Genetic Polymorphisms of MnSOD Modify the Impacts of Environmental Melamine on Oxidative Stress and Early Kidney Injury in Calcium Urolithiasis Patients

Environmental melamine exposure increases the risks of oxidative stress and early kidney injury. Manganese superoxide dismutase (MnSOD), glutathione peroxidase, and catalase can protect the kidneys against oxidative stress and maintain normal function. We evaluated whether their single-nucleotide polymorphisms (SNPs) could modify melamine’s effects. A total of 302 patients diagnosed with calcium urolithiasis were enrolled. All patients provided one-spot overnight urine samples to measure their melamine levels, urinary biomarkers of oxidative stress and renal tubular injury. Median values were used to dichotomize levels into high and low. Subjects carrying the T allele of rs4880 and high melamine levels had 3.60 times greater risk of high malondialdehyde levels than those carrying the C allele of rs4880 and low melamine levels after adjustment. Subjects carrying the G allele of rs5746136 and high melamine levels had 1.73 times greater risk of high N-Acetyl-β-d-glucosaminidase levels than those carrying the A allele of rs5746136 and low melamine levels. In conclusion, the SNPs of MnSOD, rs4880 and rs5746136, influence the risk of oxidative stress and renal tubular injury, respectively, in calcium urolithiasis patients. In the context of high urinary melamine levels, their effects on oxidative stress and renal tubular injury were further increased.

Activation of Transcription Factor EB Alleviates Tubular Epithelial Cell Injury via Restoring Lysosomal Homeostasis in Diabetic Nephropathy

Disruption of lysosomal homeostasis contributes to the tubulopathy of diabetic nephropathy; however, its underlying mechanisms remain unclear. Herein, we report that decreased activity of transcription factor EB (TFEB) is responsible for the disturbed lysosome biogenesis and clearance in this pathological process. This was confirmed by the findings that insufficient lysosomal replenishment and damaged lysosomal clearance coincided with TFEB inactivation, which was mediated by mTOR hyperactivation in the renal tubular epithelial cells (TECs) of diabetic nephropathy. Furthermore, either TFEB overexpression or pharmacological activation of TFEB enhanced lysosomal clearance via promoting lysosomal biogenesis and protected TECs by reducing apoptosis in vitro. In addition, pharmacological activation of TFEB attenuated renal tubule injury, apoptosis, and inflammation in db/db mice. In conclusion, diabetes-induced mTOR activation represses TFEB function, thereby perturbing lysosomal homeostasis through impairing lysosomal biogenesis and clearance in TECs. Moreover, TFEB activation protects TECs from diabetic injuries via restoring lysosomal homeostasis.

The Effect of VPA Treatment on Radiolabeled DOTATATE Uptake: Differences Observed In Vitro and In Vivo

Background: To improve peptide receptor radionuclide therapy (PRRT), we aimed to enhance the expression of somatostatin type-2 receptors (SSTR2) in vitro and in vivo, using valproic acid (VPA). Methods: Human NCI-H69 small-cell lung carcinoma cells were treated with VPA, followed by [111In]In-DOTATATE uptake studies, RT-qPCR and immunohistochemistry analysis. Furthermore, NCI-H69 xenografted mice were treated with VPA or vehicle, followed by [177Lu]Lu-DOTATATE injection. Biodistribution studies were performed, and tissues were collected for further analysis. Results: VPA significantly increased SSTR2 expression in vitro. In animals, a statistically significant increased [177Lu]Lu-DOTATATE tumoral uptake was observed when VPA was administered eight hours before [177Lu]Lu-DOTATATE administration, but increased tumor SSTR2 expression levels were lacking. The animals also presented significantly higher [177Lu]Lu-DOTATATE blood levels, as well as an elevated renal tubular damage score. This suggests that the enhanced tumor uptake was presumably a consequence of the increased radiotracer circulation and the induced kidney damage. Conclusions: VPA increases SSTR2 expression in vitro. In vivo, the observed increase in tumoral [177Lu]Lu-DOTATATE uptake is not caused by SSTR2 upregulation, but rather by other mechanisms, e.g., an increased [177Lu]Lu-DOTATATE circulation time and renal toxicity. However, since both drugs are safely used in humans, the potential of VPA to improve PRRT remains open for investigation.

A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles

The in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and double-floxed, ensuring irreversible Cre recombinase-dependent EV reporter expression. We crossed the EV reporter mice with mice expressing Cre ubiquitously (CMV-Cre), in cardiomyocytes (αMHC-MerCreMer) and renal tubular epithelial cells (Pax8-Cre), respectively. The CD9truc-EGFP positive mice showed Cre-dependent EGFP expression, and plasma CD9truc-EGFP EVs were immunoprecipitated only from CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxαMHC-Cre mice, but not in CD9truc-EGFPxPax8-Cre and CD9truc-EGFP negative mice. In urine samples, CD9truc-EGFP EVs were detected by immunoprecipitation only in CD9truc-EGFP positive CD9truc-EGFPxCMV-Cre and CD9truc-EGFPxPax8-Cre mice, but not CD9truc-EGFPxαMHC-Cre and CD9truc-EGFP negative mice. In conclusion, our EV reporter mouse model enables Cre-dependent EV labeling, providing a new approach to studying cell-specific EVs in vivo and gaining a unique insight into their physiological and pathophysiological function.

Effects of alternative splicing events and transcriptome changes on kidney stone formation

During the development of urinary stone disease, the formation of tiny crystals that adhere to the renal tubular epithelium induces epithelial cell damage. This damage and repair of the epithelium is associated with the establishment of more crystal adhesion sites, which in turn stimulates further crystal adhesion and, eventually, stone formation. Deposited crystals typically cause changes in epithelial cell gene expression, such as transcriptome changes and alternative splicing events. Although considered important for regulating gene expression, alternative splicing has not been reported in studies related to kidney stones. To date, whether alternative splicing events are involved in the regulation of stone formation and whether crystallographic cell interactions are regulated by alternative splicing at the transcriptional level have remained unknown. Therefore, we conducted RNA sequencing and alternative splicing-related bioassays by modeling the in vitro stone environment. Many alternative splicing events were associated with crystallographic cell interactions. Moreover, these events regulated transcription and significantly affected the capacity of crystals to adhere to renal tubular epithelial cells and regulate apoptosis.

Dapagliflozin Alleviates Renal Fibrosis by Inhibiting RIP1-RIP3-MLKL-Mediated Necroinflammation in Unilateral Ureteral Obstruction

Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, offers renoprotection in diabetes. However, potential for use in nondiabetic kidney disease remains unknown. Herein, we assessed whether dapagliflozin alleviates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. After induction of UUO, rats were administered dapagliflozin daily for seven consecutive days. UUO induced significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins; these coincided with NLRP3 inflammasome activation, and subsequent development of renal fibrosis. Oxidative stress caused by UUO is tightly associated with endoplasmic reticulum stress and mitochondrial dysfunction, leading to apoptotic cell death through Wnt3α/β-catenin/GSK-3β signaling; all of which were abolished by both dapagliflozin and specific RIP inhibitors (necrostatin-1 and GSK872). In H2O2-treated HK-2 cells, dapagliflozin and RIP inhibitors suppressed overexpression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, decreased intracellular reactive oxygen species production and apoptotic cell death, whereas cell viability was improved. Moreover, activated Wnt3α/β-catenin/GSK-3β signaling was inhibited by dapagliflozin and Wnt/β-catenin inhibitor ICG-001. Our findings suggest that dapagliflozin ameliorates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/β-catenin/GSK-3β signaling in UUO.

Assessment of Hydroxyethyl Starch (6% HES 130/0.4) Kidney Storage in Critically Ill Dogs: A Post-mortem Prospective Study

Objective: Intravenous hydroxyethyl starch (HES) solutions are potentially nephrotoxic due to rapid renal tissue uptake, subsequent osmotic nephrosis, and long-lasting intracellular storage. This study aimed to investigate the severity of intracellular storage of HES in renal tissue samples from critically ill dogs receiving 6% HES 130/0.4.Materials and Methods: Fresh, post-mortem (<2 h after death) renal tissue samples were analyzed through histology, immunohistochemistry (HES 130/0.4-specific antibodies), and electron microscopy for the severity of renal tubular vacuolization (VAC), intravacuolar HES accumulation (ACC), and ultra-structure impairment. Moreover, we investigated the relationship between VAC or ACC grade and HES dose (mL/kg), duration of HES administration (h), and pre-HES plasma creatinine concentrations.Results: Histology revealed that 2/20 dogs (10%) had no, 11/20 dogs (55%) had mild, 5/20 dogs (25%) had moderate, and 2/20 dogs (10%) had severe VAC. Immunohistochemistry revealed that 5/20 dogs (25%) had no, 6/20 dogs (30%) had mild, 7/20 dogs (35%) had moderate, and 2/20 dogs (10%) had severe ACC. Both changes were predominantly found in the distal tubular epithelium of mild and moderate cases, and all tubular segments were affected in severe cases. Seven of 20 dogs (35%) had osmotic nephrosis (ON). On electron microscopy, large granules with an electron-dense content were repeatedly detected in individual cells, mainly in the distal tubules. No correlation was found between cumulative HES dose or duration of HES administration and VAC grade, ACC grade, or presence/absence of ON.Conclusion: A high percentage of dogs had renal tubular HES storage and one-third of dogs showed HES-induced ON. Short-term HES administration caused VAC and ACC, regardless of the dose or duration of administration. In contrast to previous studies, HES 130/0.4 deposits were mainly located in the renal distal tubule.

Berberine Reduces Lipid Accumulation by Promoting Fatty Acid Oxidation in Renal Tubular Epithelial Cells of the Diabetic Kidney

Abnormal lipid metabolism in renal tubular epithelial cells contributes to renal lipid accumulation and disturbed mitochondrial bioenergetics which are important in diabetic kidney disease. Berberine, the major active constituent of Rhizoma coptidis and Cortex phellodendri, is involved in regulating glucose and lipid metabolism. The present study aimed to investigate the protective effects of berberine on lipid accumulation in tubular epithelial cells of diabetic kidney disease. We treated type 2 diabetic db/db mice with berberine (300 mg/kg) for 12 weeks. Berberine treatment improved the physical and biochemical parameters of the db/db mice compared with db/m mice. In addition, berberine decreased intracellular lipid accumulation and increased the expression of fatty acid oxidation enzymes CPT1, ACOX1 and PPAR-α in tubular epithelial cells of db/db mice. The mitochondrial morphology, mitochondrial membrane potential, cytochrome c oxidase activity, mitochondrial reactive oxygen species, and mitochondrial ATP production in db/db mice kidneys were significantly improved by berberine. Berberine intervention activated the AMPK pathway and increased the level of PGC-1α. In vitro berberine suppressed high glucose-induced lipid accumulation and reversed high glucose-induced reduction of fatty acid oxidation enzymes in HK-2 cells. Importantly, in HK-2 cells, berberine treatment blocked the change in metabolism from fatty acid oxidation to glycolysis under high glucose condition. Moreover, berberine restored high glucose-induced dysfunctional mitochondria. These data suggested that berberine alleviates diabetic renal tubulointerstitial injury through improving high glucose-induced reduction of fatty acid oxidation, alleviates lipid deposition, and protect mitochondria in tubular epithelial cells.

miR-3587 Inhibitor Attenuates Ferroptosis Following Renal Ischemia-Reperfusion Through HO-1

Renal ischemia-reperfusion (IR) is frequently observed in patients who are critically ill, yet there are no reliable or effective approaches for the treatment of this condition. Ferroptosis, a form of programmed cell death, is regulated by key genes such as glutathione peroxidase 4 (GPX4) and heme oxygenase-1 (HMOX1) and participates in the injury of renal tubular epithelial cells during IR. This study aimed to investigate the miRNA-mRNA regulatory networks involved in ferroptosis following renal IR. Using bioinformatics analysis, HMOX1 was found to be significantly upregulated during the early stages of renal IR injury, and microRNA-3587 (miR-3587) was identified as a putative regulator of HMOX1. When a miR-3587 inhibitor was applied in a hypoxia-reoxygenation (HR) model system using renal tubular epithelial cells, HO-1 protein (encoded by HMOX1) expression was significantly increased relative to that observed in the HR group, with concomitant increases in GPX4 protein levels, enhanced cell viability, a reduction in malondialdehyde content, decreased Fe2+ level, and the restoration of normal mitochondrial membrane potential. Transmission electron microscopy showed a reduced or absent mitochondrial crest and a damaged mitochondrial outer membrane. Targeting of HMOX1 by miR-3587 was confirmed by luciferase reporter gene assay. In conclusion, these preliminary results indicate that inhibition of miR-3587 promotes HO-1 upregulation, thereby protecting renal tissues from IR-induced ferroptosis.