granule cells) and from neural-like cell lines (neuroblastomaglioma NG108-15) nearly doubled (reaching about 54% of total fatty acids) with respect to cell WCL, indicating their enrichment in palmitic acid-carrying lipids. The proportion of palmitic acid in detergent-resistant fractions obtained from caveolin-transfected NG108-15 cells was comparable with that obtained from caveolin-negative cells, ruling out a specific role of this protein in recruiting palmitoylated lipid species. The enrichment in palmitic acid was remarked also in membrane fractions isolated from non-neuronal cell lines (A431) using either detergents or detergent-free techniques. Lipid fractionation and mass spectrometry experiments show that palmitic acid-rich phosphatidylcholine species are responsible of the peculiar fatty acid composition of these fractions. All together these results suggest that the enrichment in palmitic acid-rich phosphatidylcholine species is a common feature of neural and non-neural cell lines and may play a major role in the biogenesis of membrane domains.

Background Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. Methods We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). Results A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. Conclusions ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful for future functional approaches aimed to study genes or pathways involved in ccRCC etiopathogenesis and to identify novel clinical markers or therapeutic targets. Moreover, SNP array technology proved to be a powerful tool to better define the cell composition and homogeneity of RCC primary cultures.

The effects of streptozotocin-induced diabetes on the various forms of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase), phosphorylated/dephosphorylated and thiolic/disulphide, were studied in rat liver. Animals were treated twice with 65 mg/kg intraperitoneally of streptozotocin to induce diabetes and sacrificed after 5 days. The relative amounts of the four possible forms of the enzyme were determined in control and diabetic rats. As determined from the total activity, and in agreement with previous reports, the enzyme protein was significantly decreased in streptozotocin-treated rats. However, the percentage of the active thiolic dephosphorylated form was higher in these animals than in controls, suggesting a response of the liver to the decrease both total and specific activity of HMGCoA reductase.