Rat Glomeruli Research Articles

Black Garlic Supplementation and Glomerular Protection in Hyperuricemic Rats: A Study on Kidney Health Prevention

Introduction: Hyperuricemia is a significant risk factor for the development of chronic kidney disease. Excessive uric acid can deposit in the kidneys, triggering inflammatory reactions and oxidative stress, leading to structural damage in the glomeruli. Black garlic, with its antioxidant and bioactive compounds, has the potential to protect the kidneys from hyperuricemia-induced damage. Our objective is to evaluate the effect of black garlic supplementation on the histopathological features of glomeruli in a hyperuricemic rat model. Methods: This study was an analytical observational study using Biological Remnant Materials (BRM) from a previous study. The sample consisted of 30 male Sprague-Dawley rats divided into 5 groups: disease control, drug control (allopurinol), and 3 black garlic dose groups (240 mg/day, 480 mg/day, and 960 mg/day). Data were collected through histopathological examination of kidney tissue using the glomerular damage scoring method (0-3). Data analysis was performed using one-way ANOVA and post hoc LSD tests. Results: Black garlic demonstrated protective effects on the histopathological features of glomeruli in hyperuricemic rats. The 240 mg/day dose showed the most significant improvement in kidney health, reducing glomerular damage scores compared to the disease control group (p < 0.05). These findings suggest that black garlic can serve as a preventive agent against kidney complications caused by hyperuricemia. Conclusion: Black garlic provides protective effects on glomerular histopathology in hyperuricemic rats, with the 240 mg/day dose being the most effective. These results suggest potential use for black garlic in preventing kidney complications in hyperuricemia.

Rutin reduces inflammation and fibrosis via TGF-β/SMAD pathways in IgA nephropathy induced rats.

Rutin is a flavonoid glycoside obtained from the plant Ruta graveolens. It was known to have immunosuppressant activities. This study was focused on effect of rutin against immunoglobulin A (IgA) nephropathy. IgA nephropathy was induced in Sprague-Dawley rats with various inducing agents described in text. During the later part of the induction phase, rutin was administered. Control group rats did not receive any treatment or inducing agent, induced group rats received only the inducing agents, whereas treatment group received the inducing agents as well as rutin. During the study, various biochemical parameters pertaining to kidney function were evaluated and also, the expression of proteins and cytokines responsible for inflammation and fibrosis were assessed. The effect of rutin in IgA nephropathy was promising as treatment with rutin reduced the deposition of IgA in the glomeruli of rats. Along with this we also tried to establish the probable mechanism of action of rutin and based on the summary of the results it was concluded that rutin reduced the inflammation and fibrosis related to IgA nephropathy by inhibiting the TGF-β/SMAD pathways and ultimately reducing the expression of α-smooth muscle actin (α-SMA). Comprehending all the above consideration, it may be safely said that that rutin alleviated inflammation and also fibrosis mediated by IgA, by suppressing the transforming growth factor-β (TGF-β) activities through suppressor of mothers against decapentaplegic pathways and reduced the epithelial-to-mesenchymal transition by downregulating the α-SMA which is a marker for fibrosis.

Magnolol Inhibits High Fructose-Induced Podocyte Inflammation via Downregulation of TKFC/Sp1/HDAC4/Notch1 Activation

Background/Objectives: High fructose has been implicated as an important trigger of kidney inflammation in patients and experimental models. Magnolol, isolated from Magnolia officinalis, has an anti-inflammatory effect, but its protective role in podocytes remains underexplored. This study explored the protective effects and underlying mechanism of magnolol against high fructose-induced podocyte inflammation. Methods: The effects of magnolol on high fructose-induced podocyte inflammation were assessed in male Sprague Dawley rats administered 10% (w/v) fructose water for 12 weeks and heat-sensitive human podocyte cell lines (HPCs) exposed to 5 mM fructose. Podocyte foot processes were examined using transmission electron microscopy. The expression levels of nephrin, podocin, tumor necrosis factor-α (TNF-α), Notch1 intracellular domain (NICD1), triokinase/FMN cyclase (TKFC), specificity protein 1 (Sp1) and histone deacetylase 4 (HDAC4) were determined by Western blot, immunofluorescence and real-time quantitative polymerase chain reaction (qRT-PCR). The chromatin immunoprecipitation (ChIP) assay was performed to evaluate the interaction between Sp1 and the promoter region of HDAC4. Results: Magnolol mitigated the impairment of glomerular filtration function in high fructose-fed rats. Besides, it significantly alleviated the inflammatory responses in glomeruli and HPCs, evidenced by decreased protein levels of TNF-α and NICD1. Increased protein levels of TKFC, Sp1 and HDAC4 were observed in high fructose-stimulated HPCs and rat glomeruli. TMP195, an HDAC4 inhibitor, reduced TNF-α and NICD1 protein levels in high fructose-exposed HPCs. The increased Sp1 was shown to associate with the promoter region of HDAC4, promoting HDAC4 protein expression in high fructose-exposed HPCs. The knockdown of TKFC in HPCs by TKFC siRNA decreased Sp1, HDAC4 and NICD1 protein levels, alleviating podocyte inflammatory response. Furthermore, magnolol inhibited TKFC/Sp1/HDAC4/Notch1 activation in vivo and in vitro. Conclusions: Magnolol attenuated high fructose-induced podocyte inflammation possibly through the suppression of TKFC/Sp1/HDAC4/Notch1 activation, providing new evidence for its potential role in podocyte protection.

Siwu tablet attenuates high fructose-induced glomerular podocyte senescence in rats through increasing Nup155 to promote INO80 mRNA nuclear export

Ethnopharmacological relevanceSiwu tablet (SWT), derived from a traditional Chinese medicinal formula named Siwu decoction, is widely used for blood deficiency syndrome. Siwu decoction and its derived formulas have been proven to improve renal anemia and prevent senescence. Whether SWT prevents glomerular podocyte senescence and the underlying molecular mechanism remains unknow. Aim of the studyTo elucidate the protective effect and possible mechanism of SWT on glomerular podocyte senescence. Material and methodsLiquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to characterize components of SWT. Male Sprague-Dawley rats were given 10% fructose drinking water for 16 weeks. SWT (810 and 1620 mg/kg) was administered orally for the last 8 weeks. The assays of senescence-associated beta-galactosidase (SA-β-gal) staining, immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot as well as enzyme linked immunosorbent assay were performed to evaluate rat glomerular podocyte senescence. The mRNA and protein levels of nucleoporin 155 (Nup155) and inositol requiring mutant 80 (INO80) in rat glomeruli were detected by qRT-PCR, Western blot and immunofluorescence. Foot processes and nuclear pore complexes (NPCs) of rat glomerular podocytes were visualized by transmission electron microscopy. ResultsOne hundred and fifty-nine components were preliminarily identified in SWT. The results of animal experiments showed that SWT decreased the activity of SA-β-gal, protein levels of p16, p21, p53 and phosphorylated histone H2AX (γ-H2AX), and mRNA levels of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) in glomeruli of high fructose-fed rats. As expected, SWT increased renal cortex erythropoietin mRNA expression and serum erythropoietin concentration in this animal model. SWT reduced urine albumin-to-creatinine ratio and serum levels of uric acid, creatinine and blood urea nitrogen, and recovered glomerular structure injury in high fructose-fed rats. It up-regulated mRNA and protein levels of Nup155 and the number of podocyte NPCs, and subsequently reinforced mRNA nuclear export and protein expression of INO80 in rat glomeruli under high fructose stimulation. ConclusionsSWT ameliorates glomerular podocyte senescence in high fructose-fed rats possibly by increasing Nup155 to promote INO80 mRNA nuclear export.

Abstract P213: Mechanisms of Renin-Angiotensin System-Mediated Nitric Oxide Signaling in Podocytes

Introduction: The renin-angiotensin system (RAS) is a crucial cardiovascular and renal function regulator. Podocytes, glomerular epithelial cells that play a pivotal role in maintaining the renal filtration barrier, are key targets for RAS peptides. Our findings showed that RAS can promote calcium (Ca 2+ ) and nitric oxide (NO) signaling in podocytes. Moreover, a decrease in NO bioavailability directly correlates with glomerular filtration barrier damage, Ca 2+ overload, and the development of salt-sensitive hypertension. We hypothesized that RAS-mediated NO production is crucial for Ca 2+ control and cytoskeletal rearrangements in podocytes, contributing to glomerular filtration regulation. Methods: We performed live confocal imaging on human podocytes and freshly isolated rat glomeruli to detect the production of NO (DAF-FM) and Ca 2+ (Fluo-8). RAS peptides (Ang II, III, IV, 1-7, 1-9, and TRV120027) were used to stimulate cells, and NO and Ca 2+ production were measured. Blockers for Ang II Type 1 (AT1R), Ang II Type 2 (AT2R), and MAS receptors were used to identify the pathways involved in NO production. The dynamic changes in podocyte cytoskeleton mediated by RAS-NO intracellular signaling were detected using rhodamine-phalloidin staining and scanning ion microscopy. The NOS isoform-specific blockers were used to determine the source of NO in podocytes. ANOVA with post-hoc was used for comparisons. Results: Angiotensins II and III generate the most profound increases in NO levels in podocytes. Ang II, Ang III, and Ang 1-7 promote NO production by activating AT2R (n ≥ 20, p < 0.01). Interestingly, the increase in NO modulates AT1R- and β-arrestin-mediated Ca 2+ signaling (n ≥ 20, p < 0.05), suggesting the role of the NO/AT2R axis in Ca 2+ control in podocytes. NO production correlates with podocyte volume increase, and scanning ion microscopy experiments further confirmed that AT2R activation and corresponding NO elevation are responsible for the protrusion of podocyte's lamellipodia. Finally, we found that in podocytes, NOS1 is responsible for NO bioavailability, accounting for 69±5% of the total NO response, while the remaining NO levels are generated by NOS2 (39±5%). Conclusion: Our data show that RAS activation promotes NO production in podocytes, mainly via the AT2R pathway. NO is crucial for Ca 2+ control in podocytes and lamellipodia formation, suggesting the essential role of NO in glomerular filtration barrier function.

Renin-angiotensin system-mediated nitric oxide signaling in podocytes.

Nitric oxide (NO) is widely recognized for its role in regulating renal function and blood pressure. However, the precise mechanisms by which NO affects renal epithelial cells remain understudied. Our previous research has shown that NO signaling in glomerular podocytes can be initiated by Angiotensin II (ANG II) but not by ATP. This study aims to elucidate the crucial interplay between the renin-angiotensin system (RAS) and NO production in podocytes. To conduct our research, we used cultured human podocytes and freshly isolated rat glomeruli. A variety of RAS peptides were used, alongside confocal microscopy, to detect NO production and NO/Ca2+ cross talk. Dynamic changes in the podocyte cytoskeleton, mediated by RAS-NO intracellular signaling, were observed using fluorescent labeling for F-actin and scanning probe microscopy. The experiments demonstrated that ANG II and ANG III generated high levels of NO by activating the angiotensin II type 2 receptor (AT2R). We did not detect functional MAS receptor presence in podocytes, and the moderate NO response to ANG 1-7 was also mediated through AT2R. Furthermore, NO production impacted intracellular Ca2+ signaling and correlated with an increase in podocyte volume and growth. Scanning probe experiments revealed that AT2R activation and the corresponding NO generation are responsible for the protrusion of podocyte lamellipodia. Taken together, our data indicate that AT2R activation enhances NO production in podocytes and subsequently mediates changes in Ca2+ signaling and podocyte volume dynamics. These mechanisms may play a significant role in both physiological and pathophysiological interactions between the RAS and podocytes.NEW & NOTEWORTHY The renin-angiotensin system plays a crucial role in the production of intracellular nitric oxide within podocytes. This mechanism operates through the activation of the angiotensin II type 2 receptor, leading to dynamic modifications in intracellular calcium levels and the actin filament network. This intricate process is vital for linking the activity of angiotensin receptors to podocyte function.

Loss of Nup155 promotes high fructose-driven podocyte senescence by inhibiting INO80 mRNA nuclear export

IntroductionPodocyte senescence causes podocyte loss and glomerulopathy. Excessive fructose intake is a risk factor for podocyte injury. However, whether high fructose promotes podocyte senescence remains unknown. ObjectivesTo explore the pathological mechanism by which high fructose drives podocyte senescence and find natural compounds to alleviate podocyte senescence. MethodsPodocyte senescence was characterized with senescence-associated beta-galactosidase (SA-β-gal) staining, Western blot, real-time quantitative polymerase chain reaction (qRT-PCR), comet assay and immunofluorescence. Proteomics analysis was performed to identify differentially expressed proteins in high fructose-exposed podocytes. Podocyte nuclear pore complexes (NPCs) and foot processes were observed by transmission electron microscopy. The mRNA and protein levels of nucleoporin 155 (Nup155) and inositol requiring mutant 80 (INO80) were detected by qRT-PCR, Western blot and immunofluorescence. Virtual screening was conducted to find natural compounds that target Nup155. ResultsHigh fructose increased SA-β-gal activity, protein level of p53, p21, p16 and phosphorylated histone H2AX (γ-H2AX), as well as mRNA expression of interleukin-1β (IL-1β), IL-6 and tumor necrosis factor α (TNF-α) in rat glomeruli and podocytes. Proteomic analysis unraveled a crucial molecule Nup155, which was decreased in high fructose-induced podocyte senescence. Meanwhile, the number of podocyte NPCs was also decreased in vivo and in vitro. Consistently, high fructose suppressed nuclear export of INO80 mRNA, thereby down-regulated INO80 protein expression in podocyte senescence. Deletion of Nup155 inhibited INO80 mRNA nuclear export to induce podocyte senescence, whereas overexpression of Nup155 or INO80 alleviated high fructose-induced podocyte senescence. Ferulic acid was found to up-regulate Nup155 by both direct binding to stabilize Nup155 protein and enhancing its transcription, to promote INO80 mRNA nuclear export in the mitigation of high fructose-caused podocyte senescence. ConclusionHigh fructose induces podocyte senescence by decreasing Nup155 to inhibit INO80 mRNA nuclear export. Ferulic acid targeting Nup155 may be a potential strategy to prevent high fructose-induced podocyte senescence.

The Protective Effects of Acteoside and Total Glycosides of the Leaves of Rehmannia on Three Types of Glomerular Structural Cells Under High Glucose and Inflammatory Stimulation

ABSTRACT Background: The challenge of chronic glomerulonephritis necessitates innovative strategies for preventing renal function deterioration. Acteoside (ACT), the primary bioactive compound of total glycosides of the leaves of Rehmannia (DHYZG), has been demonstrated availability and safety in reducing proteinuria, showing promising prospects in the treatment of kidney disease. This study aimed to elucidate the impact of ACT and DHYZG on glomerular structural cells and key proteins of rat glomeruli under several injury states. Methods: Employing 3-(4,5)-dimethylthiazolyl(-Z-Y1)-3,5-diphenyltetrazolium bromide (MTT) and Western blot methodologies, the investigation assessed varying concentrations of ACT and clinical concentration of DHYZG on cell viability and intracellular biomarkers. Specifically, the study explored the influence of ACT on connective tissue growth factor (CTGF), transforming growth factor-β (TGF-β), matrix metalloproteinase-2 (MMP-2), and MPP in rat mesangial cells, endothelial cells, and podocytes stimulated by lipopolysaccharide (LPS), high glucose, and interleukin-1β (IL-1β). Additionally, the impact of high glucose on angiotensin II (Ang II) expression in endothelial cells was investigated. Results: ACT and DHYZG demonstrated a protective effect on all cell types, and ACT administration shows a significant dose-dependent response. These compounds attenuated the expression of CTGF and TGF-β in LPS-stimulated mesangial cells and reduced the expression of MMP-2 and MMP-9 in all three cell types following IL-1β stimulation. Moreover, the high glucose-induced expression of Ang II in endothelial cells was mitigated. Conclusion: ACT and DHYZG exhibit a pronounced protective effect on glomerular cells, with ACT being the primary contributor to the therapeutic efficacy of DHYZG. Notably, ACT demonstrates a significant superiority over DHYZG in terms of both proliferation and cytokine expression in glomerular endothelial cells.

#457 Sparsentan has direct effects on the glomerular capillary wall to attenuate increased permeability after exposure to nephrotic syndrome plasma

Abstract Background and Aims The glomerular endothelial glycocalyx (eGlx), a luminal layer of proteoglycans, glycoproteins and glycolipids, forms the first part of the glomerular filtration barrier (GFB). Nephrotic syndrome (NS) describes a group of pathologies of the renal glomerulus that result in proteinuria and is associated with glomerular endothelial dysfunction. Current treatments are broad and non-specific. Sparsentan is a single-molecule dual endothelin type-A and angiotensin II type 1 receptor antagonist that has received accelerated approval in the United States for the reduction of proteinuria in adults with IgA nephropathy at high risk of disease progression. Our validated glomerular permeability assay directly measures the albumin permeability (Ps’alb)of capillary loops within individually trapped glomeruli [1]. This ex vivo assay is independent of haemodynamic factors and tubular albumin handling – factors known to affect urine protein concentrations. This work examines whether sparsentan could reduce glomerular albumin permeability in NS, by preserving the eGlx to maintain the GFB. Method Human NS plasma samples were collected under ethical consent from 3 patients that had undergone plasma exchange from periods when they were in relapse (RL), or subsequent remission (RM). Adult male Sprague Dawley rats (175–200 g) were perfused with 4% Ringer BSA solution. Glomeruli were isolated on ice by graded sieving from the cortex of each kidney. Glomeruli were incubated, in the presence of AF488-conjugated BSA (AF488-BSA, 30 μg/ml) and R18 (36.5 μg/ml), with 10% plasma from NS patients for 1 hour, and simultaneously treated with sparsentan (0.1 μM, 1 μM and 10 μM) or vehicle. The glomerular Ps’alb assay was used to measure changes in albumin permeability. We applied a fluorescence profile peak-to-peak confocal imaging technique to treated glomeruli to assess glomerular eGlx thickness [2]. Results Human NS patients in RL had a significantly greater proteinuria compared to RM (RL, 10, 000 ± 1, 354 mg/g; RM, 95.3 ± 59.7 mg/g, P = 0.017). Incubation of rat glomeruli with RL plasma induced a significant increase in Ps’alb (RM+vehicle, 6.0 × 10−7 ± 0.12 × 10−7 cm/s; RL+vehicle, 10.9 × 10−7 ± 0.84 × 10−7 cm/s, P < .001) with a significant reduction in glomerular eGlx thickness (RM+vehicle, 210 ± 21.2 nm; RL+vehicle, 109 ± 7.9 nm, P = .027), compared to rat glomeruli incubated with paired RM plasma. Sparsentan-treated RL incubated glomeruli were protected from both the increase in Ps’alb (RL+spars 10 µM, 6.3 × 10−7 ± 0.19 × 10−7 cm/s, P < .001) and the loss in glomerular eGlx (RL+spars 10 µM, 222 ± 8.5 nm, P = .013), to a level comparable to RM incubated glomeruli. The effect of sparsentan on Ps’alb was dose dependent (RL+spars 1 µM, 8.0 × 10−7 ± 0.46 × 10−7 cm/s; RL+spars 0.1 µM, 9.6 × 10−7 ± 0.22 × 10−7 cm/s) with Ps’alb changes correlating inversely with glomerular eGlx thickness (r² = 0.75, P < .0001). In RM glomeruli, sparsentan alone had no effect on Ps’alb compared with vehicle (RM+spars 10 µM, 6.2 × 10−7 ± 0.25 × 10−7 cm/s, P = .590), suggesting no effect on otherwise healthy capillaries. Conclusion We have shown that dual inhibition of endothelin and angiotensin receptors, with sparsentan, preserves the glomerular eGlx resulting in normalised glomerular permeability in NS. These findings suggest that the direct action of sparsentan on the GFB could help maintain barrier integrity in NS, in particular by glycocalyx protection.

Sirt7/HIC1 complex participates in hyperglycaemia-mediated EndMT via modulation of SDC1 expression in diabetic kidney disease and metabolic memory.

Diabetic kidney disease (DKD), a primary microvascular complication arising from diabetes, may result inend-stage renal disease. Epigenetic regulation of endothelial mesenchymal transition (EndMT) has been recently reported to exert function in metabolic memory and DKD. Here, we investigated the mechanism which Sirt7 modulated EndMT in human glomerular endothelial cells (HGECs) in the occurrence of metabolic memory in DKD. Lower levels of SDC1 and Sirt7 were noted in the glomeruli of both DKD patients and diabetes-induced renal injury rats, as well as in human glomerular endothelial cells (HGECs) with high blood sugar. Endothelial-to-mesenchymal transition (EndMT) was sustained despite the normalization of glycaemic control. We also found that Sirt7 overexpression associated with glucose normalization promoted the SDC1 expression and reversed EndMT in HGECs. Furthermore, the sh-Sirt7-mediated EndMT could be reversed by SDC1 overexpression. The ChIP assay revealed enrichment of Sirt7 and H3K18ac in the SDC1 promoter region. Furthermore, hypermethylated in cancer 1 (HIC1) was found to be associated with Sirt7. Overexpression of HIC1 with normoglycaemia reversed high glucose-mediated EndMT in HGECs. The knockdown of HIC1-mediated EndMT was reversed by SDC1 upregulation. In addition, the enrichment of HIC1 and Sirt7 was observed in the same promoter region of SDC1. The overexpressed Sirt7 reversed EndMT and improved renal function in insulin-treated diabetic models. This study demonstrated that the hyperglycaemia-mediated interaction between Sirt7 and HIC1 exerts a role in the metabolic memory in DKD by inactivating SDC1 transcription and mediating EndMT despite glucose normalization in HGECs.

Defective insulin-stimulated equilibrative nucleoside transporter-2 activity and altered subcellular transporter distribution drive the loss of adenosine homeostasis in diabetic kidney disease progression

AimProgression of diabetic nephropathy (DN) is linked to the dysregulated increase of adenosine and altered signaling properties. A major contribution to the maintenance of physiological extracellular adenosine levels relies on cellular uptake activity through plasma membrane nucleoside transporters. Because kidney cells are responsive to insulin, this study aims to determine how DN affects insulin regulation of the equilibrative nucleoside transporter-2 (ENT2). MethodsHuman Podocytes and rat glomeruli were used to study ENT2 regulation. The effects of diabetes and insulin on ENT2 mediated transport activity were determined measuring the fraction of total adenosine uptake in sodium-free medium which is inhibitable by hypoxanthine. Alterations in ENT2 subcellular distribution were assessed in the kidney of people affected with DN and diabetic rats. The consequences of impaired ENT2 activity on the kidney were evaluated using dipyridamole in an animal model. ResultsInsulin upregulates ENT2 uptake activity by increasing the Vmax, thus counteracting decreased adenosine uptake due to high d-glucose and achieving extracellular adenosine homeostasis. Insulin promoted ENT2 translocation to the plasma membrane dependent on PI3-kinase/Akt signaling and actin cytoskeleton integrity. However, in diabetic rats, the insulin-mediated induction of ENT2 activity was lost. Additionally, reduced Akt activation in response to insulin correlated with decreased ENT2 distribution at the plasma membrane. Kidney tissues from diabetic rats and human DN biopsies showed ENT2 redistribution to an intracellular pattern, evidencing dysfunctional adenosine uptake. Through ENT inhibition, we evidenced increased proteinuria and induced alpha-smooth muscle actin as a result of profibrotic activation of cells in the kidney. ConclusionDeficient insulin regulation of ENT2 activity contributes to chronically high adenosine levels and glomerular alterations that underline diabetic kidney disease progression.

Protease-Activated Receptor 1-Mediated Damage of Podocytes in Diabetic Nephropathy.

Increased urinary serine proteases are associated with diabetic nephropathy. During the development of diabetic nephropathy in type 2 diabetes, the elevation of serine proteases could overstimulate protease-activated receptor 1 (PAR1). PAR1 signaling is involved in the development of DN via TRPC6-mediated intracellular calcium signaling. This study provides fundamental knowledge that can be used to develop efficient therapeutic approaches targeting serine proteases or corresponding PAR pathways to prevent or slow the progression of diabetes-associated kidney diseases.

Obesity theranostics using nanoemulsions of probiotics and local herbs

Obesity is a polygenic disorder which has become a global epidemic in recent years. Aim of the present study was to assess the theranostic potential of probiotics (Lactobacillus bulgaricus, Bifidobacterium) and local herbs (fenugreek seeds, aloe vera) on the body weight, biochemical (liver and kidney functions) and histology of some internal organs (liver, kidney, ovary, small intestine) in obese rats. In present study, nanoemulsions of probiotics and local herbs were formulated by high energy method and characterized by FTIR and UV analysis. One hundred and sixty (160) female wistar rats were divided into sixteen groups. A high-fat diet was given to induce obesity in them. Obese rats were treated with different doses of probiotics, local herbs and their combination. Weekly body weight was monitored. Rats were dissected after fifteen weeks; blood and organs were harvested for biochemical analysis and histology. Results demonstrated a protective effect of nanoemulsion of probiotics and local herbs on the central vein, glomerulus, villi and normal ovulatory function of obese rats. Rats treated with a combination of probiotics and local herbs (fenugreek seeds, aloe vera) exhibited improved levels of bilirubin (4 mg/dl to15 mg/dl), AST from (110 U/L to 18 U/L) and ALT from (52 U/L to 10U/L). Similar renoprotective effects were recorded on the overall renal function tests (RFT). A combination of probiotics and local herbs in post treatment rats improved urea (63 mg/dl to 22 mg/dl) and creatinine (0.2 mg/dl to 0.8 mg/dl) levels. It was therefore evident that a combination of probiotics and local herbs has the potential to reverse the effects of obesity on the biochemical parameters and histological architecture of liver, kidney, small intestine and ovary. The overall results indicated that probiotics have positive effects of their own, but when combined with local herbs, they produced highly effective results in obese rats and therefore can be used as a complementary option in obese individuals.

A2BAR Antagonism Decreases the Glomerular Expression and Secretion of Chemoattractants for Monocytes and the Pro-Fibrotic M2 Macrophages Polarization during Diabetic Nephropathy.

Some chemoattractants and leukocytes such as M1 and M2 macrophages are known to be involved in the development of glomerulosclerosis during diabetic nephropathy (DN). In the course of diabetes, an altered and defective cellular metabolism leads to the increase in adenosine levels, and thus to changes in the polarity (M1/M2) of macrophages. MRS1754, a selective antagonist of the A2B adenosine receptor (A2BAR), attenuated glomerulosclerosis and decreased macrophage-myofibroblast transition in DN rats. Therefore, we aimed to investigate the effect of MRS1754 on the glomerular expression/secretion of chemoattractants, the intraglomerular infiltration of leukocytes, and macrophage polarity in DN rats. Kidneys/glomeruli of non-diabetic, DN, and MRS1754-treated DN rats were processed for transcriptomic analysis, immunohistopathology, ELISA, and in vitro macrophage migration assays. The transcriptomic analysis identified an upregulation of transcripts and pathways related to the immune system in the glomeruli of DN rats, which was attenuated using MRS1754. The antagonism of the A2BAR decreased glomerular expression/secretion of chemoattractants (CCL2, CCL3, CCL6, and CCL21), the infiltration of macrophages, and their polarization to M2 in DN rats. The in vitro macrophages migration induced by conditioned-medium of DN glomeruli was significantly decreased using neutralizing antibodies against CCL2, CCL3, and CCL21. We concluded that the pharmacological blockade of the A2BAR decreases the transcriptional expression of genes/pathways related to the immune response, protein expression/secretion of chemoattractants, as well as the infiltration of macrophages and their polarization toward the M2 phenotype in the glomeruli of DN rats, suggesting a new mechanism implicated in the antifibrotic effect of MRS1754.

#5018 EFFECTS OF CANDESARTAN COMPARED TO LOSARTAN ON INHIBITING MESANGIAL EXPANSION IN DIABETIC MODEL RATS RECEIVING ROSMARINIC ACID

Abstract Background and Aims Oxidative stress and activation of the renin-angiotensin system (RAS) due to chronic hyperglycemia are the pathogenesis of diabetic nephropathy (DN). Until now, therapy for DN has not shown satisfactory results. Rosmarinic acid (RA) which has antioxidant, anti-inflammatory, anti-fibrosis effects, and ARB which can inhibit RAS activation may have a synergistic effect to prevent DN progression. This study aims to determine the effect of inhibiting mesangial expansion in diabetic rat models after being given a combination of RA and ARB (candesartan or losartan). Method True experimental laboratory trial with randomized post-test only control group method on Rattus norvegicus strain Wistar rats. Diabetic rats were prepared by giving a high-fat diet (60% fat calories) and a single injection of 40 mg/kg streptozotocin. Mice were divided into 4 groups, each consisting of 4 rats: negative control (healthy rats); positive control (placebo diabetic rats); diabetic rats that received RA and candesartan; diabetic rats that received RA and losartan. The rat glomerulus was evaluated to assess mesangial expansion using ImageJ 1.48 software after 8 weeks of treatment by a pathologist blindly. Data analysis with One-way ANOVA and Tukey's post hoc tests. Results The results of the percentage of the mesangial matrix in the positive control group (81.49% ± 1.45) were higher than the negative control group (58.82% ± 0.99). The results of the percentage of the mesangial matrix in the RA + candesartan treatment group (61.56% ± 0.35) and the RA + losartan treatment group (62.28% ± 0.77) showed lower results than the positive control group, with the One-way ANOVA test results showing significant differences (p: 0.000). Tukey's post hoc tests showed the positive control group that received a high-fat diet and induced diabetes with STZ significantly increased mesangial expansion compared to negative control (MD: [22.66]; 95% CI: [20.62], [24.72]; p: 0.000). The RA + candesartan treatment group significantly inhibited mesangial expansion compared to the positive control (MD: [-19.92]; 95% CI: [-21.97], [-17.87]; p: 0.000), as well as the RA + losartan treatment group (MD: [-19.21]; 95%CI: [-21.26], [-17.16]; p: 0.000). The RA + candesartan treatment group showed a better effect in inhibiting mesangial expansion compared to the RA + losartan treatment group, but not significantly (p: 0.735). Conclusion The combination of rosmarinic acid with candesartan or losartan has the effect of inhibiting mesangial expansion in diabetic rats.

Phosphorylation of PACSIN2 at S313 Regulates Podocyte Architecture in Coordination with N-WASP.

Changes in the dynamic architecture of podocytes, the glomerular epithelial cells, lead to kidney dysfunction. Previous studies on protein kinase C and casein kinase 2 substrates in neurons 2 (PACSIN2), a known regulator of endocytosis and cytoskeletal organization, reveal a connection between PACSIN2 and kidney pathogenesis. Here, we show that the phosphorylation of PACSIN2 at serine 313 (S313) is increased in the glomeruli of rats with diabetic kidney disease. We found that phosphorylation at S313 is associated with kidney dysfunction and increased free fatty acids rather than with high glucose and diabetes alone. Phosphorylation of PACSIN2 emerged as a dynamic process that fine-tunes cell morphology and cytoskeletal arrangement, in cooperation with the regulator of the actin cytoskeleton, Neural Wiskott-Aldrich syndrome protein (N-WASP). PACSIN2 phosphorylation decreased N-WASP degradation while N-WASP inhibition triggered PACSIN2 phosphorylation at S313. Functionally, pS313-PACSIN2 regulated actin cytoskeleton rearrangement depending on the type of cell injury and the signaling pathways involved. Collectively, this study indicates that N-WASP induces phosphorylation of PACSIN2 at S313, which serves as a mechanism whereby cells regulate active actin-related processes. The dynamic phosphorylation of S313 is needed to regulate cytoskeletal reorganization.

Podocyte-specific deletion of ubiquitin carboxyl-terminal hydrolase L1 causes podocyte injury by inducing endoplasmic reticulum stress.

Ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a unique component of the ubiquitin-proteasome system (UPS), which has multiple activities in maintaining intracellular ubiquitin levels. We previously reported the aberrant low expression of UCHL1 in podocytes of non-immune complex-mediated glomerulonephritis, and recent studies indicate that anti-UCHL1 antibody was responsible for the refractory minimal change disease (MCD), but the specific effect of UCHL1 to the podocytopathy has not been determined. Therefore, we generated podocyte-specific UCHL1 gene knockout (UCHL1cre/cre) rats model. Podocyte-specific UCHL1 knockout rats exhibited severe kidney damage, including segmental/global glomerulosclerosis, kidney function damage and severe proteinuria, compared with littermate control. Subsequently, by carrying out mass spectrometry analysis of isolated glomeruli of rats, abnormal protein accumulation of ECM-receptor Interaction was found in UCHL1cre/cre rats. Mechanistic studies in vivo and in vitro revealed that aberrant protein accumulation after UCHL1 deficiency induced endoplasmic reticulum (ER) stress, unfolded protein reaction (UPR) to reduce the protein level of podocyte skeleton proteins, and CHOP mediated apoptosis as well, which related to the dysfunction of the ubiquitin-proteasome system with decreased free monomeric ubiquitin level, thereby affecting protein ubiquitination and degradation. In addition, inhibition of ER stress by 4-PBA could attenuate the degree of ER stress and podocyte dysfunction. Our study indicates that UCHL1 is a potential target for preventing podocytes injury in some non-immune complex-mediated glomerulopathy.

L-glutamine supplementation reduced morphological damage in the renal glomerulus of rats with Walker-256 tumor.

To evaluate the effects of the experimental subcutaneous Walker-256 tumor and L-glutamine supplementation, an antioxidant, on the glomerular morphology of rats. Twenty Wistar rats were distributed into four groups (n = 5): control (C); control treated with 2% L-glutamine (CG); rats with Walker-256 tumor (WT); and rats with Walker-256 tumor treated with 2% L-glutamine (WTG). Renal histological samples were submitted to periodic acid-Schiff and Masson's Trichrome staining to analyze glomerular density, morphometry of glomerular components and glomerulosclerosis; and to immunohistochemistry for fibroblast growth factor-2 (FGF-2). WT showed 50% reduction in body mass gain and cachexia index > 10%, while WTG demonstrated reduction in cachexia (p < 0.05). WT revealed reduction of glomerular density, increase in the glomerular tuft area, mesangial area, matrix in the glomerular tuft, decrease in the urinary space and synechia, and consequently higher glomerulosclerosis (p < 0.05). L-glutamine supplementation in the WTG improved glomerular density, and reduced glomerular tuft area, urinary space, mesangial area, and glomerulosclerosis compared to WT(p < 0.05). WT showed higher collagen area and FGF-2 expression compared to C (p < 0.05). WTG presented lower collagen fibers and FGF-2 expression compared to WT (p < 0.05). L-glutamine supplementation reduced cachexia and was beneficial for glomerular morphology of the rats, as well as it reduced kidney damage and improved the remaining glomeruli morphology.

Ntrk1 promotes mesangial cell proliferation and inflammation in rat glomerulonephritis model by activating the STAT3 and p38/ERK MAPK signaling pathways

BackgroundMesangial proliferative glomerulonephritis (MsPGN) accounts for a main cause of chronic kidney disease (CKD), chronic renal failure and uremia. This paper aimed to examine the effect of Ntrk1 on MsPGN development, so as to identify a novel therapeutic target for MsPGN.MethodsThe MsPGN rat model was constructed by single injection of Thy1.1 monoclonal antibody via the tail vein. Additionally, the Ntrk1 knockdown rat model was established by injection of Ntrk1-RNAi lentivirus via the tail vein. Periodic acid-schiff staining and immunohistochemistry (IHC) were performed on kidney tissues. Moreover, the rat urinary protein was detected. Mesangial cells were transfected and treated with p38 inhibitor (SB202190) and ERK inhibitor (PD98059). Meanwhile, the viability and proliferation of mesangial cells were analyzed by cell counting kit-8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine assays. Gene expression was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western-blot (WB) assays.ResultsThe proliferation of mesangial cells was enhanced in glomerulus and Ki67 expression was up-regulated in renal tubule of MsPGN rats. The urine protein level increased in MsPGN rats. Pro-inflammatory factors and Ntrk1 expression were up-regulated in glomerulus of MsPGN rats. Ntrk1 up-regulation promoted the viability, proliferation, expression of pro-inflammatory factors and activation of the STAT3, p38 and ERK signaling pathways in mesangial cells. Ntrk1 knockdown reduced mesangial cell proliferation, urine protein, pro-inflammatory factors, activation of STAT3, p38 and ERK signaling pathways in glomerulus, and decreased Ki67 expression in renal tubule of MsPGN rats. Treatment with SB202190 and PD98059 reversed the effect of Ntrk1 on promoting the viability, proliferation and inflammatory response of mesangial cells.ConclusionNtrk1 promoted mesangial cell proliferation and inflammation in MsPGN rats by activating the STAT3 and p38/ERK MAPK signaling pathways.

Effect of Co-culture of mesenchymal stem cell and glomerulus endothelial cell to promote endothelialization under optimized perfusion flow rate in whole renal ECM scaffold.

In recent era, many researches on implantable bio-artificial organs has been increased owing to large gap between donors and receivers. Comprehensive organ based researches on perfusion culture for cell injury using different flow rate have not been conducted at the cellular level. The present study investigated the co-culture of rat glomerulus endothelial cell (rGEC) and rat bone marrow mesenchymal stem cells (rBMSC) to develop micro vascularization in the kidney scaffolds culturing by bioreactor system. To obtain kidney scaffold, extracted rat kidneys were decellularized by 1% sodium dodecyl sulfate (SDS), 1% triton X-100, and distilled water. Expanded rGECs were injected through decellularized kidney scaffold artery and cultured using bioreactor system. Vascular endothelial cells adhered and proliferated on the renal ECM scaffold in the bioreactor system for 3, 7 and 14 days. Static, 1​ml/min and 2​ml/min flow rates (FR) were tested and among them, 1​ml/min flow rate was selected based on cell viability, glomerulus character, inflammation/endothelialization proteins expression level. However, the flow injury was still existed on primary cell cultured at vessel in kidney scaffold. Therefore, co-culture of rGEC​+​rBMSC found suitable to possibly solve this problem and resulted increased cell proliferation and micro-vascularization in the glomerulus, reducing inflammation and cell death which induced by flow injury. The optimized perfusion rate under rGEC​+​rBMSC co-culture conditions resulted in enhanced endocellularization to make ECM derived implantable renal scaffold and might be useful as a way of treatment of the acute renal failure.