Model Of Chronic Kidney Disease Research Articles

#3680 CALPAIN INHIBITION PREVENTS FIBROSIS IN PROGRESSIVE CHRONIC KIDNEY DISEASE

Abstract Background and Aims Kidney fibrosis is one of the main pathological processes of chronic kidney disease (CKD), although the pathogenesis of renal scar formation is not fully understood. Calpains (CAPN) are intracellular cysteine ​​proteases that play a key role in multiple biological processes linked to tissue damage and repair mechanisms, such as fibrosis and epithelial-mesenchymal transition. However, CAPN contribution in CKD genesis and progression remains to be fully elucidated. The aim of this study was to investigate the possible role of CAPN in the development of CKD and renal fibrosis in an experimental model of CKD induced by adenine and in human kidney proximal tubular cells (HK-2). Method C57BL/6 wild-type mice were fed a 0.2% adenine-rich diet (WTA) for 5 days and 2 weeks to induce tubulointerstitial damage resembling what occurs in human CKD. Mice with a standard diet were used as controls (WT). WT and 5 days WTA mice were injected intraperitoneally for 5 consecutive days with a dose of calpain inhibitor III (20 mg/kg). In vitro experiments were performed in HK-2 cells in the presence or absence of TGF-β (1 ng/ml, different times) and calpain inhibitor III (50 µM). Mice renal function was assessed by measuring plasma BUN and creatinine. Interstitial fibrosis was evaluated in kidney samples embedded in paraffin by Sirius red staining of the collagen renal cortex content. Fibrosis markers were determined by RT-qPCR. CAPN localization in the kidney was assessed by immunofluorescence. CAPN 2 and 5 protein content were analyzed by western blot. CAPN activity was determined by fluorescence assays. All data were analyzed using non-parametric statistics for comparisons, applying the Kruskal-Wallis with Mann-Whitney post-test. A value of p<0.05 was considered statistically significant. Results Our results show a progressive worsening of renal function and structure in adenine-fed mice, with increased BUN, creatinine and COL I mRNA expression being significantly higher at 2 weeks of treatment. CAPN 2 and 5 protein content was significantly higher in animals that received adenine for 2 weeks compared to control, with a statistically significant increased renal CAPN activity. Our results point out that CAPN 2 and 5 content in renal tissue increases as CKD and fibrosis progress. To verify this potential relationship, we treated kidney tubular cells, where we observed a greater calpain expression compared to other kidney cells, with TGF-β to induce fibrosis. This treatment increased COL I mRNA expression, as well as CAPN 2 and 5 levels in HK-2 cells. However, calpain inhibitor III administration prevents increased COL I expression, both in vitro and in vivo. Conclusion We suggest a direct relationship between the cellular content of CAPN and the renal fibrosis observed in CKD, being involved in the genesis or progression of kidney disease. Thus, effective CAPN blockade or downregulation could be helpful as a therapeutic strategy to prevent CKD.

#5517 IN CHRONIC KIDNEY DISEASE, IRON DEFICIENCY-INDUCED ANEMIA IS ASSOCIATED WITH HIGHER LEVELS OF FGF23 THAT ARE NOT REDUCED BY IRON ADMINISTRATION

Abstract Background and Aims Iron (Fe) deficiency significantly contributes to anemia in chronic kidney disease (CKD). High FGF23 levels, commonly found in CKD, are related to adverse outcomes in patients. In the normal setting, an association between Fe deficiency and FGF23 has been observed. However, it is unknown whether Fe deficiency-induced anemia might contribute to the excessive FGF23 levels in CKD. Thus, this study was intended to evaluate the interplay between Fe deficiency, CKD, and FGF23. Method Male Wistar rats with normal renal function received either standard (40 ppm Fe) or Fe-deficient (4 ppm Fe) diets for 6 weeks to induce anemia. Then, several normal and all the anemic rats were switched to an adenine-enriched (0.6%) diet to induce renal damage. Groups of anemic-uremic animals were treated with oral (ferric citrate, FC and ferrous sulfate, FS) or intravenous (ferric carboxymaltose, FCx) Fe at a dose of 9 mg/kg for 5 weeks. Hematological and mineral metabolism parameters were determined at sacrifice. All experimental protocols were approved by the Ethics Committee for Animal Research of the University of Córdoba. Results Plasma and serum biochemistries are shown in Table 1. CKD per se was associated with derangements in hematological parameters. The presence of Fe deficiency-induced anemia further decreased these parameters, being statistically significant in the case of serum Fe (P = 0.003). Iron therapy improved hematocrit, hemoglobin, and Fe levels, although statistical significance for all of them was only reached when FC was administered (P = 0.012, P = 0.038, and P = 0.002, respectively). As expected, CKD rats exhibited disturbances in mineral metabolism. Interestingly, uremic and anemic rats showed higher intact (P = 0.039) and c-terminal (P = 0.048) FGF23 levels when compared with CKD group. Despite normalization of serum Fe, intact FGF23 further increased following administration of FS (P = 0.022), and same trend was observed with all the compounds. However, cFGF23 levels remained unchanged in all the Fe-treated groups. Conclusion The presence of Fe-induced anemia in an experimental model of CKD is associated with further increases in the levels of both intact and c-terminal FGF23. Thus, other factors distinct than serum Fe might influence FGF23 levels. Our results support the need for investigating the precise mechanisms underlying the relationship between Fe and FGF23 to optimize the clinical management of anemia, particularly in the context of renal dysfunction.

#5904 COMPARATIVE TRANSCRIPTOMICS OF THREE CANONICAL MOUSE MODELS OF CHRONIC KIDNEY DISEASE

Abstract Background and Aims A range of mouse models are available in preclinical chronic kidney disease (CKD) research but differ in their disease etiology and pathological hallmarks. To enable better selection of the mouse CKD model optimal for preclinical drug discovery studies, we characterized the kidney transcriptome signature of three well-established models of CKD induced by unilateral ureter obstruction (UUO), unilateral ischemic reperfusion injury (uIRI) and adenine-supplemented diet feeding (ADI). Method Male C57BL/6JRj mice were used in all studies. UUO or sham surgery was performed in 9 weeks old mice, which were terminated two weeks post-surgery. uIRI or sham surgery was performed in 10 weeks old mice terminated 6 weeks post-surgery. 12 weeks old mice received an adenine-supplemented diet or a control diet for 6 weeks. Endpoints included plasma biochemistry, kidney histology and RNA sequencing on kidney samples. Results Compared to corresponding controls, plasma urea was only increased in ADI mice. All three models presented with increased inflammation (F4/80, UUO>ADI>uIRI) and fibrosis (COl1A1, UUO = uIRI>ADI) in the histological analysis. Compared to corresponding controls, all three models demonstrated substantial increases in differential expressed genes (DEGs, UUO, n = 12,046; uIRI, n = 12,236; ADI, n = 11,468), with significant overlap in transcriptome signatures between the models. Gene expression markers of inflammation (e.g., Ccl2, Cd68 and Il1b), fibrogenesis (e.g., Tgfb1, Serpine1 and Col1a1) and kidney injury (e.g., Havcr1, Lcn2 and Spp1) supported histological findings in the three models. Interestingly, several molecular targets pursued in CKD drug discovery were significantly regulated, however with different directionality, in UUO (upregulated Atg7, Bax, Ednra/b, Gli2, Ifng, Shh; downregulated Glp1r, Nr3c2, Ptch1, Slc5a2), uIRI (upregulated Ednra, Gli2, Glp1r, Ifng, Nr3c2; downregulated Agt, Egfr, Slc5a2), and ADI mice (upregulated Agt, Atg7, Bax, Ednra; downregulated Ednrb, Egfr, Flt1, Nr3c2, Ptch1, Slc5a2). Conclusion UUO, uIRI and ADI mouse models demonstrate histopathological hallmarks of CKD, characterized by increased macrophage infiltration and fibrosis, which was corroborated by their individual transcriptome signatures. Despite similarities in histological phenotype, the models were distinguished based on kidney transcriptome changes, with several current drug targets regulated in a model-specific fashion. In conclusion, the current work will enable researchers to select the mouse model optimal for profiling individual preclinical test drugs with therapeutic potential in CKD. Also, our data may serve to enable further development of CKD mouse models with improved clinical translatability.

#3330 INFLAMMATION SECONDARY TO CHRONIC KIDNEY DISEASE AND PERITONEAL DIALYSIS ACCELERATES ATHEROSCLEROSIS IN A NEW MOUSE MODEL

Abstract Background and Aims Inflammation is an established contributor to accelerated atherosclerosis, which is highly prevalent in people with chronic kidney disease (CKD) and even more so in those treated with peritoneal dialysis (PD). We therefore hypothesised that PD-induced systemic inflammation is essential in the development of atherosclerosis. To study this we developed a novel mouse model of CKD and PD accelerated atherosclerosis. Method We performed a 5/6ths nephrectomy in ApoE deficient “black 6” mice, fed these mice a high-cholesterol diet (HCD), and intraperitoneally administered 3.86% Physioneal daily for 67 days. There were three groups of mice: control (HCD-only), CKD+HCD, and CKD+HCD+PD, with relevant shams. Tissues were harvested twelve weeks after the nephrectomy. Assessments of residual kidney function, atherosclerotic plaque pathology and immunology, and systemic immune responses were performed. Results Atherosclerotic plaque size in the aortic arch was larger in the PD group compared to the control, increasing from 83,779 μm2 ±18,430 to 109,973 μm2 ±25,602 (p = 0.0349), whilst plaque size was unchanged in the CKD group. In contrast, both the CKD and PD groups displayed altered plaque stability. There was less collagen in the plaque vs control, decreasing from 43.54% ±10.65 to 29.71% ±9.43 and 29.73% ±10.18 respectively. Additionally, fibrous cap thickness decreased in both groups vs control. The plaques from only the PD group showed infiltration by T-cells compared to control, with no changes in macrophage content in either the CKD or PD group. In the spleen in the CKD and PD groups, naïve CD4 cells increased and effector CD4 cells decreased compared to control, with the latter change also occurring in the circulation. Central memory CD4 cells, terminally differentiated Th1 cells, Th17 cells, and ‘vascular homing’ cells all increased in the PD group compared to control mice. These homing cells also increased in circulation. The PD group showed decreases in the spleen of regulatory T-cells, effector Th1 cells, and effector CD8 cells. Notably, all of these changes were absent from the mesenteric lymph node. There was a large drop in monocytes in the spleen and a loss of immature and mature dendritic cells in the PD group. Conclusion Combining CKD, a high-cholesterol diet, and exposure to PD fluids in ApoE−/-mice successfully generated a new model for cardiovascular disease in human PD patients. This new model is in a widespread and well-understood mouse line representative of human atherosclerosis. Here we demonstrate the pronounced effects of CKD and PD on the innate and adaptive immune response which increases atherosclerotic burden and leads to larger more vulnerable plaques

#5485 CHRONIC KIDNEY DISEASE NOR STROMAL CELL-DERIVED FACTOR 1Α PEPTIDE (SDF-1A) BIO-FUNCTIONALIZATION ALTER RODENT VASCULAR IN SITU TISSUE FORMATION

Abstract Background and Aims The systemic pro-inflammatory environment of chronic kidney disease (CKD) could influence neo-tissue formation of in situ tissue engineered vascular access grafts. Here, we studied in-graft tissue formation and inflammation in a rat CKD model. Moreover, we explored graft bio-functionalization with stromal cell-derived factor 1α peptides (SDF-1α), a progenitor cell chemotactic which may improve cell engraftment. Method Pristine or SDF-1α bio-functionalized vascular grafts (1,2 mm ID, 2 cm length, 250–300 μm wall thickness) were created from biodegradable, polycarbonate-bisurea (PC-BU) electrospun meshes. Abdominal aorta interposition grafts were implanted in female Sprague Dawley rats (n = 53) that underwent sham surgery or CKD induction by 5/6th nephrectomy. Explanations and analyses were performed after two (n = 25) or twelve (n = 9) weeks. Results At two weeks, survival and graft patency were 100%. Explant cellularity and collagen content were not significantly different between sham and CKD rats and were not influenced by SDF-1α. Endothelial cell coverage (RECA+) and smooth muscle cell presence (αSMA+) were visually similar irrespective of disease or bio-functionalization. Elastin+ (p = 0,11), pan-(CD68+, p = 0,08) and anti-inflammatory macrophage (CD163+, p = 0,52) surface area, as well as (anti-)inflammatory gene expression were not significantly different between groups. The twelve-week CKD-group was taken out of experiment prematurely due to rapid disease progression. In addition, preliminary death due to vascular rupture occurred in four animals (n = 2 pristine and n = 2 SDF-1α) in the twelve-week sham animals. While patent, all remaining sham explants at twelve weeks (n = 9) showed severe vascular dilatation and calcifications (Von Kossa+). No differences in inflammatory and neo-tissue markers were found between twelve-week sham pristine and SDF-1α grafts or between explants over time. Conclusion CKD did not significantly alter inflammation or tissue formation of in situ tissue engineered vascular grafts. Additionally, SDF-1α bio-functionalization did not significantly alter cell engraftment or tissue formation. Mechanical stability of the graft appears to be the primary driver of neo-tissue formation. Future emphasis on in vitro to in vivo translation should be on the fine balance between tissue formation on one hand and fiber resorption on the other hand.

Naïve or Engineered Extracellular Vesicles from Different Cell Sources: Therapeutic Tools for Kidney Diseases.

Renal pathophysiology is a multifactorial process involving different kidney structures. Acute kidney injury (AKI) is a clinical condition characterized by tubular necrosis and glomerular hyperfiltration. The maladaptive repair after AKI predisposes to the onset of chronic kidney diseases (CKD). CKD is a progressive and irreversible loss of kidney function, characterized by fibrosis that could lead to end stage renal disease. In this review we provide a comprehensive overview of the most recent scientific publications analyzing the therapeutic potential of Extracellular Vesicles (EV)-based treatments in different animal models of AKI and CKD. EVs from multiple sources act as paracrine effectors involved in cell-cell communication with pro-generative and low immunogenic properties. They represent innovative and promising natural drug delivery vehicles used to treat experimental acute and chronic kidney diseases. Differently from synthetic systems, EVs can cross biological barriers and deliver biomolecules to the recipient cells inducing a physiological response. Moreover, new methods for improving the EVs as carriers have been introduced, such as the engineering of the cargo, the modification of the proteins on the external membrane, or the pre-conditioning of the cell of origin. The new nano-medicine approaches based on bioengineered EVs are an attempt to enhance their drug delivery capacity for potential clinical applications.

Intrinsic TGF-β signaling attenuates proximal tubule mitochondrial injury and inflammation in chronic kidney disease

Excessive TGF-β signaling and mitochondrial dysfunction fuel chronic kidney disease (CKD) progression. However, inhibiting TGF-β failed to impede CKD in humans. The proximal tubule (PT), the most vulnerable renal segment, is packed with giant mitochondria and injured PT is pivotal in CKD progression. How TGF-β signaling affects PT mitochondria in CKD remained unknown. Here, we combine spatial transcriptomics and bulk RNAseq with biochemical analyses to depict the role of TGF-β signaling on PT mitochondrial homeostasis and tubulo-interstitial interactions in CKD. Male mice carrying specific deletion of Tgfbr2 in the PT have increased mitochondrial injury and exacerbated Th1 immune response in the aristolochic acid model of CKD, partly, through impaired complex I expression and mitochondrial quality control associated with a metabolic rewiring toward aerobic glycolysis in the PT cells. Injured S3T2 PT cells are identified as the main mediators of the maladaptive macrophage/dendritic cell activation in the absence of Tgfbr2. snRNAseq database analyses confirm decreased TGF-β receptors and a metabolic deregulation in the PT of CKD patients. This study describes the role of TGF-β signaling in PT mitochondrial homeostasis and inflammation in CKD, suggesting potential therapeutic targets that might be used to mitigate CKD progression.

Novel biomarkers to predict chronic kidney disease and hypertension at 3 and 12 months post-cisplatin.

e22003 Background: Urine kidney injury biomarkers measured during cisplatin therapy may help identify patients at risk for adverse long-term kidney outcomes. In children treated for cancer, we examined associations of urine tubular injury biomarkers collected during two cisplatin cycles, with chronic kidney disease (CKD) and hypertension at 3 and 12 months post-cisplatin. Methods: We analyzed data from the Applying Biomarkers to Minimize Long-Term Effects of Childhood/Adolescent Cancer Treatment (ABLE) Nephrotoxicity Study: a twelve-center prospective cohort of 159 children receiving cisplatin. Urine tubular injury biomarkers (by ELISA: neutrophil gelatinase-associated lipocalin [NGAL]; kidney injury molecule-1 [KIM-1]; tissue inhibitor of metalloproteinase-2 [TIMP-2]; insulin-like growth factor-binding protein-7 [IGFBP-7]) were measured at three timepoints (pre-infusion; 12-24 hours post-infusion; discharge after cisplatin cycle) during an early cisplatin cycle (EarlyCisP: first or second cycle of therapy) and a later cycle (LateCisP: last or second-to-last cycle). Area under the curve (AUC) for biomarkers to predict CKD (estimated glomerular filtration rate below normal or urine albumin/creatinine above normal for age, per international definition) and hypertension (3 blood pressures; per American Academy of Pediatrics guidelines) at 3 and 12 months post-cisplatin was calculated. AUC change from adding biomarkers to a published clinical prediction model for 3-month outcomes was evaluated (DeLong method). Results: 156 patients were available for analysis (mainly solid tumors). 3-month (median 90 days) outcomes were 52/118 (44%) CKD; 17/125 (14%) hypertension. 12-month (median 335 days) outcomes were 47/118 (40%) CKD; 15/125 (12%) hypertension. AUCs for all biomarkers to predict 3 and 12-month CKD and hypertension were poor to modest (best AUC = 0.70, 95% CI 0.60-0.80 for pre-infusion EarlyCisP TIMP-2*IGFBP-7 to predict 3-month hypertension). Biomarkers did not improve the clinical prediction model for 3-month CKD (AUC change not statistically significant with vs. without biomarkers). Adding pre-infusion EarlyCisP NGAL and TIMP-2*IGFBP-7 to the clinical prediction model for 3-month hypertension led to AUC change from 0.81 (0.72-0.91) to 0.89 (0.83-0.95) (p < 0.05). Conclusions: Tubular injury biomarkers we studied were individually not strong predictors of 3 or 12-month post-cisplatin kidney outcomes. Adding biomarkers to existing clinical prediction models may help predict post-therapy hypertension development or identify higher kidney-risk patients.

THE HIGH-ENERGY DIET/STREPTOZOTOCIN TREATED MUNICH WISTAR FRÖMTER RATS: A NEW EXPERIMENTAL MODEL OF DIABETES INDUCTION IN ESTABLISHED KIDNEY DISEASE

Objective: Chronic kidney disease (CKD) is especially prevalent in patients with diabetes mellitus (DM). We aim to induce diabetes in the Munich Wistar Frömter (MWF) rat, a genetic model of established CKD. Design and method: 16-week-old male MWF rats (n = 10 rats/group) were fed standard chow (MWF) or a high fat/high-sucrose diet for 6 weeks together with streptozotocin administration (STZ; 15mg/Kg i.p.) at the start of dietary exposure (MWF-D). Results: Water intake, urine excretion, GTT and HOMA-IR index evidenced the development of diabetes in MWF-D rats. Albuminuria was higher in MWF with no further effect of diabetes. However, renal expression of Kim-1 and Ngal, markers of renal damage, AT1R, AT2R and Collagen 1A1 were significantly higher in MWF-D rats. Perirenal adipose tissue (PRAT) amount was significantly higher in MWF-D rats and exhibited an upregulation of TNF-alpha and profibrotic factors (TGF-beta and Collagen 1A1), together with a downregulation of IL-10. Although blood pressure was not further increased by the induction of diabetes, heart weight and left ventricular wall thickness were higher in MWF-D, coincident with a reduction in systolic volume, but without a change in the ejection fraction. Conclusions: We propose the MWF-D rat as an experimental model of NDKD and suggest a strong relation between alterations in PRAT and increased renal damage.

A single administration of FGF2 after renal ischemia-reperfusion injury alleviates post-injury interstitial fibrosis.

Despite lack of clinical therapy in acute kidney injury (AKI) or its progression to chronic kidney disease (CKD), administration of growth factors shows great potential in the treatment of renal repair and further fibrosis. At an early phase of AKI, administration of exogenous fibroblast growth factor 2 (FGF2) protects against renal injury by inhibition of mitochondrial damage and inflammatory response. Here, we investigated whether this treatment attenuates the long-term renal interstitial fibrosis induced by ischemia-reperfusion (I/R) injury. Unilateral renal I/R with contralateral nephrectomy was utilized as an in vivo model for AKI and subsequent CKD. Rats were randomly divided into four groups: Sham-operation group, I/R group, I/R-FGF2 group and FGF2-3D group. These groups were monitored for up to 2 months. Serum creatinine, inflammatory response and renal histopathology changes were detected to evaluate the role of FGF2 in AKI and followed renal interstitial fibrosis. Moreover, the expression of vimentin, α-SMA, CD31 and CD34 were examined. Two months after I/R injury, the severity of renal interstitial fibrosis was significantly attenuated in both of I/R-FGF2 group and FGF2-3D group, compared with the I/R group. The protective effects of FGF2 administration were associated with the reduction of high-mobility group box 1 (HMGB1)-mediated inflammatory response, the inhibition of transforming growth factor beta (TGF-β1)/Smads signaling-induced epithelial-mesenchymal transition and the maintenance of peritubular capillary structure. A single dose of exogenous FGF2 administration 1 h or 3 days after reperfusion inhibited renal fibrogenesis and thus blocked the transition of AKI to CKD. Our findings provided novel insight into the role of FGF signaling in AKI-to-CKD progression and underscored the potential of FGF-based therapy for this devastating disease.

Paeoniflorin suppresses kidney inflammation by regulating macrophage polarization via KLF4-mediated mitophagy

BackgroundMacrophages M1 polarization involved in the process of renal inflammatory injury, is a well-established hallmark of chronic kidney disease (CKD). Paeoniflorin (PF), a water-soluble monoterpene glycoside extracted from Paeonia lactiflora, revealed renal anti-inflammatory activities in our previous study. However, the potential molecular mechanism of PF on CKD remains unknown. PurposeThe present study aims to investigate the regulation of PF on macrophage polarization in CKD. MethodsA CKD model was established by cationic bovine serum albumin and a murine macrophage cell line RAW264.7 induced with lipopolysaccharide (LPS) were used to clarify the underlying mechanisms of PF in CKD. ResultsResults showed that PF exhibited favorable protective effects on CKD model mice by promoting renal function, ameliorating renal pathological injury and podocyte damage. Furthermore, PF inhibited the infiltration of M1 macrophage marker CD68 and iNOS in kidney tissue, but increased the proportion of M2 macrophage marker CD206. In RAW264.7 cells stimulated with LPS, the levels of cytokines including IL-6, IL-1β, TNF-α, MCP-1 were lessened under PF treatment, while the levels of Arg1, Fizz1, IL-10 and Ym-1 were augmented. These results indicated that PF promoted macrophage polarization from M1 to M2 in vivo and in vitro. More importantly, PF repaired the damaged mitochondria through increasing mitochondrial membrane potential and reducing ROS accumulation. The mitophagy-related proteins PINK1, Parkin, Bnip3, P62 and LC3 were up-regulated by PF, accompanied by the incremental expressions of Krüppel-like transcription factor 4 (KLF4). Moreover, the promotion of mitophagy and inhibition of M1 macrophage polarization owing to PF were reversed by mitophagy inhibitor Mdivi-1 or silencing KLF4. ConclusionOverall, PF suppressed renal inflammation by promoting macrophage polarization from M1 to M2 and inducing mitophagy via regulating KLF4. It is expected to provide a new strategy for exploring the effects of PF in treating CKD.

Fufang Shenhua tablet inhibits renal fibrosis by inhibiting PI3K/AKT

BackgroundFufang Shenhua tablet (SHT), a traditional Chinese medicine compound, has been utilized in the clinical management of chronic kidney disease (CKD) for a long time. Nevertheless, the fundamental active constituents and potential mechanism of action remain unclear. Thus, the objective of this study was to investigate the renoprotective effect of SHT on residual renal tissue in CKD model rats and to explore its primary efficacious components and their underlying mechanism. MethodsAfter a 12-week period of SHT treatment through gavage in a 5/6 nephrectomized animal model of CKD, we evaluated the body weight, renal function, and renal pathological changes. Furthermore, the expression levels of fibronectin (FN), collagen I (COL-1), α-smooth muscle actin (α-SMA), and vimentin in renal tissues were assessed. In addition, network pharmacology analysis and molecular docking were utilized to predict the primary active components, potential therapeutic targets, and intervention pathways through which SHT could potentially exert its anti-kidney fibrosis effects. Subsequently, these predictions were validated in renal tissues of rats with CKD and in transforming growth factor β1 (TGF-β1)-induced HK-2 cells. ResultsSHT significantly improved renal function and reduced renal pathological damage and fibrosis in CKD model rats. Network pharmacological analysis identified 62 active components in SHT, with quercetin ranked first, and 105 protein targets shared by SHT and CKD. Based on the protein‒protein interaction network (PPI) and the SHT-CKD-pathway network, AKT1, MYC, IL2, and VEGFA were identified as key targets. Furthermore, GO and KEGG pathway enrichment analyses indicated that the renoprotective effect of SHT on CKD was closely associated with the PI3K/AKT signaling pathway. Molecular docking results demonstrated that the main active components of SHT had a strong binding affinity to the hub genes. During experimental validation, SHT hindered the activity of the PI3K/AKT signaling pathway in the renal tissue of CKD model rats. Furthermore, activation of the PI3K/AKT signaling pathway was correlated with a modified fibrotic phenotype in rats with 5/6 nephrectomy-induced CKD and TGF-β1-induced HK-2 cells. Conversely, SHT and quercetin curtailed the activation of the PI3K/AKT signaling pathway and inhibited the formation of renal fibrosis, thus indicating that the PI3K/AKT signaling pathway is the basis of the antifibrotic effects of SHT. Ultimately, administration of the PI3K/AKT agonist 740Y-P counteracted the fibrotic phenotype of TGF-β1-induced HK-2 cells induced by SHT. ConclusionsIn this investigation, we employed a fusion of systems pharmacology and in vivo and in vitro experiments to elucidate the mechanism of SHT's antifibrotic properties via obstruction of the PI3K/AKT signaling pathway. Additionally, we surmised that AKT may be the principal target of SHT for the management of CKD and that quercetin may be its efficacious component. We have thus identified SHT as a promising drug for the amelioration of renal fibrosis and the progression of CKD.

A kidney-brain neural circuit drives progressive kidney damage and heart failure

Chronic kidney disease (CKD) and heart failure (HF) are highly prevalent, aggravate each other, and account for substantial mortality. However, the mechanisms underlying cardiorenal interaction and the role of kidney afferent nerves and their precise central pathway remain limited. Here, we combined virus tracing techniques with optogenetic techniques to map a polysynaptic central pathway linking kidney afferent nerves to subfornical organ (SFO) and thereby to paraventricular nucleus (PVN) and rostral ventrolateral medulla that modulates sympathetic outflow. This kidney-brain neural circuit was overactivated in mouse models of CKD or HF and subsequently enhanced the sympathetic discharge to both the kidney and the heart in each model. Interruption of the pathway by kidney deafferentation, selective deletion of angiotensin II type 1a receptor (AT1a) in SFO, or optogenetic silence of the kidney-SFO or SFO-PVN projection decreased the sympathetic discharge and lessened structural damage and dysfunction of both kidney and heart in models of CKD and HF. Thus, kidney afferent nerves activate a kidney-brain neural circuit in CKD and HF that drives the sympathetic nervous system to accelerate disease progression in both organs. These results demonstrate the crucial role of kidney afferent nerves and their central connections in engaging cardiorenal interactions under both physiological and disease conditions. This suggests novel therapies for CKD or HF targeting this kidney-brain neural circuit.

Glomerular Hyperfiltration and Inflammation Promote Glomerular Injury in a CKD Model Induced by Reduced Kidney Mass

Renal autoregulation maintains renal blood flow and glomerular filtration rate (GFR) in a wide range of renal perfusion pressure (RPP). This mechanism is impaired in diabetes and chronic kidney disease (CKD), such that even modest increases in arterial pressure (AP) are transmitted to glomerular capillaries resulting in glomerular injury. Elevated RPP drives renal immune cell infiltration ( Hypertens. 76: 849-858, 2020). We showed that hypertensive mechanical stretch and T cell cytokine interleukin (IL)-17A synergistically promote vascular fibrosis ( Wu, Circ. Res. 114: 616-625, 2014). However, the mechanisms by which hemodynamic and inflammatory stimuli cause CKD remain poorly understood. We sought to investigate the contributions of glomerular hyperfiltration (GHF) and inflammation on glomerular damage in a mouse CKD model induced by reduced kidney mass (RKM). This model is characterized by impaired autoregulation, early onset hypertension and subsequent lymphocytic inflammation, albuminuria and glomerulosclerosis. We hypothesize that the initial rise in AP post RKM surgery promotes kidney injury through both GHF and renal inflammation. Eight-week old male 129/S6 mice were implanted with radiotelemeters. After a 10-day recovery, mice underwent sham surgery or RKM which involved uninephrectomy and partial renal artery ligation in the remaining kidney, such that the functional renal mass is reduced by ~ 2/3. AP, urine albumin, and transcutaneous GFR were measured at baseline, day (D) 3, D7, D14, D21, D28, and D56 after RKM.Immediately after RKM, GFR dropped to 30% of sham levels (Mean ± SEM in μL/min/100 g: RKM 279 ± 59 vs Sham 913 ± 61, n = 4 – 6, p < 0.05, two-way ANOVA RM), consistent with the extent of renal mass ablation. The mean AP of RKM mice was identical to sham controls at baseline (115 ± 2 vs 116 ± 2 mmHg) but increased by 29 mmHg by D5 (144 ± 8 vs 112 ± 2 mmHg, p < 0.05), and remained significantly elevated through D56, albeit a declining trend. Compared to GFR of sham mice (100%), the GFR of RKM mice gradually increased to 36% at D3, 43% at D7 and 69% at D14. By D21 post surgery, the GFR of RKM mice rose to 71% of sham (800 ± 106 vs 1121 ± 47 μL/min/100 g, p > 0.05) and plateaued through day 28 (69% of sham), suggesting that the early rise in AP and impaired autoregulation may have caused GHF in the RKM mice. The adaptive increase in GFR post RKM surgery was associated with a trend towards increased renal leukocytes, monocyte/macrophages, CD3 T cells and CD4/CD8 subsets at D14 and significantly elevated albuminuria at D28 (832 ± 227 vs 341 ± 15 μg/day, p < 0.05), implying hyperfiltration-induced renal inflammation and glomerular damage. Importantly, the GFR declined to 57% of sham levels by day 56 (597 ± 86 vs 1047 ± 80 μL/min/100 g, p < 0.05), suggesting progressive glomerular injury. We conclude that impaired autoregulation exposes the kidney to both inflammation and GHF, causing glomerular injury, albuminuria, and CKD progression. This work is supported by a NIDDK K01 DK126792 and a University of Rochester Environmental Health Science Center Pilot award (as part of a NIEHS P30ES001247) to JW. This is the full abstract presented at the American Physiology Summit 2023 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.

Evolution of hypoxia and hypoxia-inducible factor asparaginyl hydroxylase regulation in chronic kidney disease.

The roles of hypoxia and hypoxia inducible factor (HIF) during chronic kidney disease (CKD) are much debated. Interventional studies with HIF-α activation in rodents have yielded contradictory results. The HIF pathway is regulated by prolyl and asparaginyl hydroxylases. While prolyl hydroxylase inhibition is a well-known method to stabilize HIF-α, little is known about the effect asparaginyl hydroxylase factor inhibiting HIF (FIH). We used a model of progressive proteinuric CKD and a model of obstructive nephropathy with unilateral fibrosis. In these models we assessed hypoxia with pimonidazole and vascularization with three-dimensional micro-computed tomography imaging. We analysed a database of 217 CKD biopsies from stage 1 to 5 and we randomly collected 15 CKD biopsies of various severity degrees to assess FIH expression. Finally, we modulated FIH activity in vitro and in vivo using a pharmacologic approach to assess its relevance in CKD. In our model of proteinuric CKD, we show that early CKD stages are not characterized by hypoxia or HIF activation. At late CKD stages, some areas of hypoxia are observed, but these are not colocalizing with fibrosis. In mice and in humans, we observed a downregulation of the HIF pathway, together with an increased FIH expression in CKD, according to its severity. Modulating FIH in vitro affects cellular metabolism, as described previously. In vivo, pharmacologic FIH inhibition increases the glomerular filtration rate of control and CKD animals and is associated with decreased development of fibrosis. The causative role of hypoxia and HIF activation in CKD progression is questioned. A pharmacological approach of FIH downregulation seems promising in proteinuric kidney disease.

Effects of a High-Protein Diet on Kidney Injury under Conditions of Non-CKD or CKD in Mice.

Considering the prevalence of obesity and global aging, the consumption of a high-protein diet (HPD) may be advantageous. However, an HPD aggravates kidney dysfunction in patients with chronic kidney disease (CKD). Moreover, the effects of an HPD on kidney function in healthy individuals are controversial. In this study, we employed a remnant kidney mouse model as a CKD model and aimed to evaluate the effects of an HPD on kidney injury under conditions of non-CKD and CKD. Mice were divided into four groups: a sham surgery (sham) + normal diet (ND) group, a sham + HPD group, a 5/6 nephrectomy (Nx) + ND group and a 5/6 Nx + HPD group. Blood pressure, kidney function and kidney tissue injury were compared after 12 weeks of diet loading among the four groups. The 5/6 Nx groups displayed blood pressure elevation, kidney function decline, glomerular injury and tubular injury compared with the sham groups. Furthermore, an HPD exacerbated glomerular injury only in the 5/6 Nx group; however, an HPD did not cause kidney injury in the sham group. Clinical application of these results suggests that patients with CKD should follow a protein-restricted diet to prevent the exacerbation of kidney injury, while healthy individuals can maintain an HPD without worrying about the adverse effects.

Icariin, the main prenylflavonoid of Epimedii Folium, ameliorated chronic kidney disease by modulating energy metabolism via AMPK activation

Ethnopharmacological relevanceEpimedii Folium is a famous traditional Chinese medicine (TCM) widely used in classic formulas, Chinese patent drugs and health care products for treating kidney diseases. Therefore, we speculated that icariin, its main component, might also have a good therapeutic effect on chronic kidney disease (CKD). Aim of studyTo investigate the efficacy and potential mechanism of icariin on CKD. Materials and methodsA CKD model was established by intragastric administration of adenine (200 mg/kg/d) to adult male SD rats for 28 consecutive days. TGF-β1-induced fibrotic HK-2 cells were applied to establish the renal fibrosis model in vitro. Biochemical determination, pathological staining, flow cytometry and ELISA were performed to preliminarily evaluate the renoprotection of icariin. The intervention effect of icariin on renal fibrosis progression was assessed by cell stiffness determination and multiple immunological methods. The potential mechanism of icariin on CKD was revealed by means of 1H NMR metabolomics, qRT-PCR and Western blotting analysis. ResultsIcariin at the dosage of 100 mg/kg/d and 200 mg/kg/d markedly ameliorated rat renal function in a dose-dependent manner. Based on renal pathological features, the mechanism of icariin intervention in CKD was initially revealed by metabolomics, which was closely related to energy metabolism pathways. Furthermore, the detection results of AMPK and related factors in its mediated signaling pathways indicated that icariin exerted a therapeutic effect on CKD by attenuating inflammation and oxidative stress responses and retarding renal fibrosis progression through regulating AMPK/SIRT1/NF-κB and AMPK/ACC signaling pathways. ConclusionIt was the first time to demonstrate that icariin could treat adenine-induced CKD by modulating energy metabolism via AMPK activation in a dose-dependent manner.

Redox Signaling in Chronic Kidney Disease-Associated Cachexia.

Redox signaling alterations contribute to chronic kidney disease (CKD)-associated cachexia. This review aims to summarize studies about redox pathophysiology in CKD-associated cachexia and muscle wasting and to discuss potential therapeutic approaches based on antioxidant and anti-inflammatory molecules to restore redox homeostasis. Enzymatic and non-enzymatic systems of antioxidant molecules have been studied in experimental models of kidney diseases and patients with CKD. Oxidative stress is increased by several factors present in CKD, including uremic toxins, inflammation, and metabolic and hormone alterations, leading to muscle wasting. Rehabilitative nutritional and physical exercises have shown beneficial effects for CKD-associated cachexia. Anti-inflammatory molecules have also been tested in experimental models of CKD. The importance of oxidative stress has been shown by experimental studies in which antioxidant therapies ameliorated CKD and its associated complications in the 5/6 nephrectomy model. Treatment of CKD-associated cachexia is a challenge and further studies are necessary to investigate potential therapies involving antioxidant therapy.

Biomarker identification and pathway analysis of Astragalus membranaceus and Curcuma zedoaria couplet medicines on adenine-induced chronic kidney disease in rats based on metabolomics.

Background: Chronic kidney disease (CKD) is usually insidious, and most affected individuals are asymptomatic until the disease becomes advanced. The effective treatment of CKD would rely on the incorporation of multidisciplinary approaches. Astragalus membranaceus (AM) and Curcuma zedoaria (CZ) have been widely used in the treatment of CKD. However, the mechanism of AM and CZ in the treatment of CKD is still unclear. Methods: This study was designed to evaluate the effects of AM and CZ on adenine-induced rats and to investigate the underlying mechanism by using metabolomic analysis. Addition of 0.75% adenine to the diet of rats for 3weeks induced the animal model of CKD. The rats in the treatment group were treated with AM and CZ (2.1g/kg/day) for 4weeks. Blood and kidney samples were collected for biochemical and histological examination. Ultra-high-performance liquid chromatography/Q Exactive HFX mass spectrometer (UHPLC-QE-MS) was applied to analyze metabolic profiling variations in the kidney. Results: The results showed that AM and CZ could significantly reduce serum creatinine (Scr) and blood urea nitrogen (BUN) levels in CKD rats and alleviate renal pathological injury. By comparing the endogenous components of the normal group and the model group in positive ion mode and negative ion mode, a total of 365 and 155 different metabolites were screened, respectively. A total of 117 and 73 metabolites with significantly different expressions were identified between model group and AM and CZ group in positive ion mode and negative ion mode, respectively. The pivotal pathways affected by AM and CZ included nicotinate and nicotinamide metabolism, and glycine, serine and threonine metabolism. Furthermore, significant changes in metabolites in CKD rats after AM and CZ therapies were observed, including L-Threonine, D-pantothenic acid, and nicotinamide. Moreover, we found that AM and CZ significantly reduced renal fibrosis and inflammation in CKD rats, which may be related to the regulation of SIRT1/JNK signaling pathway. Conclusion: In conclusion, AM and CZ significantly reduced renal fibrosis and inflammation in CKD rats, which may be related to the regulation of SIRT1/JNK signaling pathway. Furthermore, L-Threonine, D-pantothenic acid, and nicotinamide may be potential biomarkers for the progression and treatment of CKD.

Phosphate induces inflammation and exacerbates injury from cigarette smoke in the bronchial epithelium

An elevation in serum phosphate—also called hyperphosphatemia—is associated with reduced kidney function in chronic kidney disease (CKD). Reports show CKD patients are more likely to develop lung disease and have poorer kidney function that positively correlates with pulmonary obstruction. However, the underlying mechanisms are not well understood. Here, we report that two murine models of CKD, which both exhibit increased serum levels of phosphate and fibroblast growth factor (FGF) 23, a regulator of phosphate homeostasis, develop concomitant airway inflammation. Our in vitro studies point towards a similar increase of phosphate-induced inflammatory markers in human bronchial epithelial cells. FGF23 stimulation alone does not induce a proinflammatory response in the non-COPD bronchial epithelium and phosphate does not cause endogenous FGF23 release. Upregulation of the phosphate-induced proinflammatory cytokines is accompanied by activation of the extracellular-signal regulated kinase (ERK) pathway. Moreover, the addition of cigarette smoke extract (CSE) during phosphate treatments exacerbates inflammation as well as ERK activation, whereas co-treatment with FGF23 attenuates both the phosphate as well as the combined phosphate- and CS-induced inflammatory response, independent of ERK activation. Together, these data demonstrate a novel pathway that potentially explains pathological kidney-lung crosstalk with phosphate as a key mediator.