Pathogenesis Of Diabetic Kidney Disease Research Articles

Integrating serum pharmacochemistry and network pharmacology to explore potential compounds and mechanisms of Alpiniae oxyphyllae fructus in the treatment of cellular senescence in diabetic kidney disease.

Diabetic kidney disease (DKD), one of the microvascular complications in patients with diabetes mellitus, is a common cause of end-stage renal disease. Cellular senescence is believed to be an essential participant in the pathogenesis of DKD. Although there is evidence that Alpiniae oxyphyllae fructus (AOF) can ameliorate DKD progression and organismal senescence, its ability to ameliorate renal cellular senescence in DKD as well as active components and molecular mechanisms remain to be explored. This study aimed to investigate the role of AOF in the treatment of cellular senescence in DKD and to explore its active components and potential molecular mechanisms. The pharmacological efficacy of AOF in ameliorating cellular senescence in DKD was assessed by establishing DKD mouse models and HK-2 cells under high glucose stress. UHPLC-QTOF-MS was used to screen the active compounds in AOF, which were used in conjunction with network pharmacology to predict the molecular mechanism of AOF in the treatment of cellular senescence in DKD. In vivo experiments showed that AOF reduced GLU, mAlb, Scr, BUN, MDA, SOD levels, and ameliorated renal pathological damage and renal cell senescence in DKD mice. In vitro experiments showed that AOF-containing serum improved the decline in HK-2 cell viability and alleviated cellular senescence under high glucose intervention. The results of the UHPLC-QTOF-MS screened 26 active compounds of AOF. The network pharmacological analyses revealed that Cubebin, 2',6'-dihydroxy-4'-methoxydihydrochalcone, Chalcone base + 3O,1Prenyl, Batatasin IV, and Lucidenolactone were the five core compounds and TP53, SRC, STAT3, PIK3CA, and AKT1 are the five core targets of AOF in the treatment of DKD. Molecular docking simulation results showed that the five core compounds had good binding ability to the five core targets. Western blot validated the network pharmacological prediction results and showed that AOF and AOF-containing serum down-regulate the expression of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT. Our study shows that AOF may delay the development of cellular senescence in DKD by down-regulating the levels of TP53, and phosphorylation of SRC, STAT3, PIK3CA, and AKT.

Elevated RHAMM as a biomarker for predicting diabetic kidney disease in patients with type 2 diabetes.

Diabetic kidney disease (DKD) poses a significant challenge globally as a complication of diabetes. Hyaluronan (HA), a critical non-sulfated glycosaminoglycan in the extracellular matrix, plays a pivotal role in the progression of DKD. This study assesses the predictive significance of HA's corresponding receptor, RHAMM (receptor for HA-mediated motility), in DKD pathogenesis in type 2 diabetes (T2DM) patients. Enzyme-linked immunosorbent assays were utilized to measure plasma and urine levels of HA, CD44 and RHAMM in 99 diabetic patients. Immunohistochemistry staining was employed to examine HA deposition, CD44 and RHAMM expressions from 18 biopsy-proven DKD patients. Spearman correlation analysis, linear regression and receiver operating characteristic (ROC) analysis were conducted to establish associations between plasma HA, CD44 and RHAMM levels, and clinical parameters in DKD patients with T2DM. Elevated plasma and urine HA, CD44 and RHAMM levels were notably observed in the severe renal dysfunction group. Plasma RHAMM exhibited positive correlations with HA (r=0.616, P<.001) and CD44 (r=0.220, P<.001), and a negative correlation with estimated glomerular filtration rate (eGFR) (r=-0.618, P<.001). After adjusting for other potential predictors, plasma RHAMM emerged as an independent predictor of declining eGFR (β=-0.160, P<.05). Increased HA, CD44 and RHAMM levels in kidney biopsies of DKD patients were closely associated with heightened kidney injury. The ROC curve analysis highlighted an area under the curve (AUC) of 0.876 for plasma RHAMM, indicating superior diagnostic efficacy compared to CD44 in predicting DKD pathogenesis. The combined AUC of 0.968 for plasma RHAMM, HA and CD44 also suggested even greater diagnostic potential for DKD pathogenesis. These findings provide initial evidence that elevated RHAMM levels predict DKD pathogenesis in T2DM patients. The formation of a triple complex involving HA, CD44 and RHAMM on the cell surface shows promise as a targetable biomarker for early intervention to mitigate severe renal dysfunctions.

Uncovering the Role of Anoikis-Related Genes in Modulating Immune Infiltration and Pathogenesis of Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is an intricate complication of diabetes with limited treatment options. Anoikis, a programmed cell death activated by loss of cell anchorage to the extracellular matrix, participated in various physiological and pathological processes. Our study aimed to elucidate the role of anoikis-related genes in DKD pathogenesis. Differentially expressed genes (DEGs) associated with anoikis in DKD were identified. Weighted gene co-expression network analysis (WGCNA) was conducted to identify DKD-correlated modules and assess their functional implications. A diagnostic model for DKD was developed using LASSO regression and Gene set variation analysis (GSVA) was performed for enrichment analysis. Experimental validation was employed to validate the significance of selected genes in the progression of DKD. We identified 10 anoikis-related DEGs involved in key signaling pathways impacting DKD progression. WGCNA highlighted the yellow module's significant enrichment in immune response and regulatory pathways. Correlation analysis further revealed the association between immune infiltration and anoikis-related DEGs. Our LASSO regression-based diagnostic model demonstrated a well-predictive efficacy with seven identified genes. GSVA indicated that gene function in the high-risk group was primarily associated with immune regulation. Further experimental validation using diabetic mouse models and data analysis in the single-cell dataset confirmed the significance of PYCARD and SFN in DKD progression. High glucose stimulation in RAW264.7 and TCMK-1 cells showed significantly increased expression levels of both Pycard and Sfn. Co-expression analysis demonstrated distinct functions of PYCARD and SFN, with KEGG pathway analysis showing significant enrichment in immune regulation and cell proliferation pathway. In conclusion, our study provides valuable insights into the molecular mechanisms involved in DKD pathogenesis, specifically highlighting the role of anoikis-related genes in modulating immune infiltration. These findings suggest that targeting these genes may hold promise for future diagnostic and therapeutic approaches in DKD management.

MTORC1 signaling and diabetic kidney disease.

Diabetic kidney disease (DKD) represents the most lethal complication in both type 1 and type 2 diabetes. The disease progresses without obvious symptoms and is often refractory when apparent symptoms have emerged. Although the molecular mechanisms underlying the onset/progression of DKD have been extensively studied, only a few effective therapies are currently available. Pathogenesis of DKD involves multifaced events caused by diabetes, which include alterations of metabolisms, signals, and hemodynamics. While the considerable efficacy of sodium/glucose cotransporter-2 (SGLT2) inhibitors or angiotensin II receptor blockers (ARBs) for DKD has been recognized, the ever-increasing number of patients with diabetes and DKD warrants additional practical therapeutic approaches that prevent DKD from diabetes. One plausible but promising target is the mechanistic target of the rapamycin complex 1 (mTORC1) signaling pathway, which senses cellular nutrients to control various anabolic and catabolic processes. This review introduces the current understanding of the mTOR signaling pathway and its roles in the development of DKD and other chronic kidney diseases (CKDs), and discusses potential therapeutic approaches targeting this pathway for the future treatment of DKD.

Lactate drives epithelial-mesenchymal transition in diabetic kidney disease via the H3K14la/KLF5 pathway

High levels of urinary lactate are an increased risk of progression in patients with diabetic kidney disease (DKD). However, it is still unveiled how lactate drive DKD. Epithelial-mesenchymal transition (EMT), which is characterized by the loss of epithelial cells polarity and cell-cell adhesion, and the acquisition of mesenchymal-like phenotypes, is widely recognized a critical contributor to DKD. Here, we found a switch from oxidative phosphorylation (OXPHOS) toward glycolysis in AGEs-induced renal tubular epithelial cells, thus leading to elevated levels of renal lactic acid. We demonstrated that reducing the lactate levels markedly delayed EMT progression and improved renal tubular fibrosis in DKD. Mechanically, we observed lactate increased the levels of histone H3 lysine 14 lactylation (H3K14la) in DKD. ChIP-seq & RNA-seq results showed histone lactylation contributed to EMT process by facilitating KLF5 expression. Moreover, KLF5 recognized the promotor of cdh1 and inhibited its transcription, which accelerated EMT of DKD. Additionally, nephro-specific knockdown and pharmacological inhibition of KLF5 diminished EMT development and attenuated DKD fibrosis. Thus, our study provides better understanding of epigenetic regulation of DKD pathogenesis, and new therapeutic strategy for DKD by disruption of the lactate-drived H3K14la/KLF5 pathway.

Integrative Analysis by Mendelian Randomization and Large-Scale Single-Cell Transcriptomics Reveals Causal Links between B Cell Subtypes and Diabetic Kidney Disease

Introduction: The increasing incidence of diabetic kidney disease (DKD) and the challenges in its management highlight the necessity for a deeper understanding of its pathogenesis. While recent studies have underscored the substantial impact of circulating immunity on the development of diabetic microvascular complications such as retinopathy and neuropathy, research on circulating immunity in DKD remains limited. Methods: This study utilized Mendelian randomization analysis to explore the potential independent causal relationships between circulating immune cells and DKD pathogenesis. Additionally, a combination of single-cell disease relevance score (scDRS) and immune cell infiltration analysis was employed to map the circulating immunity landscape in DKD patients. Results: Ten immune traits, including 5 of B cells, 2 of T cells, 2 of granulocytes, and one of monocytes, were defined to be associated with the pathogenesis of DKD. Notably, IgD-CD27-B cell Absolute Count (IVW: OR, 1.102 [1.023–1.189], p = 0.011) and IgD-CD24-B cell Absolute Count (IVW: OR, 1.106 [1.030–1.188], p = 0.005) were associated with promoting DKD pathogenesis, while CD24+CD27+B cell %B cell (IVW: OR, 0.943 [0.898–0.989], p = 0.016) demonstrated a protective effect against DKD onset. The presence of B cell-activating factor receptor (BAFF-R) on CD20−CD38−B cell (IVW: OR, 0.946 [0.904–0.989], p = 0.015) and BAFF-R on IgD-CD38+B cell (IVW: OR, 0.902 [0.834–0.975], p = 0.009) also indicated a potential role in preventing DKD. scDRS analysis revealed that two main subsets of B cells, naïve B and memory B cells, had a higher proportion of DKD-related cells or a higher scDRS score of DKD phenotype, indicating their strong association with DKD. Furthermore, immune infiltrate deconvolution analysis showed a notable decrease in the circulating memory B cells and class-switched memory B cells in DKD patients compared to those of DM patients without DKD. Conclusion: Our study revealed the causal relations between circulating immunity and DKD susceptibility, particularly highlighted the potential roles of B cell subtypes in DKD development. Further studies addressing the related mechanisms would broaden the current understanding of DKD pathogenesis.

Glycolytic lactate in diabetic kidney disease.

Lactate elevation is a well-characterized biomarker of mitochondrial dysfunction, but its role in diabetic kidney disease (DKD) is not well defined. Urine lactate was measured in patients with type 2 diabetes (T2D) in 3 cohorts (HUNT3, SMART2D, CRIC). Urine and plasma lactate were measured during euglycemic and hyperglycemic clamps in participants with type 1 diabetes (T1D). Patients in the HUNT3 cohort with DKD had elevated urine lactate levels compared with age- and sex-matched controls. In patients in the SMART2D and CRIC cohorts, the third tertile of urine lactate/creatinine was associated with more rapid estimated glomerular filtration rate decline, relative to first tertile. Patients with T1D demonstrated a strong association between glucose and lactate in both plasma and urine. Glucose-stimulated lactate likely derives in part from proximal tubular cells, since lactate production was attenuated with sodium-glucose cotransporter-2 (SGLT2) inhibition in kidney sections and in SGLT2-deficient mice. Several glycolytic genes were elevated in human diabetic proximal tubules. Lactate levels above 2.5 mM potently inhibited mitochondrial oxidative phosphorylation in human proximal tubule (HK2) cells. We conclude that increased lactate production under diabetic conditions can contribute to mitochondrial dysfunction and become a feed-forward component to DKD pathogenesis.

#930 Elevated glucose in kidney organoids induces epithelial detachment driven by proinflammatory cytokines

Abstract Background and Aims Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease affecting 40% of diabetics. The pathogenesis of DKD is linked with inflammation, altered metabolism, and hemodynamic change but the relationship between these factors is not well understood. Proteinuria and albuminuria are common features, suggesting that impairments in the renal glomerular filtration barrier may be among the primary disease initiators. Limited DKD models are available, which has hampered our disease understanding and the ability to develop new therapies. Therefore, creating an in-vitro model that harbors multiple renal cell types organized in their natural complex arrangement becomes an attractive option to fill in this gap. Here, we conducted a systematic investigation of the effects of high glucose on kidney organoids, with the goal of understanding whether these cultures can reflect specific features relevant to DKD. Method To evaluate the effect of high glucose, kidney organoids were differentiated until maturation (day 21) in standard glucose and then switched for six days to concentrations of 5.5, 11, 33 and 100 mM. Then, to test the effect of proinflammatory cytokines on the injury phenotypes we switched organoids, at day 21, in control glucose condition (11 mM) ± TNFα or high glucose (33 mM) ± inhibitors (TNFα). Morphology was analysed via immunostaining, LDH and live/dead assay were used for toxicity, qPCR and western blotting to investigate differences in gene and protein expression, and mannose was used as an osmotic control. scRNA-seq of organoid in 11 vs 33 mM was conducted to gain insight into the molecular mechanism(s) contributing to the high glucose. Results Organoids remain well preserved in glucose up to 11 mM whereas organoids cultured in 33 and 100 mM showed a ∼30% reduction in overall organoid number between days 3 and 6. Moreover, in high glucose (33 and 100 mM), we observed an increase in the number of small, single PODXL+ cells surrounding the main organoid body, suggesting detachment from the podocyte clusters that normally form in organoids (Fig. A). To quantify it, we divided the total PODXL+ area into two sub-regions, classified as intact area and detached area, and calculated their ratio (Fig. B). Results indicated a 2-fold reduction of the intact vs detached area between 11 and 33 mM (p = 0.0011), suggesting that organoids undergo a spreading process under the influence of high glucose (Fig. C). Comparison of equimolar glucose vs mannose, live/dead and LDH assays identify a non-osmotic and non-apoptotic cause of the injury. ScRNA-seq analysis identified TNF-α as an upregulated pathway, which was confirmed with qPCR (p = 0.0131). Comparison of organoids at 11mM ± TNFα indicated that TNFα addition sensitizes podocytes to detachment at lower glucose levels, specifically, we identified a 4-fold reduction of the PODXL+ intact vs detached area at 11 mM ± TNFα (p &amp;lt; 0.0001), Fig. D. To further validate these findings and investigate possible therapeutic strategies, organoids were cultured with 33 mM glucose with TNF-α inhibitor and we identified a protective effect of the inhibitor which prevented the podocyte spreading phenotype at 33 mM glucose (Fig. E). Conclusion Our experiments identify an injury phenotype under high glucose concentrations, in kidney organoids, characterized by podocyte detachment from the main organoid body. Moreover, no osmotic or toxicity effect was observed. Importantly, this phenotype resembles disease features identified in diabetic patients, where podocyte loss and detachment from the glomerular basement membrane is associated with pathophysiology. We identified a correlation between the injury phenotype, characterized by epithelial detachment, and proinflammatory cytokine upregulation, which is driven by high glucose, and we have shown it can be targeted therapeutically. Therefore, this new experimental model in-vitro offers up new insights into the pathophysiology of diabetes and its complications, and represents a new platform to screen for therapeutics.

#1172 Identification of phenyl sulfate producer phenotypes by an oral tyrosine challenge test: a microbiota-based personalized nutritional approach

Abstract Background and Aims Phenyl sulfate (PS) has been implicated in the pathogenesis of diabetic kidney disease (DKD). PS is converted from phenol, a gut microbial metabolite of dietary tyrosine, and sulfated into PS by aryl sulfotransferases in the host liver. We hypothesized that the effects of a particular diet on metabolic responses might differ from person to person. In the present study, we aimed to characterize various PS producer phenotypes using an oral tyrosine challenge test (TCT). Method We developed the TCT to determine the individual PS producing capacity. Plasma samples were collected at baseline after overnight fasting, and were then collected at 4, 8, 12, 24, 36 and 48 hours after oral administration of L-tyrosine at a dose of 100 mg/kg. PS concentrations were measured by high performance liquid chromatography. The gut microbiome was assessed by metagenomic shotgun sequencing. Results A total of 48 healthy volunteers were enrolled to undergo the TCT. The mean age of the participants was 32 ± 8 years; 50% were men, and 50% were vegetarians. The time-concentration curves of PS were similar among the participants, with the maximal increase of PS at the 24th hour (Fig. A). Participants were stratified into tertiles according to the PS levels at the 24th hour after the TCT. The 16 participants in the highest tertile were defined as high-PS producers, whereas those in the lowest tertile were defined as low-PS producers (Fig. B). We found that there was no difference in age, gender, dietary habit, BMI, eGFR, and baseline plasma PS levels between the 2 groups. However, the high-PS producers had higher levels of cholesterol, triglycerides, and low-density lipoprotein cholesterol. In addition, distinct differences in gut microbial composition were identified. At the species level, the relative abundance of Alistipes dispar, Clostridium sp. C5-48, and Butyricimonas faecihominis was markedly increased in the high-PS producers (Fig. C). Conclusion We found high PS variability in the response to a TCT, suggesting that universal dietary recommendations may have limited utility in reducing PS levels. Further studies are warranted to investigate the potential clinical applications of the TCT in patients with DKD.

#71 Dysregulation of ITGAM and ITGB2 in the pathogenesis of diabetic kidney disease—results from bioinformatics analysis and in vitro studies

Abstract Background and Aims Activated neutrophils can release web-like chromatin structures known as neutrophil extracellular traps (NETs), but their roles in the immunopathogenesis of diabetic kidney disease (DKD) remains elusive. Method The characteristic NETs associated targets of differentially expressed genes (DEGs) from human kidney biopsy datasets of patients with DKD (GSE30528 and GSE30529) were identified by machine learning method (LASSO and SVM-RFE). The genes expression was further validated using kidney biopsy dataset of GSE142025. The most abundant single cell of the targets in DKD was further selected by single-nucleus RNA sequencing datasets (GSE131882). Predictive pathway network of targeted genes was constructed using GENEMANIA database. The expressions and potential signaling pathway of these candidate genes were validated using flow cytometry and ELISA. Results Three predicting NETs related genes (ITGAM, ITGB2 and TLR7), identified by machine learning algorithms, which all showed significant upregulation in both glomerular and tubulointerstitial compartments in human DKD kidney samples. Single-cell analysis suggested that ITGAM, ITGB2 and TLR7 were most enriched in leucocytes. DKD patients (n = 18) showed significantly higher activated neutrophils as well as increased expression of ITGAM and ITGB2 compared to healthy controls (HCs, n = 9). DKD patients also exhibited higher serum levels of IL6 but lower IL-10 than HCs (all p &amp;lt; 0.01). The significant network of IL10, IL6, ITGAM and ITGB2 was generated. This network might be associated with leukocyte activation, macrophage activation, JAK-STAT pathway, acute inflammatory response and lymphocyte immune response (all p &amp;lt; 0.05). Conclusion Aberrant ITGAM and ITGB2 expressions in activated neutrophils contribute to DKD pathogenesis and may serve as novel therapeutic targets for DKD.

A potential defensive role of TIM-3 on T lymphocytes in the inflammatory involvement of diabetic kidney disease.

The aberrant mobilization and activation of various T lymphocyte subpopulations play a pivotal role in the pathogenesis of diabetic kidney disease (DKD), yet the regulatory mechanisms underlying these processes remain poorly understood. Our study is premised on the hypothesis that the dysregulation of immune checkpoint molecules on T lymphocytes disrupts kidney homeostasis, instigates pathological inflammation, and promotes DKD progression. A total of 360 adult patients with DKD were recruited for this study. The expression of immune checkpoint molecules on T lymphocytes was assessed by flow cytometry for peripheral blood and immunofluorescence staining for kidney tissue. Single-cell sequencing (scRNA-seq) data from the kidneys of DKD mouse model were analyzed. Patients with DKD exhibited a reduction in the proportion of CD3+TIM-3+ T cells in circulation concurrent with the emergence of significant albuminuria and hematuria (p=0.008 and 0.02, respectively). Conversely, the incidence of infection during DKD progression correlated with an elevation of peripheral CD3+TIM-3+ T cells (p=0.01). Both univariate and multivariate logistic regression analysis revealed a significant inverse relationship between the proportion of peripheral CD3+TIM-3+ T cells and severe interstitial mononuclear infiltration (OR: 0.193, 95%CI: 0.040,0.926, p=0.04). Immunofluorescence assays demonstrated an increase of CD3+, TIM-3+ and CD3+TIM-3+ interstitial mononuclear cells in the kidneys of DKD patients as compared to patients diagnosed with minimal change disease (p=0.03, 0.02 and 0.002, respectively). ScRNA-seq analysis revealed decreased gene expression of TIM3 on T lymphocytes in DKD compared to control. And one of TIM-3's main ligands, Galectin-9 on immune cells showed a decreasing trend in gene expression as kidney damage worsened. Our study underscores the potential protective role of TIM-3 on T lymphocytes in attenuating the progression of DKD and suggests that monitoring circulating CD3+TIM3+ T cells may serve as a viable strategy for identifying DKD patients at heightened risk of disease progression.

Chinese botanical drugs targeting mitophagy to alleviate diabetic kidney disease, a comprehensive review.

Diabetic kidney disease (DKD) is one of the chronic microvascular complications caused by diabetes, which is characterized by persistent albuminuria and/or progressive decline of estimated glomerular filtration rate (eGFR), and has been the major cause of dialysis around the world. At present, although the treatments for DKD including lifestyle modification, glycemic control and even using of Sodium-glucose cotransporter 2 (SGLT2) inhibitors can relieve kidney damage caused to a certain extent, there is still a lack of effective treatment schemes that can prevent DKD progressing to ESRD. It is urgent to find new complementary and effective therapeutic agents. Growing animal researches have shown that mitophagy makes a great difference to the pathogenesis of DKD, therefore, exploration of new drugs that target the restoration of mitophagy maybe a potential perspective treatment for DKD. The use of Chinese botanical drugs (CBD) has been identified to be an effective treatment option for DKD. There is growing concern on the molecular mechanism of CBD for treatment of DKD by regulating mitophagy. In this review, we highlight the current findings regarding the function of mitophagy in the pathological damages and progression of DKD and summarize the contributions of CBD that ameliorate renal injuries in DKD by interfering with mitophagy, which will help us further explain the mechanism of CBD in treatment for DKD and explore potential therapeutic strategies for DKD.

Mammalian target of rapamycin mediates expression and activity of ADAM 17 in diabetic kidney disease

Purpose: Previous data have illustrated a role for the matrix metalloprotease A Disintegrin And Metalloprotease 17 (ADAM17), known to cleave growth factors and cytokines, in renal cell injury in diabetes. The goal of this study was to identify upstream regulators of ADAM17 in the cascade of events contributing to extracellular matrix accumulation in diabetic nephropathy. Rationale: Diabetic kidney disease is a serious complication faced by type 1 and type 2 diabetic patients alike. Albuminuria and extracellular matrix accumulation are prominent features of the disease and this accumulation of extracellular matrix is a contributing factor to renal fibrosis and decline in renal function. The mechanisms involved in the pathogenesis of diabetic kidney disease have not been completely identified. Methodology: Age and weight-matched Sprague Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). Type 1 diabetes was induced through tail vein injection of streptozotocin. After rats were determined to be diabetic by blood glucose measurement, rapamycin treatments were administered intraperitoneally for two months. Both kidneys were removed and frozen in liquid nitrogen for microscopy and experimental analyses or formalin fixed for morphometric imaging at the experimental endpoint. Kidney cortex homogenates were used for western blot analyses and enzymatic activity assays. Findings: Using the mTOR complex 1 inhibitor rapamycin, it was determined that increased ADAM17 enzymatic activity and ADAM17 protein expression is dependent on mTORC1 in streptozotocin-induced type 1 diabetic rats. Inhibition of mTORC1 with rapamycin abrogated the increase in collagen IV α 2 protein expression observed in diabetic rat cortex. Additionally, this study is the first to provide evidence that mTOR complex 1 activates ADAM17 contributing to extracellular matrix accumulation in diabetic nephropathy. Studies are continuing looking at the molecular mechanisms involved in mTOR activation of ADAM17 resulting in downstream effects in cultured human proximal tubular epithelial cells. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activin A Contribution to Diabetic Kidney Disease Injury and Effects of Activin A Antagonism In-Vitro

Diabetic kidney disease (DKD) is the worldwide leader in end-stage kidney failure and has limited treatment options. Cellular senescence abundance and inflammation contribute to DKD pathogenesis. We previously demonstrated that activin A, an inflammatory mediator of pro-fibrotic kidney injury and cellular senescence, is increased in DKD and correlates with DKD injury and kidney function. We hypothesized that activin A contributes to DKD injury causing pro-fibrotic, pro-inflammatory, and cellular senescence effects in vitro, and that antagonism of activin A is achieved with follistatin. An in vitro model of DKD injury was applied with high-glucose (HG) and transforming growth factor-β (TGFβ) exposure for 24 hours in U-937-derived macrophages and renal epithelial (HK2) cells. Prior to exposure, U-937 cells were converted to M0 macrophages with phorbol myristate acetate (PMA). To examine the potential of reducing activin A release by DKD injured cells, follistatin, an activin A antagonist, was added to the media of cells at the time of injury for 24 hours. Additionally, an activin A neutralizing antibody was added to the collected supernatant of DKD injured cells. Activin A, senescence (p16), pro-fibrotic (collagen I), and pro-inflammatory (monocyte chemoattractant protein [MCP]-1, tumor necrosis factor [TNF]-alpha, interleukin [IL]-6) markers were measured through qPCR, ELISA (activin A), and immunofluorescence staining (activin A). DKD injury in renal epithelial cells increased protein expression of activin A, increased gene expression of activin A, p16, and collagen I, but did not significantly increase MCP-1 expression at 24 hour exposure (Figure 1). Furthermore, DKD injury led to increased activin A release in conditioned medium vs. control renal epithelial cells. Administration of follistatin reduced activin A release by renal epithelial cells. Application of neutralizing antibody reduced activin A levels in collected medium after cell injury. After DKD injury in U-937 derived macrophages, activin A gene expression was increased. Injured macrophages had increased gene expression of TNF-alpha and IL-6, demonstrating a pro-inflammatory phenotype. In conclusion, diabetic kidney injury increases activin A release, inflammation markers, and senescence markers in kidney cells and macrophages, therein contributing to downstream effects that further propagate disease. As shown, follistatin antagonism reduces activin A release by injured cells. Additional studies are needed to explore activin A as a promising therapeutic target and follistatin as a possible treatment in DKD. NIH 5TL1TR002380-07, AG076537, DK123492, DK109134. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unraveling the interplay of ferroptosis and immune dysregulation in diabetic kidney disease: a comprehensive molecular analysis

BackgroundDiabetic kidney disease (DKD) is a primary microvascular complication of diabetes with limited therapeutic effects. Delving into the pathogenic mechanisms of DKD and identifying new therapeutic targets is crucial. Emerging studies reveal the implication of ferroptosis and immune dysregulation in the pathogenesis of DKD, however, the precise relationship between them remains not fully elucidated. Investigating their interplay is pivotal to unraveling the pathogenesis of diabetic kidney disease, offering insights crucial for targeted interventions and improved patient outcomes.MethodsIntegrated analysis, Consensus clustering, Machine learning including Generalized Linear Models (GLM), RandomForest (RF), Support Vector Machine (SVM) and Extreme Gradient Boosting (xGB), Artificial neural network (ANN) methods of DKD glomerular mRNA sequencing were performed to screen DKD-related ferroptosis genes.CIBERSORT, ESTIMATE and ssGSEA algorithm were used to assess the infiltration of immune cells between DKD and control groups and in two distinct ferroptosis phenotypes. The ferroptosis hub genes were verified in patients with DKD and in the db/db spontaneous type 2 diabetes mouse model via immunohistochemical and Western blotting analyses in mouse podocyte MPC5 and mesangial SV40-MES-13 cells under high-glucose (HG) conditions.ResultsWe obtained 16 differentially expressed ferroptosis related genes and patients with DKD were clustered into two subgroups by consensus clustering. Five ferroptosis genes (DUSP1,ZFP36,PDK4,CD44 and RGS4) were identified to construct a diagnostic model with a good diagnosis performance in external validation. Analysis of immune infiltration revealed immune heterogeneity between DKD patients and controls.Moreover, a notable differentiation in immune landscape, comprised of Immune cells, ESTIMATE Score, Immune Score and Stromal Score was observed between two FRG clusters. GSVA analysis indicated that autophagy, apoptosis and complement activation can participate in the regulation of ferroptosis phenotypes. Experiment results showed that ZFP36 was significantly overexpressed in both tissue and cells while CD44 was on the contrary.Meanwhile,spearman analysis showed both ZFP36 and CD44 has a strong correlation with different immune cells,especially macrophage.ConclusionThe regulation of the immune landscape in DKD is significantly influenced by the focal point on ferroptosis. Newly identified ferroptosis markers, CD44 and ZFP36, are poised to play essential roles through their interactions with macrophages, adding substantial value to this regulatory landscape.

Recent Advances in the Management of Diabetic Kidney Disease: Slowing Progression.

Diabetic kidney disease (DKD) is a major cause of chronic kidney disease (CKD), and it heightens the risk of cardiovascular incidents. The pathogenesis of DKD is thought to involve hemodynamic, inflammatory, and metabolic factors that converge on the fibrotic pathway. Genetic predisposition and unhealthy lifestyle practices both play a significant role in the development and progression of DKD. In spite of the recent emergence of angiotensin receptors blockers (ARBs)/angiotensin converting enzyme inhibitor (ACEI), sodium-glucose cotransporter 2 (SGLT2) inhibitors, and nonsteroidal mineralocorticoid receptors antagonists (NS-MRAs), current therapies still fail to effectively arrest the progression of DKD. Glucagon-like peptide 1 receptor agonists (GLP-1RAs), a promising class of agents, possess the potential to act as renal protectors, effectively slowing the progression of DKD. Other agents, including pentoxifylline (PTF), selonsertib, and baricitinib hold great promise as potential therapies for DKD due to their anti-inflammatory and antifibrotic properties. Multidisciplinary treatment, encompassing lifestyle modifications and drug therapy, can effectively decelerate the progression of DKD. Based on the treatment of heart failure, it is recommended to use multiple drugs in combination rather than a single-use drug for the treatment of DKD. Unearthing the mechanisms underlying DKD is urgent to optimize the management of DKD. Inflammatory and fibrotic factors (including IL-1, MCP-1, MMP-9, CTGF, TNF-a and TGF-β1), along with lncRNAs, not only serve as diagnostic biomarkers, but also hold promise as therapeutic targets. In this review, we delve into the potential mechanisms and the current therapies of DKD. We also explore the additional value of combing these therapies to develop novel treatment strategies. Drawing from the current understanding of DKD pathogenesis, we propose HIF inhibitors, AGE inhibitors, and epigenetic modifications as promising therapeutic targets for the future.

Secretory leukocyte protease inhibitor as a novel predictive biomarker in patients with diabetic kidney disease.

Secretory leukocyte protease inhibitor (SLPI) is a multifunctional protein involved in the chronic inflammatory process, implicated in the pathogenesis of diabetic kidney disease (DKD). However, its potential as a diagnostic and prognostic biomarker of DKD has yet to be evaluated. This study explored the clinical utility of SLPI in the diagnosis and prognosis of renal endpoint events in patients with DKD. A multi-center cross-sectional study comprised of 266 patients with DKD and a predictive cohort study comprised of 120 patients with stage IV DKD conducted between December 2016 and January 2022. The clinical parameters were collected for statistical analysis, a multivariate Cox proportional hazards model was used to evaluate the independent risk factors for renal endpoints. Serum SLPI levels gradually increased with DKD progression (p<0.01). A significant correlation was observed between serum SLPI levels and renal function in patients with DKD. The mean follow-up duration in this cohort study was 2.32 ± 1.30 years. Multivariate Cox regression analysis showed SLPI levels≥51.61ng/mL (HR=2.95, 95% CI[1.55, 5.60], p<0.01), 24h urinary protein levels≥3500 mg/24h (HR=3.02, 95% CI[1.66, 5.52], p<0.01), Alb levels<30g/l (HR=2.19, 95% CI[1.12, 4.28], p<0.05), HGB levels<13g/dl (HR=3.18, 95% CI[1.49, 6.80], p<0.01), and urea levels≥7.1 mmol/L (HR=8.27, 95% CI[1.96, 34.93], p<0.01) were the independent risk factors for renal endpoint events in DKD patients. Serum SLPI levels increased with DKD progression and were associated with clinical parameters of DKD. Moreover, elevated SLPI levels showed potential prognostic value for renal endpoint events in individuals with DKD. These findings validate the results of previous studies on SLPI in patients with DKD and provide new insights into the role of SLPI as a biomarker for the diagnosis and prognosis of DKD that require validation.

New insights into the role of immunity and inflammation in diabetic kidney disease in the omics era.

Diabetic kidney disease (DKD) is becoming the leading cause of chronic kidney disease, especially in the industrialized world. Despite mounting evidence has demonstrated that immunity and inflammation are highly involved in the pathogenesis and progression of DKD, the underlying mechanisms remain incompletely understood. Substantial molecules, signaling pathways, and cell types participate in DKD inflammation, by integrating into a complex regulatory network. Most of the studies have focused on individual components, without presenting their importance in the global or system-based processes, which largely hinders clinical translation. Besides, conventional technologies failed to monitor the different behaviors of resident renal cells and immune cells, making it difficult to understand their contributions to inflammation in DKD. Recently, the advancement of omics technologies including genomics, epigenomics, transcriptomics, proteomics, and metabolomics has revolutionized biomedical research, which allows an unbiased global analysis of changes in DNA, RNA, proteins, and metabolites in disease settings, even at single-cell and spatial resolutions. They help us to identify critical regulators of inflammation processes and provide an overview of cell heterogeneity in DKD. This review aims to summarize the application of multiple omics in the field of DKD and emphasize the latest evidence on the interplay of inflammation and DKD revealed by these technologies, which will provide new insights into the role of inflammation in the pathogenesis of DKD and lead to the development of novel therapeutic approaches and diagnostic biomarkers.

PPARα/δ dual agonist H11 alleviates diabetic kidney injury by improving the metabolic disorders of tubular epithelial cells

Diabetic kidney disease (DKD) is responsible for nearly half of all end-stage kidney disease and kidney failure is a major driver of mortality among patients with diabetes. To date, few safe and effective drugs are available to reverse the decline of kidney function. Kidney tubules producing energy by fatty acid metabolism are pivotal in development and deterioration of DKD. Peroxisome proliferator-activated receptors (PPARs), comprising PPARα, PPARδ and PPARγ play a senior role in the pathogenesis of DKD for their functions in glycemic control and lipid metabolism; whereas systemic activation of PPARγ causes serious side-effects in clinical settings. Compound H11 was a potent PPARα and PPARδ (PPARα/δ) dual agonist with potent and well-balanced PPARα/δ agonistic activity and a high selectivity over PPARγ. In this study, the potential therapeutic effects of compound H11 were determined in a db/db mouse model of diabetes. Expressions of PPARα and PPARδ in nuclei of tubules were markedly reduced in diabetes. Transcriptional changes of tubular cells showed that H11 was an effective PPARα/δ dual agonist taking effects both in vivo and in vitro. Systemic administration of H11 showed glucose tolerance and lipid metabolic benefits in db/db mice. Moreover, H11 treatment exerted protective effects on diabetic kidney injury. In addition to fatty acid metabolism, H11 also regulated diabetes-induced metabolic alternations of branch chain amino acid degradation and glycolysis. The present study demonstrated a crucial role of H11 in regulation of energy homeostasis and metabolism in glucose-treated tubular cells. Overall, compound H11 holds therapeutic promise for DKD.

GLP-1 receptor agonist attenuates tubular cell ferroptosis in diabetes via enhancing AMPK-fatty acid metabolism pathway through macropinocytosis

Kidney tubules are mostly responsible for pathogenesis of diabetic kidney disease. Actively reabsorption of iron, high rate of lipid metabolism and exposure to concentrated redox-active compounds constructed the three main pillars of ferroptosis in tubular cells. However, limited evidence has indicated that ferroptosis is indispensable for diabetic tubular injury. Glucagon-like peptide-1 receptor agonist (GLP-1RA) processed strong benefits on kidney outcomes in people with diabetes. Moreover, GLP-1RA may have additive effects by improving dysmetabolism besides glucose control and weight loss. Therefore, the present study aimed at exploring the benefits of exendin-4, a high affinity GLP-1RA on kidney tubular dysregulation in diabetes and the possible mechanisms involved, with focus on ferroptosis and adenosine 5′-monophosphate-activated protein kinase (AMPK)-mitochondrial lipid metabolism pathway. Our data revealed that exendin-4 treatment markedly improved kidney structure and function by reducing iron overload, oxidative stress, and ACSL4-driven lipid peroxidation taken place in diabetic kidney tubules, along with reduced GPX4 expression and GSH content. AMPK signaling was identified as the downstream target of exendin-4, and enhancement of AMPK triggered the transmit of its downstream signal to activate fatty acid oxidation in mitochondria and suppress lipid synthesis and glycolysis, and ultimately alleviated toxic lipid accumulation and ferroptosis. Further study suggested that exendin-4 was taken up by tubular cells via macropinocytosis. The protective effect of exendin-4 on tubular ferroptosis was abolished by macropinocytosis blockade. Taken together, present work demonstrated the beneficial effects of GLP-1RA treatment on kidney tubular protection in diabetes by suppressing ferroptosis through enhancing AMPK-fatty acid metabolic signaling via macropinocytosis.