Chronic Kidney Injury Research Articles

Dapagliflozin alleviates renal inflammation and protects against diabetic kidney diseases, both dependent and independent of blood glucose levels.

Diabetic kidney disease (DKD) has become the leading cause of end-stage renal disease worldwide. Therefore, efforts to understand DKD pathophysiology and prevent its development at the early phase are highly warranted. Here, we analyzed kidneys from healthy mice, diabetic mice, and diabetic mice treated with the sodium-glucose cotransporter 2 inhibitor dapagliflozin using ATAC and RNA sequencing. The findings were verified at the protein levels and in cultured cells. Our combined method of ATAC and RNA sequencing revealed Csf2rb, Btla, and Isg15 as the key candidate genes associated with hyperglycemia, azotemia, and albuminuria. Their protein levels were altered together with multiple other inflammatory cytokines in the diabetic kidney, which was alleviated by dapagliflozin treatment. Cell culture of immortalized renal tubular cells and macrophages unraveled that dapagliflozin could directly effect on these cells in vitro as an anti-inflammatory agent independent of glucose concentrations. We further proved that dapagliflozin attenuated ischemia/reperfusion-induced chronic kidney injury and renal inflammation in mice. Overall, our data emphasize the importance of inflammatory factors to the pathogenesis of DKD, and provide valuable mechanistic insights into the renoprotective role of dapagliflozin.

Elucidating the role of nicotinamide N-methyltransferase-p53 axis in the progression of chronic kidney disease.

Chronic kidney disease (CKD) is a significant global health issue characterized by progressive loss of kidney function. Renal interstitial fibrosis (TIF) is a common feature of CKD, but current treatments are seldom effective in reversing TIF. Nicotinamide N-methyltransferase (NNMT) has been found to increase in kidneys with TIF, but its role in renal fibrosis is unclear. Using mice with unilateral ureteral obstruction (UUO) and cultured renal interstitial fibroblast cells (NRK-49F) stimulated with transforming growth factor-β1 (TGF-β1), we investigated the function of NNMT in vivo and in vitro. We performed single-cell transcriptome sequencing (scRNA-seq) on the kidneys of mice and found that NNMT increased mainly in fibroblasts of UUO mice compared to sham mice. Additionally, NNMT was positively correlated with the expression of renal fibrosis-related genes after UUO injury. Knocking down NNMT expression reduced fibroblast activation and was accompanied by an increase in DNA methylation of p53 and a decrease in its phosphorylation. Our findings suggest that chronic kidney injury leads to an accumulation of NNMT, which might decrease p53 methylation, and increase the expression and activity of p53. We propose that NNMT promotes fibroblast activation and renal fibrosis, making NNMT a novel target for preventing and treating renal fibrosis.

A Clinical Trial of Comprehensive Regenerative Treatment for Acute and Chronic Kidney Injury by Autologous Granulocyte-Colony Stimulating Factor (G-CSF)-Mobilized Peripheral Blood-Derived CD34+ Cell Administration

A Clinical Trial of Comprehensive Regenerative Treatment for Acute and Chronic Kidney Injury by Autologous Granulocyte-Colony Stimulating Factor (G-CSF)-Mobilized Peripheral Blood-Derived CD34+ Cell Administration

Environmental Circadian Disruption Accelerates 5/6 Nephrectomy-Induced Chronic Kidney Injury in Mice

Environmental Circadian Disruption Accelerates 5/6 Nephrectomy-Induced Chronic Kidney Injury in Mice

A New Method for Estimating Glomerular Filtration Rate in Rhesus Macaques (Macaca mulatta).

Late effects of total- or partial-body irradiation include chronic kidney injury (CKI), which increases morbidity and mortality. Glomerular filtration rate (GFR) is the gold standard measure of kidney function. Renal function markers, such as blood urea nitrogen (BUN) and serum creatinine (Cr), may not be higher than reference ranges until 50% or more of nephrons are affected. Currently available methods to measure GFR are difficult and expensive, requiring multiple blood draws or timed urine collections, but their use can provide a framework for the development of simpler GFR estimates. The measurement of iohexol clearance is a validated tool used to determine GFR in veterinary patients. In this study, we aimed to determine if the Schwartz formula as used in human pediatric medicine can estimate GFR in rhesus macaques. We hypothesized that iohexol-GFR would correlate with the Schwartz formula-estimated GFR (eGFR) in irradiated and non-irradiated rhesus macaques. Twelve rhesus macaques [age 5-14 years (mean 7 years); 5 females, 7 males] with a range of BUN levels were selected for comparison to 4 non-irradiated controls (2 females, 2 males). Irradiated animals were divided by BUN into 3 groups: BUN ≤20 mg/dL (n = 4), BUN >20-24 mg/dL (n = 4), and BUN ≥25 mg/dL (n = 4). Baseline serum chemistry and urinalysis were used to assess renal function. For measurement of GFR, macaques were maintained under general anesthesia and received an intravenous injection of iohexol (2 mL/kg, 300 mg I/mL). Whole blood was collected at 10, 30, 60 and 90 min post-iohexol injection. Plasma iohexol concentrations were determined by mass spectrometry. GFR was calculated from the peak iohexol concentration and trapezoidal area under the curve (tAUC). The iohexol-GFR significantly correlated with the Schwartz formula-eGFR. In macaques with renal irradiation doses below 6 Gy, GFR was higher for males than females. GFR was lower in macaques with renal irradiation doses greater than 6 Gy compared to macaques with renal doses less than 6 Gy. We conclude that use of the Schwartz formula can provide a rapid, non-invasive, cost-effective, and accurate estimation of GFR to aid in the clinical assessment of renal function in irradiated rhesus macaques.

Endothelial cell activation and glycocalyx shedding - potential as biomarkers in patients with lupus nephritis.

Lupus nephritis (LN) is a common and severe manifestation of systemic lupus erythematosus and an important cause of acute and chronic kidney injury. Early diagnosis of LN and preventing relapses are key to preserving renal reserve. However, due to the complexity and heterogeneity of the disease, clinical management remains challenging. Kidney biopsy remains the gold standard for confirming the diagnosis of LN and subsequent assessment of kidney histopathology, but it is invasive and cannot be repeated frequently. Current clinical indicators of kidney function such as proteinuria and serum creatinine level are non-specific and do not accurately reflect histopathological changes, while anti-dsDNA antibody and C3 levels reflect immunological status but not kidney injury. Identification of novel and specific biomarkers for LN is prerequisite to improve management. Renal function deterioration is associated with changes in the endothelial glycocalyx, a delicate gel-like layer located at the interface between the endothelium and bloodstream. Inflammation induces endothelial cell activation and shedding of glycocalyx constituents into the circulation. This review discusses the potential role of soluble glycocalyx components as biomarkers of active LN, especially in patients in whom conventional serological and biochemical markers do not appear helpful.

Myocardial Remodeling in Early Chronic Kidney Disease—Mineral and Bone Disorder Model with Low Bone Turnover

Chronic kidney disease—mineral and bone disorder (CKD-MBD) plays a significant role in causing cardiovascular morbidity and mortality related to CKD. CKD-MBD has been studied during advanced stages when changes in inorganic phosphate (Pi) and its hormonal regulation are obvious. The initial phases of myocardial remodeling (MR) in early CKD-MBD remain poorly understood. We induced mild CKD-MBD in spontaneously hypertensive rats using 3/4 nephrectomy. Animals were fed standard chow, containing 0.6% phosphate. In each animal, we analyzed indices of chronic kidney injury, bone turnover and Pi exchange, and assessed the myocardial histology and gene expression profile. Applied CKD-MBD models corresponded to human CKD S1-2 with low bone turnover and without an increase in systemic Pi-regulating factors (parathyroid hormone and fibroblast growth factor 23). In mild CKD-MBD models, we found MR features characterized by cardiomyocyte hypertrophy, interstitial and perivascular fibrosis, intramyocardial artery media thickening, along with alterations in Ppp3ca, Mapk1, Jag1, Hes1, Ptch1, Numb, Lgr4 and Bmp4 genes. Among other genes, the down-regulation of Jag1 was most tightly associated with either myocardial hypertrophy or fibrosis. Myocardial alterations concurrently occurred with mild CKD-MBD and comprised fibrosis preceding cardiomyocyte hypertrophy. The histological features of MR were associated with myocardial P accumulation in settings of low bone turnover, prior to a response of systemic Pi-regulating factors and with alterations in calcineurin, ERK1/2, Notch, BMP and Hedgehog genes.

Mesenchymal stem cell‐derived extracellular vesicles in treatment against renal fibrosis

AbstractBackgroundRenal fibrosis (RF) is the main pathological pathway of acute kidney injury and chronic kidney disease. Its main pathological feature is extracellular matrix aggregation in the renal stroma and glomerulus. There is currently no effective treatment to delay or treat RF. Mesenchymal stem cells (MSCs) are multipotent somatic stem cells that can direct differentiation, direct cell‐cell interactions and have secretory activity. The most important secretory activity of MSCs is mediated by extracellular vesicles (EVs). At present, there is increasing evidence that EVs produced by MSCs may be used in regenerative methods of tissue repair.AimThe goal of this review is to present the latest efficacy of MSC‐EVs in the prevention, treatment and improvement of RF caused by acute and chronic kidney injury and reveals the molecular mechanism of MSCs in various models.Results and discussionMSC‐EVs show potential for RF by regulating autophagy, inhibiting apoptosis, countering oxidation, suppressing inflammation, reducing EMT and modulating angiogenesis.ConclusionMSC‐EVs show potential for treating RF. However, more studies establishing their safety, efficiency and specific mechanisms in the context of RF are still needed before further clinical application.

Acute and chronic kidney injury during therapy for pediatric acute leukemia: A report from the Leukemia Electronic Abstraction of Records Network (LEARN).

Children with acute leukemia are at increased risk of kidney injury. Using electronic health record data from three centers between 2010 and 2018, this study retrospectively described acute kidney injury (AKI) and chronic kidney disease (CKD) prevalence in children with acute lymphoblastic or myeloid leukemia (ALL, AML) using Common Terminology Criteria for Adverse Events (CTCAE) and Kidney Disease Improving Global Outcomes (KDIGO) definitions. AKI during therapy was 25% (ALL) and 32% (AML) using CTCAE, versus 84% (ALL) and 74% (AML) using KDIGO. CKD prevalence was low and Grade 1/Stage 2. Further investigation is needed to optimally define kidney injury in acute leukemia.

Omega-3 Fatty Acids Attenuate Renal Fibrosis via AMPK-Mediated Autophagy Flux Activation.

The unilateral ureteral obstruction (UUO) injury model is well-known to mimic human chronic kidney disease, promoting the rapid onset and development of kidney injury. ω3-poly unsaturated fatty acids (PUFAs) have been observed to protect against tissue injury in many disease models. In this study, we assessed the efficacy of ω3-PUFAs in attenuating UUO injury and investigated their mechanism of action. The immortalized human proximal tubular cells human kidney-2 (HK2) were incubated for 72 h with docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) in various concentrations, in the presence or absence of transforming growth factor (TGF)-β. DHA/EPA reduced the epithelial-mesenchymal transition in the TGF-β-treated HK2 cells by enhancing autophagy flux and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. C57BL/6 mice were divided into four groups and treated as follows: sham (no treatment, n = 5), sham + ω3-PUFAs (n = 5), UUO (n = 10), and UUO + ω3-PUFAs (n = 10). Their kidneys and blood were harvested on the seventh day following UUO injury. The kidneys of the ω3-PUFAs-treated UUO mice showed less oxidative stress, inflammation, and fibrosis compared to those of the untreated UUO mice. Greater autophagic flux, higher amounts of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II, Beclin-1, and Atg7, lower amounts of p62, and higher levels of cathepsin D and ATP6E were observed in the kidneys of the omega-3-treated UUO mice compared to those of the control UUO mice. In conclusion, ω3-PUFAs enhanced autophagic activation, leading to a renoprotective response against chronic kidney injury.

Pathway from Acute Kidney Injury to Chronic Kidney Disease: Molecules Involved in Renal Fibrosis.

Acute kidney injury (AKI) is one of the main conditions responsible for chronic kidney disease (CKD), including end-stage renal disease (ESRD) as a long-term complication. Besides short-term complications, such as electrolyte and acid-base disorders, fluid overload, bleeding complications or immune dysfunctions, AKI can develop chronic injuries and subsequent CKD through renal fibrosis pathways. Kidney fibrosis is a pathological process defined by excessive extracellular matrix (ECM) deposition, evidenced in chronic kidney injuries with maladaptive architecture restoration. So far, cited maladaptive kidney processes responsible for AKI to CKD transition were epithelial, endothelial, pericyte, macrophage and fibroblast transition to myofibroblasts. These are responsible for smooth muscle actin (SMA) synthesis and abnormal renal architecture. Recently, AKI progress to CKD or ESRD gained a lot of interest, with impressive progression in discovering the mechanisms involved in renal fibrosis, including cellular and molecular pathways. Risk factors mentioned in AKI progression to CKD are frequency and severity of kidney injury, chronic diseases such as uncontrolled hypertension, diabetes mellitus, obesity and unmodifiable risk factors (i.e., genetics, older age or gender). To provide a better understanding of AKI transition to CKD, we have selected relevant and updated information regarding the risk factors responsible for AKIs unfavorable long-term evolution and mechanisms incriminated in the progression to a chronic state, along with possible therapeutic approaches in preventing or delaying CKD from AKI.

Effect of Intestinal Flora on Hyperuricemia-Induced Chronic Kidney Injury in Type 2 Diabetic Patients and the Therapeutic Mechanism of New Anti-Diabetic Prescription Medications.

This article examined the current research on hyperuricemia (HUA) exacerbating diabetic kidney damage and novel anti-diabetic medications for treating these people. Hyperuricemia and type 2 diabetes (T2D), both of which are frequent metabolic disorders, are closely connected. Recent studies have shown that hyperuricemia can increase kidney injury in T2D patients by aggravating insulin resistance, by activating the renin-angiotensin-aldosterone system (RAAS), and by stimulating inflammatory factors, and the diversity, distribution, and metabolites of intestinal flora. Considering this, there are just a few of the research examining the effect of hyperuricemia on diabetic kidney injury via intestinal flora. Through the gut-kidney axis, intestinal flora primarily influences renal function. The primary mechanism is that variations in diversity, distribution, and metabolites of intestinal flora led to alterations in metabolites (such as short-chain fatty acids, Indoxyl sulfate and p-cresol sulfate, Trimethylamine N-oxide TMAO). This article reviewed the research and investigates the association between hyperuricemia and T2D, as well as the influence of hyperuricemia on diabetic kidney injury via intestinal flora. In addition, the current novel antidiabetic drugs are discussed, and their characteristics and mechanisms of action are reviewed. These novel antidiabetic drugs include SGLT2 inhibitors, GLP-1 receptor agonists, DDP-4 inhibitors, glucokinase (GK) enzyme activators (GK agonists), and mineralocorticoid receptor antagonists (MRA). Recent studies suggest that these new anti-diabetic medications may have a therapeutic effect on hyperuricemia-induced kidney impairment in diabetes patients via various mechanisms. Some of these medications may reduce blood uric acid levels, while others may improve kidney function by attenuating the overstimulation of RAAS or by decreasing insulin resistance and inflammation in the kidneys. These novel antidiabetic medicines may have a multifaceted approach to treating hyperuricemia-induced kidney impairment in diabetic patients; nevertheless, additional study is required to establish their efficacy and comprehend their specific mechanisms.

Nephroprotective Effects of Cardamonin on Renal Ischemia Reperfusion Injury/UUO-Induced Renal Fibrosis.

Acute kidney injury and chronic renal fibrosis are intractable pathological processes to resolve, yet limited strategies are able to effectively address them. Cardamonin (CAD) is a flavonoid with talented antioxidant, anti-inflammatory capacity, and satisfactory biosafety. In our study, animal and cellular models of renal ischemia/reperfusion (I/R) and unilateral ureteral obstruction (UUO) were successfully constructed to confirm whether CAD confers protective effects and underlying mechanisms. Animal experiments demonstrated that CAD application (100 mg/kg) distinctly ameliorated tissue damage and improved renal function. Meanwhile, the continuous oral administration of CAD after UUO surgery efficiently inhibited renal fibrosis as confirmed by hematoxylin-eosin (H&E), Sirius red, and Masson staining as well as the downregulated mRNA and protein expression of collagen I, α-smooth muscle actin (α-SMA), collagen III, and fibronectin. Interestingly, in transforming growth factor β1 (TGF-β1)-stimulated and hypoxia/reoxygenation (H/R)-exposed human kidney-2 (HK-2) cells, protective effects of CAD were again authenticated. Meanwhile, we performed bioinformatics analysis and constructed the "ingredient-target-pathway-disease" network to conclude that the potential mechanisms of CAD protection may be through the regulation of oxidative stress, inflammation, apoptosis, and mitogen-activated protein kinase (MAPK) pathway. Furthermore, experimental data validated that CAD evidently decreased the reactive oxygen species (ROS) production and malondialdehyde (MDA) content while depressing the mRNA and protein expression of inflammatory markers (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and Il-1β) and inhibiting apoptosis as evidenced by decreased levels of P53, BAX, cleaved caspase-3, and apoptotic rate in renal I/R and UUO models. In addition, the impact of CAD on restraining oxidative stress and inflammation was attributed to its ability to elevate antioxidant enzyme activities including catalase, superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2) and to inhibit the inflammation-associated MARK/nuclear factor-κB (MAPK/NF-κB) signaling pathway. In conclusion, cardamonin restored the antioxidative capacity to block oxidative stress and suppressed the MAPK/NF-κB signaling pathway to alleviate inflammatory response, thus mitigating I/R-generated acute kidney injury/UUO-induced renal fibrosis in vivo and in vitro, which indicated the potential therapeutic advantage of cardamonin in attenuating acute and chronic kidney injuries.

Determination of serum KIM-1 in patients with chronic kidney injury

Background: Chronic kidney disease (CKD) affects more than 800 million people worldwide and is one of the leading non-communicable causes of death. Despite being a latent issue, once renal damage has started, the disease can rapidly progress to an advanced stage. Aims and Objectives: Currently, the most commonly used markers for the diagnosis of renal disease are non-specific and insensitive. As a result, the goal of the current study is to investigate whether KIM-1 could be a precise and sensitive biomarker for identifying early kidney injury in CKD patients. Materials and Methods: This case–control study recruited 155 participants from the Index Medical College Hospital v Research Centre, Indore, Madhya Pradesh, based on inclusion and exclusion criteria. 150 non-CKD subjects matched for age and sex were also taken from the hospital. The levels of KIM-1 were compared between CKD and non-CKD participants. Serum creatinine, urea, and creatinine clearance were also measured. Results: The levels of KIM-1 were substantially higher in CKD patients than in non-CKD participants. In addition, a negative relationship between KIM-1 and creatinine clearance was observed with a P<0.05. Conclusion: KIM-1 is a precise and sensitive kidney injury biomarker that can identify early kidney injury in CKD and contribute to the progression of interstitial fibrosis in kidney disease.

Identification of cell division cycle protein 20 in various forms of acute and chronic kidney injury in mice.

Tubular epithelial cell fate following exposure to various types of injurious stimuli can be decided at distinct cell cycle checkpoints. One such checkpoint occurs during mitosis, known as the spindle assembly checkpoint, and is tightly regulated through the actions of cell division cycle protein 20 (CDC20). Due to our paucity of knowledge about the role of CDC20 in the kidney, the present study was designed to investigate the expression levels and distribution of CDC20 within the kidney and how pharmacological inhibition of CDC20 function affects kidney recovery using various rodent models of kidney injury. CDC20 is normally detected in distal tubules, but upon injury by either cisplatin administration or ureter obstruction, CDC20 accumulation is considerably elevated. Blockade of CDC20 activity using a selective pharmacological inhibitor, Apcin, lowered serum creatinine, tubular damage, and DNA injury following acute kidney injury compared with vehicle-treated mice. In unilateral ureteral obstruction, Apcin reduced tissue kidney injury molecule-1 levels, sirius red staining, and tubulointerstitial α-smooth muscle actin staining in the tissue. The findings in the present study demonstrated that elevations in CDC20 levels in the kidney are associated with kidney injury and that inhibition of CDC20 can alleviate and reverse some of the pathological effects on the architecture and function of kidney.NEW & NOTEWORTHY To our knowledge, this is the first study to characterize the expression and localization of cell division cycle 20 protein (CDC20) in normal and acute, and chronically injured kidneys. Tubular epithelial cell damage was markedly reduced through the administration of a selective inhibitor of CDC20, Apcin. This study provides new evidence that CDC20 can be induced in damaged kidney cells and negatively impact the recovery of the kidney following acute kidney injury.

#5333 IMPROVING AUTOPHAGY FLUX BY TFEB ACTIVATION WITH CERIA NANOPARTICLES ATTENUATED CHRONIC KIDNEY INJURY IN CELLULAR AND ANIMAL MODELS OF FABRY DISEASE

Abstract Background and Aims Fabry disease (FD) is a lysosome storage disease (LSD) characterized by significantly reduced intracellular autophagy function. This contributes to the progression of intracellular pathologic signaling and can lead to organ injury. Phospholipid–polyethyleneglycol-capped Ceria-Zirconia antioxidant nanoparticles (PEG-CZNPs) have been reported to enhance autophagy flux. We accessed the action mechanisms of PEG-CZNPs in autophagy regulation and checked the effect on chronic kidney injury in cellular and animal models of FD. Method PEG-CZNPs were synthesized using a non-hydrolytic sol-gel reaction method. HK-2 cells were transfected with α-galactosidase A (α-GLA) shRNA for permanent cellular model of FD. For in-vivo study 4-week-old male B6;129-Glatm1Kul/J mice were treated for 48 weeks with 10mg/kg of PEG-CZNPs twice per week via intraperitoneal injection. PCR, immunoblotting, immunoflouresce assay, electron microscopy analysis, ICP-MS, biochemical and histological analysis were done Results TFEB tanslocated to the nucleus by treatment with PEG-CZNPs. Autophagy flux was evaluated with chloroquine. Autophagy flux was enhanced by PEG-CZNPs treatment. To show whether TFEB plays the important role in autophagy flux, we transfected HK-2 cells with siTFEB. Autophagy flux significantly decreased after knockdown of TFEB with PEG-CZNPs treatment. We next assessed upper signaling pathway of TFEB by PEG-CZNPs. TFEB dephosphorylation was independent of both mTOR and ERK but GSK3ß signaling pathway showed massive impact on TFEB dephosphoryation by PEG-CZNPs. PEG-CZNPs decrease intracellular globotriaosyceramide (Gb3) accumulation and decreased the levels of Collagen type IV, αSMA and MMP9 expression in cellular model of FD. Gb3 levels were significantly reduced in the kidney tissues and the levels of Fibronectin, Collagen type 4 and αSMA was decreased by PEG-CZNPs in animal model of FD. Conclusion These results suggested PEG-CZNPs promote autophagy flux through GSK3ß -TFEB signaling pathways, showed the beneficial effect on renal fibrosis in cellular and animal models of FD. It thus provided a new insights of the potential therapeutics on FD.

Hypoxia controls expression of kidney-pathogenic MUC1 variants.

The interplay between genetic and environmental factors influences the course of chronic kidney disease (CKD). In this context, genetic alterations in the kidney disease gene MUC1 (Mucin1) predispose to the development of CKD. These variations comprise the polymorphism rs4072037, which alters splicing of MUC1 mRNA, the length of a region with variable number of tandem repeats (VNTR), and rare autosomal-dominant inherited dominant-negative mutations in or 5' to the VNTR that causes autosomal dominant tubulointerstitial kidney disease (ADTKD-MUC1). As hypoxia plays a pivotal role in states of acute and chronic kidney injury, we explored the effects of hypoxia-inducible transcription factors (HIF) on the expression of MUC1 and its pathogenic variants in isolated primary human renal tubular cells. We defined a HIF-binding DNA regulatory element in the promoter-proximal region of MUC1 from which hypoxia or treatment with HIF stabilizers, which were recently approved for an anti-anemic therapy in CKD patients, increased levels of wild-type MUC1 and the disease-associated variants. Thus, application of these compounds might exert unfavorable effects in patients carrying MUC1 risk variants.

#4033 RAPID COMPLEMENT INHIBITION WITH THE C5 INHIBITOR CROVALIMAB: A TIMING ANALYSIS USING ANIMAL MODEL AND COMPOSER TRIAL DATA

Abstract Background and Aims Atypical haemolytic uraemic syndrome (aHUS) is a life-threatening disease, characterised by acute kidney injury, thrombocytopenia, and microangiopathic haemolytic anaemia due to complement dysregulation. Currently approved treatments for aHUS include the anti-complement C5 monoclonal antibodies eculizumab and ravulizumab. Although currently approved therapeutic options are effective at both inhibiting complement-mediated thrombotic microangiopathy (TMA) and improving renal function, the regular intravenous (IV) infusion dosing regimens impose a significant treatment burden, particularly in paediatric patients (pts) with aHUS, who may need dialysis in the future due to chronic kidney injury and would benefit from preservation of vascular access. Crovalimab is a novel monoclonal antibody developed against complement C5, engineered to allow for small volume subcutaneous (SC) self-injection every 4 weeks, in a weight-based dosing regimen [1]. In the adaptive Phase I/II COMPOSER trial (NCT03157635) evaluating crovalimab in pts with paroxysmal nocturnal haemoglobinuria (PNH), that, like aHUS, is a complement disorder characterised by uncontrolled complement activation, crovalimab has shown maintained disease control and was well tolerated after a median exposure of 3 years. Further, a Phase III study evaluating crovalimab in previously untreated pts met its primary efficacy endpoints [2]. The efficacy and safety of crovalimab are currently being evaluated in adult and paediatric pts with aHUS, either treatment-naive or switching from another complement inhibitor, in the ongoing Phase III single-arm COMMUTE-a and COMMUTE-p trials [3]. Due to the rapidly progressing nature of aHUS, pts experiencing a TMA require rapid suppression of complement activation upon diagnosis to shut down the complement cascade and avoid further kidney deterioration and organ damage. Here, data from in vivo models and the COMPOSER trial were used to determine the time to complete complement inhibition after first crovalimab IV dose. Method Crovalimab's ability to suppress C5 function and complement activity rapidly was initially studied in in vivo models of cynomolgus monkeys. This study assessed the pharmacokinetics and pharmacodynamics of crovalimab in these monkeys after a single IV or SC dose. As part of this study, four animals per group were evaluated with single 4 mg/kg IV and 20 mg/kg IV doses. Crovalimab was also evaluated in the four-part, Phase I/II COMPOSER trial. Part 2 (n = 10) and Part 4A (n = 8) enrolled pts with PNH who were naive to complement inhibition. Pts in Part 2 received crovalimab 375 mg IV on Day 1, 500 mg IV on Day 8, 1000 mg IV on Day 22 and 170 mg SC weekly from Day 36 for 20 weeks. Pts in Part 4A received an optimised crovalimab dosing regimen of 1000 mg IV on Day 1, 340 mg SC on Days 2, 8, 15, and 22 and 680 mg SC Q4W from Day 29 onwards for 20 weeks. Crovalimab concentration, free C5 and complement activity were measured using validated assays in cynomolgus monkeys and in pts with PNH. Results Compared with baseline values in cynomolgus monkeys, a single crovalimab IV dose of 4 mg/kg reduced mean free C5 concentration by 99.6% and terminal complement activity by 81.4%, within 5 minutes after administration. In treatment-naive pts from COMPOSER Parts 2 and 4, mean free C5 concentration dropped to below 1 μg/mL, indicating a high level of target engagement within 1–6 hours from the first IV dose (Figure 1). Correspondingly, inhibition of terminal complement activity was reached within 1 hour, with values near or below the lower limit of quantification (10 U/mL; Figure 2). Complete complement blockade was generally maintained long-term, up to Week 20, in both Parts 2 and 4, regardless of dose. Conclusion Crovalimab induced a complete, rapid, and sustained blockade of terminal complement activity in both non-human primates and pts with PNH, within hours from first dose. The dosing schedule of crovalimab included an initial IV loading dose, allowing for a rapid onset of action, followed by a convenient long-term SC maintenance regimen.

#5790 IMPAIRED FATTY ACID METABOLISM PERPETUATES LIPOTOXICITY ALONG THE TRANSITION TO CHRONIC KIDNEY INJURY

Abstract Background and Aims Understanding the cell-intrinsic mechanisms contributing to the maintenance of a dysfunctional cellular state in chronic kidney disease (CKD) and identifying therapeutic targets are research priorities in renal medicine. A key contributor to chronic kidney histological damage is acute kidney injury, especially ischemia-reperfusion injury (IRI). Persistent cell-extrinsic perturbations generated upon IRI, for example hypoxia, impair the energetic metabolism of Proximal Tubular Cells (PTC) participating in the process of transition from acute to chronic kidney injury. Here, we propose to investigate the PTC intrinsic factors involved in the perpetuation of an impaired cellular state which contribute to CKD progression Method We combined single nucleus transcriptomic, metabolomic and lipidomic approaches in experimental models and patient cohorts to investigate the molecular bases of the progression to chronic kidney allograft injury initiated by IRI. Results The urinary metabolome of kidney transplant recipients with chronic allograft injury and who experienced severe IRI was significantly enriched with long chain fatty acids (FA). We identified a renal FA-related gene signature with low levels of Cpt2 and Acsm5 and high levels of Acsl4 and Acsm5 associated with IRI, transition to chronic injury, and established CKD in mouse models and kidney transplant recipients. The findings were consistent with the presence of Cpt2-, Acsl4+, Acsl5+, Acsm5- PTC failing to recover from IRI as identified by single nucleus RNA sequencing. In vitro experiments indicated that endoplasmic reticulum (ER) stress contributes to CPT2 repression, which, in turn, promotes lipids accumulation, drives profibrogenic epithelial phenotypic changes, and activates the unfolded protein response. Conclusion ER stress through CPT2 inhibition and lipid accumulation, engages an auto-amplification loop leading to lipotoxicity and self-sustained cellular stress. Thus, IRI imprints a persistent FA metabolism disturbance in the proximal tubule sustaining the progression to chronic kidney allograft injury.

#3078 MILD EXPERIMENTAL CKD IS ASSOCIATED WITH MYOCARDIAL FIBROSIS AND NOTCH/HEDGEHOG SIGNALING DYSREGULATION

Abstract Background and Aims Chronic kidney disease (CKD) is an independent cardiovascular risk factor. The initial mechanisms of myocardial remodeling (MR) in CKD are poorly understood. The major developmental signaling pathways Notch, Hedgehog, Wnt, and Bmp seem to be involved in many crucial steps of MR (cardiomyocytes survival and regeneration, fibrotic response, angiogenesis), while their role in MR due to CKD was not previously studied. Phosphate (Pi) is essential for cellular signaling and its redistribution to the cardiovascular compartment in CKD could be a one of the mechanisms of MR. Here we investigated the involvement of Pi/PPi transport, and reactivation of developmental signaling pathways in myocardial alterations associated with mild CKD. Method We induce mild CKD by 3/4 nephrectomy in adult male SHRs (SHR-Nx, n=8) with normal phosphate intake (0.6%) and 2-month follow-up. Controls were sham-operated SHR (SHR-Sham, n=8) and Wistar Kyoto rats (WKY-Sham, n=8). We analyzed chronic kidney injury and Pi exchange parameters, serum intact parathyroid hormone (PTH), intact fibroblast growth factor 23 (FGF23), myocardial phosphorus content (ICP-AES), histomorphometry, PiT1 and PiT2 expression (IHC), and mRNA expression of Slc20a1, Slc20a2, Ankh, Mapk1, Mapk3, Bmp2, Bmp4, Ctnnb1, Fzd2, Sfrp2, Dkk1, Wif1, Numb, Notch1, Jag1, Hes1, Lgr4, Tnfrsf11b, Tnfsf11, Ptch1. Results Systolic blood pressure and myocardial mass index (MMI) were higher in SHRs vs WKY-Sham (Table 1). Decrease in creatinine clearance by 30% and an increase in albuminuria corresponded to mild CKD in SHR-Nx model (Table 1). In SHR-Nx, myocardial interstitial fibrosis was significantly higher (Figure 1), while MMI and cardiomyocyte diameter did not differ compared to SHR-Sham (Table 1). Myocardial alterations were accompanied by the increase in myocardium phosphorus content (Figure 1), down-regulation in PPi-exporter Ankh, and up-regulation in Hes1 and decline in Ptch1 mRNA expression (Figure 2). We found no changes in Slc20a1 and Slc20a2 mRNA levels (Figure 2). An appearance of PiT2-positive fibroblasts-like cells in myocardial interstitium was apparent in SHR-Nx rats (data not shown). Conclusion Myocardial alterations in mild CKD are manifested in an increase in its phosphorus content, interstitial fibrosis, and dysregulation in genes likely related to the induction of the myofibroblasts and extracellular matrix remodeling.