CELASTROL AMELIORATES DIABETIC KIDNEY DISEASE BY SUPPRESSING NOX2-MEDIATED FERROPTOSIS SIGNALING

Diabetic kidney disease (DKD), a leading microvascular complication of diabetes mellitus, is driven by incompletely understood pathomechanisms. Emerging evidence implicates ferroptosis—an iron-dependent, regulated form of cell death—in the onset and progression of DKD. Celastrol, a bioactive quinone methide purified from Tripterygium wilfordii, exerts robust anti-inflammatory and antioxidant effects and has been reported to ameliorate experimental DKD, although its precise molecular targets remain elusive. The objective of this study is to quantify the expression of ferroptosis-related biomarkers and NADPH oxidase 2 (NOX2) in human diabetic kidney tissue and a validated rat model of DKD, and thereby delineate the regulatory impact of celastrol on ferroptotic signaling in diabetic nephropathy.

This study integrated clinical specimen analysis with validation in an animal model.  

(1) Collection of human renal tissue samples: Renal tissue samples were collected from patients who underwent renal biopsy and were histopathologically diagnosed with stage II–III DKD at our hospital between March and July 2024 (n = 3). Adjacent non-tumorous renal tissues from patients undergoing radical nephrectomy for renal cell carcinoma (RCC-ANT, n = 3) served as controls. Hematoxylin–eosin (HE) staining was used to evaluate histo-architecture, Perls staining to visualize iron deposition, and transmission electron microscopy (TEM) to assess mitochondrial ultrastructure in tubular epithelial cells, with particular attention to mitochondrial size, cristae integrity, and outer-membrane integrity.  

(2) Animal studies: Male Sprague–Dawley rats received a single intraperitoneal injection of streptozotocin (STZ, 25 mg/kg) to induce DKD. Once hyperglycaemia and albuminuria were confirmed, animals were randomized into DKD vehicle group and celastrol-treatment group (DKD + Celastrol); age-matched non-diabetic rats served as normal controls (NC). Vehicle or celastrol was administered daily for 4 weeks. Thereafter, metabolic and renal phenotypes—body weight, kidney index (KI), blood glucose, serum creatinine (Scr), blood urea nitrogen (BUN), and urinary albumin-to-creatinine ratio (UACR)—were quantified. Renal tissue was subjected to HE staining, Perls staining, TEM, ROS fluorimetry, Western blot, and immunofluorescence to determine the effect of celastrol on NOX2 and ferroptosis-related signalling cascades.

Compared with RCC-ANT controls, renal biopsies from DKD patients exhibited markedly increased iron deposition, indicative of overt iron overload. TEM revealed early ferroptotic ultrastructural changes in tubular epithelial cells—mitochondrial swelling, indistinct outer membranes, and disappearance of cristae. In the animal study, DKD rats displayed lower body weight and significantly higher random blood glucose, UACR, and KI than normal controls. Four weeks of celastrol treatment reduced UACR and KI, whereas random blood glucose, Scr, and BUN remained unchanged. Histologically, celastrol attenuated tubular dilation, diminished Perls-positive iron deposits, and ameliorated mitochondrial injury. Western blot and immunofluorescence showed that DKD kidneys had elevated NOX2 and suppressed glutathione peroxidase-4 (GPX4) expression; both alterations were reversed by celastrol. Concordantly, dihydroethidium (DHE) staining demonstrated that celastrol markedly curtailed renal ROS accumulation.

This study establishes that ferroptosis is an integral driver of DKD. The natural quinone-methide celastrol markedly attenuates albuminuria and renal structural injury in DKD by silencing NOX2, restoring GPX4 expression, curbing ROS overproduction, and re-establishing intracellular iron homeostasis, thereby blocking ferroptosis in tubular epithelial cells. These findings provide a mechanistic rationale for advancing celastrol as a therapeutic agent against DKD.