Hypoxic human proximal tubular epithelial cells undergo ferroptosis and elicit an NLRP3 inflammasome response in CD1c+ dendritic cells.

Inflammasomes are multiprotein platforms responsible for the release of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. Mouse studies have identified inflammasome activation within dendritic cells (DC) as pivotal for driving tubulointerstitial fibrosis and inflammation, the hallmarks of chronic kidney disease (CKD). However, translation of this work to human CKD remains limited. Here, we examined the complex tubular cell death pathways mediating inflammasome activation in human kidney DC and, thus, CKD progression. Ex vivo patient-derived proximal tubular epithelial cells (PTEC) cultured under hypoxic (1% O2) conditions modelling the CKD microenvironment showed characteristics of ferroptotic cell death, including mitochondrial dysfunction, reductions in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and increases in lipid peroxidation by-product 4-hydroxynonenal (4-HNE) compared with normoxic PTEC. The addition of ferroptosis inhibitor, ferrostatin-1, significantly reduced hypoxic PTEC death. Human CD1c+ DC activated in the presence of hypoxic PTEC displayed significantly increased production of inflammasome-dependent cytokines IL-1ß and IL-18. Treatment of co-cultures with VX-765 (caspase-1/4 inhibitor) and MCC950 (NLRP3 inflammasome inhibitor) significantly attenuated IL-1ß/IL-18 levels, supporting an NLRP3 inflammasome-dependent DC response. In line with these in vitro findings, in situ immunolabelling of human fibrotic kidney tissue revealed a significant accumulation of tubulointerstitial CD1c+ DC containing active inflammasome (ASC) specks adjacent to ferroptotic PTEC. These data establish ferroptosis as the primary pattern of PTEC necrosis under the hypoxic conditions of CKD. Moreover, this study identifies NLRP3 inflammasome signalling driven by complex tubulointerstitial PTEC-DC interactions as a key checkpoint for therapeutic targeting in human CKD.

Adenine overload induces ferroptosis in human primary proximal tubular epithelial cells.

The pathogenesis of crystal nephropathy involves deposition of intratubular crystals, tubular obstruction and cell death. The deposition of 8-dihydroxyadenine (DHA) crystals within kidney tubules, for instance, is caused by a hereditary deficiency of adenine phosphoribosyl transferase in humans or adenine overload in preclinical models. However, the downstream pathobiological patterns of tubular cell attrition in adenine/DHA-induced nephropathy remain poorly understood. In this study, we investigated: (i) the modes of adenine-induced tubular cell death in an experimental rat model and in human primary proximal tubular epithelial cells (PTEC); and (ii) the therapeutic effect of the flavonoid baicalein as a novel cell death inhibitor. In a rat model of adenine diet-induced crystal nephropathy, significantly elevated levels of tubular iron deposition and lipid peroxidation (4-hydroxynonenal; 4-HNE) were detected. This phenotype is indicative of ferroptosis, a novel form of regulated necrosis. In cultures of human primary PTEC, adenine overload-induced significantly increased mitochondrial superoxide levels, mitochondrial depolarisation, DNA damage and necrotic cell death compared with untreated PTEC. Molecular interrogation of adenine-stimulated PTEC revealed a significant reduction in the lipid repair enzyme glutathione peroxidase 4 (GPX4) and the significant increase in 4-HNE compared with untreated PTEC, supporting the concept of ferroptotic cell death. Moreover, baicalein treatment inhibited ferroptosis in adenine-stimulated PTEC by selectively modulating the mitochondrial antioxidant enzyme superoxide dismutase 2 (SOD2) and thus, suppressing mitochondrial superoxide production and DNA damage. These data identify ferroptosis as the primary pattern of PTEC necrosis in adenine-induced nephropathy and establish baicalein as a potential therapeutic tool for the clinical management of ferroptosis-associated crystal nephropathies (e.g., DHA nephropathy, oxalate nephropathy).

T-Cell Expression and Release of Kidney Injury Molecule-1 in Response to Glucose Variations Initiates Kidney Injury in Early Diabetes.

Half of the mortality in diabetes is seen in individuals <50 years of age and commonly predicted by the early onset of diabetic kidney disease (DKD). In type 1 diabetes, increased urinary albumin-to-creatinine ratio (uACR) during adolescence defines this risk, but the pathological factors responsible remain unknown. We postulated that early in diabetes, glucose variations contribute to kidney injury molecule-1 (KIM-1) release from circulating T cells, elevating uACR and DKD risk. DKD risk was assigned in youth with type 1 diabetes (n = 100; 20.0 ± 2.8 years; males/females, 54:46; HbA1c 66.1 [12.3] mmol/mol; diabetes duration 10.7 ± 5.2 years; and BMI 24.5 [5.3] kg/m2) and 10-year historical uACR, HbA1c, and random blood glucose concentrations collected retrospectively. Glucose fluctuations in the absence of diabetes were also compared with streptozotocin diabetes in apolipoprotein E -/- mice. Kidney biopsies were used to examine infiltration of KIM-1-expressing T cells in DKD and compared with other chronic kidney disease. Individuals at high risk for DKD had persistent elevations in uACR defined by area under the curve (AUC; uACRAUC0-10yrs, 29.7 ± 8.8 vs. 4.5 ± 0.5; P < 0.01 vs. low risk) and early kidney dysfunction, including ∼8.3 mL/min/1.73 m2 higher estimated glomerular filtration rates (modified Schwartz equation; Padj < 0.031 vs. low risk) and plasma KIM-1 concentrations (∼15% higher vs. low risk; P < 0.034). High-risk individuals had greater glycemic variability and increased peripheral blood T-cell KIM-1 expression, particularly on CD8+ T cells. These findings were confirmed in a murine model of glycemic variability both in the presence and absence of diabetes. KIM-1+ T cells were also infiltrating kidney biopsies from individuals with DKD. Healthy primary human proximal tubule epithelial cells exposed to plasma from high-risk youth with diabetes showed elevated collagen IV and sodium-glucose cotransporter 2 expression, alleviated with KIM-1 blockade. Taken together, these studies suggest that glycemic variations confer risk for DKD in diabetes via increased CD8+ T-cell production of KIM-1.

Oxidative stress and inflammasome activation in human rhabdomyolysis-induced acute kidney injury.

Acute kidney injury (AKI) is a life-threatening complication of rhabdomyolysis. The pathophysiological mechanisms of rhabdomyolysis-induced AKI (RIAKI) have been extensively studied in the murine system, yet clinical translation of this knowledge to humans is lacking. In this study, we investigated the cellular and molecular pathways of human RIAKI. Renal biopsy tissue from a RIAKI patient was examined by quantitative immunohistochemistry (Q-IHC) and compared to healthy kidney cortical tissue. We identified myoglobin casts and uric acid localised to sites of histological tubular injury, consistent with the diagnosis of RIAKI. These pathological features were associated with tubular oxidative stress (4-hydroxynonenal staining), regulated necrosis/necroptosis (phosphorylated mixed-lineage kinase domain-like protein staining) and inflammation (tumour necrosis factor (TNF)-α staining). Expression of these markers was significantly elevated in the RIAKI tissue compared to the healthy control. A tubulointerstitial inflammatory infiltrate accumulated adjacent to these sites of RIAKI oxidative injury, consisting of macrophages (CD68), dendritic cells (CD1c) and T lymphocytes (CD3). Foci of inflammasome activation were co-localised with these immune cell infiltrate, with significantly increased staining for adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain) and active caspase-1 in the RIAKI tissue compared to the healthy control. Our clinical findings identify multiple pathophysiological pathways previously only reported in murine RIAKI, providing first evidence in humans linking deposition of myoglobin and presence of uric acid to tubular oxidative stress/necroptosis, inflammasome activation and necroinflammation.

Role of inflammation and inflammasome activation in human bile cast nephropathy.

Bile cast nephropathy (BCN) is an underdiagnosed cause of acute kidney injury (AKI). The precise pathogenesis of bilirubin tubular toxicity remains unknown. The aim of this study is to explore the cellular and molecular pathophysiology of human BCN. Paraffin-embedded sections of renal biopsy tissue from a BCN patient were stained by immunohistochemistry (IHC) for oxidative stress (4-hydroxynonenal), immune cell subpopulations, including dendritic cells (CD1c), macrophages (CD68) and T cells (CD3), and inflammasome activation by staining for active-caspase-1 and the inflammasome adaptor protein, ASC (apoptosis-associated speck-like protein containing a caspase activation and recruitment domain). Quantitative analyses of IHC staining were compared to healthy renal cortical tissue. We identified yellow to brown granular casts within the BCN case, consistent with the presence of bile pigment. The presence of bile pigment was associated with strong tubular 4-hydroxynonenal staining intensity, a marker of oxidative stress. Diffuse tubulointerstitial inflammatory cell infiltrate was detected, with elevated CD1c, CD68 and CD3 staining. Foci of inflammasome activity were co-localized with this intense immune cell infiltration, with increased active-caspase-1 and ASC staining. Our findings are the first to suggest that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving AKI pathobiology. SUMMARY AT A GLANCE The report suggests that bile casts may lead to oxidative stress and trigger the inflammasome signalling cascade, leading to interstitial inflammation and driving bile cast nephropathy pathobiology.

Underlying Histopathology Determines Response to Oxidative Stress in Cultured Human Primary Proximal Tubular Epithelial Cells.

Proximal tubular epithelial cells (PTEC) are key players in the progression of kidney diseases. PTEC studies to date have primarily used mouse models and transformed human PTEC lines. However, the translatability of these models to human kidney disease has been questioned. In this study, we investigated the phenotypic and functional response of human primary PTEC to oxidative stress, an established driver of kidney disease. Furthermore, we examined the functional contribution of the underlying histopathology of the cortical tissue used to generate our PTEC. We demonstrated that human primary PTEC from both histologically 'normal' and 'diseased' cortical tissue responded to H2O2-induced oxidative stress with significantly elevated mitochondrial superoxide levels, DNA damage, and significantly decreased proliferation. The functional response of 'normal' PTEC to oxidative stress mirrored the reported pathogenesis of human kidney disease, with significantly attenuated mitochondrial function and increased cell death. In contrast, 'diseased' PTEC were functionally resistant to oxidative stress, with maintenance of mitochondrial function and cell viability. This selective survival of 'diseased' PTEC under oxidizing conditions is reminiscent of the in vivo persistence of maladaptive PTEC following kidney injury. We are now exploring the impact that these differential PTEC responses have in the therapeutic targeting of oxidative stress pathways.

Pigment Nephropathy: Novel Insights into Inflammasome-Mediated Pathogenesis.

Pigment nephropathy is an acute decline in renal function following the deposition of endogenous haem-containing proteins in the kidneys. Haem pigments such as myoglobin and haemoglobin are filtered by glomeruli and absorbed by the proximal tubules. They cause renal vasoconstriction, tubular obstruction, increased oxidative stress and inflammation. Haem is associated with inflammation in sterile and infectious conditions, contributing to the pathogenesis of many disorders such as rhabdomyolysis and haemolytic diseases. In fact, haem appears to be a signalling molecule that is able to activate the inflammasome pathway. Recent studies highlight a pathogenic function for haem in triggering inflammatory responses through the activation of the nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome. Among the inflammasome multiprotein complexes, the NLRP3 inflammasome has been the most widely characterized as a trigger of inflammatory caspases and the maturation of interleukin-18 and -1ß. In the present review, we discuss the latest evidence on the importance of inflammasome-mediated inflammation in pigment nephropathy. Finally, we highlight the potential role of inflammasome inhibitors in the prophylaxis and treatment of pigment nephropathy.