Jurong Yang

ATP-P2X4 signaling mediates NLRP3 inflammasome activation: A novel pathway of diabetic nephropathy

Tubulointerstitial inflammation plays a key role in the development of diabetic nephropathy (DN). Cytokines in the IL-1 family are the key pro-inflammatory cytokines of tubulointerstitial inflammation. Extracellular ATP can cause P2X receptors to activate the NOD-like receptor 3 (NLRP3) inflammasome and cause IL-1β and IL-18 maturation and release. We investigated the role of ATP-P2X4 signaling in NLRP3 inflammasome activation and renal interstitial inflammation characteristic of DN. Ex vivo studies, P2X4 showed increased expression in renal tubule epithelial cells in patients with nephropathy due to type 2 diabetes compared to those in the control group. Linear correlation analysis shows that P2X4 expression was positively related with urine IL-1β and IL-18 levels. Moreover, P2X4 expression was co-localized with NLRP3, IL-1β, and IL-18 expression. In vitro culture experiments showed NLRP3 protein expression, cleavage of caspase-1 and IL-1β, and release of IL-1β, IL-18 and ATP in HK-2 cells significantly increased after high glucose stimulation. However, apyrase, which consumes extracellular ATP, completely blocked the changes caused by high glucose. The P2 receptor antagonist suramin, P2X receptor antagonist TNP-ATP, P2X4 selective antagonist 5-BDBD, and P2X4 gene silencing attenuated NLRP3 expression, cleavage of caspase-1 and IL-1β, and release of IL-1β and IL-18 induced by high glucose. Taken together, these results suggest that ATP-P2X4 signaling mediates high glucose-induced activation of the NLRP3 inflammasome, regulates IL-1 family cytokine secretion, and causes the development of tubulointerstitial inflammation in DN.

Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling.

Premature senescence is a key process in the progression of diabetic nephropathy (DN). In our study, we hypothesized that receptors for advanced glycation end-products (RAGE) mediate endoplasmic reticulum (ER) stress to induce premature senescence via p21 signaling activation in diabetic nephropathy. Here, we demonstrated that elevated expression of RAGE, ER stress marker glucose-regulated protein 78 (GRP78), and cell-cycle regulator p21 was all positively correlated with enhanced senescence-associated-β-galactosidase (SA-β-gal) activity in DN patients. In addition, the fraction of SA-β-gal or cells in the G0G1 phase were enhanced in cultured mouse proximal tubular epithelial cells (PTECs) and the expression of RAGE, GRP78 and p21 was up-regulated by advanced glycation end-products (AGEs) in a dose- and time-dependent manner. Interestingly, ER stress inducers or RAGE overexpression mimicked AGEs induced-premature senescence, and this was significantly suppressed by p21 gene silencing. However, RAGE blocking successfully attenuated AGEs-induced ER stress and p21 expression, as well as premature senescence. Moreover, ER stress inducers directly caused p21 activation, premature senescence, and also enhanced RAGE expression by positive feedback. These observations suggest that RAGE promotes premature senescence of PTECs by activation of ER stress-dependent p21 signaling.

Ischemia-reperfusion induces renal tubule pyroptosis via the CHOP-caspase-11 pathway

The apoptotic or necrotic death of renal tubule epithelial cells is the main pathogenesis of renal ischemia-reperfusion-induced acute kidney injury (AKI). Pyroptosis is a programmed cell death pathway that depends on the activation of the caspase cascade and IL-1 cytokine family members. However, the role of pyroptosis in AKI induced by ischemia-reperfusion remains unclear. In this study, we found that the levels of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1β, were significantly increased after 6 h of renal ischemia-reperfusion injury (IRI) and peaked at 12 h after IRI. Enhanced pyroptosis was accompanied by elevated renal structural and functional injury. Similarly, hypoxia-reoxygenation injury (HRI) also induced pyroptosis in renal tubule epithelial NRK-52E cells, which was characterized by increased pore formation and elevated lactate dehydrogenase release. In addition, obvious upregulation of the endoplasmic reticulum (ER) stress biomarkers glucose-regulated protein 78 and C/EBP homologous protein (CHOP) preceded the incidence of pyroptosis in cells treated with IRI or HRI. Pretreatment with a low dose of tunicamycin, an inducer of ER stress, relieved IRI-induced pyroptosis and renal tissue injury. Silencing of CHOP by small interfering RNA significantly decreased HRI-induced pyroptosis of NRK-52E cells, as evidenced by reduced caspase-11 activity and IL-1β generation. Therefore, we conclude that pyroptosis of renal tubule epithelial cells is a key event during IRI and that CHOP-caspase-11 triggered by overactivated ER stress may be an essential pathway involved in pyroptosis.

Impact of ER stress-regulated ATF4/p16 signaling on the premature senescence of renal tubular epithelial cells in diabetic nephropathy

Premature senescence is an important event during diabetic nephropathy (DN) progression. Here, we investigated the role of endoplasmic reticulum (ER) stress-regulated activation of transcription factor 4 (ATF4)/p16 signaling in the premature senescence of renal tubular epithelial cells (RTECs) during DN development. In the renal tissues of Type 2 DN patients, we detected an increased number of senescent cells; elevated deposition of advanced glycation end products (AGEs); upregulated expression of ER stress marker, glucose-regulated protein 78; as well as overexpression of ATF4 and p16. Similarly, these phenomena were also observed in cultured mouse RTECs following AGE treatment. Interestingly, AGE-induced p16 expression and premature senescence were successfully attenuated by ER stress inhibitor and ATF4 gene silencing. Moreover, AGE-induced premature senescence was mimicked by ER stress inducers and ATF4 overexpression, while suppressed by p16 gene silencing. In addition, ER stress inducers can augment ATF4 expression. Therefore, our results demonstrate that the ER stress-regulated ATF4/p16 pathway is involved in the premature senescence of RTECs during DN progression.

Anticubilin Antisense RNA Ameliorates Adriamycin-Induced Tubulointerstitial Injury in Experimental Rats

This study was designed to determine the effects of in vivo anticubilin antisense RNA on the uptake of albumin in tubules and on the tubulointerstitial injury in adriamycin-induced proteinuric rats. Adriamycin-treated rats were subjected to intrarenal delivery of adenoviral vectors encoding empty plasmid, cubilin sense RNA expression vector pAd-CUB or anticubilin antisense RNA expression vector pAd-ACUB on day 3. On days 14 and 28, half of the rats in each group were randomly selected to be killed, and blood samples, kidney tissues and 24-hour urine were collected. The diseased rats treated with pAdEasy-ACUB showed a 60% decrease in serum creatinine and glomerular filtration rate. Interestingly, the anticubilin antisense treatment led to a marked increase in albuminuria. Antisense treatment attenuated the histologic changes on both day 14 and day 28. The antisense treatment induced more than 60% recovery of adriamycin-induced injury, accompanied with 85% knockdown in the expression of cubilin protein and markedly decreased albumin deposition. Adriamycin induced an increase in the expression of monocyte chemoattractant protein-1, transforming growth factor-&bgr; and regulated on activation in normal T-cell expressed and secreted and the number of infiltrating cells, which was reversed by the antisense treatment. Anticubilin antisense RNA delivered by an adenoviral vector ameliorates albuminuria-induced glomerulosclerosis and tubulointerstitial damage in adriamycin nephrotic rats, indicating that cubilin could be a potential therapeutic target in proteinuric nephropathy.