AMP-activated protein kinase α2 contributes to acute and chronic hyperuricemic nephropathy via renal urate deposition in a mouse model.

Hyperuricemia can induce acute and chronic kidney damage, but the pathological mechanism remains unclear. The potential role of AMP-activated protein kinase (AMPK) α2 in hyperuricemia-induced renal injury was investigated in this study. Acute and chronic hyperuricemic nephropathy was induced by administering intraperitoneal injections of uric acid and oxonic acid to AMPK α2 knockout and wild-type mice. Changes in renal function, histopathology, inflammatory cell infiltration, renal interstitial fibrosis, and urate deposition were analyzed. In both acute and chronic hyperuricemic nephropathy mouse models, knockout of AMPK α2 significantly reduced serum creatinine levels and renal pathological changes. The tubular expression of kidney injury molecule-1 was also reduced in hyperuricemic nephropathy mice deficient in AMPK α2. In addition, knockout of AMPK α2 significantly suppressed the infiltration of renal macrophages and progression of renal interstitial fibrosis in mice with chronic hyperuricemic nephropathy. Knockout of AMPK α2 reduced renal urate crystal deposition, probably through increasing the expression of the uric acid transporter, multidrug resistance protein 4. In summary, AMPK α2 is involved in acute and chronic hyperuricemia-induced kidney injury and may be associated with increased urate crystal deposition in the kidney.

Metformin improves renal injury of MRL/lpr lupus-prone mice via the AMPK/STAT3 pathway.

OBJECTIVE: Lupus nephritis (LN) is a major complication and cause of death among patients with SLE. This research used in vivo and in vitro experiments to explore the therapeutic potential of metformin in kidney injury from LN-induced inflammation. METHODS: In vivo study, 8-week-old MRL/MpJ-Faslpr/J (MRL/lpr) mice were randomly divided into two groups (n=12 each): daily administration of 0.3 mg/mL metformin in drinking water and control (water only). Body weight and urinary samples were measured biweekly. Mice were sacrificed after 8-week treatment to harvest serum, lymph nodes, spleen and kidneys. In vitro study, human kidney-2 (HK-2) cells were pretreated with 1 mM metformin for 1 hour and then stimulated with 20 µg/mL lipopolysaccharides (LPS) or 10 ng/mL tumour necrosis factor-α (TNF-α) for another 48 hours. Protein was collected for subsequent analysis. RESULTS: We found that metformin administration improved renal function in MRL/lpr lupus-prone mice, measured by decreased urea nitrogen and urinary proteins. Metformin reduced immunoglobulin G and complement C3 deposition in glomeruli. The treatment also downregulated systemic and renal inflammation, as seen in decreased renal infiltration of F4/80-positive macrophages and reduced splenic and renal MCP-1 (monocyte chemoattractant protein-1) and TNF-α, and renal IL-1ß (interleukin 1ß) expression. Metformin administration decreased renal expression of necroptosis markers p-RIPK1 (phosphorylated receptor-interacting protein kinase 1) and p-MLKL, along with tubular injury marker KIM-1 (kidney injury molecule-1) in lupus mice. In addition, metformin alleviated the necroptosis of HK-2 cells stimulated by LPS and TNF-α, evidencing by a decrease in the expression of necroptosis markers p-RIPK1, p-RIPK3 and p-MLKL, and the inflammasome-related markers NLRP3 (NLR family pyrin domain containing 3), ASC (apoptosis-associated speck-like protein containing a CARD), caspase-1. Mechanistically, metformin treatment upregulated p-AMPK (phosphorylated AMP-activated protein kinase) and downregulated p-STAT3 (phosphorylated signal transducer and activator of transcription 3) expression in the kidneys. Moreover, AMPKα2 knockdown abolished the protective effects of metformin in vitro. CONCLUSIONS: Metformin alleviated kidney injury in LN though suppressing renal necroptosis and inflammation via the AMPK/STAT3 pathway.

Hydroxychloroquine administration exacerbates acute kidney injury complicated by lupus nephritis.

BACKGROUND: Hydroxychloroquine (HCQ) has been recommended as a basic treatment for lupus nephritis (LN) during this decade based on its ability to improve LN-related renal immune-mediated inflammatory lesions. As a classical lysosomal inhibitor, HCQ may inhibit lysosomal degradation and disrupt protective autophagy in proximal tubular epithelial cells (PTECs). Therefore, the final renal effects of HCQ on LN need to be clarified. METHOD: HCQ was administered on spontaneous female MRL/lpr LN mice with severe proteinuria daily for 4 weeks. Moreover, the MRL/lpr mice with proteinuric LN were subjected to cisplatin-induced or unilateral ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) after 2 weeks of HCQ preadministration. RESULTS: As expected, HCQ treatment increased the survival ratio and downregulated the levels of serum creatinine in the mice with LN, ameliorated renal lesions, and inhibited renal interstitial inflammation. Unexpectedly, HCQ preadministration significantly increased susceptibility to and delayed the recovery of AKI complicated by LN, as demonstrated by an increase in PTEC apoptosis and expression of the tubular injury marker KIM-1 as well as the retardation of PTEC replenishment. HCQ preadministration suppressed the proliferation of PTECs by arresting cells in G1/S phase and upregulated the expression of cell cycle inhibitors. Furthermore, HCQ preadministration disrupted the PTEC autophagy-lysosomal pathway and accelerated PTEC senescence. CONCLUSION: HCQ treatment may increase susceptibility and delay the recovery of AKI complicated by LN despite its ability to improve LN-related renal immune-mediated inflammatory lesions. The probable mechanism involves accelerated apoptosis and inhibited proliferation of PTECs via autophagy-lysosomal pathway disruption and senescence promotion.

Cyclosporine A blocks autophagic flux in tubular epithelial cells by impairing TFEB-mediated lysosomal function.

Cyclosporine A (CsA) is an immunosuppressor widely used for the prevention of acute rejection during solid organ transplantation. However, severe nephrotoxicity has substantially limited its long-term usage. Recently, an impaired autophagy pathway was suggested to be involved in the pathogenesis of chronic CsA nephrotoxicity. However, the underlying mechanisms of CsA-induced autophagy blockade in tubular cells remain unclear. In the present study, we observed that CsA suppressed the activation and expression of transcription factor EB (TFEB) by increasing the activation of mTOR, in turn promoting lysosomal dysfunction and autophagy flux blockade in tubular epithelial cells (TECs) in vivo and in vitro. Restoration of TFEB activation by Torin1-mediated mTOR inhibition significantly improved lysosomal function and rescued autophagy pathway activity, suppressing TEC injury. In summary, targeting TFEB-mediated autophagy flux represents a potential therapeutic strategy for CsA-induced nephrotoxicity.

Autophagy Inhibition Sensitizes Renal Tubular Epithelial Cell to G1 Arrest Induced by Transforming Growth Factor beta (TGF-ß).

BACKGROUND Cell cycle arrest and autophagy have been demonstrated to be involved in various transforming growth factor (TGF)-ß-mediated phenotype alterations of tubular epithelial cells (TECs) and tubulointerstitial fibrosis. But the relationship between cell cycle arrest and the autophagy induced by TGF-ß has not been explored well. MATERIAL AND METHODS The effects of autophagy inhibition on TGF-ß-induced cell cycle arrest in TECs were explored in vitro. Human kidney-2 (HK-2) cells were stimulated by TGF-ß with or without a combined treatment of autophagy inhibitor chloroquine (CQ) or bafilomycin A1 (Baf). RESULTS Autophagy inhibition by CQ or Baf promotes the suppression of growth in TGF-ß-treated HK-2 cells, as detected by the Cell Counting Kit-8 (CCK-8) method. In addition, CQ or Baf stimulation enhances G1 arrest in TGF-ß treated HK-2 cells, as investigated using propidium iodide (PI) staining and flow cytometry, which was further confirmed by a decrease in the expression of phosphorylated retinoblastoma protein (p-RB) and cyclin-dependent kinase 4 (CDK4). The upregulation of p21 induced by CQ or Baf may mediate an enhanced G1 arrest in TGF-ß treated HK-2 cells. Western blot analysis showed that TGF-ß-induced expression of extracellular matrix fibronectin was notably upregulated in the presence of autophagy inhibitors. CONCLUSIONS Inhibition of autophagy sensitizes the TECs to G1 arrest and proliferation suppression induced by TGF-ß that contributes to the induction of tubulointerstitial fibrosis.

Protective effect of forsythoside B against lipopolysaccharide-induced acute lung injury by attenuating the TLR4/NF-κB pathway.

Acute lung injury (ALI), which is mainly triggered by infection, pneumonia, vasculitis, and sepsis, has no specific and effective therapy except for primary supportive treatment or bedside care. Excessive inflammation caused by innate immune cells is the major characteristic of ALI. Forsythoside B, a phenylethanoside compound, possesses good antioxidant and anti-bacterial properties in vivo and in vitro. In this study, the therapeutic potential of forsythoside B and its mechanism of action were investigated in a lipopolysaccharide (LPS)-induced ALI mouse model. The results showed that LPS-induced edema exudation and lung pathological changes in mice were significantly suppressed by forsythoside B pre-treatment. Furthermore, it also attenuated lung inflammation caused by LPS stimulation, evidenced by decreased inflammatory cell infiltration and down-regulated expression of cytokines, chemokines, and inducible enzymes. The anti-inflammation property of forsythoside B was confirmed in vitro using LPS-stimulated RAW 264.7 macrophages. Moreover, it alleviated LPS-induced inflammation by inhibiting the activation of TLR4/NF-κB signaling pathway in vivo and in vitro. In conclusion, the results demonstrated that forsythoside B protects against LPS-induced ALI by attenuating inflammatory cell infiltration and suppressing TLR4/NF-κB-mediated lung inflammation. Therefore, it might be a potential therapeutic agent for ALI caused by sepsis.

Accelerated Glomerular Cell Senescence in Experimental Lupus Nephritis.

BACKGROUND The aim of this study was to determine whether senescence in renal glomeruli is involved in lupus nephritis (LN); the expression of senescence-associated ß-galactosidase (SA-ß-Gal) and its association with glomerular lesions were investigated in a mouse model of LN. MATERIAL AND METHODS Eighteen MRL/lpr mice with severe proteinuria were randomly divided into 2 equal groups and intraperitoneally injected with dexamethasone (DEX) or saline; 4 age-matched mice with mild proteinuria served as controls. Serum creatinine and urinary protein levels were analyzed, and kidney histological changes were observed by periodic acid-Schiff and Sirius Red staining. SA-ß-Gal was detected via histochemistry. Glomerular expression of collagen IV, α-SMA, and nephrin was analyzed by immunohistochemistry, and glomerular complement C3 deposition was tested by immunofluorescence. The relationships between SA-ß-Gal expression and renal function or glomerular lesion markers were determined by Spearman's correlation analysis. RESULTS Mice with severe proteinuria exhibited glomerular segmental sclerosis and endothelial cell proliferation. DEX administration suppressed these lesions but had no significant effect on 24-hour urinary protein levels. The elevated glomerular expression of SA-ß-Gal in proteinuric mice was attenuated by DEX treatment. In addition, DEX treatment markedly downregulated glomerular C3 deposition and collagen IV and α-SMA expression, while significantly increasing nephrin expression. Furthermore, SA-ß-Gal expression was positively correlated with urinary protein levels and expression of α-SMA. CONCLUSIONS Accelerated senescence of glomerular cells may contribute to glomerular injury in LN.