Kailong Li

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.

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.

Albumin Overload Induces Apoptosis in Renal Tubular Epithelial Cells through a CHOP-Dependent Pathway

The proteinuria-induced apoptosis of proximal tubular cells (PTCs) plays a crucial role in renal tubulointerstitial injury in chronic kidney disease. Recent studies have shown that endoplasmic reticulum (ER) stress is involved in proteinuria-induced apoptosis of PTCs. Our study showed that albumin overload led to ER stress, CCAAT/enhancer-binding protein-homologous protein (CHOP), and PKR-like kinase (PERK) activation and to apoptosis of PTCs in proteinuria patients. The apoptotic index of proximal renal tubular cells in the nephrotic kidneys was about 13-fold higher than that in control kidneys. The increased tubular expression of GRP78, ORP150, and CHOP and nuclear localization of CHOP in nephrotic kidneys were also detected. The expression of GRP78, CHOP, PERK, and phosphorylated PERK increased proportionately with HSA overload in a dose- and time-dependent manner. Knockdown of CHOP by siRNA significantly reduced the HSA-induced apoptosis of HKC. The expression of PERK did not significantly change, but the phosphorylation of PERK increased. Furthermore, knockdown of PERK significantly inhibited HSA-induced CHOP expression, suppressing apoptosis in HKCs by 2.48-fold compared to controls. Overexpression of CHOP enhanced the apoptosis of HKC induced by albumin, no significant difference was observed in the expression of PERK, whereas the phosphorylation of PERK decreased. Our data indicated that proteinuria induces ER stress in renal tubular cells, which may subsequently lead to tubular damage through a PERK-CHOP-dependent pathway. This ER stress-induced apoptosis pathway may contribute to renal tubulointerstitial injury by proteinuria in chronic kidney disease.

Mutual activation of CD4+ T cells and monocytes mediated by NKG2D-MIC interaction requires IFN-gamma production in systemic lupus erythematosus.

The activating receptor NKG2D is mainly expressed by human CD8(+) T cells and NK cells but normally absent on CD4(+) T cells. However, a subset of autoreactive NKG2D(+)CD4(+) T cells has been found to exist in some autoimmune disease such as rheumatoid arthritis (RA) and to participate in the imbalance of immune response and inflammation. Up to date this observation has been extended to some autoimmune diseases such as RA and Crohns disease and the mechanism underlying the presence of this type of NKG2D(+)CD4(+) T cells has not been delineated yet. In this study, we found that a substantial proportion of CD4(+) T cells expressed NKG2D in the PBMC of SLE patients. We also found that monocytes in SLE aberrantly expressed the NKG2D ligand of MHC class I chain-related (MIC) molecules and membrane-bound IL-15 (mIL-15) at the cell surface. When cultured with the sera from SLE patients, the monocytes from healthy volunteers could be induced to express MIC and mIL-15. However, this induced expression of MIC and mIL-15 could be blocked with anti-IFN-gamma receptor (anti-IFN-gammaR) antibody. We further demonstrated that NKG2D could be induced on normal CD4(+) T cells either cocultured with monocytes from patients with SLE, or monocytes from healthy volunteers but pretreated with IFN-gamma. Moreover, Th1 cytokines were found to be produced by NKG2D(+)CD4(+) T cells in the coculture system. By transwell assay, we found that both NKG2D expression and Th1 cytokines production depended on the cell-cell contact. These results indicate that the elevated sera IFN-gamma may be responsible for MIC and mIL-15 induction on monocytes in SLE; mIL-15 on monocytes contribute to NKG2D receptor induction on a subset of CD4(+) T cells. Moreover, CD14(+) monocytes promote NKG2D(+)CD4(+) T cells activation through the NKG2D-MIC engagement in the pathogenesis of SLE.

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.

Optineurin-mediated mitophagy protects renal tubular epithelial cells against accelerated senescence in diabetic nephropathy

Premature senescence is a key process in the progression of diabetic nephropathy (DN). Premature senescence of renal tubular epithelial cells (RTEC) in DN may result from the accumulation of damaged mitochondria. Mitophagy is the principal process that eliminates damaged mitochondria through PTEN-induced putative kinase 1 (PINK1)-mediated recruitment of optineurin (OPTN) to mitochondria. We aimed to examine the involvement of OPTN in mitophagy regulation of cellular senescence in RTEC in the context of DN. In vitro, the expression of senescence markers P16, P21, DcR2, SA-β-gal, SAHF, and insufficient mitophagic degradation marker (mitochondrial P62) in mouse RTECs increased after culture in 30 mM high-glucose (HG) conditions for 48 h. Mitochondrial fission/mitophagy inhibitor Mdivi-1 significantly enhanced RTEC senescence under HG conditions, whereas autophagy/mitophagy agonist Torin1 inhibited cell senescence. MitoTempo inhibited HG-induced mitochondrial reactive oxygen species and cell senescence with or without Mdivi-1. The expression of PINK1 and OPTN, two regulatory factors for mitophagosome formation, decreased significantly after HG stimulation. Overexpression of PINK1 did not enhance mitophagosome formation under HG conditions. OPTN silencing significantly inhibited HG-induced mitophagosome formation, and overexpression of OPTN relieved cellular senescence through promoting mitophagy. In clinical specimens, renal OPTN expression was gradually decreased with increased tubulointerstitial injury scores. OPTN-positive renal tubular cells did not express senescence marker P16. OPTN expression also negatively correlated with serum creatinine levels, and positively correlated with eGFR. Thus, OPTN-mediated mitophagy plays a crucial regulatory role in HG-induced RTEC senescence in DN. OPTN may, therefore, be a potential antisenescence factor in DN.

Urinary DcR2 is a novel biomarker for tubulointerstitial injury in patients with diabetic nephropathy

Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients who were diagnosed by renal biopsy. uDcR2 levels were measured by ELISA, and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2 and renal functional parameters were evaluated. Receiver operating characteristics (ROC) curve analyzed area under the curve (AUC) of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers; senescent markers p16, p21, and senescence-associated β-galactosidase (SA-β-gal); and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics and were associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was coexpressed with p16 and p21, and nearly more than one-half of tubular DcR2 was positive for SA-β-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs. Methods Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied. Results Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001). Conclusion p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.