Models Of Diabetic Kidney Disease Research Articles

Tonabersat suppresses priming/activation of the NOD-like receptor protein-3 (NLRP3) inflammasome and decreases renal tubular epithelial-to-macrophage crosstalk in a model of diabetic kidney disease

BackgroundAccompanied by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, aberrant connexin 43 (Cx43) hemichannel-mediated ATP release is situated upstream of inflammasome assembly and inflammation and contributes to multiple secondary complications of diabetes and associated cardiometabolic comorbidities. Evidence suggests there may be a link between Cx43 hemichannel activity and inflammation in the diabetic kidney. The consequences of blocking tubular Cx43 hemichannel-mediated ATP release in priming/activation of the NLRP3 inflammasome in a model of diabetic kidney disease (DKD) was investigated. We examined downstream markers of inflammation and the proinflammatory and chemoattractant role of the tubular secretome on macrophage recruitment and activation.MethodsAnalysis of human transcriptomic data from the Nephroseq repository correlated gene expression to renal function in DKD. Primary human renal proximal tubule epithelial cells (RPTECs) and monocyte-derived macrophages (MDMs) were cultured in high glucose and inflammatory cytokines as a model of DKD to assess Cx43 hemichannel activity, NLRP3 inflammasome activation and epithelial-to-macrophage paracrine-mediated crosstalk. Tonabersat assessed a role for Cx43 hemichannels.ResultsTranscriptomic analysis from renal biopsies of patients with DKD showed that increased Cx43 and NLRP3 expression correlated with declining glomerular filtration rate (GFR) and increased proteinuria. In vitro, Tonabersat blocked glucose/cytokine-dependant increases in Cx43 hemichannel-mediated ATP release and reduced expression of inflammatory markers and NLRP3 inflammasome activation in RPTECs. We observed a reciprocal relationship in which NLRP3 activity exacerbated increased Cx43 expression and hemichannel-mediated ATP release, events driven by nuclear factor kappa-B (NFκB)-mediated priming and Cx43 hemichannel opening, changes blocked by Tonabersat. Conditioned media (CM) from RPTECs treated with high glucose/cytokines increased expression of inflammatory markers in MDMs, an effect reduced when macrophages were pre-treated with Tonabersat. Co-culture using conditioned media from Tonabersat-treated RPTECs dampened macrophage inflammatory marker expression and reduced macrophage migration.ConclusionUsing a model of DKD, we report for the first time that high glucose and inflammatory cytokines trigger aberrant Cx43 hemichannel activity, events that instigate NLRP3-induced inflammation in RPTECs and epithelial-to-macrophage crosstalk. Recapitulating observations previously reported in diabetic retinopathy, these data suggest that Cx43 hemichannel blockers (i.e., Tonabersat) may dampen multi-system damage observed in secondary complications of diabetes.

Proteomic and lipidomic analysis of the mechanism underlying astragaloside IV in mitigating ferroptosis through hypoxia-inducible factor 1α/heme oxygenase 1 pathway in renal tubular epithelial cells in diabetic kidney disease

Ethnopharmacological relevanceThe limitations of modern medicine in mitigating the pathological process of diabetic kidney disease (DKD) necessitate novel, precise, and effective prevention and treatment methods. Huangqi, the root of Astragalus membranaceus Fisch. ex Bunge has been used in traditional Chinese medicine for various kidney ailments. Astragaloside IV (AS-IV), the primary pharmacologically active compound in A. membranaceus, is involved in lipid metabolism regulation; however, its potential in ameliorating renal damage in DKD remains unexplored. Aim of the studyTo elucidate the specific mechanism by which AS-IV moderates DKD progression. Materials and methodsA murine model of DKD and high glucose-induced HK-2 cells were treated with AS-IV. Furthermore, multiomics analysis, molecular docking, and molecular dynamics simulations were performed to elucidate the mechanism of action of AS-IV in DKD, which was validated using molecular biological methods. ResultsAS-IV regulated glucose and lipid metabolism in DKD, thereby mitigating lipid deposition in the kidneys. Proteomic analysis identified 12 proteins associated with lipid metabolism regulated by AS-IV in the DKD renal tissue. Additionally, lipid metabolomic analysis revealed that AS-IV upregulated and downregulated 4 beneficial and 79 harmful lipid metabolites, respectively. Multiomics analysis further indicated a positive correlation between the top-ranked differential protein heme oxygenase (HMOX)1 and the levels of various harmful lipid metabolites and a negative correlation with the levels of beneficial lipid metabolites. Furthermore, enrichment of both ferroptosis and hypoxia-inducible factor (HIF)-1 signaling pathways during the AS-IV treatment of DKD was observed using proteomic analysis. Validation results showed that AS-IV effectively reduced ferroptosis in DKD-affected renal tubular epithelial cells by inhibiting HIF-1α/HMOX1 pathway activity, upregulating glutathione peroxidase-4 and ferritin heavy chain-1 expression, and downregulating acyl-CoA synthetase long-chain family member-4 and transferrin receptor-1 expression. Our findings demonstrate the potential of AS-IV in mitigating DKD pathology by downregulating the HIF-1α/HMOX1 signaling pathway, thereby averting ferroptosis in renal tubular epithelial cells. ConclusionsAS-IV is a promising treatment strategy for DKD via the inhibition of ferroptosis in renal tubular epithelial cells. The findings of this study may help facilitate the development of novel therapeutic strategies.

Chlorogenic acid alleviates renal fibrosis by reducing lipid accumulation in diabetic kidney disease through suppressing the Notch1 and Stat3 signaling pathway

Aims Abnormal renal lipid metabolism causes renal lipid deposition, which leads to the development of renal fibrosis in diabetic kidney disease (DKD). The aim of this study was to investigate the effect and mechanism of chlorogenic acid (CA) on reducing renal lipid accumulation and improving DKD renal fibrosis. Methods This study evaluated the effects of CA on renal fibrosis, lipid deposition and lipid metabolism by constructing in vitro and in vivo models of DKD, and detected the improvement of Notch1 and Stat3 signaling pathways. Molecular docking was used to predict the binding between CA and the extracellular domain NRR1 of Notch1 protein. Results In vitro studies have shown that CA decreased the expression of Fibronectin, α-smooth muscle actin (α-SMA), p-smad3/smad3, alleviated lipid deposition, promoted the expression of carnitine palmitoyl transferase 1 A (CPT1A), and inhibited the expression of cholesterol regulatory element binding protein 1c (SREBP1c). The expression of Notch1, Cleaved Notch1, Hes1, and p-stat3/stat3 were inhibited. These results suggested that CA might reduce intercellular lipid deposition in human kidney cells (HK2) by inhibiting Notch1 and stat3 signaling pathways, thereby improving fibrosis. Further, in vivo studies demonstrated that CA improved renal fibrosis and renal lipid deposition in DKD mice by inhibiting Notch1 and stat3 signaling pathways. Finally, molecular docking experiments showed that the binding energy of CA and NRR1 was −6.6 kcal/mol, which preliminarily predicted the possible action of CA on Notch1 extracellular domain NRR1. Conclusion CA reduces renal lipid accumulation and improves DKD renal fibrosis by inhibiting Notch1 and stat3 signaling pathways.

Acupuncture improve proteinuria in diabetic kidney disease rats by inhibiting ferroptosis and epithelial-mesenchymal transition

ObjectiveTo explore the mechanism of acupuncture to relieve diabetic proteinuria in Diabetic Kidney Disease (DKD). MethodsA total of 10 male Sprague-Dawley rats were randomly selected as the negative control group (NC), and a further 30 rats were fed a high-fat diet (HFD) and intraperitoneal streptozotocin (STZ). The DKD model rats in the model group (DKD) and the acupuncture group (DKD + Acu) were randomly assigned. After 4 weeks of intervention, collected urine, peripheral blood, and renal tissues from all rats, and assessed blood urea nitrogen, serum creatinine, triglyceride, 24-h urine protein quantification, and blood glucose, left kidney weight, kidney body ratio index, then observe changes in renal histology in the rats. The renal cortex tissues of three rats from each group were sent for transcriptomic analysis. According to the results of transcriptomic analysis, various kits were used to detect SOD, MDA, GSH, GSH-px, and iron concentration. The expression levels of GPX4 and System Xc-, members of the antioxidative stress pathway, and TfR 1, SLC39A14, FTH 1, and SLC40A1, involved in iron metabolism, in the kidney tissues were measured by western blotting and reverse transcription-quantitative PCR. The expression of mesenchymal phenotype markers and podocyte-specific markers were evaluated by immunofluorescence. ResultsAcupuncture promoted the levels of GSH, GSH-px, and SOD, decreased the level of MDA (P < 0.05), promoted the expression of GPX4 and System Xc- (P < 0.05), decreased the expression of TfR1 and SLC39A14 (P < 0.01), and increased the expression of FTH 1 and SLC40A1 (P < 0.05), inhibited the expression of TGF-β1, desmin, FSP-1, and α-SMA (P < 0.05), promoted the expression of Nephrin, Podocin, and CD2AP (P < 0.05). ConclusionImproving the ability of podocytes to prevent oxidative stress and restoring iron ion homeostasis, can improve Ferroptosis and block epithelial-mesenchymal transition, improve podocyte injury, restore filtration function, and reduce proteinuria in DKD rats.

#2584 Mapping Progression of DKD in ReninAAV UNx db/db mice utilizing time-series RNA sequencing

Abstract Background and Aims The Renin adeno-associated virus (AAV) uninephrectomized (UNx) db/db mouse model of diabetic kidney disease (DKD) recapitulates hallmarks of DKD with hypertension, including albuminuria and glomerulosclerosis. Despite previous characterization of the model, gaps persist in our understanding regarding temporal patterns of gene expression and pathophysiology in the glomeruli. This longitudinal study characterizes isolated glomeruli from ReninAAV UNx db/db mice during progression of DKD with hypertension. Method Female db/db (BKS.Cg-Dock7m +/+ Leprdb/J) received a single i.v. dose of LacZAAV (control) or ReninAAV. Untreated db/m mice (BKS.Cg-Dock7m +/+ Leprdb/J) served as healthy controls. One week after AAV injection, ReninAAV mice underwent UNx, recovered for three weeks, and were subsequently randomized and stratified into 4 groups according to fed blood glucose and body weight. ReninAAV UNx mice were terminated at 4, 8, 12 and 16 weeks (w4-w16) after UNx. Control groups were terminated at 16 weeks. Endpoints included urine and plasma biochemistry, kidney histology (i.e. Collagen 1a1 and Glomerulosclerosis) and RNA sequencing of glomeruli isolated by laser capture microdissection. Results ReninAAV UNx db/db mice showed progressive kidney fibrosis (Col1a1%-area) and albuminuria (uACR) compared to LacZAAV db/db and db/m mice across all timepoints investigated. Furthermore, a significant increase in glomerulosclerosis was observed from w12 and onwards compared to LacZAAV and db/m mice. When compared to LacZAAV mice, more than 1,700 glomerular differentially regulated genes and more than 250 uniquely regulated genes were found at each timepoint, including markers of fibrosis, inflammation and glomerular injury. Several glomerular drug targets exhibited time-specific regulated in ReninAAV UNx db/db mice (e.g. Slc5a2, Egfr, Agtr1a). Regulated genes were segmented into nine clusters with distinct temporal patterns. Among these, a continuous increase in expression of genes associated with extracellular matrix (ECM), small molecule transport and temporal stimulation of metabolic pathways. Finally, a correlation analysis revealed strong associations between ECM and ECM-related pathways vs. glomerulosclerosis and albuminuria. Conclusion The ReninAAV UNx db/db mouse model of hypertensive DKD demonstrates a progressive increase in fibrosis and albuminuria paralleled by dynamic changes in glomerular gene expression signatures. Notably, the identification of temporal patterns of gene expression and pathway regulation provides novel insights into DKD pathophysiology and progression. This underscores the ReninAAV UNx db/db mouse as an applicable model of human DKD in preclinical target and drug discovery.

#2267 IL-33 Inhibition with tozorakimab for diabetic kidney disease: a randomized, placebo-controlled phase 2b study

Abstract Background and Aims In patients with type 2 diabetes (T2D) and chronic kidney disease (CKD), persistent, low-grade inflammation is a significant risk factor for adverse kidney outcomes. Previously, we identified interleukin-33 (IL-33) as an over-expressed inflammatory cytokine in kidney biopsies from patients with CKD and showed a significant benefit of inhibiting IL-33 signaling in a rodent model of diabetic kidney disease (DKD). Here, we investigate the therapeutic potential of tozorakimab, a high-affinity IL-33-neutralizing immunoglobulin G1 monoclonal antibody, in patients with DKD. Method FRONTIER-1 (NCT04170543) was a phase 2b, randomized, double-blind, placebo-controlled, multicenter study to evaluate the efficacy, safety, pharmacokinetics (PK), and immunogenicity of tozorakimab in patients with DKD; defined as T2D with an estimated glomerular filtration rate (eGFR) of 25-75 mL/min/1.73 m2, a urinary albumin-to-creatinine ratio (UACR) of 100-3,000 mg/g, and on angiotensin-converting-enzyme inhibitor (ACEi) or angiotensin-receptor blocker (ARB) for &amp;gt; 6 weeks before the treatment period. Participants were randomly assigned to four different doses of tozorakimab or volume-matched placebo, administered subcutaneously every 28 days for 24 weeks. The randomization process was stratified based on region and the use of SGLT2 inhibitors (SGLT2i). All participants received 10 mg of dapagliflozin Days 85–168, discontinuing existing SGLT2i if applicable. The primary objective of the study was to evaluate the effect of tozorakimab on UACR. A sample size of 565 patients provided at least 80% power to detect a 30% reduction in the change from baseline to week 24 in UACR between each tozorakimab treatment group and placebo. Efficacy endpoints were evaluated in the Per Protocol Population (PPP; initiated dapagliflozin treatment), whereas safety and tolerability were assessed for all dosed participants. Results Between 11 December 2019 and 16 May 2023, we assessed 1,575 patients for eligibility in Argentina, Canada, Chile, Peru, South Korea, Japan, and the United States. 599 (38%) participants were assigned to tozorakimab or placebo. Owing to the COVID-19 pandemic, the study was temporarily halted between March and June 2020. This precautionary measure resulted in the discontinuation of 26 participants. Upon resuming, the study incorporated significant modifications to adapt to the pandemic environment. 573 participants (Full Analysis Population) were randomized after the study was restarted: 29.7% were female; their mean age (±SD) was 67 years (±10); mean eGFR was 48 mL/min/1.73 m2 (±15); and the geometric mean UACR was 460 mg/g (coefficient of variation = 151%). Most participants were taking an ACEi (30%) or ARB (65%), and 28% an SGLT2i. 139 participants received placebo, while 96, 91, 95, and 152 participants received 30, 60, 120, and 300 mg tozorakimab, respectively. The baseline characteristics were balanced across treatment groups, and significant target engagement was observed in all dose arms. Tozorakimab was generally well tolerated and the incidences of adverse events (Aes), serious Aes, discontinuations, and death were similar to placebo. Initial evaluation identified 476 participants in the PPP with 109, 81, 80, 72, and 134 on placebo, 30, 60, 120, and 300 mg tozorakimab, respectively. The primary efficacy analysis demonstrated an inhibition of IL-33 signaling but failed to establish a statistically significant difference in UACR reduction between placebo and treatment groups. The median %-change from baseline was –22% (–46%, 24%) in the placebo group and ranged from –23% to –25% in the tozorakimab groups at week 24. Similarly, no significant effect on eGFR was observed. Following study completion, Good Clinical Practice concerns arose at one clinical trial site with 15 participants. The investigation of these issues is ongoing, but significant alterations to the study results are not anticipated. Conclusion This study did not demonstrate a clinically meaningful reduction in UACR with tozorakimab treatment in patients with T2D and CKD. Further investigation is warranted to clarify the role of IL-33 in DKD.

#1206 Combination therapy with Lisinopril and Dapagliflozin rescues GFR decline and glomerular damage in the KKAy mouse model of diabetic kidney disease

Abstract Background and Aims Male KKAy mice develop diabetic kidney disease (DKD) with progressive decline of GFR upon high fat diet feeding which is accelerated by the vasoconstrictor LNNA. In this study, efficacy of combination therapy with clinical relevant therapeutics in the form of an ACE-inhibitor (Lisinopril) and SGLT2-inhibitor (Dapagliflozin) on renal function and histopathology was investigated. Method Male KKAy mice underwent uninephrectomy (UNX). After recovery mice received high fat diet (45% LARD) and 50 mg/L LNNA in drinking water (wk0). At week 4, Lisinopril (2.5 mg/kg/day in drinking water) and at week 8 Dapagliflozin (5 and 20 mg/kg/day in diet) were started. Body weight, blood glucose, food and water intake and albuminuria were determined regularly. GFR was measured transdermally by FITC-sinistrin clearance. Mice were terminated at week 16 and renal histology was scored by a team of renal histopathologists. Non-induced and induced non-treated mice were used as controls. Results Treatment of KKAy mice on HFD+LNNA with Dapagliflozin reduced blood glucose immediately. Combination therapy with Lisinopril and Dapagliflozin (5 mg/kg/day) rescued the progressive GFR decline to levels seen in non-induced chow-controls (p &amp;lt; 0.05 vs UNX+HFD+LNNA). Pathology showed that the percentage of healthy glomeruli increased from 14% to 26% after combination therapy (Dapa 5). Interstitial fibrosis and tubular atrophy were significantly reduced by combination therapy (p &amp;lt; 0.05 vs UNX+HFD+LNNA). Conclusion Male KKAy mice on a HFD and LNNA developed DKD resulting in CKD. Combination therapy with Lisinopril and Dapagliflozin rescued GFR decline and reduced glomerular damage and interstitial fibrosis and tubular atrophy This indicates the clinical relevance of the model which can be used to study compound efficacy in both early and more advanced stages of DKD.

#1311 Cardiac damage in a multifactorial DKD/CKD mouse model resembles HFpEF and can be reduced by standard-of-care treatment

Abstract Background and Aims We developed a diet-induced hypertension-accelerated mouse model of diabetic kidney disease characterized by progressive loss of GFR resulting in chronic kidney disease. Since cardiovascular disease is the major cause of death in CKD, we characterized the functional and structural cardiac damage in this model. Additionally, we studied the efficacy of combination therapy with an ACE-inhibitor (Lisinopril) and SGLT2-inhibitor (Dapagliflozin) on cardiac histopathology. Method Male KKAy mice underwent uninephrectomy. After recovery mice received high fat diet (45% LARD) and drinking water with or without 50 mg/L LNNA (wk0). At 12 weeks, imaging was performed to study cardiac function and mice were terminated at week 13. In the intervention study, at week 4, lisinopril (2.5 mg/kg/day; drinking water) and at week 8 dapagliflozin (5 and 20 mg/kg/day; foodadmix) treatment were started. At week 16 mice were terminated and lung and heart weight and cardiac histology were determined. Results Upon termination, macroscopic evaluation of the hearts showed extensive scar tissue formation on the outside of the left ventricle. Histological evaluation showed the presence left ventricular hypertrophy, coronary calcification and myocardial fibrosis in male KKAy mice with UNX, HFD and LNNA. KKAy mice with UNX and HFD but without LNNA also showed myocardial fibrosis, monocyte infiltration and focal mineralization. Imaging showed preserved ejection fraction, a significant reduction, increased left ventricle posterior wall thickness and decrease left ventricle inner diameter. Treatment with combination therapy reduced lung wet weight, significantly reduced heart weight and significantly decreased cardiac fibrosis. Macroscopically, less scar tissue was observed after treatment. Conclusion This multifactorial mouse model shows cardiac damage on a background of hypertension, diabetes, renal dysfunction and obesity. Functional measurement including preserved ejection fraction, left ventricle hypertrophy, diastolic dysfunction and increased fibrosis resembling the clinical HFpEF phenotype. Combination therapy with Lisinopril and Dapagliflozin reduced cardiac weight and cardiac fibrosis. This indicates cardiac involvement in the DKD mouse model which confirms that this multifactorial model is clinically relevant.

Romosozumab rescues impaired bone mass and strength in a murine model of diabetic kidney disease

As international incidence of diabetes and diabetes-driven comorbidities such as chronic kidney disease (CKD) continue to climb, interventions are needed that address the high-risk skeletal fragility of what is a complex disease state. Romosozumab (Romo) is an FDA-approved sclerostin inhibitor that has been shown to increase bone mineral density and decrease fracture rates in osteoporotic patients with mild to severe CKD, but its effect on diabetes-weakened bone is unknown. We aimed to test Romo's performance in a model of combined diabetes and CKD. 6-week old male C57BL/6 mice were randomly divided into control (CON) and disease model (STZ-Ad) groups, using a previously established streptozotocin- and adenine-diet-induced model. After 16 weeks of disease induction, both CON and STZ-Ad groups were subdivided into two treatment groups and given weekly subcutaneous injections of 100 μL vehicle (phosphorus buffered saline, PBS) or 10 mg/kg Romo. Mice were euthanized after 4 weeks of treatment via cardiac exsanguination and cervical dislocation. Hindlimb bones and L4 vertebrae were cleaned of soft tissue, wrapped in PBS-soaked gauze and stored at 20C. Right tibiae, femora, and L4s were scanned via microcomputed tomography; tibiae were then tested to failure in 4-pt bending while L4s were compression tested. Romo treatment significantly increased cortical and trabecular bone mass in both STZ-Ad and CON animals. These morphological improvements created corresponding increases in cortical bending strength and trabecular compression strength, with STZ-Ad treated mice surpassing vehicle CON mice in all trabecular mechanics measures. These results suggest that Romo retains its efficacy at increasing bone mass and strength in diabetic kidney disease.

Is the Zucker Diabetic Fat Rat an Appropriate Model of Diabetic Kidney Disease: A Discussion on Hydronephrosis

Introduction: Kidney disease is a significant concern for global healthcare, particularly in individuals with diabetes. Experimental research into the pathology of diabetes is dependent on the utilisation of appropriate animal models. The Zucker Diabetic Fat (ZDF) rat has a Leptin receptor deficiency and is a commonly used model of type 2 diabetes. Here, we examine the relevance of this model for the investigation of diabetic kidney disease, with a particular focus on vascular imaging. The Zucker Lean (ZL) rat is a non-diabetic strain of the same rat lineage and will be used as a control. Method: This dataset used the ZDF rat (n=13) and the Zucker Lean (ZL) rat (n=16). Individuals were scanned at ages of 12 weeks (ZDF n=4, ZL n=4), 22 weeks (ZDF n=4, ZL n=5), and 40 weeks (ZDF n=5, ZL n=7). During open laparotomy, rats were maintained on a respirator under general anaesthesia via isoflurane and blood pressure was monitored via a central catheter. During surgery, a blood glucose measurement was taken from the tail vein and samples of arterial blood and urine were collected. The contrast medium Microfil (MV122, Flow Tech Inc., Carver, MA) was injected post-mortem via the renal artery and excised kidneys were scanned for 11 hours in a ZEISS XRadia 410 Versa μCT scanner (Carl Zeiss Microscopy GmbH, Jena, Germany). Results: As expected, the ZDF rats developed significant hyperglycaemia from 12 weeks, significant increases in albumin creatinine ratio (ACR) from 22 weeks, and a significant decrease in glomerular filtration rate (GFR) from 40 weeks. Concurrent hydronephrosis affecting more than 10% of total kidney volume was detected in 51% of all rats (group prevalence rates were ZDF=69% and ZL=38%). The percent of the kidney mass affected by hydronephrosis was significantly higher in ZDF than ZL (ANOVA row factor p=0,029*). Age had no significant impact on the size of the hydronephrotic cavity. There was a significant correlation between the degree of hyperglycaemia and the size of the hydronephrotic cavity (mm) (r=0.57, p=0,0014**). ACR, GFR and urine excretion rate were unaffected by hydronephrosis. Hydronephrosis did not have a significant impact on vascular density in the remaining kidney tissue (p=0.53). Conclusion: The high prevalence of hydronephrosis development in the ZDF rat questions its appropriateness to model human diabetic kidney disease, as hydronephrosis is not commonly seen in diabetic patients. While vascular density was preserved in the remaining kidney tissue, the huge loss of vascular tissue caused by hydronephrotic erosion means this model is limited for all renal imaging applications. Both ZDF and ZL rats develop hydronephrosis suggesting this may be a genetic condition of their common lineage. However, the conditions' increased severity in the ZDF rat indicates that the deficit in the leptin receptor contributes to disease pathology. The significant relationship between hyperglycaemia and hydronephrosis size suggests the two conditions are interacting, raising questions about whether the underlying molecular mechanisms of this model of diabetic kidney disease have any relevance for translational research. Funded by European Research Council Synergy grant SURE, project no. 854796. 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.

Systemic administration of miR-451 improves autophagy response in an accelerated mouse model of diabetic kidney disease

Diabetic kidney disease (DKD) is a common and severe microvascular complication of both type 1 and type 2 diabetes mellitus. It is the most common cause of end stage renal disease in the United States. Autophagy is a highly conserved pathway that recycles damaged organelles and macromolecules to maintain intracellular homeostasis. There is mounting evidence highlighting the role of autophagy in diabetic kidney disease. MicroRNAs (miRNA) affect the transcription of several genes involved in the development and progression of DKD, regulating key pathways and processes, including fibrotic signaling and autophagy. Thus, the dysregulation of miRs exacerbates kidney injury in the diabetic setting. Previous work from our group suggests that chronic systemic inhibition of miR-451 exacerbates fibrosis in the kidney of an obese insulin resistant mouse model (tallyHo/Jng), indicating a potential renoprotective role for miR-451 in the diabetic kidney. However, few studies have investigated this protective role of miR-451 in the diabetic kidney. This study aimed to understand the renoprotective effects of miR-451 in an accelerated mouse model of DKD. BTBR ob/ob mice rapidly develop morphological renal lesions characteristic of both early and advanced human DKD, with proteinuria typically beginning at 4-weeks of age. 12-week old male black and tan, brachyuric (BTBR) ob/ob mice and BTBR wild-type mice were treated with commercially available miR-451 mimics (2mg/kg/bw; n=3/treatment group; treatment groups: WT, BTBR ob/ob, WT+miR-451, and BTBR ob/ob+miR-451) 1X per week for 3-consective weeks. Spot urine was collected weekly until the end of study. Random blood glucose was measured by tail prick weekly. Mice were humanely euthanized, and kidneys and blood harvested at the end of the treatment period. Western blotting of whole kidney homogenate was performed following a standard protocol. Data was analyzed using students t-test or ANOVA, where appropriate, p&lt;0.05 considered significant. Albumin: creatinine ratio was four times higher (p=0.01) in BTBR ob/ob mice beginning at 5 weeks. Treatment with miR-451 mimics had no significant effect on body weight in either group. Blood glucose was significantly higher in miR-451 treated and untreated BTBR ob/ob mice at 12-weeks (425± 33.1 mg/dL; p=0.04) and 13-weeks (383± 25.3 mg/dL; p=0.007). However, blood glucose decreased significantly from week 13 (554.7± 10.8 mg/dL) to week 14(289± 13.3 mg/dL; p=0.0002) in BTBR ob/ob miR-451 treated mice. Western blot analysis of MiR-451 mimic treated BTBR ob/ob and WT mice whole kidney homogenate revealed a 91% (p=0.02) and 95% (p=0.01) reduction, respectively, in YWHAZ (a predicted miR-451 target). Conversely, western blot analysis of autophagy related proteins revealed a 68% (p=0.01) increase in ATG101 and a 44% increase in Beclin-1 in miR-451 mimic treated BTBR ob/ob mice. Collectively, the findings of this study suggest that miR-451 may offer protection in the diabetic kidney via autophagic signaling and improved blood glucose control. GHUCCTS KL2 Scholar Program (Award Number: KL2TR001432) The Charles and Mary Latham Fund Howard University Department of Medicine Start-Up Funds; Howard University Bridge Fund and Pilot Study Awards. 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.

Polysaccharides from Dendrobium officinale delay diabetic kidney disease interstitial fibrosis through LncRNA XIST/TGF-β1

PurposeRenal interstitial fibrosis is a pathological manifestation of the progression of diabetic kidney disease (DKD). Dendrobium officinale polysaccharides (DOP), one of the major active components of Dendrobium officinale, have hypoglycemic and hypolipidemic effects and are used clinically to treat diabetes. However, the role of DOP in delaying DKD progression remains unclear. This study aimed to explore the potential mechanisms by which DOP delays DKD renal interstitial fibrosis. MethodsUsing db/db mice as a model of DKD, we administered DOP by gavage and observed its therapeutic effectiveness. Employing ASO technology, we knocked down lncRNA XIST expression in kidney tissues and detected the expression of lncRNA XIST, TGF-β1, and renal interstitial fibrosis-related molecules. ResultsDOP was primarily composed of monosaccharides, with 91.57% glucose and 1.41% mannose, forming a spheroid-like structure. It has a high polydispersity index with an Mw/Mn of 6.146, and the polysaccharides are mainly connected by 4-Man(p) and 4-Glc(p) linkages. In the kidneys of db/db mice, lncRNA XIST and TGF-β1 are highly expressed; however, their expression is significantly reduced after gastric infusion with DOP, and upon knockdown of lncRNA XIST, it might delay the progression of renal interstitial fibrosis in DKD. ConclusionDOP may delay the progression of DKD renal interstitial fibrosis through the regulation of the LncRNA XIST/TGF-β1 related fibrotic pathway. This provides a new perspective for clinical strategies to delay the progression of DKD renal interstitial fibrosis.

Set7 Methyltransferase and Phenotypic Switch in Diabetic Glomerular Endothelial Cells.

Hyperglycaemia influences the development of glomerular endothelial cell damage and nowhere is this more evident than in the progression of diabetic kidney disease (DKD). While the Set7 lysine methyltransferase is a known hyperglycaemic sensor, its role in endothelial cell function in the context of DKD remains poorly understood. Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and shRNA inhibition of Set7. Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of single cell RNA-seq (scRNA-seq) data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of GEN cell populations through transcriptional regulation of IGFBP5 (Insulin growth factor binding protein 5). Chromatin immunoprecipitation assays confirmed the expression of the IGFBP5 gene was associated with mono- and di-methylation of histone H3 lysine 4 (H3K4me1/2). The generalisability was investigated in human renal and circulating hyperglycaemic cells exposed to TGFβ1. We show that the highly selective Set7 inhibitor, PFI-2, attenuated indices associated with renal cell damage and mesenchymal transition; specifically (i) reactive oxygen species production, (ii) IGFBP5 gene regulation, and (iii) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely correspond in human GEN cells with PFI-2 inhibition or Set7 shRNA silencing. Set7 regulates the phenotypic endothelial-mesenchymal transition (EDMT) switch and suggest that targeting the lysine methyltransferase could protect glomerular cell injury in DKD.

Endothelial NOX5 Obliterates the Reno-Protective Effect of Nox4 Deletion by Promoting Renal Fibrosis via Activation of EMT and ROS-Sensitive Pathways in Diabetes.

Chronic hyperglycemia induces intrarenal oxidative stress due to the excessive production of reactive oxygen species (ROS), leading to a cascade of events that contribute to the development and progression of diabetic kidney disease (DKD). NOX5, a pro-oxidant NADPH oxidase isoform, has been identified as a significant contributor to renal ROS in humans. Elevated levels of renal ROS contribute to endothelial cell dysfunction and associated inflammation, causing increased endothelial permeability, which can disrupt the renal ecosystem, leading to progressive albuminuria and renal fibrosis in DKD. This study specifically examines the contribution of endothelial cell-specific human NOX5 expression in renal pathology in a transgenic mouse model of DKD. This study additionally compares NOX5 with the previously characterized NADPH oxidase, NOX4, in terms of their relative roles in DKD. Regardless of NOX4 pathway, this study found that endothelial cell-specific expression of NOX5 exacerbates renal injury, albuminuria and fibrosis. This is attributed to the activation of the endothelial mesenchymal transition (EMT) pathway via enhanced ROS formation and the modulation of redox-sensitive factors. These findings underscore the potential therapeutic significance of NOX5 inhibition in human DKD. The study proposes that inhibiting NOX5 could be a promising approach for mitigating the progression of DKD and strengthens the case for the development of NOX5-specific inhibitors as a potential therapeutic intervention.

Nephroprotective effect of HZF by upregulating IκBα protein expression in DKD mice

HEZHIFNAG (HZF) is a traditional Chinese prescription used to treat diabetes. In this study, a mouse model of diabetic kidney disease (DKD) induced by alloxan was used, and the random blood glucose of diabetic mice decreased significantly after the intervention of HZF, and the morphological results showed that HZF improved the pathological damage of glomeruli, reduced the degree of fibrosis of kidney tissue, and increased the expression level of IKBα in the kidney of diabetic mice, suggesting that HZF has a protective effect on the DKD mice, and the mechanism of action may be related to the up-regulation of IKB-α expression.

Tangshen Formula alleviates inflammatory injury against aged diabetic kidney disease through modulating gut microbiota composition and related amino acid metabolism

Diabetic kidney disease (DKD) is leading causes and one of the fastest growing causes of chronic kidney disease worldwide, and leads to high morbidity and mortality. Emerging evidences have revealed gut microbiota dysbiosis and related metabolism dysfunction play a dominant role in DKD progression and treatment through modulating inflammation. Our previous studies showed that Tangshen Formula (TSF), a Chinese herbal prescription, exhibited anti-inflammatory effect on DKD, but underlying mechanism that involved gut microbiota and related metabolism in aged model remained obscure. Here, BTBR ob/ob mice were used to establish aged DKD model, and 16S rRNA sequence and untargeted metabolomic analyses were employed to investigate the correlation between colonic microbiota and serum metabolism. The aged ob/ob mice exhibited obvious glomerular and renal tubule injury and kidney function decline in kidney, while TSF treatment significantly attenuated these abnormalities. TSF also exhibited potent anti-inflammatory effect in aged ob/ob mice indicating by reduced proinflammatory factor IL-6 and TNF-α, MCP-1 and COX-2 in serum, kidney and intestine, which suggested the involvement of gut microbiota with TSF effect. The 16S rDNA sequencing of the colonic microbiome and untargeted serum metabolomics analysis revealed significant differences in gut microbiota structure and serum metabolomic profiles between WT and ob/ob mice. Notably, TSF treatment reshaped the structure of gut microbiota and corrected the disorder of metabolism especially tryptophan metabolism and arginine biosynthesis. TSF increased Anaeroplasma and Barnesiella genera and decreased Romboutsia, Akkermansia, and Collinsella genera, and further elevated tryptophan, 5-hydroxyindoleacetate, glutamic acid, aspartate and reduced 4-hydroxy-2-quinolinecarboxylic acid, indole-3-acetic acid, xanthurenic acid, glutamine. Further correlation analysis indicated that disturbed gut microbiota was linked to tryptophan metabolism and arginine biosynthesis to regulate inflammation in aged DKD. Our data revealed that TSF attenuated renal inflammation by modulating gut microbiota and related amino acid metabolism in aged DKD model, highlighting gut microbiota and related metabolism functioned as potential therapeutic target for DKD in elderly patients.

Qi-dan-dihuang decoction ameliorates renal fibrosis in diabetic rats via p38MAPK/AKT/mTOR signaling pathway.

Qi-dan-dihuang decoction (QDD) has been used to treat diabetic kidney disease (DKD), but the underlying mechanisms are poorly understood. This study reveals the mechanism by which QDD ameliorates DKD. The compounds in QDD were identified by high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry (HPLC-Q-TOF-MS). Key targets and signaling pathways were screened through bioinformatics. Nondiabetic Lepr db/m mice were used as control group, while Lepr db/db mice were divided into model group, dapagliflozin group, 1% QDD-low (QDD-L), and 2% QDD-high (QDD-H) group. After 12 weeks of administration, 24 h urinary protein, serum creatinine, and blood urea nitrogen levels were detected. Kidney tissues damage and fibrosis were evaluated by pathological staining. In addition, 30 mmol/L glucose-treated HK-2 and NRK-52E cells to induce DKD model. Cell activity and migration capacity as well as protein expression levels were evaluated. A total of 46 key target genes were identified. Functional enrichment analyses showed that key target genes were significantly enriched in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, in vivo and in vitro experiments confirmed that QDD ameliorated renal fibrosis in diabetic mice by resolving inflammation and inhibiting the epithelial-mesenchymal transition (EMT) via the p38MAPK and AKT-mammalian target of rapamycin (mTOR) pathways. QDD inhibits EMT and the inflammatory response through the p38MAPK and AKT/mTOR signaling pathways, thereby playing a protective role in renal fibrosis in DKD.

Mechanism of ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix regulates HIF-1α signaling pathway mediated by renal hypoxia to ameliorate renal fibrosis in DKD rats

This study explored the mechanism of the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix in ameliorating renal fibrosis in the rat model of diabetic kidney disease(DKD) based on the expression of hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF) and HIF-1α/platelet-derived growth factor(PDGF)/platelet-derived growth factor receptor(PDGFR) signaling pathways in the DKD rats. After 1 week of adaptive feeding, 50 male SPF-grade Wistar rats were randomized into a blank group(n=7) and a modeling group. After 24 h of fasting, the rats in the modeling group were subjected to intraperitoneal injection of streptozocin and fed with a high-sugar and high-fat diet to establish a DKD model. After modeling, the rats were randomly assigned into model(n=7), low-dose ultrafiltration extract(n=7), medium-dose ultrafiltration extract(n=7), irbesartan(n=8), and high-dose ultrafiltration extract(n=8) groups. After intervention by corresponding drugs for 12 weeks, the general conditions of the rats were observed. The body weights and blood glucose levels of the rats were measured weekly, and the 24 h urinary protein(24hUP) was measured at the 6th and 12th weeks of drug administration. After the last drug administration, the renal function indicators were determined. Masson staining was employed to observe the pathological changes of the renal tissue. The expression of prolyl hydroxylase domain 2(PHD2) and HIF-1α in the renal tissue was detected by immunohistochemistry(IHC). Real-time qPCR was employed to determine the mRNA levels of PHD2, VEGF, PDGF, and PDGFR in the renal tissue. Western blot was employed to determine the protein levels of HIF-1α, VEGF, PDGF, and PDGFR in the renal tissue. The results showed that compared with the model group, drug administration lowered the levels of glycosylated serum protein(GSP), aerum creatinine(Scr), and blood urea nitrogen(BUN) in a dose-dependent manner(P&lt;0.05 or P&lt;0.01) and mitigated the pathological changes in the renal tissue. Furthermore, drug administration up-regulated mRNA level of PHD2(P&lt;0.05 or P&lt;0.01), down-regulated the mRNA levels of VEGF, PDGF, and PDGFR(P&lt;0.05 or P&lt;0.01) and the protein levels of HIF-1α, VEGF, PDGF, and PDGFR(P&lt;0.01) in the renal tissue, and increased the rate of PHD2-positive cells(P&lt;0.01). In conclusion, the ultrafiltration extract of Angelicae Sinensis Radix and Hedysari Radix effectively alleviated the renal fibrosis in DKD rats by inhibiting the expression of key proteins in the HIF-1α signaling pathway mediated by renal hypoxia and reducing extracellular matrix(ECM) deposition.

Co-differential genes between DKD and aging: implications for a diagnostic model of DKD.

Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus (DM) that is closely related to aging. In this study, we found co-differential genes between DKD and aging and established a diagnostic model of DKD based on these genes. Differentially expressed genes (DEGs) in DKD were screened using GEO datasets. The intersection of the DEGs of DKD and aging-related genes revealed DKD and aging co-differential genes. Based on this, a genetic diagnostic model for DKD was constructed using LASSO regression. The characteristics of these genes were investigated using consensus clustering, WGCNA, functional enrichment, and immune cell infiltration. Finally, the expression of diagnostic model genes was analyzed using single-cell RNA sequencing (scRNA-seq) in DKD mice (model constructed by streptozotocin (STZ) injection and confirmed by tissue section staining). First, there were 159 common differential genes between DKD and aging, 15 of which were significant. These co-differential genes were involved in stress, glucolipid metabolism, and immunological functions. Second, a genetic diagnostic model (including IGF1, CETP, PCK1, FOS, and HSPA1A) was developed based on these genes. Validation of these model genes in scRNA-seq data revealed statistically significant variations in FOS, HSPA1A, and PCK1 gene expression between the early DKD and control groups. Validation of these model genes in the kidneys of DKD mice revealed that Igf1, Fos, Pck1, and Hspa1a had lower expression in DKD mice, with Igf1 expression being statistically significant. Our findings suggest that DKD and aging co-differential genes are significant in DKD diagnosis, providing a theoretical basis for novel research directions on DKD.

SKP alleviates the ferroptosis in diabetic kidney disease through suppression of HIF-1α/HO-1 pathway based on network pharmacology analysis and experimental validation.

Diabetic kidney disease (DKD) represents a microvascular complication of diabetes mellitus. Shenkang Pills (SKP), a traditional Chinese medicine formula, has been widely used in the treatment of DKD and has obvious antioxidant effect. Ferroptosis, a novel mode of cell death due to iron overload, has been shown to be associated with DKD. Nevertheless, the precise effects and underlying mechanisms of SKP on ferroptosis in diabetic kidney disease remain unclear. The active components of SKP were retrieved from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Protein-protein interaction (PPI) network and Herb-ingredient-targets gene network were constructed using Cytoscape. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted utilizing the Metascape system database. Additionally, an in vivo model of DKD induced by Streptozotocin (STZ) was established to further investigate and validate the possible mechanisms underlying the effectiveness of SKP. We retrieved 56 compounds and identified 223 targets of SKP through the TCMSP database. Key targets were ascertained using PPI network analysis. By constructing a Herb-Ingredient-Targets gene network, we isolated the primary active components in SKP that potentially counteract ferroptosis in diabetic kidney disease. KEGG pathway enrichment analysis suggested that SKP has the potential to alleviate ferroptosis through HIF signaling pathway, thereby mitigating renal injury in DKD. In animal experiments, fasting blood glucose, 24h urine protein, urea nitrogen and serum creatine were measured. The results showed that SKP could improve DKD. Results from animal experiments were also confirmed the efficacy of SKP in alleviating renal fibrosis, oxidative stress and ferroptosis in DKD mice. These effects were accompanied by the significant reductions in renal tissue expression of HIF-1α and HO-1 proteins. The mRNA and immunohistochemistry results were the same as above. SKP potentially mitigating renal injury in DKD by subduing ferroptosis through the intricacies of the HIF-1α/HO-1 signaling pathway.