Global Analysis Reveals the Complexity of the Human Glomerular Extracellular Matrix

The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.The glomerulus is a sophisticated organelle comprising unique cellular and extracellular matrix (ECM) components. Fenestrated capillary endothelial cells and overlying podocytes are separated by a specialized glomerular basement membrane (GBM), and these three components together form the filtration barrier. Mesangial cells and their associated ECM, the mesangial matrix, exist between adjacent capillary loops and maintain the three-dimensional organization of the capillary bundle. In turn, the parietal epithelial cells and ECM of Bowman’s capsule enclose this network of capillaries. Cells adhere to ECM proteins by adhesion receptors, and these interactions are required to maintain intact barrier function of the glomerulus.^1,2In addition to operating as a signaling platform, ECM provides a structural scaffold for adjacent cells and has a tissue-specific molecular composition.^3,4 Candidate-based investigations of glomerular ECM have focused on the GBM and shown that it resembles the typical basal lamina found in multicellular organisms, containing a core of glycoproteins (collagen IV, laminins, and nidogens) and heparan sulfate proteoglycans (agrin, perlecan, and collagen XVIII).⁵ Mesangial and parietal cell ECMs have been less well investigated; nonetheless, they are also thought to contain similar core components in addition to other glycoproteins, including fibronectin.^6,7 Thus, the glomerulus consists of a combination of condensed ECM within the GBM and Bowman’s capsule and loose ECM supporting the mesangial cells.The ECM compartments in the glomerulus are thought to be distinct and exhibit different functional roles. The GBM is integral to the capillary wall and therefore, functionally linked to glomerular filtration.⁵ Mutations of tissue-restricted isoforms of collagen IV (COL4A3, COL4A4, and COL4A5) and laminin (LAMB2), which are found in the GBM, cause significant barrier dysfunction and ultimately, renal failure.^8,9 Less is understood about the functions of mesangial and parietal cell ECMs, although expansion of the mesangial compartment is a histologic pattern seen across the spectrum of glomerular disease.¹⁰Compositional investigation of the distinct glomerular ECM compartments is limited by the technical difficulties of separation. Early investigations of GBM constituents used the relative insolubility of ECM proteins to facilitate separation from cellular proteins in the glomerulus but did not separate the GBM from mesangial and parietal cells ECMs.^11,12 Recently, studies incorporating laser microdissection of glomerular sections have been coupled with proteomic analyses.^13,14 These studies report both cellular and ECM components and typically require pooled material from glomerular sections to improve protein identification. The ability of laser microdissection to separate glomerular ECM compartments has not yet been tested; however, this approach will be limited by the amount of protein that is possible to retrieve. To achieve good coverage of ECM proteins within a tissue, proteomic studies need to combine a reduction in sample complexity with maximal protein quantity. Currently, the inability to separate glomerular ECM compartments in sufficient quantity is a limitation that prohibits proteomic studies of these structures; however, for other tissues, proteomic analysis of ECM has been achieved by enrichment of ECM combined with sample fractionation.¹⁵Although the composition of the ECM in other tissues has been addressed using proteomic approaches,¹⁵ studies of glomerular ECM to date have used candidate-based technologies. These studies have identified key molecular changes during development and disease and highlighted the compositional and organizational dynamics of glomerular ECM. Nonetheless, the extracellular environment within the glomerulus is the setting for a complex series of interactions between both structural ECM proteins and ECM-associated proteins, such as growth factors^16–18 and proteases,¹⁹ which together provide a specialized niche to support glomerular cell function. Therefore, to interrogate this complexity effectively, a systems-level understanding of glomerular ECM is required. To address the need for a global analysis of the extracellular environment within the glomerulus, we used mass spectrometry (MS)-based proteomics of glomerular ECM fractions to define the human glomerular ECM proteome.     

Human milk fortifier: An occult cause of bowel obstruction in extremely premature neonates

Background

Human milk fortifier (HMF) is used in neonatal units throughout North America to facilitate growth of preterm infants. Little data is available on the gastrointestinal side effects and potential adverse events. The purpose of this paper was to present a series of infants presenting with bowel obstruction associated with HMF.

Methods

Cases of HMF obstruction were collected between January 2010 and December 2012. Charts were reviewed and relevant data was collected.

Results

During the study period, 7 premature infants presented with bowel obstruction secondary to intestinal concretions of HMF. All babies were premature with gestational ages from 25 to 27 weeks. Birth weight was less than 1000 grams in all patients. Patients presented with feeding intolerance, bilious aspirates, abdominal distension, and obstipation. Four of the patients presented with acute deterioration and required urgent surgical intervention.

Conclusions

HMF is an important source of nutritional support in infants, which is felt to be safe. We present a series of infants where its use has resulted in significant complications. HMF should be used with caution in infants, especially those with a history of necrotizing enterocolitis. Further research should examine the calcium, protein, and fatty acid concentration tolerable in the gastrointestinal tract of infants.