Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

The need for osteocyte cultures is well known to the community of bone researchers; isolation of primary osteocytes is difficult and produces low cell ...

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Filtrin (NEPH3/KIRREL2) is a recently characterized member of the nephrin-like proteins of the immunoglobulin superfamily, and it has been suggested to participate in the maintenance of the glomerular filtration barrier in the kidney. In this study, the gene and protein expression of filtrin were examined in patients with acquired proteinuric diseases.

Expression of filtrin in human glomerular diseases.

Recently we reported a possible role for secretory IgA (SIgA) in IgA nephropathy (IgAN), as suggested by increased serum levels in patients with active disease and accumulation of SIgA in a glomerular eluate. Therefore, we attempted to find support for these findings by analysis of the presence of SIgA in biopsies of IgAN patients.

Demonstration of secretory IgA in kidneys of patients with IgA nephropathy.

Although metabolic derangement plays a central role in diabetic nephropathy, a better understanding of secondary mediators of injury may lead to new therapeutic strategies. Expression of macrophage migration inhibitory factor (MIF) is increased in experimental diabetic nephropathy, and increased tubulointerstitial mRNA expression of its receptor, CD74, has been observed in human diabetic nephropathy. Whether CD74 transduces MIF signals in podocytes, however, is unknown. Here, we found glomerular and tubulointerstitial CD74 mRNA expression to be increased in Pima Indians with type 2 diabetes and diabetic nephropathy. Immunohistochemistry confirmed the increased glomerular and tubular expression of CD74 in clinical and experimental diabetic nephropathy and localized glomerular CD74 to podocytes. In cultured human podocytes, CD74 was expressed at the cell surface, was upregulated by high concentrations of glucose and TNF-alpha, and was activated by MIF, leading to phosphorylation of extracellular signal-regulated kinase 1/2 and p38. High glucose also induced CD74 expression in a human proximal tubule cell line (HK2). In addition, MIF induced the expression of the inflammatory mediators TRAIL and monocyte chemoattractant protein 1 in podocytes and HK2 cells in a p38-dependent manner. These data suggest that CD74 acts as a receptor for MIF in podocytes and may play a role in the pathogenesis of diabetic nephropathy.

The MIF receptor CD74 in diabetic podocyte injury.

Podocytes possess the complete machinery for glutamatergic signaling, raising the possibility that neuron-like signaling contributes to glomerular function. To test this, we studied mice and cells lacking Rab3A, a small GTPase that regulates glutamate exocytosis. In addition, we blocked the glutamate ionotropic N-methyl-d-aspartate receptor (NMDAR) with specific antagonists. In mice, the absence of Rab3A and blockade of NMDAR both associated with an increased urinary albumin/creatinine ratio. In humans, NMDAR blockade, obtained by addition of ketamine to general anesthesia, also had an albuminuric effect. In vitro, Rab3A-null podocytes displayed a dysregulated release of glutamate with higher rates of spontaneous exocytosis, explained by a reduction in Rab3A effectors resulting in freedom of vesicles from the actin cytoskeleton. In addition, NMDAR antagonism led to profound cytoskeletal remodeling and redistribution of nephrin in cultured podocytes; the addition of the agonist NMDA reversed these changes. In summary, these results suggest that glutamatergic signaling driven by podocytes contributes to the integrity of the glomerular filtration barrier and that derangements in this signaling may lead to proteinuric renal diseases.

Podocyte glutamatergic signaling contributes to the function of the glomerular filtration barrier.

Adducins are cytoskeletal actin-binding proteins (alpha, beta, gamma) that function as heterodimers and heterotetramers and are encoded by distinct genes. Experimental and clinical evidence implicates alpha- and beta-adducin variants in hypertension and renal dysfunction. Here, we have addressed the role of alpha- and beta-adducin on glomerular function and disease using beta-adducin null mice, congenic substrains for alpha- and beta-adducin from the Milan hypertensive (MHS) and Milan normotensive (MNS) rats and patients with IgA nephropathy. Targeted deletion of beta-adducin in mice reduced urinary protein excretion, preceded by an increase of podocyte protein expression (phospho-nephrin, synaptopodin, alpha-actinin, ZO-1, Fyn). The introgression of polymorphic MHS beta-adducin locus into MNS (Add2, 529R) rats was associated with an early reduction of podocyte protein expression (nephrin, synaptopodin, alpha-actinin, ZO-1, podocin, Fyn), followed by severe glomerular and interstitial lesions and increased urinary protein excretion. These alterations were markedly attenuated when the polymorphic MHS alpha-adducin locus was also present (Add1, 316Y). In patients with IgA nephropathy, the rate of decline of renal function over time was associated to polymorphic beta-adducin (ADD2, 1797T, rs4984) with a significant interaction with alpha-adducin (ADD1, 460W, rs4961). These findings suggest that adducin genetic variants participate in the development of glomerular lesions by modulating the expression of specific podocyte proteins.

alpha- and beta-Adducin polymorphisms affect podocyte proteins and proteinuria in rodents and decline of renal function in human IgA nephropathy.

Glomerular diseases account for the majority of cases with chronic renal failure. Several genes have been identified with key relevance for glomerular function. Quite a few of these genes show a specific or preferential mRNA expression in the renal glomerulus. To identify additional candidate genes involved in glomerular function in humans we generated a human renal glomerulus-enriched gene expression dataset (REGGED) by comparing gene expression profiles from human glomeruli and tubulointerstitium obtained from six transplant living donors using Affymetrix HG-U133A arrays. This analysis resulted in 677 genes with prominent overrepresentation in the glomerulus. Genes with 'a priori' known prominent glomerular expression served for validation and were all found in the novel dataset (e.g. CDKN1, DAG1, DDN, EHD3, MYH9, NES, NPHS1, NPHS2, PDPN, PLA2R1, PLCE1, PODXL, PTPRO, SYNPO, TCF21, TJP1, WT1). The mRNA expression of several novel glomerulus-enriched genes in REGGED was validated by qRT-PCR. Gene ontology and pathway analysis identified biological processes previously not reported to be of relevance in glomeruli of healthy human adult kidneys including among others axon guidance. This finding was further validated by assessing the expression of the axon guidance molecules neuritin (NRN1) and roundabout receptor ROBO1 and -2. In diabetic nephropathy, a prevalent glomerulopathy, differential regulation of glomerular ROBO2 mRNA was found.In summary, novel transcripts with predominant expression in the human glomerulus could be identified using a comparative strategy on microdissected nephrons. A systematic analysis of this glomerulus-specific gene expression dataset allows the detection of target molecules and biological processes involved in glomerular biology and renal disease.

Systematic analysis of a novel human renal glomerulus-enriched gene expression dataset.

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study, we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and β, IL-1R1, and vascular endothelial growth factor A, and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, by decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs, and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not anti-neutrophil cytoplasmic Ab-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1R antagonist, downregulation of vascular endothelial growth factor A, and glomerular and blood vessel decreased capillary density, compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.

The detrimental effects of IFN-α on vasculogenesis in lupus are mediated by repression of IL-1 pathways: potential role in atherogenesis and renal vascular rarefaction.

Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells.

Nephrin expression in adult rodent central nervous system and its interaction with glutamate receptors.

[Synaptic-like signals at the filtration barrier: the role of nephrin].

The relative contribution of genetic factors and dietary patterns to glomerular damage in healthy individuals and prediabetic conditions is currently unclear. All Rab3A knockout (KO) mice spontaneously develop macroalbuminuria, but only male mice exhibit a glucose-intolerant phenotype, thus making the model suitable to examine the impact of a diet on preexisting podocyte damage.

Proteinuria and glomerular damage in Rab3A knockout mice chronically fed a high-glucose diet.

Podocyte injury and resulting albuminuria are hallmarks of diabetic nephropathy, but targeted therapies to halt or prevent these complications are currently not available. Here, we show that the immune-related molecule B7-1/CD80 is a critical mediator of podocyte injury in type 2 diabetic nephropathy. We report the induction of podocyte B7-1 in kidney biopsy specimens from patients with type 2 diabetes. Genetic and epidemiologic studies revealed the association of two single nucleotide polymorphisms at the B7-1 gene with diabetic nephropathy. Furthermore, increased levels of the soluble isoform of the B7-1 ligand CD28 correlated with the progression to ESRD in individuals with type 2 diabetes. In vitro, high glucose conditions prompted the phosphatidylinositol 3 kinase-dependent upregulation of B7-1 in podocytes, and the ectopic expression of B7-1 in podocytes increased apoptosis and induced disruption of the cytoskeleton that were reversed by the B7-1 inhibitor CTLA4-Ig. Podocyte expression of B7-1 was also induced in vivo in two murine models of diabetic nephropathy, and treatment with CTLA4-Ig prevented increased urinary albumin excretion and improved kidney pathology in these animals. Taken together, these results identify B7-1 inhibition as a potential therapeutic strategy for the prevention or treatment of diabetic nephropathy.

Deborah Mattinzoli

Renal Research Laboratory

Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Deborah Mattinzoli

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Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico

Application of Retinoic Acid to Obtain Osteocytes Cultures from Primary Mouse Osteoblasts

Renal Research Laboratory