Model Of Chronic Kidney Disease Research Articles

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-β1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

FINERENONE PREVENTS RENAL AND VASCULAR DAMAGE IN A NOVEL MODEL OF TYPE 1 DIABETES MELLITUS

Objective: The non-steroidal mineralocorticoid receptor antagonist (MRA), finerenone (FIN) improves renal and cardiovascular outcomes in patients with chronic kidney disease (CKD) in type 2 diabetes (T2D). We explored the effects of FIN in a novel model of CKD in type 1 diabetes (T1D). Design and method: We employed the Munich Wistar Frömter (MWF) rat model of non-diabetic CKD with mild hypertension and albuminuria to generate a novel model of CKD with T1D. This was achieved by an intra-peritoneal injection of streptozotocin (15 mg/kg) and additional exposure to a high-fat/high-sucrose diet. Oral treatment with FIN (10 mg/kg/day in rat chow) in diabetic animals (MWF-DKD-FIN) was compared to a group of diabetic rats receiving no treatment (MWF-DKD-Control) and a group of non-diabetic untreated MWF rats (MWF). Dietary exposure with or without pharmacological treatment was maintained for 6 weeks (n = 7–10 animals per group). Results: After 6 weeks, MWF-DKD-Control and MWF-DKD-FIN exhibited significantly elevated blood glucose levels (271.7 ± 67.1 mg/dl and 266.3 ± 46.8 mg/dl) as compared to MWF (110.3 ± 4.4 mg/dl; p < 0.05). MWF-DKD-Control showed a 10-fold higher expression of renal damage markers Kim-1 and Ngal compared to MWF. This upregulation was significantly suppressed in MWF-DKD-FIN. FIN treatment resulted in a significant reduction of blood pressure and pulse wave velocity compared to MWF-DKD-Control (p < 0.05). Vascular function studies showed an improvement in endothelial function in MWF-DKD-FIN compared to MWF-DKD-Control due to a reduction in pro-contractile prostaglandins, oxidative stress, and inflammatory cytokines (including IL-1, IL-6, TNFalpha and TGFbeta). FIN induced no changes in blood glucose levels, glucose tolerance or the increase in HOMA index compared to MWF-DKD-Control. Conclusions: Our data show that treatment with FIN improves renal and vascular damage in a new rat model of CKD with T1D. These beneficial effects associated with a reduction in inflammation and oxidative stress and was independent from changes in glucose homeostasis.

Segregating the effects of ferric citrate-mediated iron utilization and FGF23 in a mouse model of CKD.

Ferric citrate (FC) is an approved therapy for chronic kidney disease (CKD) patients as a phosphate (Pi) binder for dialysis‐dependent CKD, and for iron deficiency anemia (IDA) in non‐dialysis CKD. Elevated Pi and IDA both lead to increased FGF23, however, the roles of iron and FGF23 during CKD remain unclear. To this end, iron and Pi metabolism were tested in a mouse model of CKD (0.2% adenine) ± 0.5% FC for 6 weeks, with and without osteocyte deletion of Fgf23 (flox‐Fgf23/Dmp1‐Cre). Intact FGF23 (iFGF23) increased in all CKD mice but was lower in Cre+ mice with or without FC, thus the Dmp1‐Cre effectively reduced FGF23. Cre+ mice fed AD‐only had higher serum Pi than Cre− pre‐ and post‐diet, and the Cre+ mice had higher BUN regardless of FC treatment. Total serum iron was higher in all mice receiving FC, and liver Tfrc, Bmp6, and hepcidin mRNAs were increased regardless of genotype; liver IL‐6 showed decreased mRNA in FC‐fed mice. The renal 1,25‐dihydroxyvitamin D (1,25D) anabolic enzyme Cyp27b1 had higher mRNA and the catabolic Cyp24a1 showed lower mRNA in FC‐fed mice. Finally, mice with loss of FGF23 had higher bone cortical porosity, whereas Raman spectroscopy showed no changes in matrix mineral parameters. Thus, FC‐ and FGF23‐dependent and ‐independent actions were identified in CKD; loss of FGF23 was associated with higher serum Pi and BUN, demonstrating that FGF23 was protective of mineral metabolism. In contrast, FC maintained serum iron and corrected inflammation mediators, potentially providing ancillary benefit.

Enhancing the Therapeutic Potential of Mesenchymal Stromal Cell-Based Therapies with an Anti-Fibrotic Agent for the Treatment of Chronic Kidney Disease.

Chronic kidney disease (CKD) affects 1 in 10 members of the general population, placing these patients at an increasingly high risk of kidney failure. Despite the significant burden of CKD on various healthcare systems, there are no effective cures that reverse or even halt its progression. In recent years, human bone-marrow-derived mesenchymal stromal cells (BM-MSCs) have been recognised as a novel therapy for CKDs, owing to their well-established immunomodulatory and tissue-reparative properties in preclinical settings, and their promising safety profile that has been demonstrated in patients with CKDs from several clinical trials. However, renal fibrosis (scarring), a hallmark of CKD, has been shown to impair the viability and functionality of BM-MSCs post-transplantation. This has suggested that BM-MSCs might require a pre-treatment or adjunct therapy that can enhance the viability and therapeutic efficacy of these stromal cells in chronic disease settings. To address this, recent studies that have combined BM-MSCs with the anti-fibrotic drug serelaxin (RLX), have demonstrated the enhanced therapeutic potential of this combination therapy in normotensive and hypertensive preclinical models of CKD. In this review, a critical appraisal of the preclinical data available on the anti-fibrotic and renoprotective actions of BM-MSCs or RLX alone and when combined, as a treatment option for normotensive vs. hypertensive CKD, is discussed.

TREM-2 mediates dendritic cell-induced NO to suppress Th17 activation and ameliorate chronic kidney diseases.

Chronic kidney disease (CKD) is a global public health issue. CKD is caused by the infiltration of various myeloid cell types into renal tissue, resulting in renal fibrosis and tubular atrophy. Unilateral ureteral obstruction (UUO) surgery in mice is a model of CKD and characterized by high expression of the anti-inflammatory receptor, Triggering receptor expressed on myeloid cells 2 (TREM-2), on myeloid cells in affected kidneys. Here, we show that iNOS expression and nitric oxide (NO) induction were decreased in Trem-2-/- bone marrow-derived DCs (BMDCs) and in Trem-2 knockdown DC2.4 cells stimulated in vitro with LPS. The nitration of RORγt was decreased in T cells co-cultured with LPS-stimulated Trem-2-/- BMDCs, enhancing IL-17 production. UUO-treated Trem-2-/- mice displayed aggravated renal pathogenesis accompanied by greater neutrophil infiltration and enhanced Th17 cells differentiation, phenotypes that could be rescued by the administration of L-arginine (a biological precursor of NO). Our data identify a key mechanism underlying TREM-2-mediated NO to modulate the cellular crosstalk between dendritic cells, Th17, and neutrophils. Furthermore, we also reveal TREM-2 as a potential novel target for the development of anti-inflammatory drugs in CKD treatment. KEY MESSAGES: The expression of TREM-2 is increased in nephritis TREM-2+ DCs maintain NO production to negatively regulate Th17 differentiation The severe pathologies of nephritis can be rescued by L-arginine supplementation.

Mitophagy induced by UMI-77 preserves mitochondrial fitness in renal tubular epithelial cells and alleviates renal fibrosis.

Renal fibrosis is the final common outcome of chronic kidney disease (CKD), which remains a huge challenge due to a lack of targeted treatment. Growing evidence suggests that during the process of CKD, the integrity and function of mitochondria in renal tubular epithelial cells (TECs) are generally impaired and strongly connected with the progression of renal fibrosis. Mitophagy, a selective form of autophagy, could remove aberrant mitochondria to maintain mitochondrial homeostasis. Deficiency of mitophagy has been reported to aggravate renal fibrosis. However, whether induction of mitophagy could alleviate renal fibrosis has not been stated. In this study, we explored the effect of mitophagy activation by UMI-77, a compound recently verified to induce mitophagy, on murine CKD model of unilateral ureteral obstruction (UUO) in vivo and TECs in vitro. In UUO mice, we found the changes of mitochondrial damage, ROS production, transforming growth factor (TGF)-β1/Smad pathway activation, as well as epithelial-mesenchymal transition phenotype and renal fibrosis, and these changes were ameliorated by mitophagy enhancement using UMI-77. Moreover, TEC apoptosis, nuclear factor (NF)-κB signaling activation, and interstitial inflammation after UUO were significantly mitigated by augmented mitophagy. Then, we found UMI-77 could effectively and safely induce mitophagy in TECs in vitro, and reduced TGF-β1/Smad signaling and downstream profibrotic responses in TGF-β1-treated TECs. These changes were restored by a mitophagy inhibitor. In conclusion, we demonstrated that mitophagy activation protected against renal fibrosis through improving mitochondrial fitness, downregulating TGF-β1/Smad signaling and alleviating TEC injuries and inflammatory infiltration in kidneys.

Metabolomics analysis of human plasma reveals decreased production of trimethylamine N-oxide retards the progression of chronic kidney disease.

Chronic kidney disease (CKD) is a global public health problem and one of the leading causes of all-cause mortality. However, the pathogenic mechanisms and intervention methods for CKD progression are not fully understood. Plasma from patients with uraemia and from healthy controls (n = 30 per group) was analysed with LC-MS/MS-based non-targeted metabolomics to identify potential markers of uraemia. These potential markers were validated in the same cohort and a second cohort (n = 195) by quantitative analysis of the markers, using LC-MS/MS. The most promising marker was identified by correlation analysis and further validated using HK-2 cells and mouse models. Trimethylamine N-oxide (TMAO) was identified as a promising marker among the 18 potential markers found in the first cohort, and it was optimally correlated with renal function of CKD patients in the second cohort. Treatment of HK-2 cells with TMAO decreased cell viability and up-regulated expression of α-smooth muscle actin. In mice, a TMAO-containing diet decreased kidney mass and increased protein expression of α-smooth muscle actin. Also, control of TMAO production by inhibiting its biosynthetic pathway with 3,3-dimethyl-1-butanol or disrupting gut microbiota function with an antibiotic cocktail, attenuated renal injury in a murine model of CKD. Our data show that decreased TMAO production could be a new strategy to attenuate the progression of renal injury in CKD.

MO056: Alteration of Glycocalyx on Endothelium of Peritubular Capillaries in CKD

Abstract BACKGROUND AND AIMS The glycocalyx is a carbohydrate-rich gel-like mesh, which covers the luminal surface of cells, including the endothelium. The glycocalyx is involved in many regulatory functions of the endothelium, including vascular permeability. In chronic kidney disease (CKD), peritubular capillaries undergo anatomical, structural and functional alterations such as rarefaction, increased tortuosity and increased permeability. Here, we hypothesized that the glycocalyx of peritubular capillaries might be affected in CKD and investigated morphological and ultrastructural pathological alterations of the glycocalyx in different murine CKD models and human kidney tissue. METHOD We stained the glycocalyx of peritubular capillaries in kidney tissue of murine and human CKD specimen using different fluorescently labeled plant-derived lectins. Next, we established the Lanthanum Dysprosium Glycosamino Glycan adhesion (LaDy GAGa) staining technique to visualize the ultrastructure of the glycocalyx using transmission electron microscopy and to perform quantitative analyses. Finally, we analyzed the expression and regulation of glycocalyx components in primary murine endothelial cells. RESULTS Fluorescence stainings using different lectins with high affinity to components of the renal glycocalyx revealed a reduced binding to the endothelium in CKD. We found a similar change in a human kidney tissue. The LaDy GAGa staining technique visualized the ultrastructure of the glycocalyx and enabled quantitive analyses. We found a significant reduction of the glycocalyx thickness and density in two different models of renal fibrosis and CKD, i.e. unilateral ureteral obstruction and ischemia–reperfusion injury. Additionally, mRNA expression of proteins involved in glycocalyx biology, synthesis and turnover, i.e. syndecan 1 and glypican 1, which are main components of the glycocalyx, and exostosin 2, involved in the synthesis of the glycocalyx, were significantly upregulated in endothelial cells isolated from murine CKD models. CONCLUSION Visualization of glycocalyx using specific ultrastructural analyses allows qualitative and quantitative analyses, revealing significant pathological alterations in the glycocalyx of peritubular capillaries in CKD.

MO587: Chronic Kidney Disease Induces Endotoxin-Related Activation of The Innate Immune System

Abstract BACKGROUND AND AIMS Chronic inflammation is common in patients with chronic kidney disease (CKD) and is associated with poor outcomes. Although the etiology is only partly understood, the gut–kidney axis is considered an important contributor [1]. Lipopolysaccharide (LPS), also known as endotoxin, is a well-characterized pyrogen found in the exterior cell membrane of most of the gram-negative bacteria. It plays an important role in promoting intestinal inflammatory responses. When absorbed through the intestinal epithelium, LPS induces inflammation by activating macrophages and monocytes. Due to short half-life and semi-quantitative assay characteristics of the Limulus Amebocyte Lysate (LAL) assay, direct quantification of LPS is not suitable to quantify activation of the innate immune system. Lipoprotein-binding protein (LBP), a more stable biomarker, is an acute phase protein produced mostly by the hepatocytes and intestinal epithelial cells and is an essential component of an effective innate immune response to LPS. Circulating LBP significantly enhances the sensitivity of CD14 + cells (mostly monocytes and macrophages) to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14 receptor. Levels of LBP peak in serum shortly after endotoxemia and remain increased up to 72 h later. An increased plasma LBP indicates gut epithelial barrier dysfunction [2]. Whether LBP is altered by CKD is not known. METHOD We analyzed the effects of CKD on LBP plasma concentrations. We used samples from the Leuven mild-to-moderate CKD cohort (NCT00441623). To study causality, we used animal models of experimentally induced CKD. To exclude model-related bias, two different rat models of CKD, i.e. 5/6th nephrectomy and adenine supplementation, were used. LBP was measured using commercially available ELISA kits (Hycultbiotech, The Netherlands). RESULTS In a cohort of 460 patients with CKD, we found a significant increase in LBP levels across the different stages of CKD (ANOVA, P: 0.001). When analyzed as a continuous variable, LBP is significantly inversely correlated with eGFR (P < 0.001; Spearman, r: −0.221), and positively correlated with CRP (P < 0.001; Spearman, r: 0.592). During a median follow-up of 56 (IQR, 53–59) months, 70 patients died, with more deaths observed among patients with LBP in higher tertiles [tertiles 1–3: 11, 22, and 37 events, respectively (Fig. 1)]. In univariate Cox proportional hazard analysis, plasma LBP was significantly associated with mortality [hazard ratio (HR) of 1.032; 95% confidence interval (95% CI): 1.014– 1.046; P < 0.001). This association remained significant in multivariate models with adjustment for age, sex and BMI. In two different rat models, induction of CKD resulted in a significant increase in LBP (P < 0.001). In these animals, we observed a significant inverse association between eGFR (and measured creatinine clearance) and LBP concentrations (P < 0.001; Spearman, r: −0.460). CONCLUSION Patients with CKD have higher levels of LBP, with higher levels of plasma LBP present in patients with more advanced CKD. Plasma LBP is an independent predictor of mortality. Experimentally induced CKD equally results in significantly increased LBP. Our data suggest that CKD leads to increased passage of LPS across the intestinal barrier. These findings strengthen the hypothesis of the gut–kidney axis as a source of chronic inflammation.

MO432: The ESRD Progression Delayed by Transplanting 3D Printed Omental Patch

Abstract BACKGROUND AND AIMS Renal fibrosis is one of the pathological hallmarks of chronic kidney disease (CKD) that can further progress to end-stage renal diseases (ESRD). Haemodialysis and kidney transplantation are the only feasible treatments of ESRD. In this study, a new treatment with autologous omental tissue was assessed to delay the progression of renal fibrosis. The omental tissue, a fatty structure, has been widely used in surgical procedures due to its immunologic, angiogenic characteristics and anti-fibrotic factors that enhance tissue repairs, cell proliferation and regeneration in a damaged tissue. Finally, we hypothesized that 3D printed omental patch can prevent renal fibrosis and help regenerate the damaged kidney in the animal model. The 3D printing can print cells and extracellular matrix uniformly to promote cell survival and differentiation during transplantation. The printing also can adjust the patch stiffness, providing a suitable environment for kidney recovery. METHOD Animal models of CKD were randomly divided into four groups: (A) sham-operated, (B) sham-operated + omental patch, (C) unilateral ureteral obstruction (UUO)-operated (Control) and (D) UUO-operated + omental patch. Micronized omental tissue as bio-ink was 3D printed in the form of a regular patch deformation. Then, the 3D printed omental patch was transplanted subcapsular layer of the kidney. All of the patches used in those groups were transplanted after 3 days of modelling. Kidneys were assessed histologically [Masson's Trichrome (MT), Sirius Red], by immunohistochemistry (α-SMA), molecular work (Bulk RNA sequencing, western blot) after 2 weeks of transplantation. RESULTS Omental patch treated group has been shown histologically less tubulointerstitial fibrosis and tubule atrophy by MT, Sirius Red staining. Particularly, the TGF beta mRNA expression level of the 3D printed omental treated group was decreased in the kidney, whereas the HGF level was significantly increased. Also, in terms of western blotting, pro-inflammatory and renal fibrosis markers (TGF-b, α-SMA) were all decreased. Moreover, in bulk RNA sequencing, we confirmed that STAT 3 kidney repair pathway and TF-beta signalling. From all those assessments we suggest that the induction of myofibroblasts and collagen deposition are all inhibited in the omental patch treated kidney model. The autologous 3D omental patch can prevent tubular atrophy and decreased tubulointerstitial fibrosis and this further delays the progression of ESRD. CONCLUSION The study demonstrated that the omental patch delays fibrosis and repairs tubule structure in the damaged kidney by providing protective effects to kidney disease. These studies indicate the feasibility of the use of the autologous omental patch in chronic kidney disease therapeutic effects and new treatment of renal replacement therapy (RRT).

MO550: Vascular Cells Senescence and Modifications in NRF2 Pathway in a Model of URaEMIC Vascular Calcification

Abstract BACKGROUND AND AIMS Vascular dysfunction including vascular calcification (VC), is a consequence of ageing and chronic kidney disease (CKD) that increase the risk of cardiovascular events. Accumulation of senescent cells in arteries can lead to structural and functional alterations and contributes to increased arterial stiffness, reduced compliance and impaired contractility. We tested the hypothesis that senescent cells in the arterial wall are involved in the pathophysiology of vascular calcification in a rat model of CKD and VC induced by a high phosphate diet and vitamin D supplementation. METHOD Eight-week-old Sprague–Dawley rats underwent subtotal nephrectomy (SNx, n = 18), or no surgery (Control, n = 6). Eight weeks after, control animals (n = 6) and part of the SNx group (n = 6) were maintained on the standard diet while the remaining rats underwent a 4-week VC-diet, where a standard diet was supplemented with high phosphate and 1-hydroxy-vitamin D at a dose of 0.1 µg/day (SNx 0.1 group, n = 6) or 0.4 µg/day (SNx 0.4 group, n = 6). At the end of the protocol, renal function, blood pressure and VC (von Kossa) were studied. mRNA and protein expressions of cellular senescence markers (p16, p21, yH2AX), antioxidants (NRF2, Nqo-1), VC inducers/inhibitors and osteoblastic transition drivers were assessed by qPCR, immunohistochemistry (IHC), or immunofluorescence (IF). Correlations between mRNA or protein levels and VC were computed for the VC-diets animals (n = 12). RESULTS A decrease in creatinine clearance (P < 0.05), increase in urinary protein excretion (P < 0.05) and tubulointerstitial fibrosis were observed in the three SNx groups. VC-diets induced vascular calcification in thoracic aorta and aortic arch associated with an increase in pulse pressure (P < 0.01). NRF2 mRNA and protein expression increased in the calcified groups (P < 0.05) and were positively correlated with VC (Figure 1). In addition, mRNA expression of NRF2 target Nqo-1 was strongly correlated to VC (P < 0.01). p16 mRNA (P < 0.05) and protein (IHC, P < 0.01) levels were correlated to VC; a similar expression pattern was observed for p21 protein (P < 0.05) (Figure 1). The number of yH2AX positive cells increased in calcified groups (IF, P < 0.05) and some of the positive cells for p16 were co-labelled with yH2AX (Figure 1). CONCLUSION In our rat model of induced VC associated with CKD, NRF2 and downstream targets expression was increased in thoracic aorta, confirming the link between NRF2 and VC. This might be a response mechanism as observed with other ‘protective’ factors with increased mRNA expression in this model (data not presented here). Furthermore, we observed that cellular senescence was clearly associated with VC. The co-labelling for p16 and yH2AX in some vascular cells in the media layer, exclusively observed in calcified aortas, is another clue that highlights the link between VC and vascular cell senescence.

MO421: Chronic Kidney Disease is Associated With Cardiac Downregulation of Vascular Endothelial Growth Factor a (VEGFA) and Development of Diastolic Dysfunction: Epigenetics and Micrornas

Abstract BACKGROUND AND AIMS Patients with chronic kidney disease (CKD) have a higher risk to develop heart failure with preserved ejection fraction (HFpEF). We showed in a swine model of CKD with diastolic dysfunction and cardiac remodelling that these cardiac features are associated with blunted cardiac mRNA and protein expression of vascular endothelial growth factor (VEGF), a pro-angiogenic cytokine that plays important roles in cardiac vascular development, morphogenesis and contractility. Hence, we aim to identify potential underlying mechanisms of cardiac VEGF downregulation in CKD. METHOD CKD and normal pigs (n = 3 each) were studied for 14 weeks. Cardiac morphology and function (echocardiography) were quantified in vivo and microvascular density (3D micro-CT) ex vivo. Then, cardiac micro-RNA (miRNA) and 5-hydroxymethylcytosine (5-hmC) profiles of VEGFA gene were examined by hydroxymethylated DNA immunoprecipitation and miRNA sequencing (hMeDIP-seq and miRNA-seq, respectively) and validated by qPCR and Western blotting. RESULTS Blunted renal function in CKD pigs associated with left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction with pEF (Figure 1A) and reduced subendocardial microvascular density (Figure 1B) compared to normal. miRNA-seq showed that mir-183 was the only miRNA capable of targeting VEGFA and upregulated in CKD hearts (Figure 1C), accompanied by downregulation of VEGFA gene and VEGF protein (Figure 1D), whereas hMeDIP-seq revealed reduced VEGFA 5hmC levels in the hearts of CKD versus normal pigs (Figure 1E). CONCLUSION Our study suggests that miRNA-modulation and cardiac epigenetic changes in VEGF might contribute to cardiac damage and the development of HF in CKD. These observations may assist in developing novel therapies, like miRNA antagonists or epigenetic modulators, to preserve VEGF signalling and protect the heart in patients with CKD.

NaCl Intake Promotes Greater Increases in Blood Pressure Than NaHCO3 in Rats with Chronic Kidney Disease

Sodium bicarbonate (NaHCO3) is used in patients with chronic kidney disease (CKD) to treat metabolic acidosis and slow CKD progression. One potential adverse effect of NaHCO3 use in patients with CKD is the increase in dietary sodium (Na+) intake, which may worsen both fluid retention and hypertension in this already vulnerable population. Previous data, however, indicate that anions other than Cl‐ coupled to Na+ may not lead to increases in volume retention and blood pressure. These data raise the possibility that ingestion of NaHCO3 in patients with CKD may be relatively benign. The effects of NaHCO3 loading on fluid retention and blood pressure, however, have not been directly investigated in a model of CKD.We hypothesized that NaHCO3 loading promotes similar levels of Na+ retention and hypertension as NaCl loading in a rat model of CKD. We utilized 21, 12‐week‐old male Sprague Dawley rats that underwent sham (n=6), 2/3 (n=7), or 5/6 nephrectomy (n=8, Nx). In order to limit intra‐animal variability, we utilized a crossover study design. After 4 weeks of recovery from surgery, rats were matched by renal mass reduction and assigned to one of two groups. One group was started on a 0.1M NaHCO3 (n=11) drinking water solution while the other was started on 0.1M NaCl (n=10) in the drinking water for 2 weeks before a wash‐out (tap water for 2 weeks). The first group was then started on 0.1M NaCl while the other received 0.1M NaHCO3 for 2 weeks. Rats were maintained in metabolic cages during the initiation of treatments to allow for daily measurements of urine output, fluid, and food ingestion. Urine Na+ content was measured by atomic absorption spectrometry. Mean arterial pressure was measured in separate group with telemetry over 2 weeks of treatment with NaHCO3 or NaCl.Following the initiation of treatment, there was a significantly greater increase in urinary Na+following NaCl treatment (1.18 ± 0.09 mmol vs 5.74 ± 0.39 mmol) compared to NaHCO3 treatment (1.23 ± 0.06 mmol vs 4.82 ± 0.40 mmol; Two‐Way ANOVA, PRx=0.006). However, there was no significant difference in total retained Na+ following NaCl (2.03 ± 0.73 mmol) and NaHCO3 treatment (2.19 ± 0.62 mmol; unpaired t‐test, P=0.87). There was also no significant difference in Na+ retention between the different levels of Nx after the initiation of NaCl (Two‐Way ANOVA, PLevelofNx=0.50) and NaHCO3 treatments (Two‐Way ANOVA, PLevelofNx=0.53).Despite a similar amount of Na+ retention, we found that 0.1M NaCl resulted in greater increases in mean arterial pressure over 2 weeks in 5/6 Nx rats compared to 0.1M NaHCO3 (RM Two‐Way ANOVA, PInteraction=0.02, PSubjects<0.0001; n=4/5). The change in mean arterial pressure from baseline was greater in 5/6 Nx rats treated with NaCl (40.5 ± 5.6 mmHg) compared to NaHCO3 (20.9 ± 0.82 mmHg; unpaired t‐test, P=0.03).We conclude NaHCO3 supplementation promotes Na+ and fluid retention and increases in blood pressure in rats with CKD, however, blood pressure responses are less than that observed with NaCl. Our data indicate that NaHCO3 loading in patients with CKD is unlikely to be benign. As NaHCO3 loading can promote increases in blood pressure, caution is warranted with chronic NaHCO3 use in patients with CKD.

Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in the Absence and Presence of Chronic Kidney Disease

BackgroundChronic Kidney Disease (CKD) is a public health epidemic that is associated with elevated serum levels of phosphate (hyperphosphatemia) as well as skeletal muscle atrophy, but this interconnection is poorly understood. Elevated phosphate (Pi) has direct effects on smooth muscle cells and induces vascular calcification. Pi also induces the production of the hormone fibroblast growth factor (FGF) 23 in bone. We wanted to test if Pi induces atrophy and/or FGF23 expression in skeletal muscle cells. Furthermore, we analyzed skeletal muscle on a functional, histological and molecular level in three models of hyperphosphatemia – two CKD models, i.e. mice with global deletion of collagen 4a3 (Col4a3‐/‐) and wildtype mice receiving an adenine‐rich diet, as well as wildtype mice on a high Pi diet with normal kidney function. Finally, we determined the effect of a low Pi diet on skeletal muscle in Col4a3‐/‐ mice.MethodsC2C12 myotubes were treated with 1‐5 mM Pi for 24 hours, followed by qPCR expression analysis of atrophy genes (atrogenes), including MT1, Trim63, and Fbox32, as well as FGF23. Furthermore, we studied Col4a3‐/‐ mice receiving normal chow or a 0.2% Pi diet at 10 weeks of age. We also analyzed C57Bl/6 mice receiving an adenine‐rich (0.2%) diet for 14 weeks or a 3% Pi diet for 3 months. We analyzed grip strength, hindlimb area by MRI, muscle wet weight, cross‐sectional area of individual muscle fibers immuno‐labeled with anti‐laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR and of FGF23 by qPCR and ELISA.ResultsPi treatments increased the expression levels of atrogenes as well as FGF23 in C2C12 myotubes. In the three mouse models, grip strength and cross‐sectional area of myofibers were significantly reduced, and the expression levels of atrogenes were significantly elevated when compared to respective controls. Furthermore, we detected elevations in the mRNA and protein levels of FGF23 in the hindlimb muscles of all models. Additionally, the two CKD models showed significant reductions in muscle weight and hindlimb area. Administration of a 0.2% Pi diet protected Col4a3‐/‐ mice from developing skeletal muscle atrophy.ConclusionElevated Pi induces myotube atrophy and FGF23 expression in vitro. Mouse models with hyperphosphatemia not only develop skeletal muscle atrophy, but also produce FGF23 in skeletal muscle tissue in the presence and absence of CKD, and a low Pi diet protects the skeletal muscle in CKD mice. Pharmacological approaches targeting Pi uptake or excretion, or inhibition of Pi’s direct actions on tissues might alleviate various CKD‐associated pathologies. Future studies need to determine whether skeletal muscle‐derived FGF23 contributes to tissue injury or is protective against Pi‐induced damage.

Loss of Cingulin‐like 1 in Dahl Salt‐Sensitive Rats Leads to Reduced Renal Injury and Blood Pressure

Hypertension is both a cause and effect of chronic kidney disease (CKD) and impacts a significant proportion of patients, with a worldwide prevalence of 10‐15%. Previous genetic studies involving the Dahl salt‐sensitive (SS) rat, a model of hypertensive CKD, identified multiple genetic loci linked to kidney injury, including a region on chromosome (Chr) 8. Subsequently, an S.SHR(8) congenic (i.e. transfer of kidney injury resistant SHR genome onto the SS background) demonstrated significantly reduced proteinuria and improved renal function compared to SS control. To identify the gene/s responsible, genome sequencing and bioinformatics analysis identified cingulin‐like 1 (Cgnl1) as a putative gene linked to kidney injury on Chr. 8. Cgnl1 is known to localize to adherens and tight cell‐cell junctions, mediating junction assembly via regulating the activity of the small GTPases such as RhoA and Rac1. A SS‐Cgnl1−/− knockout (KO) was developed to test the hypothesis that loss of Cgnl1 will result in a significant attenuation of kidney injury compared to SS (WT). Both on low and high‐salt (LS=0.3% or HS=2.0% NaCl), KO animals (n=6) demonstrated reduced proteinuria compared to WT animals (n=6) for the entirety of the experiment (HS; KO= 28.7 ± 2.9 versus WT= 161.4 ± 33.1 mg/24hr at week 15, p<0.0001). To further validate Cgnl1’s effect on blood pressure and kidney injury, a temporal analysis (6‐12 weeks) using telemetry and additional renal measurements were conducted on WT and KO animals. On HS, KO animals (n=6) demonstrated reduced proteinuria compared to WT (n=6) for the entirety of the experiment (52.1 ± 7.2 versus 395.9 ± 29.8 mg/24hr at week 12, p<0.0001). While HS fed WT animals exhibited a significant increase in BP compared to LS WT (MAP=197.0 ± 5.0 versus 142.8 ± 3.5 mmHg, p<0001), KO remained unchanged (MAP= 127.6 ± 2.7 and 133.9 ± 3.7 mmHg respectively, NS). Kidney pathology measures, including glomerular and tubular injury were all significantly improved (all p<0.05) in KO compared to WT. Nephron number was measured with no significant difference between WT and KO in young animals, but HS fed WT demonstrated a significant loss of nephrons compared to KO. Bulk RNAseq of kidneys from WT and KO identified Cgnl1 as top downregulated gene with altered expression of many known genes linked with Cgnl1 compared to WT, along downregulation of genes associated with renal injury, mainly reflective of physiological differences from week12‐15. To better identify genes involved in onset of injury, single nuclei RNAseq was performed at week 4 (before physiological differences) to evaluate impact of Cgnl1 in distinct cell populations initiating injury. Additional work is also currently being done on HEK293‐ Cgnl1 knockout cell lines for more mechanistic study of Cgnl1 and pathways. In total, the molecular work, along with the physiological characterization of the SS‐Cgnl1−/−, is expected to provide insight into the role of Cgnl1 in the onset and progression of hypertensive kidney disease and identify new therapeutic targets.

Apelinergic System: an in‐vitro Investigation of Inflammatory & Oxidative Stress in Chronic Kidney Disease

Over 37 million US adults have Chronic Kidney Disease (CKD). Of that population–35% are Black. With 4x higher predisposition to developing renal failure due to comorbidities (i.e. hypertension and diabetes), there is a dire need to address both preventative and therapeutic options in this population. Besides dialysis and transplant as reactionary treatments of CKD, vitamin E—an antioxidant—has been given to those in late stage CKD. Our study aims to address mechanisms behind CKD pathology. The 5/6 nephrectomy (5/6Nx) rat CKD model provides the opportunity to isolate the effects of impaired renal function without significant confounding comorbidities. A hallmark feature of both rat and human CKD is adverse vascular remodeling which is often preceded by endothelial cell (EC) dysfunction. With respect to EC dysfunction, inflammatory and oxidative imbalances are suspected to be involved. Our previous transcriptomic analysis of kidney tissues from the 5/6Nx rat identified a significant increase in apelin receptor (APLNR) expression compared to sham controls. In addition, the data revealed several pro‐inflammatory cytokines (IL‐6 and IL‐1β) preceding APLNR transcript increases. Treatment of Normal Rat Kidney epithelial (NRK) cells with IL‐6 caused a significant reduction in apelin (APLN) ligand, while IL‐1β augmented APLNR transcript levels. After further study of the 3 pathways, a common downstream target, protein kinase B (AKT), arose. Interestingly enough, AKT is responsible for various vascular cascades including interaction with well‐known reactive oxygen species (ROS) producer–NADPH Oxidase 4 (NOX4). Hydrogen Peroxide (H2O2), a ROS product of NOX4, coordinates vasoactivity through AKT signaling and has overlapping function with apelinergic (APJ) system in the vasculature. Due to the inflammatory and oxidative nature of CKD, we want to address the relationship between H2O2 and the APJ system. We hypothesize that pretreating NRK cells with APLN prior to administration of H2O2 will have 2 effects: oxidant attenuation and antioxidant augmentation. NRK cells were pretreated with 2.5ng/mL apelin or 30mg/mL vitamin E 48‐hours prior to addition of 20mM H2O2. At study termination, cells were collected/quantified using the Bradford protein assay. Protein expression was measured via Western blots against catalase, an antioxidant marker, and GPx, an oxidant protein. One‐Way ANOVA and Tukey Post‐Hoc test were used to establish statistical significance (p<0.05). Catalase: no significant expression changes after APLN or vitamin E treat (p = 0.10), but significant increases after H2O2 treat (p = 0.05) compared to control. GPx: significant increases in expression after APLN treat compared to [control (p=0.0002), vitamin E (p=0.04), and H2O2 + APLN (p=0.0001)] and vitamin E compared to H2O2 + Vitamin E treat (p=0.01). Data presented as mean ± SEM (n=3). These studies assessed functionality of the APJ system in an in‐vitro model of CKD. Both of the inflammatory cytokines are regulators of APJ system transcription, which is relevant for patients in a chronic inflammatory state. These studies also highlight the interaction between APJ system and oxidative stressors. Results here begin to lay the foundation for future studies involving the APJ system as a viable target for assessing CKD pathology.

Spatial transcriptomics and single cell sequencing identifies a unique injury associated resident macrophage subpopulation and reveals differential response to injury

Abstract Acute kidney injury (AKI) is a growing concern in the U.S. and globally. Despite recovery of kidney function, patients remain at increased risk for developing chronic kidney disease (CKD). The cause of this increased risk is unknown and biomarkers to determine kidney injury are severely lacking. A potential candidate is the network of kidney resident macrophages interwoven throughout the tissue, and specifically a subpopulation of kidney injury associated macrophages (KIA) we first identified to appear after AKI and lack MHC II expression. KIA cells were localized within the tissue using Visium Spatial Transcriptomics and flow cytometry. Comparison of KIA cell kinetics between AKI and CKD indicated a correlation between the presence of KIA cells and the persistence of injury. Additionally, KIA cells have localized to the sight of greatest injury following an ischemic event. As kidney function recovers, KIA cells decrease in number and return to quiescent levels by 14 days post injury. In contrast, in the severe CKD model, KIA cells persist for up to six weeks, composing approximately 20% of the total kidney resident macrophage population. A less severe version of CKD results in fewer KIA cells but similar persistence, indicating appearance of KIA cells directly correlates with injury severity. Targeting KIA cells to either enhance or negate their function is a possible therapeutic target to reduce the risk of developing CKD following AKI. As we have identified KIA cells in multiple models of kidney injury, in the future they should be investigated as a potential injury biomarker. We hypothesize that KIA cells are uniquely involved in the pathogenesis of injury and may be a key to an AKI to CKD transition. Supported by grants from the American Heart Association: (AHA 827257)

A Reproducible Mouse Model of Moderate CKD With Early Manifestations of Osteoblastic Transition of Cardiovascular System.

Chronic kidney disease (CKD) is a significant public health challenge with a substantial associated risk of mortality, morbidity, and health care expenditure. Culprits that lead to development and progression of CKD are multifaceted and heterogenous in nature. This notion underscores the need for diversification of animal models to investigate its pathophysiology, related complications, and to subsequently enable discovery of novel therapeutics. Importantly, animal models that could recapitulate complications of CKD in both genders are desperately needed. Cardiovascular disease is the most common cause of death in CKD patients that may be due in part to high prevalence of vascular calcification (VC). Using DBA/2 mice that are susceptible to development of VC, we sought to investigate the feasibility and reproducibility of a unilateral ischemia-reperfusion model followed by contralateral nephrectomy (UIRI/Nx) to induce CKD and its related complications in female and male mice. Our results demonstrate that irrespective of gender, mice faithfully displayed complications of moderate CKD following UIRI/Nx as evidenced by significant rise in serum creatinine, albuminuria, higher degree of collagen deposition, elevated expression of classic fibrotic markers, higher circulating levels of FGF-23, PTH and hepcidin. Moreover, we corroborate the osteoblastic transition of aortic smooth muscle cells and cardiomyocytes based on higher levels of osteoblastic markers namely, Cbfa-1, osteopontin, osteocalcin, and osterix. Our data confirms a viable, and consistent model of moderate CKD and its associated complications in both male and female mice. Furthermore, early evidence of osteoblastic transition of cardiovascular system in this model confirms its suitability for studying and implementing potential preventive and/or therapeutic approaches that are urgently needed in this field.

Phosphate restriction using a processed clay mineral reduces vascular pathologies and microalbuminuria in rats with chronic renal failure

BackgroundThe progression of chronic kidney disease (CKD) is associated with an increasing risk of cardiovascular morbidity and mortality due to elevated serum phosphate levels. Besides low phosphate diets and hemodialysis, oral phosphate binders are prescribed to treat hyperphosphatemia in CKD patients. This study reports on a processed clay mineral as a novel and efficient phosphate sorbent with comparable efficacy of a clinically approved phosphate binder.Methods5/6 nephrectomized rats, which develop chronic renal failure (CRF), received a high phosphate and calcium diet supplemented with either a processed Montmorillonite-Illite clay mineral (pClM) or lanthanum carbonate (LaC) for 12 weeks. Levels of plasma uremic toxins, glomerular filtration rates and microalbuminuria were determined and the histomorphology of blood vessels and smooth muscle cells was analyzed.Results5/6 nephrectomy induced an increase in plasma uremic toxins levels and progressive proteinuria. Treatment of CRF rats with pClM decreased observed vascular pathologies such as vascular fibrosis, especially in coronary vessels. The transition of vascular smooth muscle cells from a contractile to a secretory phenotype was delayed. Moreover, pClM administration resulted in decreased blood creatinine and urea levels, and increased glomerular filtration rates, reduced microalbuminuria and eventually the mortality rate in CRF rats.ConclusionOur study reveals pClM as a potent phosphate binding agent with beneficial impacts on pathophysiological processes in an animal model of CKD. pClM effectively attenuates the progression of vascular damage and loss of renal function which are the most severe consequences of chronic renal failure.

Current kidney function parameters overestimate kidney tissue repair in reversible experimental kidney disease

Although underlying mechanisms and the clinical course of kidney disease progression are well described, less is known about potential disease reversibility. Therefore, to analyze kidney recovery, we adapted a commonly used murine chronic kidney disease (CKD) model of 2,8- dihydroxyadenine (2,8-DHA) crystal-induced nephropathy to study disease recovery and efficacy of disease-modifying interventions. The recovery phase after CKD was characterized by improved kidney function after two weeks which remained stable thereafter. By contrast, even after eight weeks recovery, tubular injury and inflammation were only partially reduced, and fibrosis persisted. Deep-learning-based histologic analysis of 8,604 glomeruli and 596,614 tubular cross sections revealed numerous tubules had undergone either prominent dilation or complete atrophy, leading to atubular glomeruli and irreversible nephron loss. We confirmed these findings in a second CKD model, reversible unilateral ureteral obstruction, in which a rapid improvement of glomerular filtration rate during recovery also did not reflect the permanent histologic kidney injury. In 2,8-DHA nephropathy, increased drinking volume was highly effective in disease prevention. However, in therapeutic approaches, high fluid intake was only effective in moderate but not severe CKD and established tissue injury was again poorly reflective of kidney function parameters. The injury was particularly localized in the medulla, which is often not analyzed. Thus, recovery after crystal- or obstruction-induced CKD is characterized by ongoing tissue injury, fibrosis, and nephron loss, but not reflected by standard measures of kidney function. Hence, our data might aid in designing kidney recovery studies and suggest the need for biomarkers specifically monitoring intra-kidney tissue injury.