Kidney Tissue Research Articles

Assessment of the effect of sodium tetraborate on oxidative stress, inflammation, and apoptosis in lead-induced nephrotoxicity

Exposure to Pb, a toxic heavy metal, is a risk factor for renal damage. Borax, an essential trace element in cellular metabolism, is a naturally occurring compound found in many foods. This study investigated the effects of sodium tetraborate (ST), a source of borax, on renal oxidative stress and inflammation in rats exposed to Pb. Wistar Albino rats (n = 24) were divided into four groups: Control (0.5 mL, i.p. isotonic), Pb (50 mg/kg/day/i.p.), ST (4.0 mg/kg/day/oral), and Pb + ST groups. At the end of the five-day experimental period, kidney tissue samples were obtained and analyzed. Histopathologically, the Pb-induced damage observed in the Pb group improved in the Pb + ST group. Immunohistochemically, Pb administration increased the expression of inducible nitric oxide synthase, cyclooxygenase-2, and caspase-3. When evaluated biochemically, Pb application inhibited catalase and glutathione peroxidase (GSH-Px) enzyme activities and activated superoxide dismutase enzyme activity. An increase in malondialdehyde levels was considered an indicator of damage. ST application increases glutathione peroxidase enzyme activity and decreased malondialdehyde levels. These results indicate that ST might play a protective role against Pb-induced renal damage via the upregulation of renal tissue antioxidants and cyclooxygenase-2, inducible nitric oxide synthase, and caspase-3 immunoexpression.

The inhibitory impact of Schisandrin on inflammation and oxidative stress alleviates LPS-induced acute kidney injury.

Inflammation and oxidative stress (OS) are the major pathogenic characteristics of acute kidney injury (AKI). Studies have shown that Schisandrin (Sch) could regulate inflammatory disease. However, the function and mechanism of Sch in AKI progression are still unknown. Here, we investigated Sch's potential effects and mechanism on mice's renal damage and macrophages induced by lipopolysaccharide (LPS). Sch decreased LPS-induced inflammatory factor production while increasing the activity of related antioxidant enzymes in macrophages and mouse kidney tissues. Hematoxylin and eosin staining revealed that Sch may have the ability to profoundly inhibit inflammatory cell invasion and tissue damage caused by LPS in renal tissue. Furthermore, Western blot and immunohistochemical studies showed that Sch exerted its effects mainly through up-regulation of nuclear factor erythroid 2-related factor 2/heme oxygenase-1 and inhibition of Toll-like receptor 4‒mitogen-activated protein kinases/nuclear factor-kappa B pathways. Collectively, this study illustrates that Sch suppresses LPS-stimulated AKI by descending inflammation and OS, illuminating prospective AKI treatment options.

Unraveling the role of natriuretic peptide clearance receptor (NPR3) in glomerular diseases

Natriuretic peptides (NPs) are cardio-derived hormones that have a crucial role in maintaining cardiovascular homeostasis. Physiological effects of NPs are mediated by binding to natriuretic peptide receptors 1 and 2 (NPR1/2), whereas natriuretic peptide receptor 3 (NPR3) acts as a clearance receptor that removes NPs from the circulation. Mouse studies have shown that local NP-signaling in the kidney glomerulus is important for the maintenance of renal homeostasis. In this study we examined the expression of NPR3 in kidney tissue and explored its involvement in renal physiology and disease by generating podocyte-specific knockout mice (NPR3podKO) as well as by using an NPR3 inhibitor (NPR3i) in rodent models of kidney disease. NPR3 was highly expressed by podocytes. NPR3podKO animals showed no renal abnormalities under healthy conditions and responded similarly to nephrotoxic serum (NTS) induced glomerular injury. However, NPR3i showed reno-protective effects in the NTS-induced model evidenced by decreased glomerulosclerosis and reduced podocyte loss. In a ZSF1 rat model of diabetic kidney injury, therapy alone with NPR3i did not have beneficial effects on renal function/histology, but when combined with losartan (angiotensin receptor blocker), NPR3i potentiated its ameliorative effects on albuminuria. In conclusion, these results suggest that NPR3 may contribute to kidney disease progression.

Linagliptin Mitigates TGF-β1 Mediated Epithelial-Mesenchymal Transition in Tacrolimus-Induced Renal Interstitial Fibrosis via Smad/ERK/P38 and HIF-1α/LOXL2 Signaling Pathways.

The dipeptidyl peptidase-4 (DPP-4) inhibitors, a novel anti-diabetic medication family, are renoprotective in diabetes, but a comparable benefit in chronic non-diabetic kidney diseases is still under investigation. This study aimed to elucidate the molecular mechanisms of linagliptin's (Lina) protective role in a rat model of chronic kidney injury caused by tacrolimus (TAC) independent of blood glucose levels. Thirty-two adult male Sprague Dawley rats were equally randomized into four groups and treated daily for 28 d as follows: The control group; received olive oil (1 mL/kg/d, subcutaneously), group 2; received Lina (5 mg/kg/d, orally), group 3; received TAC (1.5 mg/kg/d, subcutaneously), group 4; received TAC plus Lina concomitantly in doses as the same previous groups. Blood and urine samples were collected to investigate renal function indices and tubular injury markers. Additionally, signaling molecules, epithelial-mesenchymal transition (EMT), and fibrotic-related proteins in kidney tissue were assessed by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, immunohistochemical and histological examinations. Tacrolimus markedly induced renal injury and fibrosis as indicated by renal dysfunction, histological damage, and deposition of extracellular matrix (ECM) proteins. It also increased transforming growth factor β1 (TGF-β1), Smad4, p-extracellular signal-regulated kinase (ERK)1/2/ERK1/2, and p-P38/P38 mitogen-activated protein kinase (MAPK) protein levels. These alterations were markedly attenuated by the Lina administration. Moreover, Lina significantly inhibited EMT, evidenced by inhibiting Vimentin and α-smooth muscle actin (α-SMA) and elevating E-cadherin. Furthermore, Lina diminished hypoxia-related protein levels with a subsequent reduction in Snail and Twist expressions. We concluded that Lina may protect against TAC-induced interstitial fibrosis by modulating TGF-β1 mediated EMT via Smad-dependent and independent signaling pathways.

Effects of organophosphates on precision-cut kidney slices

Organophosphate (OP) poisoning, both accidental and with suicidal intent, is a global medical challenge. While the primary toxicity of these pesticides is based on the inhibition of acetylcholinesterase (AChE), case reports describe patients developing OP-mediated renal insufficiency. We set out to investigate possible pathomechanisms utilizing rat precision-cut kidney slices (PCKS). Depending on the method of investigation, PCKS were observed for a maximum of 10 days. PCKS exposed to OP compounds (malaoxon, malathion, paraoxon, parathion) showed a dose-dependent loss of viability and a reduction of total protein content over the course of 10 days. A concentration of 500 µM OP showed the most differences between OP compounds. After two days of incubation parathion showed a significantly lower level of viability than malathion. The respective effects of paraoxon and malaoxon were not significantly different from the control. However, effects of OP were only observed in concentrations exceeding those that were needed to achieve significant AChE inhibition in rat kidney tissue. In addition, we observed histological changes, without inducing LDH leakage. Overall, results suggest that OP exert effects in kidney tissue, that exceed those expected from the sole inhibition of AChE and vary between compounds. Without signs of necrosis, findings call for studies that address other possible pathomechanisms, including inflammatory response, oxidative stress or activation of apoptosis to further understand the nephrotoxicity of OP compounds. Monitoring oxon concentration over time, we demonstrated reduced enzyme-inhibiting properties in the presence of PCKS, suggesting interactions between OP compound and kidney tissue.

#1181 The relationship between renal ischaemia-reperfusion injury and circadian rhythm

Abstract Background and Aims Kidney disease is a global health problem in which various aetiologies contribute to renal injury and dysfunction. Renal ischaemia-reperfusion injury (IRI) is a condition caused by early oxidative stress due to renal tissue ischaemia and a rapid immune response after reperfusion. Circadian rhythms are important regulators of metabolism and their disruption can lead to heart disease and fibrosis in various organs. Although recent reports have shed light on the impact of renal injury and disease on the renal circadian clock, the exact mechanisms by which circadian rhythm genes contribute to the progression of renal dysfunction in IRI and the onset of chronic kidney disease (CKD) are not fully understood. Method Male Wistar rats (8-9 weeks old, 250-280 g body weight) were used to establish the I/R model. To induce renal I/R injury, the left renal artery was clamped for 30 or 45 min and then removed to restore blood flow. Simultaneously, nephrectomy of the right kidney was performed; renal tissue and blood were collected 14 days after I/R. HE staining, Sirius red staining and immunostaining (F4/80 staining) were performed. Kidney tissue was RNA-seq'd and qPCR'd for circadian rhythm genes and fibrosis genes involved in kidney failure that were altered by I/R. Kidney tissue was also RNA-seq'd and qPCR'd for circadian rhythm genes and fibrosis genes involved in kidney failure. We also established an in vitro hypoxia-reoxygenation (H/R) model using single cell RPTECs and performed qPCR and Western blotting. Results In the IRI rat group, the expression of circadian repressor genes Nr1d1 and Nr1d2, which are important for reducing inflammation, decreased, while the expression of Bmal1 and Cry1, which regulate renal deterioration, increased. Using RNA-seq, we also identified the involvement of the TGFβ pathway in linking the circadian clock to kidney injury and fibrosis, and observed similar results in the H/R group using RPTEC cells in vivo. Conclusion Fibrosis and circadian rhythms were found to be closely related in renal IRI. The findings described here may help in the development of new therapeutic approaches to ameliorate the effects of renal IRI and improve the outcome of kidney disease, including kidney transplantation, offering hope to patients worldwide.

#799 M1-M2 macrophages in postmortem biopsies from patients with acute kidney injury with COVID-19 compared to pulmonary bacterial sepsis

Abstract Background and Aims AKI associated with COVID19 is highly prevalent; there is controversy if it is similar to AKI in sepsis of bacterial origin. Macrophage differentiation M1-M2 is a predictor of outcomes. Macrophage expression in kidney tissue is not known in patients with severe COVID19 compared to sepsis of bacterial origin. We compare the tissue expression of macrophage differentiation markers M1-M2, through the expression of CD163, CD68, INOS in postmortem biopsies from patients with C19-LRA and S-AKI. Method Observational, comparative, analytical study. Results 49 subjects were analyzed, 39(COVID-19) and 10(pulmonary sepsis of bacterial origin). No significant baseline differences. The degree of AKI had no difference between groups, but in COVID-19 there were severe grades 2-3, compared to bacterial sepsis p = 0.89. Histopathologically there were no differences with the degree of LTA and IFTA. NTI was more frequent in bacterial sepsis vs COVID-19 100%/12.8% (p < 0.001). When comparing macrophage expression we found that the expression of M1(INOS) was not different, the expression of M2(CD163) was higher in patients with bacterial Sepsis with 100% corresponding to severe grade vs 28.2% in patients with COVID19 p>0.001. The classification between degrees of AKI were statistically positive for patients with COVID19 and CD163 with r2: 0.26 and CD68 with r2: 0.36 with p 0.01 and 0.02 respectively. The classification with NTI was positive in COVID19 and bacterial sepsis with an r2: 0.34 for CD163, r2: 0.11 for CD68 p < 0.001. Regarding IFTA in COVID-19 and bacterial sepsis, r2: 0.12 for CD163, r2: 0.11 for CD68 (p 0.01 and 0.03 respectively). Steroid use was negatively correlated on the COVID19 with bacterial sobreinfectión + the bacterial sepsis group with the marker CD163 with r2: 0.23 p = 0.005. The association between moderate and severe positivity for CD68+ and NTI had an OR of 15.5 (CI 3.21-64.6) p = 0.001. Conclusion The 2 groups had a similar severity, AKI in COVID19 was more severe compared to bacterial Sepsis, histologically there were no differences in AKI, but there was a marked difference in the presence of NTI, this being more frequent in the bacterial sepsis group. Findings different from those reported in other bibliographies where the degree of acute kidney injury was higher in COVID19. When comparing the dominant macrophage phenotype, iNOS(M1) had no difference between the groups, on the contrary CD163(M2) was different, with severe staining in all cases with bacterial sepsis, similar to what was seen with NTI and lower degrees of clinical severity of LRA. When evaluating the correlations we found a moderate correlation between the presence of NTI and CD163(M2), mild for NTI and CD68(M0). Finding suggestive of the macrophage role especially low M2 in the context of NTI, it is important to note that those with CD163 had lower degrees of acute kidney injury, more reported as KDIGO1, which is reproducible taking into account other studies in which the CD163(M2) was associated with higher degrees of repair, while their depletion was associated with greater severity of kidney injury. With respect to the treatment established, there was a slight correlation between the use of steroids in the population with bacterial superinfection with CD163(M2), in a recent study it was shown that the use of steroids could induce the repopulation of CD163.

#2485 CellMatchR—RNA-seq based matching of kidney cell lines to kidney reference cell types

Abstract Background and Aims Cultured kidney cell lines are broadly used to model the physiology and pathophysiology of the kidney. Due to immortalization, passaging and culture conditions, a cell line's gene expression profile might differ greatly from the primary cells it was originally derived from. This might be further enhanced by experimental treatments with compounds or genetic modifications. It is therefore a relevant question if a given cell culture system is still the appropriate model for a certain disease or scientific investigations. Reasoning that RNA sequencing (RNA-seq) based transcriptomes are generated as part of many experimental setups and hence may be used to address this question, we developed and tested an RNA-seq based approach, CellMatchR. This approach matches kidney cell lines, primary cells and even tissue specimen to known kidney reference cell types to determine which primary kidney cell type is the most similar and to what degree global gene expression or the expression of selected marker genes differs. Method CellMatchR uses published murine and human kidney single cell- and tubule-level transcriptomic datasets from healthy mice and human donors as references. Single cell datasets were further processed to pseudobulk references for each of the contained cell types. Then RNA-seq data from cell lines or tissues of interest (test data) was compared to the reference (pseudo)bulk data with Spearman correlations of gene counts-per-million values (CPM) or Euclidean distance using log-transformed CPM values. Both approaches were systematically tested for various combinations of test and reference datasets across different species and using global gene expression (i.e. all genes measured in reference and test datasets) and a set of 315 manually curated, kidney cell type marker genes. Results We sequentially used different kidney tissue types, primary cells and cell lines as positive controls and matched them to our reference datasets. Spearman correlations of gene expression rankings showed the highest correlation coefficients with biologically correct reference cell types (examples in Fig. 1A-C). We found Spearman correlations to be superior to Euclidean distances as method of comparison. Analyses based on global gene expression compared to our curated set of 315 kidney cell type marker genes yielded similar results. Notably, correlation coefficients were higher and showed less variation between reference cell types when using the global gene set. Matchings across species, i.e. using murine test and human reference data or vice versa still yielded mostly correct results. However, correlation coefficients were generally lower (i.e. rho = 0.9 vs. 0.6) and varied more across reference datasets if comparisons were performed across species. Using published RNA-seq data for different cell lines of the proximal tubule (e.g. human HK-2 and opossum OKH cells) we observed low similarities with proximal tubule cells (Fig. 1D), whereas two tested cell lines of the collecting duct (mIMCD-3 and mpkCCD cells) plausibly showed the highest similarity to medullary cell types like collecting duct and Loop of Henle cells. The most similar reference cell types did not change in mIMCD-3 cells when kept in 2-dimensional vs. 3-dimensional culture conditions, and in mpkCCD cells when the osmolality of the culture medium was changed from 300 to 600 mosmol/kg. Also, a Pkd1 knockout in mIMCD-3 cells did not change the most similar reference cell type in our analyses (Fig. 1E and F). Conclusion Our CellMatchR approach uses publicly available kidney single cell and bulk RNA-seq datasets and combines these with simple, computationally fast yet effective statistical methods to determine similarities of kidney cell lines and tissue samples to reference cell types of interest. It can easily be implemented by trained users or integrated into online resources for usage with a visual interface. It relies on RNA-seq data from the cell lines of interest and hence presents a feasible complementary method to check the general similarity of cell culture models to kidney cell types and assess their stability across experimental conditions.

#2305 Mice with a PAX2 missense variant are susceptible to experimental focal segmental glomerulosclerosis (FSGS) and display impaired glomerular repair

Abstract Background and Aims Focal segmental glomerulosclerosis (FSGS) is a histopathologic lesion observed in chronic kidney disease characterized by scarring of the glomerulus due to the injury and irreversible loss of highly specialized glomerular epithelial cells called podocytes. Recent studies suggest that podocytes may be regenerated by another population of glomerular epithelial cells called parietal epithelial cells (PECs), although this remains controversial. PAX2 is a transcription factor protein that regulates glomerular development and its expression persists in adult parietal epithelial cells (PECs), postulated to serve as a reservoir for podocytes in the event of injury and loss. Our team previously found that PAX2 pathogenic missense variants account for 4% of adult FSGS. In our present study, our aim is to determine whether PEC-mediated podocyte regeneration is impaired in mice with a pathogenic Pax2 missense variant (Pax2-MV). Method FSGS was induced in wildtype and Pax2-MV mice using Adriamycin. Urine samples were collected from mice every week for 28 days to measure proteinuria. At 28 days after Adriamycin injury, kidneys were collected for histopathology, transmission electron microscopy, and immunofluorescence staining for PAX2 and podocyte marker WT-1. Glomeruli from Adriamycin-injured mice were also isolated through intra-cardiac perfusion with Dynabeads and subjected to mass-spectrometry for proteomics analysis. Proteins with statistically-significant fold changes in expression levels were further analyzed by Gene Ontology to determine biological and molecular processes involved in pathogenesis. Kidney tissue from an FSGS patient with a pathogenic PAX2 missense variant was also examined by histopathology and PAX2 immunohistochemistry. Results Compared to wildtype, Pax2-MV mice were more susceptible to worsened FSGS after Adriamycin, as demonstrated by worsened glomerular scarring, increased podocyte injury and loss, and increased proteinuria, associated with increased morality in Pax2-MV mice (Fig. 1A-B). Wildtype mice showed fewer PAX2-expressing PECs after injury, accompanied by the occurrence of PAX2/WT-1 co-expressing glomerular tuft cells (Fig. 2A-B). These suggest that in Adriamycin-injured wildtype, PAX2-expressing PECs move to the glomerular tuft, where they trans-differentiate to a podocyte fate. In contrast, Pax2-MV mice showed no changes in numbers of PAX2-expressing PECs after injury, associated with fewer PAX2/WT-1 co-expressing glomerular tuft cells compared to injured wildtype (Fig. 2A-B). These suggest that in Adriamycin-injured Pax2-MV mice, PAX2-expressing PECs are unable to move to the glomerular tuft, resulting in decreased numbers of cells undergoing podocyte trans-differentiation. A subset of ectopic PAX2-expressing glomerular tuft cells after injury was increased in Pax2-MV mice (Fig. 2A), suggesting a pathologic process given the worse outcomes observed in these mice. Consistent with findings from injured Pax2-MV mice, we found that in an FSGS patient with a pathogenic PAX2 missense variant, PAX2-expressing glomerular tuft cells overlap with areas of glomerular scarring, while in a normal human kidney biopsy, PAX2 expression is only observed in PECs but not within glomerular tuft cells (Fig. 2A). Lastly, proteomics Gene Ontology analysis of isolated glomeruli demonstrated biological and molecular processes indicative of maladaptive glomerular repair in injured Pax2-MV mice, including decreased regulation of cell shape, decreased cell-cell junction assembly and decreased actin and cadherin binding compared to wildtype (Fig. 2C). Conclusion Altogether, our findings suggest that PAX2-expressing PECs migrate to the glomerular tuft to assist in podocyte regeneration in wildtype. However, this mechanism is impaired in Pax2-MV mice, leading to worsened podocyte loss and injury and further exacerbation of glomerular disease (Fig. 2C). Our findings provide a further understanding of normal and impaired regeneration mechanisms in FSGS, and our identified pathogenic mechanisms can be further harnessed for future therapeutic development.

#1521 EZH2 promotes cyst growth in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the leading hereditary kidney disease worldwide. Epigenetic modulations of genes and proteins play important roles in ADPKD pathogenesis. In this study, the role of enhancer of zeste homolog 2 (EZH2), a histone lysine methyltransferase, was investigated in ADPKD. Method To elucidate the role of EZH2 in ADPKD pathogenesis, we employed inducible knockout models, specifically targeting Pkd1 alone (Pkd1fl/fl) or both Pkd1 and EZH2 (Pkd1fl/fl: Ezh2fl/fl) in mouse models. Molecular analyses included assessing EZH2 expression levels and H3K27 trimethylation in Pkd1 knockout cells, kidney tissues from Pkd1 knockout mice, and samples from ADPKD patients. Functional investigations involved the use of EZH2-specific inhibitors, GSK126 and EPZ-6438, to inhibit EZH2 activity. Additionally, Ezh2 knockdown was employed to assess its impact on cystogenesis in MDCK cells. The embryonic kidney cyst model was utilized to evaluate the effects of GSK126 and EPZ-6438 on cyst growth. Results We found that EZH2 expression and the trimethylation of lysine 27 on histone H3 (H3K27) were upregulated in Pkd1 knockout cells and kidney tissues of Pkd1 knockout mice and ADPKD patients. Using EZH2-specific inhibitors GSK126/EPZ-6438 and knockdown of Ezh2 both suppressed cystogenesis in MDCK cells. GSK126 and EPZ-6438 delayed cyst growth in the embryonic kidney cyst model. Double knockout of Pkd1 and Ezh2 significantly decreased the kidney volume, cyst indexes, H3K27 methylation level, and the proliferation of renal cystic epithelial cells compared with Pkd1 knockout mice. Moreover, GSK126 delayed cyst growth and protected renal function in both early- and late-onset Pkd1 conditional knockout mice. EZH2 promoted the proliferation of renal cystic epithelial cells by activating STAT3 through H3K27 methylation. Conclusion Inhibiting EZH2 emerged as a promising strategy to impede cyst growth and attenuate disease progression across various PKD models. These findings establish a molecular foundation for the potential use of EZH2-specific inhibitors in delaying ADPKD cyst growth in the future.

#966 Targeting cAMP in ADPKD by small molecule activation of long form PDE4 enzymes

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disorder, affecting over 12 million people worldwide. ADPKD is characterized by the uncontrolled growth of fluid-filled cysts in the kidney that can lead to kidney enlargement, loss of function, and associated complications. Currently, there are limited treatment options for ADPKD patients. By activating long form PDE4 enzymes and increasing cAMP hydrolysis, Mironid®’s first-in-class small molecule LoAc® compounds [1] directly target the increased kidney cAMP levels that are known to drive cyst formation in ADPKD, resulting in decreased cyst growth in vitro and in vivo. Here we present translational data demonstrating that LoAc® compound MR-L22 lowers renal cAMP after single or multiple doses and delivers efficacy in the Pkd1RC/RC mouse model of ADPKD, alongside favourable differentiation from tolvaptan, the only therapy approved for ADPKD patients. Method To demonstrate cAMP lowering in urine, male Han Wistar rats (n = 6) were dosed p.o. with 100 mg/kg MR-L22 or vehicle (1% methylcellulose, 0.2% Tween 80) either once, or on 7 consecutive days. Urine was collected on Day 1 or Day 7. To assess the ability of MR-L22 to ameliorate disease progression alone, or in combination with tolvaptan, doses of MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow), both expected to be individually sub-maximally efficacious, were administered singly, or in combination in F1 hybrid progeny Pkd1RC/RC mice (8 m and 8f/group), from 4-16 weeks of age. Urine measurements were taken after 8 weeks of dosing. Total kidney volume (TKV) was measured by MRI prior to randomisation and termination. cAMP in urine and kidney tissue were measured by ELISA. Results A 100 mg/kg oral dose of MR-L22 in rat resulted in a >40% decrease in cAMP in urine compared to vehicle. This effect was maintained over multiple days of dosing demonstrating that urinary cAMP can be used as a biomarker of LoAc® target engagement. Consistent with the mechanism of action, a small increase in urine production and commensurate decrease in urine osmolality was observed. To assess the ability of MR-L22 to ameliorate disease progression in an in vivo model of ADPKD, and to determine whether additive or synergistic effects might be seen in combination with tolvaptan, MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow) were dosed individually and in combination in the Pkd1RC/RC mouse model of ADPKD. Alongside a reduction in urinary cAMP, MR-L22 delivered reductions in TKV/Bw (Fig. 1), urinary and tissue cAMP, cystic index, terminal blood urea nitrogen (BUN) and creatinine, as well as trending reductions in urinary NGAL. Conclusion We have shown that in rat, acute or multiple dosing, of LoAc® compound MR-L22 results in a significant reduction of urinary cAMP and a small, but measurable, increase in urine production and commensurate decrease in urine osmolality. Furthermore, we have demonstrated that significantly reduced urinary and kidney cAMP levels correlate with reductions in TKV, cystic index, Kw/Bw, BUN and urinary NGAL in the Pkd1RC/RC model of ADPKD. In these studies, we have demonstrated beneficial differentiation vs tolvaptan on limiting aquaretic effects but been unable to conclusively demonstrate additive or synergistic effects of MR-L22 combined with tolvaptan due to experimental limitations; Further experiments will be needed to answer this question. Looking forwards, measurements of urine cAMP and urine osmolality will be measured as supportive of target engagement in phase 1 studies. Positive effects on TKV, seen in vivo in the Pkd1RC/RC model, will be assessed in a phase 2 clinical trial. By directly targeting cAMP, LoAc® compounds have the potential to slow renal disease progression, and to delay or prevent the need for dialysis, offering a potential new treatment option for ADPKD patients.

#162 Single-nucleus RNA-seq of biopsy-confirmed IgA nephropathy reveals cell-specific transcriptomic alteration

Abstract Background and Aims Single-nucleus RNA-seq (SnRNA-seq) is a valuable method for identifying cell-specific transcriptomic alterations in kidney cells. Our objective was to investigate SnRNA-seq data from biopsy-confirmed IgA nephropathy (IgAN) cases and identify cell-specific alterations in the transcriptome. Method We collected snap-frozen kidney biopsy tissues from 6 cases of IgA nephropathy (IgAN) and 7 nephrectomy control cases. The disease control group included 3 cases of diabetic kidney disease, 6 cases of minimal change disease, and 6 PLA2R-Ab positive membranous nephropathy cases. SnRNA-seq was performed on the kidney tissues, and kidney cells were identified in the dataset through UMAP clustering. We investigated differences in the proportion of kidney cell types and identified differentially expressed genes (DEGs). Additionally, we searched for representative gene loci previously reported in a multiethnic genome-wide association study (GWAS). Results We successfully collected transcriptomic profiles of >50 000 cells from IgAN, with a considerable enrichment of intraglomerular cells. The DEG analysis revealed a high number of DEGs in mesangial cells, fibroblasts, and vascular smooth muscle cells in the IgAN transcriptome compared to the nephrectomy and disease control cases. In the gene set enrichment analysis, the epithelial-mesenchymal transition pathway was enriched in the glomerular cells of IgAN. Furthermore, among various GWAS loci, ETS1 was the gene significantly highly expressed in the mesangial cells of IgAN, consistent across the compared control groups. Conclusion This investigation represents the largest SnRNA-Seq profiling of IgAN kidney cells to date. We identified notable enriched cell-specific pathways in IgAN. ETS1 may be a kidney-indwelling causal factor for IgAN pathophysiology, considering its notable significance in both previous GWAS results and the current SnRNA-Seq data.

#2050 APRT deficiency: insights from in vitro modelling of DHA-induced kidney injury and identification of therapeutic target

Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in renal excretion of 2,8-dihydroxyadenine (DHA) in excessive amounts, leading to kidney stones and crystal nephropathy with associated inflammation and fibrosis. The effects of other kidney stone constituents, such as calcium oxalate and uric acid, have been thoroughly studied in cell culture models, both in monolayer and three-dimensional (3D) assays. However, the effect of DHA in cell culture models has not yet been investigated. This study aimed to establish a comprehensive cell culture model to investigate DHA crystal-induced kidney injury and to identify therapeutic targets for clinical intervention. Method Three lines of kidney cells (MDCK, HK-2, and HEK293) were utilized in monolayer and 3D assays, employing both liquid-liquid interface (LLI) and “on-top” of Matrigel culture models. DHA exposure, matching the quantities found in the urine of untreated patients with APRT deficiency, was used in conjunction with siRNA-mediated APRT gene knockdown to assess the changes in cellular phenotypes. Assessments included cell viability and migration assays, RT-PCR, western blotting, phase-contrast microscopy, and immunostaining. Paraffin-embedded kidney tissue samples from patients with APRT deficiency and healthy controls were obtained from the Landspitali National University Hospital Biobank (Icelandic IRB permit: VSN 21-117-V1) and phenotypically analyzed using immunohistochemistry (IHC) staining for collagen I (Col I), collagen III (Col III), and APRT. Results After 72 h of incubation with DHA at concentrations of 60, 120, 240, and 480 μg/mL, the viability of all cell lines decreased. Proliferation was evaluated using a scratch assay after treatment with DHA at two different concentrations (120 and 480 μg/mL) for 24 h, and all cell lines showed decreased migration at the highest concentration. Enhanced CD44 expression was observed in both HEK293 and MDCK cells with increasing DHA concentration. In a 3D environment “on-top” of Matrigel, MDCK cells maintained polarized structures despite accumulation of DHA and did not show an increased EMT phenotype compared with TGFβ-treated cells. HEK293 and HK-2 cells formed solid colonies with DHA accumulation within the colony. MDCK cells formed a polarized cell layer grown on Transwell polyester membranes and demonstrated trans-epithelial electrical resistance (TEER) in LLI, which decreased when the cells were treated with DHA at 120 and 480 μg/mL. APRT expression was significantly reduced in all cell lines after successful knockdown. Analysis of kidney tissue specimens using IHC showed increased expression of Col III in patients with APRT deficiency compared to healthy controls. Enhanced Col I expression was also observed in these patients, which, together with Col III, was consistent with increased fibrosis. Conclusion This study revealed that incubation with varying concentrations of DHA led to decreased viability and impaired migration in all cell lines tested. Increased CD44 expression suggests potential crystal binding to renal tubular epithelial cells when exposed to increasing DHA concentrations. In a 3D environment, MDCK cells maintained polarized structures with DHA accumulation without enhancement of the EMT phenotype. Treatment with DHA resulted in a decrease in TEER, indicating a defective barrier function. Increased expression of Col III and Col I was observed in kidney tissue samples from APRT-deficient patients, suggesting increased fibrosis. Overall, these findings highlight the impact of DHA on cell viability, proliferation, EMT, and barrier function, highlighting the role of DHA in kidney fibrosis in patients with APRT-deficiency.

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

#1902 Remdesivir ameliorates ferroptotic renal injury in hamster after SARS-CoV-2 infection

Abstract Background and Aims The purpose of this study was to see how remdesivir affected renal ferroptotic damage in hamsters following a human Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. We evaluated the inhibitory effects of remdesivir on the ferroptosis of SARS-CoV-2 infection in a hamster infection model and in in vivo assays. Method To investigate the effect of SARS-CoV-2 on renal ferroptosis, we administered 2.5 × 104 p.f.u. SARS-CoV-2 via intranasal to hamster. In the initial study, animals were treated with remdesivir subcutaneously (s.c.) for 7 days beginning at various times in relation to virus challenge. After 5 days, kidney tissues were harvested. The effect of REMDESIVIR on SARS-CoV-2 infection and ferroptosis were evaluated by immunohistochemistry. Next, ferroptosis-related markers, glutathione peroxidase 4 (Gpx4), 4-hydroxynonenal (4-HNE), Acyl-CoA synthetase long chain family member 4 (Acsl4), nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (Ho-1) were evaluated by quantitative PCR. Results In hamsters, SARS-CoV-2 resulted in high viral protein expression in the renal tubules and decreased renal tubular luminal diameter in periodic acid-Schiff (PAS) stained kidney sections. Remdesivir treatment decreased SARS-CoV-2-induced viral spike protein, HO-1, ferritin heavy chain1 (FTH1), ferritin light chain (FTL), and 4-HNE expression in renal tubular epithelial cells of hamsters in a dose-dependent manner. GPX4 expression suppression after infection was ameliorated by remdesivir administration. Increased ferroptosis-related mRNA marker (Gpx4, 4-HNE, Acsl4, Nrf2, Ho-1) expression in hamster kidneys was suppressed by remdesivir treatment. Conclusion All of our data suggest that remdesivir treatment ameliorated SARS-CoV-2 infection-induced ferroptosis in the kidney in a hamster model.

#1957 Microbial and metabolomic remodeling by Sichuan dark tea-based formula improves obesity-induced renal lipid metabolism disorder in mice

Abstract Background and Aims The theory of medicine-food homology is long-standing and has been widely applied in traditional Chinese medicine. The Sichuan dark tea-based medicated dietary formula (alternatively referred to as Qing, or clarity in Chinese) is previously used for its lipid-lowering properties in apoE mice. This study further investigated the renoprotective effects of Qing on diet-induced obesity (DIO) mice and explored the specific mechanisms involved. Method Male C57BL/6 mice were randomly assigned to three groups, a control group, a DIO group, and a Qing treatment group, or the Qing group, with 8 mice in each group. The mice in the control group were given normal maintenance feed and purified water, and the other two groups were fed a high-fat diet for 12 weeks to establish the DIO model. After that, high-fat diet continued in the DIO group, while the Qing group was given Qing at the same time for 12 weeks, during which period the weight of the mice was monitored and recorded every week. The mice were sacrificed after 12 weeks. Serum samples were collected and the levels of triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin were measured to evaluate liver function. In addition, renal lipids were extracted to determine the levels of TG and TC in the kidney and periodic acid-Schiff (PAS) and oil red O stainings were performed to evaluate kidney pathological injury. Western blot was performed to determine the phosphorylated AMPK (pAMPK)/AMPK ratio in the kidney tissue. RT-qPCR and Western blot were used to determine the expression of proteins related to fatty acid oxidation and key proteins related to lipid synthesis. 16SrRNA and metabolomics were applied to analyze the gut microbiota in the intestinal contents and its metabolites. Results Compared with those of the control group, the levels of liver mass (P=0.0003), serum ALT(P < 0.0001), AST (P=0.0001), TC (P=0.0191) and TG (P=0.0101) in the model group were significantly increased. Qing treatment can effectively reduce liver mass (P=0.0316), and improve AST (P=0.0012), ALT (P=0.0027), TC (P=0.0200), TG (P=0.0499) levels of mice compared to those of the DIO group. Qing treatment inhibited the expression of lipid synthesis related genes (SREBP-1, FASN and SCD1) and promoted the expression of fatty acid oxidation related genes (CPT1A, PGC1α and PPARγ) in kidneys (Fig. 1). Obesity-triggered dysfunction of structure and composition of gut microbiota were improved by Qing (Fig. 2), where the content of short-chain fatty acids, especially isovaleric acid and propionic acid, were also restored (Fig. 3). Conclusion Our data suggested that Sichuan dark tea-based medicated dietary formula may improve renal lipid metabolism by regulating gut microbiota and the levels of intestinal short-chain fatty acids, thereby protecting obesity-related kidney injury. Isovaleric acid and propionic acid may be the metabolites key to its regulation of gut microbiota.

#163 Multiome-seq and spatial transcriptomics of refractory PLA2RAb(+) membranous nephropathy reveals importance of immunomodulatory function of B cells

Abstract Background and Aims PLA2RAb-associated membranous nephropathy (MN) is a glomerulonephritis that may require immunosuppression to achieve proteinuria remission. However, the disease course is heterogeneous, and some patients exhibit refractory or relapsing nephrotic-range proteinuria. Additional biomarker studies are necessary to dissect the molecular profiles that reflect substantial differences in treatment response and clinical courses. Multiome-seq includes the investigation of gene expression profiles and ATAC-sequencing for open-chromatin regions, which may reveal pathophysiologic gene expression biomarkers and pathways. Method We performed multiome-sequencing of peripheral blood mononuclear cells (PBMCs) from 6 refractory or relapsing primary PLA2RAb-associated MN cases, 7 PLA2RAb-associated MN cases that reached complete remission, and disease controls including 6 minimal change disease and 6 diabetic kidney disease cases. We also collected PBMCs from 7 healthy controls. Cell type clustering, comparison of cell type proportions, and differential gene expression analysis were performed. Additionally, we conducted ATAC-sequencing analysis and identified open-chromatin profiles specific to the refractory or relapsing MN group. Lastly, we investigated the glomerular spatial transcriptome using GeoMx digital spatial profiling to determine whether transcriptomic profiles differ based on the disease courses of MN in the initial kidney tissue. Results We successfully identified diverse PBMCs in the multiome-seq data. In the DEG analysis, we identified a number of cell-type-specific DEGs in the refractory or relapsing MN cases. Of these, CD83 was the top gene that was lowly expressed in the B cells of refractory/relapsing MN compared to the remission cases. In the ATAC data, among the number of differentially expressed peaks, NFKB2 was the region that consistently showed a low open chromatin profile in the B cells of CD83. When comparing the cell subtype proportions, refractory or relapsing MN cases showed low regulatory T cells in the peripheral blood. Lastly, in the kidney tissue spatial transcriptomic profiling, there were no significant DEGs between the refractory/relapsing MN and MN with remission in the glomerulus. Conclusion The clinical course or risk of remission of PLA2RAb-associated MN was more likely a result of differences in systemic immunity rather than differences in the glomerular transcriptome at the initial diagnosis. Both CD83 and NFκB2 are well-known biomarkers of activated B cells that may have immunomodulatory roles leading to the enrichment of regulatory T cells. Such a deficient immunomodulatory status in refractory/relapsing PLA2RAb-associated MN may explain the disease course.

#1316 Sirtuin 3 promotes renal gluconeogenesis in fibrotic kidneys

Abstract Background and Aims Decreased renal gluconeogenesis is currently identified as a hallmark of chronic kidney disease (CKD), which may contribute to renal fibrosis. Sirt3 is a crucial metabolic sensor that regulates ATP generation, mitochondrial adaptive response to stress, and glucose metabolism. It has been shown that Sirt3 is renal protective in fibrotic kidneys, however its underlying mechanisms is not completely known. In this study, we aimed to investigated whether Sirt3 inhibits CKD associated renal fibrosis through restoring renal gluconeogenesis. Method 1. Animal model of renal fibrosis (in vivo) For unilateral ureter obstructed (UUO) mouse model, a. Twenty-eight C57 mice were divided into four groups: sham + Vehicle, UUO + Vehicle, UUO + 3-TYP and UUO + HKL group. Mice were treated with saline or 20 mg/kg 3-TYP, an inhibitor of SIRT3 or 1 mg/kg HKL, an activator of SIRT3 daily (i.p.). b. Fourteen C57 mice were divided into two groups: Ad-Sirt 3 or Ad-LacZ (0.5 × 109 pfu/100 μL) was injected into the left renal artery before the UUO operation. Systemic glucose metabolism was evaluated by insulin and glucose tolerance tests. 7 or 14 days after operation, kidney tissues were collected and performed histopathological and molecular examination. 2. Primary murine renal tubules fibrosis (ex vivo) Primary renal tubules were isolated from C57 mouse of 4 weeks of age. 5 ng/mL TGF-β was added to culture medium for inducing fibrosis. After this Sirt3 was inhibited by 3-TYP (25 μm) or activated by HKL (2 μm) or overexpressed by adenovirus for different time periods (1, 3, 24, 48 hours). Protein or RNA samples were taken to evaluate renal fibrosis and gluconeogenesis. 3. Human renal epithelial (HK2) cells culture and treatment (in vitro) TGF-β stimulated HK2 cells were used as an in vitro model. Sirt3 was inhibited by 3-TYP or activated by HKL or overexpressed by adenovirus. Protein and RNA samples were taken to evaluate renal fibrosis and gluconeogenesis. Glucose and lactate were measured in supernatant of culture medium. Results In vivo, Masson staining showed that 3-TYP treatment increased the tubulointerstitial collagen deposition of UUO kidneys (Fig. 1A). WB results indicated that 3-TYP enhanced the increased expression of fibrotic marks (Fibronectin, Collagen-I, Vimentin) (Fig. 1B), and further reduced the expression of gluconeogenesis limitation enzyme (PCK1, FBP1) and transcription factors (PGC-1α and FOXO1) 14 days after UUO operation (Fig. 1C). Conversely, HKL treatment or overexpression of Sirt3 in UUO kidney reduced expression of fibrotic marks, increased the expression of gluconeogenesis markers (Fig. 1D-E). 3-TYP increased the lactate concentration in UUO kidneys (Fig. 1F). Insulin sensitivity and glucose clearance tests showed that treatment with 3-TYP further damaged the glucose homeostasis in UUO mice (Fig. 1G-J). Ex vivo, q-PCR results showed that the mRNA of Sirt3 was significantly decreased by time after TGF-β stimulation while the mRNA of FN, Snail was up-regulated and PCK1 and FBP1 were down-regulated (Fig. 2A-D). WB showed that 3-TYP treatment inhibited the expression of PCK1, FBP1 and PGC-1α, FOXO1 (Fig. 2E), however, HKL treatment or overexpressed of Sirt3 by adenovirus increased the expression of gluconeogenesis markers respectively in primary renal tubules (Fig. 2F). In vitro, WB showed that transfection of Sirt3 increased the expression of gluconeogenesis markers and decreased the expression of fibrotic marks of HK2 cells in a fibrotic condition (Fig. 3A-B). Moreover, 3-TYP treatment further decreased the mRNA expression of PCK1 and FBP1 in TGF-β stimulated HK2 cells (Fig. 3C-D). The increased lactate concentration and decreased glucose concentration in the culture medium of TGF-β stimulated HK2 cells were further escalated after 3-TYP treatment (Fig. 3E-F) while reversed after overexpression of Sirt3 (Fig. 3G-H). Conclusion Sirt 3 promotes renal gluconeogenesis in fibrotic kidneys through up-regulation of renal gluconeogenic enzyme PCK1 and FBP1 expression, which is associated with activation of renal metabolic transcription factors PGC-1α and FOXO1.

#2372 Investigating the effects of low-level and transient DNA damage on kidney health in a novel mouse model of aging

Abstract Background and Aims Increasing life expectancy worldwide underscores the importance of addressing the effects of aging on organ and cell function. Aging is characterized by a gradual decline in organ function that varies among individuals and is influenced by lifestyle choices and various external factors that induce DNA stress. This mismatch between kidney aging and an individual's lifespan contributes to the increase in end-stage renal disease. Among the kidney's constituent cells, the proximal tubules are frequently damaged by external factors such as ischemia and drugs. Our research has focused on DNA damage and its repair mechanisms (DDR) caused by these injuries, and we have previously shown that extensive DNA damage beyond the capacity of DDR or abnormalities in DDR cause cellular senescence in proximal tubules, resulting in incomplete renal repair after injury (Kishi et al. Sci Transl Med 2019, J Clin Invest 2019). In this study, we extend these studies and test whether acquired and mild DNA stress induces renal senescence using a novel mouse model of aging, the Inducible Changes to the Epigenome (ICE) mouse (Hayano et al. Cell. 2023). Method ICE mice were generated by mating I-PpoI STOP/+ mice with CreERT2/+ mice. I-PpoI, an endonuclease, cleaves DNA only upon administration of tamoxifen and the resulting DNA damage is subsequently repaired by the DDR. To induce transient DNA damage, 4- to 6-month-old mice were fed a tamoxifen-containing diet for 3 weeks and then housed until they reached 14 months of age. A comprehensive renal analysis was then performed, which included renal tissue evaluation, single-nucleus RNA sequencing, q-PCR, and Western blot analysis for phenotypic analysis of mouse kidneys. Results No significant histological changes were observed in the kidney tissues between the ICE mice and the control group, and the levels of interstitial fibrosis were comparable. To ensure unbiased evaluation, we performed single-nucleus RNA-seq. In ICE mice, there was an increased presence of proximal tubules with incomplete repair expressing KIM-1 and VCAM-1. In addition, ICE mice showed increased gene expression of DNA repair-related enzymes (Mgmt) in proximal tubules and evidence of cellular senescence. No differences in mitochondrial number or biosynthetic capacity were observed. Conclusion The consequences of short-term and transient DNA damage were found to have lasting effects on the kidney. In particular, significant changes were observed in the proximal tubules, where DNA instability and accelerated cellular senescence were evident, suggesting an impaired repair phenotype within the proximal tubules. This may be related to increased susceptibility to various external stimuli. For further detailed analysis, studies using proximal tubule-specific ICE mice are currently performed.

#1771 ANXA13 protects against acute kidney injury by inhibiting TGF-β/Smad3 pathway

Abstract Background and Aims Acute kidney injury (AKI) is a common clinical syndrome characterized by rapid loss of kidney function but with limited diagnostic and treatment options. Annexins (ANXA) are a superfamily of calcium-dependent and lipid-binding proteins involved in multiple intracellular and extracellular functions. As a founding member of ANXA, ANXA13 is considered the prototype of a conserved evolutionary property of the annexin superfamily and is restricted to kidney and intestinal epithelial cells. However, it is currently unclear whether ANXA13 is a key regulator of AKI. Therefore, we aimed to investigate the role of ANXA13 in AKI. Method Kidney biopsy tissues from AKI patients were collected for immunohistochemical examination and ELISA method was used to detect the concentration of ANXA13 in serum and urine. A mouse model of AKI induced by ischemia-reperfusion injury (IRI) was established to verify the role of ANXA13 in AKI. RNA sequencing was also used to explore the mechanism of ANXA13 in AKI, and this result was further confirmed using Smad3 wild-type (WT) and knockout (KO) mice. Results AKI patients had reduced serum ANXA13 levels and increased urinary ANXA13 levels compared with healthy controls. The levels of ANXA13 in both serum and urine are related to serum creatinine (Scr). In kidney tissue, ANXA13 was significantly reduced in both AKI patients and IRI-AKI mice. By overexpressing ANXA13 by specifically transferring ANXA13-expressing plasmid into IRI-AKI mice via ultrasound microbubble-mediated kidney, we found that ANXA13 reduced Scr and blood urea nitrogen (BUN) levels and renal function in IRI-AKI mice. damage. In contrast, knockdown of ANXA13 by shANXA13 plasmid significantly exacerbated renal injury. Mechanistically, we found that the protective mechanism of ANXA13 was related to the inhibition of the TGF-β/Smad3-p21 cell cycle arrest pathway, and the aggravating effect of shANXA13 was reversed in Smad3 knockout mice. In turn, activation of Smad3 also suppresses ANXA13 mRNA and protein levels in vivo and in vitro. Conclusion ANXA13 can intervene in AKI by inhibiting the TGF-β/Smad3-p21 cell cycle blocking pathway. In turn, activation of Smad3 inhibits ANXA13 expression. Therefore, ANXA13 may be a new therapeutic target for AKI.