Novel insights into NOD-like receptors in renal diseases.

Nucleotide-binding oligomerization domain-like receptors (NLRs), including NLRAs, NLRBs (also known as NAIPs), NLRCs, and NLRPs, are a major subfamily of pattern recognition receptors (PRRs). Owing to a recent surge in research, NLRs have gained considerable attention due to their involvement in mediating the innate immune response and perpetuating inflammatory pathways, which is a central phenomenon in the pathogenesis of multiple diseases, including renal diseases. NLRs are expressed in different renal tissues during pathological conditions, which suggest that these receptors play roles in acute kidney injury, obstructive nephropathy, diabetic nephropathy, IgA nephropathy, lupus nephritis, crystal nephropathy, uric acid nephropathy, and renal cell carcinoma, among others. This review summarises recent progress on the functions of NLRs and their mechanisms in the pathophysiological processes of different types of renal diseases to help us better understand the role of NLRs in the kidney and provide a theoretical basis for NLR-targeted therapy for renal diseases.

Conditional knockout of TGF-ßRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.

Rationale: TGF-ß/Smad signaling is the central mediator for renal fibrosis, however, its functional role in acute kidney injury (AKI) is not fully understood. We previously showed Smad2 protects against renal fibrosis by limiting Smad3 signaling, but details on its role in acute phase are unclear. Recent evidence showed that TGF-ß/Smad3 may be involved in the pathogenesis of AKI, so we hypothesized that Smad2 may play certain roles in AKI due to its potential effect on programmed cell death. Methods: We established a cisplatin-induced AKI mouse model with TGF-ß type II receptor or Smad2 specifically deleted from renal tubular epithelial cells (TECs). We also created stable in vitro models with either Smad2 knockdown or overexpression in human HK2 cells. Importantly, we evaluated whether Smad2 could serve as a therapeutic target in both cisplatin- and ischemic/reperfusion (I/R)-induced AKI mouse models by silencing Smad2 in vivo. Results: Results show that disruption of TGF-ß type II receptor suppressed Smad2/3 activation and attenuated renal injury in cisplatin nephropathy. Furthermore, we found that conditional knockout of downstream Smad2 in TECs protected against loss of renal function, and alleviated p53-mediated cell apoptosis, RIPK-mediated necroptosis and p65 NF-κB-driven renal inflammation in cisplatin nephropathy. This was further confirmed in cisplatin-treated Smad2 knockdown and overexpression HK2 cells. Additionally, lentivirus-mediated Smad2 knockdown protected against renal injury and inflammation while restoring renal function in established nephrotoxic and ischemic AKI models. Conclusions: These findings show that unlike its protective role in renal fibrosis, Smad2 promoted AKI by inducing programmed cell death and inflammation. This may offer a novel therapeutic target for acute kidney injury.

The role of Ca2+ in acid-sensing ion channel 1a-mediated chondrocyte pyroptosis in rat adjuvant arthritis.

Rheumatoid arthritis is an autoimmune disease with a poor prognosis. Pyroptosis is a type of proinflammatory programmed cell death that is characterised by the activation of caspase-1 and secretion of the proinflammatory cytokines interleukin (IL)-1ß/18. Previous reports have shown that pyroptosis is closely related to the development of some autoimmune diseases, such as rheumatoid arthritis. The decrease in the pH of joint fluid is a main pathogenic feature of RA and leads to excessive apoptosis in chondrocytes. Acid-sensitive ion channels (ASICs) are extracellular H+-activated cation channels that mainly influence Na+ and Ca2+ permeability. In this study, we investigated the role of Ca2+ in acid-sensing ion channel 1a-mediated chondrocyte pyroptosis in an adjuvant arthritis rat model. The expression of apoptosis-associated speck-like protein, NLRP3, caspase-1, ASIC 1a, IL-1ß and IL-18 was upregulated in the joints of rats compared with that in normal rats, but the expression of Col2a in cartilage was decreased. However, these changes were reversed by amiloride, which is an inhibitor of ASIC1a. Extracellular acidosis significantly increased the expression of ASIC1a, IL-1ß, IL-18, ASC, NLRP3 and caspase-1 and promoted the release of lactate dehydrogenase. Interestingly, Psalmotoxin-1 (Pctx-1) and BAPTA-AM inhibited these effects. These results indicate that ASIC1a mediates pyroptosis in chondrocytes from AA rats. The underlying mechanism may be associated with the ability of ASIC1a to promote [Ca2+]i and upregulate the expression of the NLRP3 inflammasome.

Noncoding RNAs in acute kidney injury.

Acute kidney injury (AKI), caused by various stimuli including ischemia reperfusion, nephrotoxic insult, and sepsis, is characterized by abrupt decline of kidney function. Till now, the molecular mechanisms for AKI have not been fully explored and the effective therapies are still lacking. Noncoding RNAs (ncRNAs), a group of biomolecules function at RNA level, are involved in a wide range of physiopathological processes including AKI. MicroRNAs (miRNAs) are the most extensively studied ncRNAs in AKI. Evidence indicated that miRNAs are altered significantly in various types of AKI. Gain-and-loss-of-function studies demonstrated that miRNAs, such as miR-24, miR-126, miR-494, and miR-687, may bind to the 3'-untranslated region of their target genes to regulate inflammation, programmed cell death, and cell cycle in the injury and repair stages of AKI, indicating their therapeutic potential in AKI. In contrast, functions of long noncoding RNAs and circular RNAs in AKI are hot topics but still largely unknown. Additionally, ncRNAs packaged in exosome can be detected in circulation and urine, they may serve as specific biomarkers for AKI. This review summarized the alteration and functional role of ncRNAs and their therapeutic potential in AKI.

Restoration of E-cadherin by PPBICA protects against cisplatin-induced acute kidney injury by attenuating inflammation and programmed cell death.

E-cadherin is a major component of tubular adherent proteins that maintain intercellular contacts and cell polarity in epithelial tissue. It is involved in pathological processes of renal cell carcinoma and fibrotic diseases via epithelial-mesenchymal transition. Although studies have shown E-cadherin is significantly downregulated in acute kidney injury (AKI), its function in AKI is unknown. Here, we evaluated cell damage and inflammation in cisplatin-stimulated tubular epithelial cell lines after disrupting E-cadherin and restoring it with PPBICA, a small molecule identified by high-throughput screening. We also determined the therapeutic potential of restoring E-cadherin in vivo. Results show cisplatin reduced E-cadherin expression both in mouse kidney and proximal tubular epithelial cell lines (mTECs). PPBICA restored E-cadherin levels, which increased cell viability while attenuating programmed cell death. This may be mediated via deactivation of the RIPK1/RIPK3 axis and decreased caspase3 cleavage. In addition, PPBICA suppressed inflammatory response in cisplatin-treated mTECs, which correlated with suppressed NF-κB phosphorylation and promoter activity. In contrast, disruption of E-cadherin promoted cell damage and inflammation. PPBICA failed to further attenuate kidney damage in E-cadherin knockdown cells, indicating that PPBICA protects against mTECs through E-cadherin restoration. We also found that peritoneal injection of PPBICA in mice prevented loss of renal function and tubular damage by suppressing NF-κB-driven renal inflammation and RIPK-regulated programmed cell death. This was driven by restoration of E-cadherin in cisplatin nephropathy. Additionally, PPBICA attenuated cisplatin-induced kidney damage in an established AKI model, indicating its therapeutic potential in the treatment of AKI. In conclusion, E-cadherin plays functional roles in tubule integrity, programmed cell death, and renal inflammation. Our results underscore the potential of E-cadherin restoration as a novel therapeutic strategy for AKI.

Wogonin protects against cisplatin-induced acute kidney injury by targeting RIPK1-mediated necroptosis.

Acute kidney injury (AKI), characterized by aggressive inflammatory responses and destruction of renal resident cells, can cause abrupt kidney dysfunction. To date, effective therapy for AKI is lacking. In this study, we evaluated the renoprotective effect of wogonin, an herbal active compound, using a cisplatin-induced AKI mouse model. In vivo results show that wogonin substantially suppressed the increased levels of serum creatinine and blood urea nitrogen (BUN) almost to the normal level. Wogonin also attenuated tubular damage, shown by PAS staining, electron microscopy and molecular analysis of KIM-1. In addition, wogonin suppressed kidney inflammation as indicated by a >60% decrease in macrophage infiltration, a >50% reduction in inflammatory cytokine production and inhibited NF-κB activation in the injured kidney. Mechanistically, molecular docking results show that wogonin effectively inhibited RIPK1 by occupying the ATP-binding pocket of the enzyme, which is a key regulator of necroptosis. Moreover, inhibition of RIPK1, or RIPK3, reversed the protective effects of wogonin in cisplatin-treated HK2 cells, indicating wogonin works in a RIPK1/RIPK3-dependent manner. Surprisingly, wogonin enhanced the anti-proliferative effect of cisplatin on human hepatoma HepG2 cells. Thus, our findings suggest wogonin may be a renoprotective adjuvant for cisplatin-based anticancer therapy.

NADPH oxidase 4 promotes cisplatin-induced acute kidney injury via ROS-mediated programmed cell death and inflammation.

The goal of this study was to elucidate the functional role of Nox4 during acute kidney injury (AKI). NADPH oxidases are a major source of reactive oxygen species (ROS) in the kidney in normal and pathological conditions. Among NADPH oxidase isoforms, NADPH oxidase4 (Nox4) is highly expressed in the kidney and has an important role in kidney diseases, such as diabetic nephropathy and renal carcinoma. We previously found that Nox4 expression significantly increased in the toxic AKI model. However, its functional role and mechanism of action in AKI are still unknown. We scavenged ROS with apocynin in vitro and in vivo and found it attenuated cisplatin-triggered renal function decline. It also alleviated programmed cell death and renal inflammation, indicating a critical role for ROS in mediating AKI. Nox4 protein and mRNA levels were substantially upregulated by cisplatin in vivo and in vitro. Nox4 knockdown alleviated cisplatin-induced cell death and inflammatory response, while Nox4 overexpression aggravated them. Moreover, N-acetyl-L-cysteine (NAC)-mediated inhibition of ROS suppressed cell injury led by Nox4 overexpression, indicating Nox4-mediated ROS generation may be the key mediator in cisplatin-induced nephrotoxicity. Mechanistically, excessive expression of Nox4 induced programmed cell death, especially RIP-mediated necroptosis. Finally, we tested whether Nox4 is a potential therapeutic target using an AKI mouse model by injecting a lentivirus-packaged Nox4 shRNA plasmid through tail vein. Disruption of Nox4 led to renal function recovery, kidney damage relief and reduced inflammation. We conclude that Nox4 aggravates cisplatin-induced nephrotoxicity by promoting ROS-mediated programmed cell death and inflammation. Thus Nox4 may serve as a potential therapeutic target in the treatment of AKI.

Protocatechuic Aldehyde Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Nox-Mediated Oxidative Stress and Renal Inflammation.

Cisplatin is a classic chemotherapeutic agent widely used to treat different types of cancers including ovarian, head and neck, testicular and uterine cervical carcinomas. However, cisplatin induces acute kidney injury by directly triggering an excessive inflammatory response, oxidative stress, and programmed cell death of renal tubular epithelial cells, all of which lead to high mortality rates in patients. In this study, we examined the protective effect of protocatechuic aldehyde (PA) in vitro in cisplatin-treated tubular epithelial cells and in vivo in cisplatin nephropathy. PA is a monomer of Traditional Chinese Medicine isolated from the root of S. miltiorrhiza (Lamiaceae). Results show that PA prevented cisplatin-induced decline of renal function and histological damage, which was confirmed by attenuation of KIM1 in both mRNA and protein levels. Moreover, PA reduced renal inflammation by suppressing oxidative stress and programmed cell death in response to cisplatin, which was further evidenced by in vitro data. Of note, PA suppressed NAPDH oxidases, including Nox2 and Nox4, in a dosage-dependent manner. Moreover, silencing Nox4, but not Nox2, removed the inhibitory effect of PA on cisplatin-induced renal injury, indicating that Nox4 may play a pivotal role in mediating the protective effect of PA in cisplatin-induced acute kidney injury. Collectively, our data indicate that PA blocks cisplatin-induced acute kidney injury by suppressing Nox-mediated oxidative stress and renal inflammation without compromising anti-tumor activity of cisplatin. These findings suggest that PA and its derivatives may serve as potential protective agents for cancer patients receiving cisplatin treatment.

Anti-fibrotic effect of wogonin in renal tubular epithelial cells via Smad3-dependent mechanisms.

Renal fibrosis, a common feature and leading cause for End Stage Renal Disease, still lacks effective therapy. In the current study, we detected and compared the anti-fibrotic effects of wogonin and wogonoside, two major components of Scutellaria baicalensis Georgi, in TGF-ß1-treated tubular epithelial cells of human and murine origins. Results consistently showed that compared with wogonoside, wogonin inhibits TGF-ß1-induced upregulated mRNA and protein levels of collagen I and α-SMA with more efficiency, which was further confirmed by the immunofluorescence results that wogonin decreased the percentage of collagen I and α-SMA positive cells in TGF-ß1-treated tubular epithelial cells. Mechanistically, wogonin mainly decreased Smad3 phosphorylation, but had marginal effect on non-canonical TGF-ß signaling pathways, such as p38 and ERK MAP Kinase. Furthermore, in the cells deficient for TGF-ß signaling or downstream Smad3, results demonstrated that even high concentration of wogonin failed to further decrease the level of collagen I and α-SMA, indicating the essential role of TGF-ß/Smad3 signaling inhibition in the therapeutic action of wogonin in TGF-ß1-stimulated tubular epithelial cells. Collectively, our results indicated that wogonin may be utilized as a potential anti-fibrotic Traditional Chinese Medicine monomer in the treatment of renal fibrosis.

Treatment of renal fibrosis by rebalancing TGF-ß/Smad signaling with the combination of asiatic acid and naringenin.

We recently showed that imbalance of TGF-ß/Smad signaling with over-activation of Smad3 but lower levels of Smad7 is a central mechanism of tissue fibrosis. In the present study, we report here that inhibition of Smad3 with naringenin (NG) and upregulation of Smad7 with asiatic acid (AA) produced an additive effect on inhibition of renal fibrosis in a mouse model of obstructive nephropathy. We found that AA, a triterpene from Centella Asiatica, functioned as a Smad7 agonist and suppressed TGF-ß/Smad3-mediated renal fibrosis by inducing Smad7. Whereas, NG, a flavonoid from grapefruits and citrus fruits, was a Smad3 inhibitor that inhibited renal fibrosis by blocking Smad3 phosphorylation and transcription. The combination of AA and NG produced an additive effect on inhibition of renal fibrosis by blocking Smad3 while upregulating Smad7. Thus, rebalancing the disorder of TGF-ß/Smad signaling by treatment with AA and NG may represent as a novel and effective therapy for chronic kidney disease associated with fibrosis.