Tubule Injury Research Articles

Network pharmacology combined with transcriptomics reveals that formononetin, a biologically component of Astragalus membranaceus (Fisch.) Bunge, inhibits the PI3K/AKT signaling pathway to improve chronic renal failure

Ethnopharmacological relevanceFormononetin (FMN), one of the main isoflavones isolated from Astragalus membranaceus (Fisch.) Bunge, has multiple pharmacological and renal-protective effects. Our previous study suggested FMN as a candidate compound for the treatment of chronic renal failure (CRF). However, the mechanism underlying the repressive effect of FMN on the development of CRF is still unknown. Aims of the studyTo investigate the protective effect of FMN on CRF using in vivo and in vitro models and elucidate the potential underlying mechanism. Materials and methodsAn in vivo model of adenine-induced CRF and an in vitro model of human proximal tubule epithelial cells (HK-2) stimulated with transforming growth factor (TGF)-β1 were used. Serum levels of renal function parameters and inflammatory cytokines were evaluated. Histological analysis was performed to determine the extent of renal injury and fibrosis. Network pharmacology and mRNA sequencing were used to explore the potential mechanism. PPI analysis and molecular docking were used to identify key targets. Polymerase chain reaction and western blotting were used to determine the mechanism underlying the effect of FMN on CRF. ResultsFMN decreased the levels of renal function biochemical markers, including serum creatinine, blood urea nitrogen, and 24 h urine protein content. Treatment with FMN improved renal tubule injury and extracellular matrix (ECM) components, including collagens I and III. In addition, FMN significantly inhibited epithelial-mesenchymal transition (EMT); decreased the expression of fibronectin, N-cadherin, vimentin, α-SMA, and TGF-β1; and restored the expression of E-cadherin. The effect of FMN on renal interstitial fibrosis contributed to decreasing the expression of PI3K, p-Akt, and interleukin (IL) 4, restoring the expression of nitric oxide synthase 3 (NOS3), and reducing the release of inflammatory cytokines (IL-1β, IL-6, and tumor necrosis factor-alpha), both in vivo and in vitro. FMN treatment improved renal function and deposition of ECM components, reduced protein levels of EMT markers in rat kidneys and HK-2 cells, decreased the release of inflammatory cytokines, and inhibited the PI3K/Akt signaling pathway. ConclusionsFMN treatment significantly reduced the release of inflammatory cytokines and inhibited the effects of the PI3K/Akt signaling pathway on the key targets IL-4 and NOS3. Our results suggest FMN therapy as a novel therapeutic strategy for treating CRF.

Absence of Kidney Tubular Injury in Patients With Acute Heart Failure With Acute Kidney Injury.

Worsening renal function (WRF) is common in hospitalized patients being treated for acute heart failure. However, discriminating clinically significant WRF remains challenging. In patients hospitalized with acute heart failure, we evaluated if blood and urine biomarkers of cardiac and kidney dysfunction were associated with adverse outcomes. We identified 175 of 927 participants in the AKINESIS study (Acute Kidney Neutrophil Gelatinase-Associated Lipocalin Evaluation of Symptomatic Heart Failure Study) who met criteria for stage 1 or 2 Kidney Disease: Improvement Global Outcomes acute kidney injury during the first 3 days of hospitalization. We measured 24 blood and urine biomarkers from specimens collected within 24 hours of meeting acute kidney injury criteria. The primary composite outcome consisted of worsening WRF (higher acute kidney injury stage), need for dialysis, or death at 30 days. Biomarkers' association with the composite outcome was assessed with logistic regression by tertiles and area under the curve (AUC). Of the 175 participants, 32 (18%) developed the primary composite outcome. Only history of chronic kidney disease was significantly different between those with and without the composite outcome. The highest tertile of plasma Gal-3 (galectin-3) and urine epidermal growth factor were associated with increased odds of the composite outcome compared with the lowest tertile in unadjusted analyses. After adjusting for serum creatinine, systolic blood pressure, and blood urea nitrogen, only the highest tertile of Gal-3 was associated with greater odds of the composite outcome (odds ratio, 4.6 [95% CI, 1.4-16.0). Gal-3 had the highest AUC (0.70 [95% CI, 0.58-0.82]), while epidermal growth factor had a lower AUC (0.63 [95% CI, 0.53-0.74]). Notably, urine biomarkers of kidney tubule injury were not associated with the composite outcome. Tubular injury does not occur in most patients with acute heart failure experiencing WRF, consistent with the functional mechanisms of WRF in this patient population. URL: https://www.clinicaltrials.gov/study/NCT01291836?term=NCT01291836&rank=1; Unique identifier: NCT01291836.

Proximal Tubule Secretory Clearance, Injury, and Kidney Viability in Cirrhosis.

Cirrhosis affects all structures of the kidney, in particular the tubules which are responsible for secretion of protein-bound metabolites and electrolyte/water homeostasis. Yet, prevailing assessments of kidney function focus solely on glomerular filtration rate (GFR) which may incompletely reflect these processes. We sought to characterize markers of tubular function, injury, and viability in patients with and without cirrhosis. We recruited outpatients undergoing liver transplantation evaluation for a collection of plasma and 24-hour urine, matching by GFR to control participants without cirrhosis. We measured urinary kidney injury molecule-1 (KIM-1), a marker of proximal tubular injury, as well as epidermal growth factor (EGF), a marker of viability necessary for tubular epithelial cell proliferation after injury. We also estimated secretory clearance by measuring several highly secreted endogenous metabolites in urine and plasma. We recruited 39 patients with cirrhosis (mean MELD-Na 17±4, Child-Pugh 8±2, eGFR 66±20 ml/min/1.73m2) and 58 GFR-matched controls without cirrhosis (eGFR 66±21 ml/min/1.73m2). Urinary KIM-1 was 4.4-fold higher than controls (95% CI:2.9-6.5), and EGF averaged 7.41-fold higher than controls (95% CI:2.15-25.53). We found that of 8 solutes, 5 had significantly greater kidney clearance in cirrhosis (1.3-2.1-fold higher): indoxyl sulfate, p-cresol sulfate, pyridoxic acid, tiglylglycine, and xanthosine. Cirrhosis was characterized by molecular signs of tubular injury in stable outpatients without acute kidney injury, accompanied by largely preserved tubular secretory clearance and greater signs of tubular viability. Within the limitations of the study, this suggests a phenotype of chronic ischemic injury but with initial preservation of tubular function in cirrhosis.

Distal convoluted tubule-specific disruption of the COP9 signalosome but not its regulatory target cullin 3 causes tubular injury.

The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl-, or total CO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.NEW & NOTEWORTHY CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury.

VCAM-1 mediates proximal tubule-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Studies in animal models have suggested a linkage between the inflammatory response to injury and subsequent nephron loss during the acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Failure of normal repair during the CKD transition correlates with de novo expression of vascular cell adhesion protein-1 (VCAM-1) by a subset of injured proximal tubule cells. This study identified the role of VCAM-1 expression in promoting the failed repair state. Single-cell transcriptome analysis of patients with AKI and CKD and whole kidney RNA and protein analyses of mouse models of CKD confirmed a marked increase of VCAM-1 expression in the proximal tubules of injured kidneys. In immortalized mouse proximal tubular cells and primary cultured renal cells (PCRCs), VCAM-1 expression was induced by proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β. Analyses of bulk RNA sequencing of TNF-α-treated primary cultured renal cells or pseudo-bulk RNA sequencing of biopsies from Kidney Precision Medicine Project datasets indicated activation of NF-κB and an enrichment of inflammatory response and cell adhesion pathways in VCAM-1-positive cells. Pharmacological inhibition of NF-κB signaling or genetic deletion of myeloid differentiation factor 88 and TIR domain-containing adapter-inducing interferon-β suppressed TNF-α- and IL-1β-induced VCAM-1 expression in vitro. TNF-α stimulation or overexpression of VCAM-1 significantly increased splenocyte adhesion to the mouse proximal tubular monolayer in culture. These results demonstrate that persistence of proinflammatory cytokines after AKI can induce NF-κB-dependent VCAM-1 expression by proximal tubule cells, mediating increased immune cell adhesion to the tubule and thus promoting further tubule injury and greater risk of progression from AKI to CKD.NEW & NOTEWORTHY We demonstrated the induction of VCAM-1 and its biological function in proximal tubules. We found that proinflammatory cytokines (TNF-α and IL-1β) significantly induced VCAM-1 expression via NF-κB signaling pathway. TNF-α treatment or overexpression of VCAM-1 in immortalized MPT cells increased CD45+ splenocyte adhesion. Pharmacological inhibition of NF-κB or genetic deletion of Vcam1 suppressed TNF-α-induced splenocyte adhesion in vitro, suggesting that VCAM-1 mediates proximal tubular-immune cell cross talk in failed tubule recovery during AKI-to-CKD transition.

Assessing Biomarkers of Porcine Kidneys under Normothermic Machine Perfusion-Can We Gain Insight into a Marginal Organ?

To identify potentially transplantable organs in a pool of marginal kidneys, 33 porcine slaughterhouse kidneys were perfused for 4 h with whole blood. During the normothermic perfusion, plasma, urine, and tissue samples were taken. Several biomarkers for tubule injury, endothelial activation, and inflammatory response were evaluated for a potential correlation with macroscopic appearance, histology, and filtration activity. Generally, biomarker levels increased during perfusion. TLR-4, EDN-1, and NGAL were not associated with any classification. In contrast, a steeper increase in NAG and IL-6 in plasma correlated with a poor macroscopic appearance at 4 h, indicating a higher inflammatory response in the kidneys with worse macroscopy early on, potentially due to more damage at the tubules. Although long-term effects on the graft could not be assessed in this setting, early observation under machine perfusion with whole blood was feasible. It allowed the assessment of kidneys under conditions comparable to reperfusion. This setting could give surgeons further insight into the quality of marginal kidneys and an opportunity to pre-treat them.

Macrophage autophagy protects against acute kidney injury by inhibiting renal inflammation through the degradation of TARM1

ABSTRACT Macroautophagy/autophagy activation in renal tubular epithelial cells protects against acute kidney injury (AKI). However, the role of immune cell autophagy, such as that involving macrophages, in AKI remains unclear. In this study, we discovered that macrophage autophagy was an adaptive response during AKI as mice with macrophage-specific autophagy deficiency (atg5 −/−) exhibited higher serum creatinine, more severe renal tubule injury, increased infiltration of ADGRE1/F4/80+ macrophages, and elevated expression of inflammatory factors compared to WT mice during AKI induced by either LPS or unilateral ischemia-reperfusion. This was further supported by adoptive transfer of atg5 −/− macrophages, but not WT macrophages, to cause more severe AKI in clodronate liposomes-induced macrophage depletion mice. Similar results were also obtained in vitro that bone marrow-derived macrophages (BMDMs) lacking Atg5 largely increased pro-inflammatory cytokine expression in response to LPS and IFNG. Mechanistically, we uncovered that atg5 deletion significantly upregulated the protein expression of TARM1 (T cell-interacting, activating receptor on myeloid cells 1), whereas inhibition of TARM1 suppressed LPS- and IFNG-induced inflammatory responses in atg5 −/− RAW 264.7 macrophages. The E3 ubiquitin ligases MARCHF1 and MARCHF8 ubiquitinated TARM1 and promoted its degradation in an autophagy-dependent manner, whereas silencing or mutation of the functional domains of MARCHF1 and MARCHF8 abolished TARM1 degradation. Furthermore, we found that ubiquitinated TARM1 was internalized from plasma membrane into endosomes, and then recruited by the ubiquitin-binding autophagy receptors TAX1BP1 and SQSTM1 into the autophagy-lysosome pathway for degradation. In conclusion, macrophage autophagy protects against AKI by inhibiting renal inflammation through the MARCHF1- and MARCHF8-mediated degradation of TARM1. Abbreviations: AKI, acute kidney injury; ATG, autophagy related; Baf, bafilomycin A1; BMDMs, bone marrow-derived macrophages; CCL2/MCP-1, C-C motif chemokine ligand 2; CHX, cycloheximide; CQ, chloroquine; IFNG, interferon gamma; IL, interleukin; IR, ischemia-reperfusion; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; LPS, lipopolysaccharide; MARCHF, membrane associated ring-CH-type finger; NC, negative control; NFKB, nuclear factor of kappa light polypeptide gene enhancer in B cells; NLRP3, NLR family, pyrin domain containing 3; NOS2, nitric oxide synthase 2, inducible; Rap, rapamycin; Wort, wortmannin; RT-qPCR, real-time quantitative polymerase chain reaction; Scr, serum creatinine; SEM, standard error of mean; siRNA, small interfering RNA; SYK, spleen tyrosine kinase; TARM1, T cell-interacting, activating receptor on myeloid cells 1; TAX1BP1, Tax1 (human T cell leukemia virus type I) binding protein 1; TECs, tubule epithelial cells; TNF, tumor necrosis factor; WT, wild type.

MicroRNA-223 alleviates inflammatory response in renal ischemia-reperfusion injury by targeting NLRP3.

We investigated the potential correlation between miR-223 and NAcHT, LRR, and PYd domain-containing protein 3 (NLRP3) in the context of renal ischemia-reperfusion injury (RIRI), which is a leading cause of acute renal failure with significant mortality rates. Additionally, miR-223 has been implicated in renal inflammation, further highlighting its relevance to this study. C57BL/6 male mice were used as RIRI models. After successful modeling, pathological examinations and serum creatinine and miR-223 levels were tested. Pro-inflammatory cytokine (IL-1β, IL-6, IL-8, NLPR3, TLR4) expression was detected in mice by western blot (kidney tissue) and enzyme-linked immunosorbent assay (serum). HK-2 cells were used for in vitro experiments. A hypoxia/reoxygenation (H/R) model was used, and miR-223 and pro-inflammatory cytokine levels were detected using PCR and western blot assays, respectively. A dual-luciferase reporter assay was conducted to confirm the binding of miR-223 to NLPR3. Next, NLRP3 was knocked down to determine whether the anti-inflammatory function of miR-223 is dependent on NLRP3. MiR-223 expression was lower in RIRI mice than in the sham operation group. The level of miR-223 negatively correlated with serum creatinine levels and the severity of tubule injury. Increased proinflammatory cytokine levels in RIRI mice were observed. In vitro, miR-223 alleviated the inflammatory response in H/R treated cells by inhibiting proinflammatory cytokines. Dual-luciferase reporter and western blot assays confirmed the binding of miR-223 to NLRP3. NLRP3 knockdown reversed the anti-inflammatory effects of miR-223 in HK-2 cells. MiR-223 plays an anti-inflammatory role in RIRI by targeting NLRP3 to repress pro-inflammatory factors.

Possible protective effects of vanillin against stress-induced seminiferous tubule injury via modulation of Nrf2 and ZO1.

Around 20% of the human population is distressed. Previous studies have looked into the relationship between restraint immobilization stress (IS) and sexual behavior in male rats. The current study aimed to provide a brief explanation of the mechanisms that generated testicular injury with chronic IS and an attempt to evaluate the mechanisms and effects of vanillin as a novel protective agent. Forty-eight adult male albino rats were divided into six groups: control, vanillin-treated, chronic 2-h IS, 2-h stressed-vanillin-treated, chronic 6-h IS, and 6-h stressed-vanillin treated. The rats were sacrificed, and blood samples were collected for biochemical study. The testes were processed for biochemical and histological study, as well as histological Johnsen score. The results showed that prolonged IS increased both corticosterone and TNF-α levels as well as decreased testosterone, luteinizing hormone, catalase, and Nrf2 levels. This effect was more pronounced after 6h of IS compared to 2h. It also induced various testicular injuries with weak ZO-1 and CD34 immunoreactions. On the contrary, vanillin improved all mentioned biochemical and histological alternations induced by stress. Additionally, computational molecular docking analyses were conducted on the compound vanillin within the active site of Zona Occludens-1 (PDB ID: 2JWE). The results demonstrated remarkable docking scores and binding affinity, corroborating its potential protective efficacy. It could be concluded that vanillin is a promising treatment alternative for protecting testicular tissue from the harmful effects of IS via its antioxidant and anti-inflammatory properties.

Suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited blood-testis barrier damage

Infertility caused by lipopolysaccharide (LPS) exposure due to infection is endangering male fertility worldwide, but the mechanism remains unclear. The blood-testis barrier (BTB) is essential for maintaining spermatogenesis and male fertility. In the present study, we showed that LPS (5.0 mg/kg) treatment markedly down-regulated the expression of BTB-related proteins, expanded the biotin penetration distance and caused histopathological injury in seminiferous tubules in mouse testes. Notably, testicular macrophage M1 polarization induced by LPS seems to be related to BTB damage, which was well confirmed by co-culture of RAW264.7 and TM4 cells in vitro. Interestingly, a low-dose LPS (0.1 mg/kg) pretreatment attenuated down-regulation of BTB-related proteins expression and histopathological injury and shorten biotin penetration distance in seminiferous tubules caused by LPS. Correspondingly, a low-dose LPS pretreatment suppresses testicular macrophage M1 polarization induced by LPS in mouse testes. Further experiments revealed that histone deacetylase 5 (HDAC5) was markedly down-regulated at 2 h and slightly down-regulated at 8 h, but up-regulated at 24 h in mouse testes after LPS treatment. Additionally, low-dose LPS pretreatment against the down-regulation of HDAC5 protein caused by LPS treatment. Notably, the suppressed testicular macrophage M1 polarization by low-dose LPS pretreatment was broken by BRD4354, a specific inhibitor of HDAC5 in vitro. These results suggest suppressed testicular macrophage M1 polarization by HDAC5 enforces insensitivity to LPS-elicited BTB damage.

TRPC6-Calpain-1 Axis Promotes Tubulointerstitial Inflammation by Inhibiting Mitophagy in Diabetic Kidney Disease

IntroductionRenal tubulointerstitial inflammation represents an effective indicator for predicting the progression of diabetic kidney disease (DKD). Mitophagy abnormality is one of the most important factors involving in tubule injury. However, the exact molecular mechanism underlying mitophagy abnormality-mediated tubulointerstitial inflammation in DKD remains poorly understood. MethodsHere, Streptozotocin-induced DKD mouse model was established and HK-2 cells treated with high-glucose (HG) served as an in vitro model. The tubular mitophagy was regulated through pharmacological Urolithin A administration. The functional effect of TRPC6 was explored using genetic interventions in vivo and in vitro. ResultsWe found that renal tubulointerstitial inflammation in DKD was closely associated with mitophagy inhibition, which was mediated by disturbance of PINK1/Parkin pathway. Mitophagy activation significantly attenuated tubular injury and tubulointerstitial inflammation. Further, it was found that TRPC6 was markedly increased in DKD and played an essential role in mitophagy inhibition through activating Calpain1. Knockdown of TRPC6 partially reversed mitophagy abnormality and consequently attenuated tubular injury and tubulointerstitial inflammation in vivo and in vitro. Finally, we found that tubular TRPC6-mediated mitophagy inhibition was blocked with BAPTA (a specific Ca2+ chelator) or Calpeptin (a specific Calpain1 inhibitor). ConclusionsOur study reveals that TRPC6-Calpain1 axis promotes tubulointerstitial inflammation in DKD by inhibiting mitophagy.

Injury-induced Foxm1 expression in the mouse kidney drives epithelial proliferation by a cyclin F-dependent mechanism.

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

Integrated network pharmacology and pharmacological investigations to discover the active compounds of Toona sinensis pericarps against diabetic nephropathy

Ethnopharmacological relevanceToona sinensis (A. Juss.) Roem. Is a deciduous woody plant native to Eastern and Southeastern Asia. Different parts of this plant have a long history of being applied as traditional medicines to treat various diseases. The fruits have been used for antidiabetic, antidiabetic nephropathy (anti-DN), antioxidant, anti-inflammatory, and other activities. Aim of the studyThe purpose of this study was to investigate the effects of EtOAc (PEAE) and n-BuOH extracts (PNBE) from T. sinensis pericarps (TSP) on kidney injury in high-fat and high-glucose diet (HFD)/streptozotocin (STZ)-induced DN mice by network pharmacology and pharmacological investigations, as well as to further discover active compounds that could ameliorate oxidative stress and inflammation, thereby delaying DN progression by regulating the Nrf2/NF-κB pathway in high glucose (HG)-induced glomerular mesangial cells (GMCs). Materials and methodsThe targets of TSP 1–16 with DN were analyzed by network pharmacology. HFD/STZ-induced DN mouse models were established to evaluate the effects of PEAE and PNBE. Six groups were divided into normal, model, PEAE100, PEAE400, PNBE100, and PNBE400 groups. Fasting blood glucose (FBG) levels, organ indices, plasma MDA, SOD, TNF-α, and IL-6 levels, as well as renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-β1 levels were determined, along with hematoxylin-eosin (H&E) and immunohistochemical (IHC) analysis of kidney sections. Furthermore, GMC activity screening combined with molecular docking was utilized to discover active compounds targeting HO-1, TNF-α, and IL-6. Moreover, western blotting assays were performed to validate the mechanism of Nrf2 and NF-κB in HG-induced GMCs. ResultsNetwork pharmacology predicted that the main targets of PEAE and PNBE in the treatment of DN include IL-6, INS, TNF, ALB, GAPDH, IL-1β, TP53, EGFR, and CASP3. Additionally, major pathways include AGE-RAGE and IL-17. In vivo experiments, treatment with PEAE and PNBE effectively reduced FBG levels and organ indices, while plasma MDA, SOD, TNF-α, and IL-6 levels, renal tissue Nrf2, HO-1, NF-κB, TNF-α, and TGF-β1 levels, and renal function were significantly improved. PEAE and PNBE significantly improved glomerular and tubule injury, and inhibited the development of DN by regulating the levels of oxidative stress and inflammation-related factors. In vitro experiments, compound 11 strongly activated HO-1 and inhibited TNF-α and IL-6. The molecular docking results revealed that compound 11 exhibited a high binding affinity towards the targets HO-1, TNF-α, and IL-6 (<−6 kcal/mol). Western blotting results showed compound 11 effectively regulated Nrf2 and NF-κB p65 protein levels, and significantly improved oxidative stress damage and inflammatory responses in HG-induced GMCs. ConclusionPEAE, PNBE, and their compounds, especially compound 11, may have the potential to prevent and treat DN, and are promising natural nephroprotective agents.

#2037 Mitochondrial fumarate promotes ischemia/reperfusion-induced tubular injury

Abstract Background and Aims Mitochondrial dysfunction, a characteristic pathological feature of renal Ischemic/reperfusion injury (I/RI), predisposes tubular epithelial cells to maintain an inflammatory microenvironment, however, the exact mechanisms through which mitochondrial dysfunction modulates the induction of tubular injury remains incompletely understood. Method ESI-QTRAP-MS/MS approach was used to characterize the targeted metabolic profiling of kidney with I/RI. Tubule injury, mitochondrial dysfunction and fumarate level were evaluated using qPCR, transmission electron microscopy, ELISA, and immunohistochemistry. Results We demonstrated that tubule injury occurs at the phase of reperfusion in murine models of I/RI. Meanwhile, the enhanced glycolysis and mitochondrial dysfunction are found to be associated with tubule injury. Further, we found that tubular fumarate, which resulted from fumarate hydratase deficiency and released from dysfunctional mitochondria, promotes tubular injury. Mechanistically, fumarate induces tubular injury through causing disturbance of glutathione (GSH) hemostasis. Suppression of GSH with buthionine sulphoximine administration could deteriorate the fumarate inhibition-mediated tubule injury recovery. Reactive oxygen species/NF-κB signaling activation plays a vital role in fumarate-mediated tubule injury. Conclusion Our studies demonstrated that the mitochondrial-derived fumarate promotes tubular epithelial cells injury in renal I/RI. This finding represents a previously unrecognized mechanism for hypoxia-induced TECs injury. Blockade of fumarate-mediated ROS/NF-κB signaling activation may serve as a novel therapeutic approach to ameliorate hypoxic tubule injury.

#1284 Proteinuria causes excessive proliferation and senescence in proximal tubular cells via receptor-mediated endocytosis

Abstract Background and Aims Proteinuria induces proximal tubule (PT) injury, oxidative stress, lysosomal dysfunction, and inflammation, which contribute to the progression of CKD. Cellular senescence and the associated secretory phenotype, where cells secrete proinflammatory and profibrotic mediators, have been linked to renal interstitial inflammation and fibrosis. Premature senescence is induced by excessive proliferation, oxidative stress, or DNA injury. In kidneys, hyperglycemia or hypertension have been reported to elicit this response. However, the direct association between proteinuria-induced PT protein overload and cellular senescence is unknown. We aimed to clarify the effect of protein overload on cellular proliferation and senescence in proteinuric mice with or without knockout (KO) of the receptors responsible for protein endocytosis, megalin and cubilin. Method Podocin-KO (proteinuric mouse model) and megalin-cubilin-podocin triple-KO mice were used in study. Kidneys from these mice were analyzed using immunohistochemical and immunofluorescent staining and the mice were injected with EdU to investigate renal proliferation. mRNA expression of different markers in renal cortex were evaluated with quantitative rt-PCR. To investigate albumin induced cellular proliferation and senescence, immortalized proximal tubular cells (RPTEC/TERT1, ATCC) were treated with human serum albumin (HSA; Sigma), fatty acid-free HSA (Sigma), and transferrin (Sigma). Western blotting (WB) and quantitative rt-PCR, of different markers were conducted and staining for senescence-associated beta-galactosidase (SA-β-gal) was performed. Results PCNA, a proliferation marker, was detected at a higher level in PTs of podocin-KO mice compared to wild-type (WT) mice, and triple-KO mice. Furthermore, we evaluated EdU DNA incorporation, in mosaic megalin and cubilin-expressing triple-KO mice. Coimmunostaining of megalin (Fig. 1-a) or cubilin (Fig. 1-b) with incorporated EdU revealed that most of the EdU-positive nuclei was observed in megalin or cubilin-positive tubules (white arrowheads). A few EdU positive nuclei was detected in megalin or cubilin-negative tubules or the interstitium (yellow arrowheads). The mRNA expression of PDGF- β, a growth factor, was more upregulated in the cortex of podocin-KO mice than in triple-KO mice. These data indicate that protein uptake via endocytic receptors induces secretion of PDGF-β from PT cells, acting in a paracrine manner potentially inducing proliferation. Additionally, immunofluorescent staining revealed that senescence markers, p21 and γ-H2AX, were detected in PTs of podocin-KO mice at a higher level than in triple-KO mice. The mRNA expression of TGF-β (profibrotic) and MCP-1 (proinflammatory) were higher in podocin-KO, than in triple-KO mice in accordance with more senescent cells in podocin KO mice. Upregulation of p21, p16, and γ-H2AX as well as positive staining for SA-β-gal in RPTEC/TERT1 cells, were only detected in HSA (containing free fatty acids)-treated cells in a dose dependent manner and not in cells treated with HSA depleted for free fatty acids or transferrin. Our results show that excessive protein reabsorption generates senescent cells in proximal tubules potentially through increased proliferation elicited by paracrine signaling. The increase in senescent cells was associated to profibrotic and proinflammatory signaling in line with a senescent secretory phenotype. Our studies in PRTEC/TERT1 confirmed our animal experiments and showed that senescence was induced only by fatty acid bound albumin. Conclusion The present study showed that proteinuria directly induced cellular senescence in PTECs via megalin/cubilin endocytosis of filtered protein. Fatty acid bound-albumin is suggested to cause the cellular senescence.

#1738 Renal endogenous hepcidin alleviates ferroptosis of collecting duct in ischemia/reperfusion induced AKI

Abstract Background and Aims Recent studies have demonstrated that urinary and serum hepcidin predicted the incidence of acute kidney injury (AKI) among critically ill patients. As an endogenous acute phase hormone, hepcidin is synthesized not only by the liver but also secreted by distal nephron. However, the autocrine impact of hepcidin secreted by distal nephron on collecting duct cells in AKI remains poorly understood. Method We determined the expression and location of hepcidin in murine kidney in different severity of bilateral ischemia/reperfusion (bIRI) induced AKI model with ischemic duration for 20 min and 32 min. The effect of renal endogenous hepcidin was explored in cultured M-1 (Murine Cortical collecting duct) exposed to hypoxia/reoxygenation (H/R), as well as in bIRI mice with collecting duct-specific overexpression of hepcidin. Transgenic mice were generated by genetic cross between Hepcidin floxed mice which knocked the gene fragment of CAG-LSL-mHamp-flag-WPRE-PolyA into H11 locus and Cdh16-cre mice. Mechanistically, RNA-seq was performed in M-1 cells under H/R condition. Results The expression of renal endogenous hepcidin was significantly elevated in bIRI32min mice compared to both bIRI20min and sham group, suggesting that augmented secretion of hepcidin was induced by more severe AKI. Interestingly, renal endogenous hepcidin was mainly localized to distal convoluted tubule and collecting tubule as indicated by its co-staining with AQP2 and Calbindin-D28k in the kidney. To investigate the role of hepcidin in collecting duct, M-1 cells were cultured under hypoxia condition. We observed remarkable ferroptosis in collecting duct cells other than apoptosis nor necroptosis. Subsequently, hepcidin knockdown further exacerbated ferroptosis under H/R condition, as evidenced by an upregulation of PTGS2 expression and downregulation of GXP4 expression, lower proportion of GSH/GSSG ratio as well as enhanced lipid peroxide accumulation and production of Fe2+. Then, RNA-seq data revealed that hepcidin knockdown promoted the enrichment of genes related to oxidative stress and glutathione metabolic pathway which may be responsible for the deterioration of ferroptosis. To investigate the role of hepcidin in vivo, bIRI induced AKI was established in Cdh16-cre+Hepcidinflox/+ mice. We observed remarkable overexpression of Hepcidin-Flag localized to collecting duct and interstium area. Impressively, Cdh16-cre+Hepcidinflox/+ mice showed ameliorated renal dysfunction adrenal tubule injury histologically. Ferroptosis of renal was alleviated in Cdh16-cre+Hepcidinflox/+ mice as confirmed by an upregulation of GPX4 expression and downregulation of PTGS2, ACSL4, FPN expression, higher proportion of GSH/GSSG ratio, as well as reduced lipid peroxide accumulation and production of Fe2+ by Lipofluo (lipid peroxides) and FerroOrange (Fe2+) staining on frozen section of kidney. Ferroptosis of the collecting duct was alleviated as indicated by downregulation of PTGS2 expression which co-staining with AQP2. To demonstrate the beneficial role of hepcidin, exogenous hepcidin was applied to bIRI induced AKI mice to strengthen the endogenous protective response. Conclusion Renal endogenous hepcidin is induced in the distal tubule and collecting duct after AKI as a protective response via alleviating ferroptosis of collecting duct. Hepcidin may represent a novel therapeutic approach to promote tubule repair by targeting ferroptosis for AKI.

#1333 SGLT2 inhibitors as a treatment for Fanconi-Bickel syndrome: a case report

Abstract Background and Aims Fanconi-Bickel syndrome is an ultra-rare genetic disease characterized by SLC2A2 mutation encoding for the basolateral glucose transporter (GLUT2) in the proximal tubule of the nephron. This defective transport ultimately leads to accumulation of glycogen and dysfunction of proximal tubule cells. This manifests clinically as the renal Fanconi syndrome and eventually kidney failure. Systemic complications of proximal tubule injury include metabolic acidosis, bone demineralization and, for some not completely elucidated reasons, dysglycemia. Method We present the case of a 43-year-old man who was diagnosed with Fanconi-Bickel syndrome (homozygous splice site mutation in SLC2A2). He had a history of infantile-onset renal Fanconi syndrome, hypercalciuria and bilateral nephrolithiasis, osteoporosis, bilateral sensorineural deafness and non-insulin dependent type 2 diabetes. At referral the patient presented with CKD G2, non-anion gap metabolic acidosis, slightly elevated serum calcium, decreased serum phosphate, suppressed PTH and hypercalciuria (Table 1), despite oral supplementation of phosphate, bicarbonate and vitamin D. We hypothesized that SGLT2i in this patient may halt the progressive proximal tubule injury by preventive glucose uptake and help the management of diabetes. Results Fractional excretion of phosphate (FEPO4) and urinary retinol-binding protein (RBP)-creatinine ratio, two markers of proximal tubular injury, were stable after 2 months of SGLT2i therapy (Fig. 1). Conclusion To date, Fanconi-Bickel syndrome does not have a specific treatment. Standard of care includes supplements of fluids and electrolytes to counterbalance proximal tubular injury and management of complications such as diabetes. SGLT2i use has been described in a mouse model of another glycogen storage disease (GSD1b), whose underlying defect is different from that of Fanconi-Bickel but presents with a similar phenotype [2]. Data showed that gliflozins prevents glycogen accumulation and restores proximal tubule cells’ function. Using the same rationale, the administration of SGLT2i in adult patients with Fanconi-Bickel syndrome may prevent further proximal damage and slow the progression towards end-stage kidney disease. In conclusion, we hypothesize a potential benefit of SGLT2i in adult patients affected by Fanconi-Bickel syndrome.

#2565 Urine amino acid profiles enlighten new prediction models for renal Fanconi syndrome and its association with eGFR level

Abstract Background and Aims Renal Fanconi syndrome (FS) presented dysfunctions of renal proximal tubular (PT) transport with or without excessive urine amino acids (AAs) excretion, which might contribute to the progression of eGFR decline. This study aimed to explore the clinical benefit of 21 urinary AAs excretion patterns in renal FS and eGFR decline. Method Liquid chromatography-tandem Mass spectrometry (LC-MS-MS) is currently quantitative and detects over 21 types of amino acids in 157 patients with renal proximal tubular dysfunctions. To evaluate the diagnostic values of urine AAs in renal FS, we established a machine learning algorithm to perform binary classification using Random Forest Regressor in Python (version 3.8.8) with a training set and a testing set (8:2). We used Randomized search to find out the best model hyperparameters, calculated feature importance for contribution evaluation, and Receiver Operating Characteristic (ROC) curves to evaluate the performance of classification algorithms. Results A model based on a machine learning algorithm of all urine AAs to diagnose renal FS established with sensitivity = 0.71, specificity = 0.73, and AUC = 0.83 (95% CI 0.648-0.957) (Fig. 1), with similar performance in combined six urine AAs (citrulline, asparagine, threonine, phenylalanine, ornithine, and proline) models. The urine proline level independently correlated with eGFR &amp;lt; 60 ml/min/1.73 m2 (bias regression coefficient 0.84, P = 0.001, OR = 2.31, 95% CI 1.41-3.80) when adjusted by age, etiology, gender, and blood urea nitrogen level. For the primary diseased diagnosis, the most susceptible urine AAs were citrulline (56.7%), proline (56.0%,) threonine (52.2%), and ornithine (45.9%) and the less susceptible ones were histidine (24.8%), aspartate (16.6%), and arginine (13.4%). Accordingly, the most common affected AA transporters were SLC6A19 (B0AT1) and etiology-specific urine AA atlas major in the eight urine AAs (isoleucine, glutamine, methionine, proline, alanine, ornithine, lysine, and arginine). Different from primary Sjӧgren syndrome patients (n = 26) with less susceptible, the patients with plasmacyte diseases (n = 8) showed elevations of almost all the urine AAs except glycine and aspartate. The connection between urine AAs excretion and hypouricemia showed gender differences, with 20 urine AAs significantly negatively correlating with serum uric acid levels in males but only five in females. Conclusion We first established a model based on the urine AA atlas detected by (LC-MS-MS) which contributed to differentiating the etiologies of renal tubular dysfunctions and associated well with eGFR. It shed light on the clinical practice in precision evaluation of tubule injury.

Zn2+ improves sepsis-induced acute kidney injury by upregulating SIRT7-mediated Parkin acetylation.

Zn2+ levels are reported to be correlated with kidney function. We explored the significance of Zn2+ in sepsis-induced acute kidney injury (SI-AKI) through the regulation of sirtuin 7 (SIRT7) activity. The sepsis rat model was established by cecal ligation and perforation (CLP) and intraperitoneally injected with ZnSO4 or SIRT7 inhibitor 97491 (SIRT7i), with renal tubular injury assessed by hematoxylin and eosin staining. In vitro, human renal tubular epithelial cells (HK-2) were induced with lipopolysaccharide to obtain a renal injury cell model, followed by ZnSO4 or SIRT7i and autophagy inhibitor (3-methyladenine) treatment. Interleukin (IL)-1β, IL-18, reactive oxygen species (ROS), Parkin acetylation level, kidney injury molecule-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) expression levels were determined. The renal tubule injury, inflammation condition, and pyroptosis-related and autophagy-related protein levels were assessed. The pyroptosis in kidney tissues and autophagosome formation were observed by transmission electron microscopy. Zn2+ alleviated renal injury in CLP rats and inhibited pyroptosis and its related protein levels by inhibiting SIRT7 activity in septic rat renal tissues. In vitro, Zn2+ increased HK-2 cell viability and reduced KIM-1, NGAL, IL-1β, IL-18, NLRP3 inflammasome, cleaved caspase-1, gasdermin D-N levels, and pyroptotic cell number. Zn2+ increased autophagosome number and LC3BII/LC3BI ratio and decreased TOM20, TIM23, P62, and mitochondrial ROS levels. Zn2+ increased Parkin acetylation by repressing SIRT7 activity. Inhibiting mitophagy partially averted Zn2+-inhibited NLRP3 inflammasome activation and apoptosis in HK-2 cells. Zn2+ upregulated Parkin acetylation by repressing SIRT7 activity to promote mitophagy and inhibit NLRP3 inflammasome activation and pyroptosis, thus improving SI-AKI.NEW & NOTEWORTHY Zn2+ upregulated Parkin acetylation by repressing sirtuin 7 activity to promote mitophagy and inhibit NLRP3 inflammasome activation and pyroptosis, thus improving sepsis-induced acute kidney injury.

RUNX3 alleviates mitochondrial dysfunction and tubular damage by inhibiting TLR4/NF-κB signalling pathway in diabetic kidney disease.

The impaired function of tubular mitochondria is critical in diabetic kidney disease (DKD) progression. RUNX3 is down-regulated in DKD models. We intend to explore the effects of RUNX3 on mitochondrial dysfunction and renal tubule injury in DKD and related mechanisms. The development of diabetes models involved injecting mice with streptozotocin while treating HK-2 cells with high glucose (HG). By using immunohistochemical techniques, the renal localizations of RUNX3 were identified. Levels of adenosine triphosphate (ATP), mitochondrial membrane potential, and biochemical index were detected by appropriate kits. Reactive oxygen species (ROS) generation was assessed with dihydroethidium and MitoSOX Red staining. Apoptosis was assessed by flow cytometry and TUNEL. RUNX3 ubiquitination was measured. RUNX3 was mainly present in renal tubules. Overexpressing RUNX3 increased Mfn2, Mfn1, ATP levels, and mitochondrial membrane potential, reduced Drp1 and ROS levels and cell apoptosis, as well as Cyt-C release into the cytoplasm. RUNX3 overexpression displayed a reduction in urinary albumin to creatinine ratio, Hemoglobin A1c, serum creatinine, and blood urea nitrogen. Overexpressing TLR4 attenuated the inhibitory effect of RUNX3 overexpression on mitochondrial dysfunction and cell apoptosis. HG promoted RUNX3 ubiquitination and SMURF2 expression. RUNX3 knockdown cancelled the inhibitory effect of SMURF2 on mitochondrial dysfunction and cell apoptosis. SMURF2 interference inhibits RUNX3 ubiquitination and TLR4/NF-κB signalling pathway, thereby alleviating renal tubule injury.