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Diabetic nephropathy (DN) is the main leading cause of chronic kidney disease worldwide. Although remarkable therapeutic advances have been made during the last few years, there still exists a high residual risk of disease progression to end-stage renal failure. To further understand the pathogenesis of tissue injury in this disease, by means of the Next-Generation Sequencing, we have studied the microRNA (miRNA) differential expression pattern in kidneys of Black and Tan Brachyury (BTBR) ob/ob (leptin deficiency mutation) mouse. This experimental model of type 2 diabetes and obesity recapitulates the key histopathological features described in advanced human DN and therefore can provide potential useful translational information. The miRNA-seq analysis, performed in the renal cortex of 22-week-old BTBR ob/ob mice, pointed out a set of 99 miRNAs significantly increased compared to non-diabetic, non-obese control mice of the same age, whereas no miRNAs were significantly decreased. Among them, miR-802, miR-34a, miR-132, miR-101a, and mir-379 were the most upregulated ones in diabetic kidneys. The in silico prediction of potential targets for the 99 miRNAs highlighted inflammatory and immune processes, as the most relevant pathways, emphasizing the importance of inflammation in the pathogenesis of kidney damage associated to diabetes. Other identified top canonical pathways were adipogenesis (related with ectopic fatty accumulation), necroptosis (an inflammatory and regulated form of cell death), and epithelial-to-mesenchymal transition, the latter supporting the importance of tubular cell phenotype changes in the pathogenesis of DN. These findings could facilitate a better understanding of this complex disease and potentially open new avenues for the design of novel therapeutic approaches to DN.

Cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), is considered a fibrotic biomarker and has been suggested as a potential therapeutic target for kidney pathologies. CCN2 is a matricellular protein with four distinct structural modules that can exert a dual function as a matricellular protein and as a growth factor. Previous experiments using surface plasmon resonance and cultured renal cells have demonstrated that the C-terminal module of CCN2 (CCN2(IV)) interacts with the epidermal growth factor receptor (EGFR). Moreover, CCN2(IV) activates proinflammatory and profibrotic responses in the mouse kidney. The aim of this paper was to locate the in vivo cellular CCN2/EGFR binding sites in the kidney. To this aim, the C-terminal module CCN2(IV) was labeled with a fluorophore (Cy5), and two different administration routes were employed. Both intraperitoneal and direct intra-renal injection of Cy5-CCN2(IV) in mice demonstrated that CCN2(IV) preferentially binds to the tubular epithelial cells, while no signal was detected in glomeruli. Moreover, co-localization of Cy5-CCN2(IV) binding and activated EGFR was found in tubules. In cultured tubular epithelial cells, live-cell confocal microscopy experiments showed that EGFR gene silencing blocked Cy5-CCN2(IV) binding to tubuloepithelial cells. These data clearly show the existence of CCN2/EGFR binding sites in the kidney, mainly in tubular epithelial cells. In conclusion, these studies show that circulating CCN2(IV) can directly bind and activate tubular cells, supporting the role of CCN2 as a growth factor involved in kidney damage progression.

Chronic kidney disease (CKD) can be considered as a clinical model for premature aging. However, non-invasive biomarkers to detect early kidney damage and the onset of a senescent phenotype are lacking. Most of the preclinical senescence studies in aging have been done in very old mice. Furthermore, the precise characterization and over-time development of age-related senescence in the kidney remain unclear. To address these limitations, the age-related activation of cellular senescence-associated mechanisms and their correlation with early structural changes in the kidney were investigated in 3- to 18-month-old C57BL6 mice. Inflammatory cell infiltration was observed by 12 months, whereas tubular damage and collagen accumulation occurred later. Early activation of cellular-senescence-associated mechanisms was found in 12-month-old mice, characterized by activation of the DNA-damage-response (DDR), mainly in tubular cells; activation of the antioxidant NRF2 pathway; and klotho downregulation. However, induction of tubular-cell-cycle-arrest (CCA) and overexpression of renal senescent-associated secretory phenotype (SASP) components was only found in 18-month-old mice. In aging mice, both inflammation and oxidative stress (marked by elevated lipid peroxidation and NRF2 inactivation) remained increased. These findings support the hypothesis that prolonged DDR and CCA, loss of nephroprotective factors (klotho), and dysfunctional redox regulatory mechanisms (NRF2/antioxidant defense) can be early drivers of age-related kidney-damage progression.

Crescentic glomerulonephritis is a devastating autoimmune disease that without early and properly treatment may rapidly progress to end-stage renal disease and death. Current immunosuppressive treatment provides limited efficacy and an important burden of adverse events. Epigenetic drugs are a source of novel therapeutic tools. Among them, bromodomain and extraterminal domain (BET) inhibitors (iBETs) block the interaction between bromodomains and acetylated proteins, including histones and transcription factors. iBETs have demonstrated protective effects on malignancy, inflammatory disorders and experimental kidney disease. Recently, Gremlin-1 was proposed as a urinary biomarker of disease progression in human anti-neutrophil cytoplasmic antibody (ANCA)-associated crescentic glomerulonephritis. We have now evaluated whether iBETs could regulate Gremlin-1 in experimental anti-glomerular basement membrane nephritis induced by nephrotoxic serum (NTS) in mice, a model resembling human crescentic glomerulonephritis. In NTS-injected mice, the iBET JQ1 inhibited renal Gremlin-1 overexpression and diminished glomerular damage, restoring podocyte numbers. Chromatin immunoprecipitation assay demonstrated BRD4 enrichment of the Grem-1 gene promoter in injured kidneys, consistent with Gremlin-1 epigenetic regulation. Moreover, JQ1 blocked BRD4 binding and inhibited Grem-1 gene transcription. The beneficial effect of iBETs was also mediated by modulation of NOTCH pathway. JQ1 inhibited the gene expression of the NOTCH effectors Hes-1 and Hey-1 in NTS-injured kidneys. Our results further support the role for epigenetic drugs, such as iBETs, in the treatment of rapidly progressive crescentic glomerulonephritis.