Inhibition of Lysine 63 Ubiquitination Prevents the Progression of Renal Fibrosis in Diabetic DBA/2J Mice.

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.

Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

The purpose of our study was to evaluate how hyperglycemia (HG) influences Lys63 protein ubiquitination and its involvement in tubular damage and fibrosis in diabetic nephropathy (DN). Gene and protein expression of UBE2v1, a ubiquitin-conjugating E2-enzyme variant that mediates Lys63-linked ubiquitination, and Lys63-ubiquitinated proteins increased in HK2 tubular cells under HG. Matrix-assisted laser desorption/ionization-time of flight/tandem mass spectrometry identified 30 Lys63-ubiquitinated proteins, mainly involved in cellular organization, such as ß-actin, whose Lys63 ubiquitination increased under HG, leading to cytoskeleton disorganization. This effect was reversed by the inhibitor of the Ubc13/UBE2v1 complex NSC697923. Western blot analysis confirmed that UBE2v1 silencing in HK2 under HG, restored Lys63-ß-actin ubiquitination levels to the basal condition. Immunohistochemistry on patients with type 2 diabetic (T2D) revealed an increase in UBE2v1- and Lys63-ubiquitinated proteins, particularly in kidneys of patients with DN compared with control kidneys and other nondiabetic renal diseases, such as membranous nephropathy. Increased Lys63 ubiquitination both in vivo in patients with DN and in vitro, correlated with α-SMA expression, whereas UBE2v1 silencing reduced HG-induced α-SMA protein levels, returning them to basal expression. In conclusion, UBE2v1- and Lys63-ubiquitinated proteins increase in vitro under HG, as well as in vivo in T2D, is augmented in patients with DN, and may affect cytoskeleton organization and influence epithelial-to-mesenchymal transition. This process may drive the progression of tubular damage and interstitial fibrosis in patients with DN.-Pontrelli, P., Conserva, F., Papale, M., Oranger, A., Barozzino, M., Vocino, G., Rochetti, M. T., Gigante, M., Castellano, G., Rossini, M., Simone, S., Laviola, L., Giorgino, F., Grandaliano, G., Di Paolo, S., Gesualdo, L. Lysine 63 ubiquitination is involved in the progression of tubular damage in diabetic nephropathy.

Urinary miRNA-27b-3p and miRNA-1228-3p correlate with the progression of Kidney Fibrosis in Diabetic Nephropathy.

Diabetic Nephropathy (DN) is a chronic complication of diabetes and the primary cause of end stage renal disease. Differential diagnosis for DN requires invasive histological investigation, thus there is need for non-invasive biomarkers to discriminate among different histological lesions in diabetic patients. With the aim to identify a pattern of differentially expressed miRNAs in kidney biopsies of DN patients, we assayed miRNA expression in kidney biopsies from DN patients, diabetic patients with membranous nephropathy and patients with normal histology. Nine miRNAs were differentially expressed among the three groups, and 2 miRNAs (miR-27b-3p and miR-1228-3p) showed interaction with an ubiquitin-conjugating E2 enzyme variant (UBE2v1). UBE2v1 mediates the formation of lysine 63-linked ubiquitin chains, a mechanism we previously showed as involved in DN kidney fibrosis. Both miRNAs were validated as down-regulated in biopsies and urines of DN patients, possibly affected by DNA methylation. Interestingly, the urinary levels of both miRNAs could also discriminate among different degrees of renal fibrosis. Finally, we showed that the combined urinary expression of both miRNAs was also able to discriminate DN patients from other glomerulonephritides in diabetic patients. In conclusion we identified two miRNAs potentially useful as candidate biomarkers of tubular-interstitial fibrosis in diabetic patients with DN.

The pathological role of the ubiquitination pathway in diabetic nephropathy.

Diabetic nephropathy (DN) is a chronic complication of type 2 diabetes and is the most frequent form of chronic kidney disease that can lead to end-stage renal disease. Different pathways, involved in oxidative stress, inflammation, fibrosis and cell death, are responsible for the pathogenesis of DN and regulate the progression of the disease. Ubiquitination is a fundamental pathway in intracellular signaling whose role is emerging in the regulation of molecular processes responsible for several human diseases. Among the conventional ubiquitination pathway, leading to proteasomal degradation of proteins, also non-conventional ubiquitination plays an important role in the regulation of intracellular signaling. Several proteasome inhibitors have been developed and tested both in humans and in animal models and show potential as promising therapeutic approaches. In this review, we focused our attention on the role of ubiquitination pathway in the principal processes involved in the pathogenesis and progression of DN.

Deregulation of autophagy under hyperglycemic conditions is dependent on increased lysine 63 ubiquitination: a candidate mechanism in the progression of diabetic nephropathy.

Diabetic nephropathy patients (DN) are characterized by increased lysine63 ubiquitination (Lys63-Ub) at the tubular level. Autophagy is deregulated under diabetic conditions, even though the molecular mechanisms and the consequences of this alteration need to be elucidated. The aim of this study was to investigate the link between Lys63-Ub and autophagy in DN and the involvement of these two processes in tubular cell fate. Immunohistochemistry of beclin-1, LC3, and p62 on kidney biopsies highlighted increased protein expression of all these autophagic factors at the tubular level in DN compared to other nephritis. Transmission electron microscopy confirmed the presence of diffuse vacuolization and autophago(lyso)somal structures in proximal tubular cells in DN. Accumulation of Lys63-Ub proteins in DN increased in accordance with the tubular damage and was associated to increased LC3 expression both in vivo and in vitro. Hyperglycemia (HG) induced LC3 and p62 protein expression in HK2 cells together with Lys63-ubiquitinated proteins, and the inhibition of HG-induced Lys63-Ub by NSC697923 inhibitor, significantly reduced both LC3 and p62 expression. Moreover, in DN, those tubules expressing LC3 showed increased caspase-3 expression, supporting the hypothesis that deregulated autophagy induces apoptosis of tubular cells. In vitro, we confirmed a tight association between impaired autophagy, Lys63-Ub, and apoptosis since Lys63-Ub inhibition by NSC697923 abrogated HG-induced cell death and LC3 silencing also blocked hyperglycemia-induced caspase-3 activation. Our data suggested that prolonged hyperglycemia in diabetic patients can impair autophagy as a consequence of Lys63-Ub protein accumulation, thus promoting intracellular autophagic vesicles increase, finally leading to tubular cell death in DN. KEY MESSAGES: In vivo autophagy is deregulated in diabetic patients with renal disease (DN). Accumulation of Lys63 ubiquitinated proteins is associated to autophagy deregulation. Accumulation of Lys63 ubiquitinated proteins correlated with apoptosis activation. Lys63 ubiquitination inhibition abrogated hyperglycemia-induced autophagy and apoptosis.