Renal and cardiac effects of the PDE9 inhibitor BAY 73-6691 in 5/6 nephrectomized rats.

It has been suggested that the novel selective phosphodiesterase 9 (PDE9) inhibitor may improve cardiac and renal function by blocking 3',5'-cyclic guanosine monophosphate (cGMP) degradation. 5/6 nephrectomized (5/6Nx) rats were used to investigate the effects of the PDE9 inhibitor (BAY 73-6691) on the heart and kidney. Two doses of BAY 73-6691 (1 mg/kg/day and 5 mg/kg/day) were given for 95 days. The 5/6Nx rats developed albuminuria, a decrease in serum creatinine clearance (Ccr), and elevated serum troponin T levels. Echocardiographic data showed that 5/6 nephrectomy resulted in increased fractional shortening (FS), stroke volume (SV), and left ventricular ejection fraction (EF). However, 95 days of PDE9 inhibitor treatment did not improve any cardiac and renal functional parameter. Histopathologically, 5/6 nephrectomy resulted in severe kidney and heart damage, such as renal interstitial fibrosis, glomerulosclerosis, and enlarged cardiomyocytes. Telmisartan attenuated renal interstitial fibrosis and glomerulosclerosis as well as improved cardiomyocyte size. However, except for cardiomyocyte size and renal perivascular fibrosis, BAY 73-6691 had no effect on other cardiac and renal histologic parameters. Pathway enrichment analysis using RNA sequencing data of kidney and heart tissue identified chronic kidney disease pathways, such as phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, complement and coagulation cascades, and nuclear factor kappa B (NF-κB) signaling pathway. PDE9i did not affect any of these disease-related pathways. Two dosages of the PDE9 inhibitor BAY 73-6691 known to be effective in other rat models have only limited cardio-renal protective effects in 5/6 nephrectomized rats.

Baicalin - 2- ethoxyethyl ester alleviates renal fibrosis by inhibiting PI3K/AKT/NF-κB signaling pathway.

With the increasing incidence of chronic kidney disease (CKD), the development of safe and effective anti-renal fibrosis drugs is particularly urgent. Recently, Baicalin has been considered to have a renal protective effect, but its bioavailability is too low. Therefore, we synthesized baicalin-2-ethoxyethyl ester (BAE) by esterification of baicalin. We hope that this experiment will demonstrate the anti-renal fibrosis effect of BAE and explain its molecular mechanism. In this study, the chronic kidney injury model of SD rats was established by 5/6 nephrectomy, and BAE was given for 28 days. The results showed that after BAE treatment, the serum creatinine and urea nitrogen levels decreased significantly, and the pathological changes in kidneys were improved. In addition, RNA-seq analysis showed that the mechanism of BAE in relieving renal fibrosis was related to the ECM receptor, PI3K/AKT signaling pathway, and inflammatory reaction. The western blotting analysis confirmed that BAE could inhibit the expression of α-SMA, TGF-ß1, p-PI3K, p-AKT, p-IκBα, and NF-κB p65. We found that BAE can inhibit the inflammatory reaction and promote the degradation of the extracellular matrix by inhibiting the activation of the PI3K/AKT/NF-κB pathway, thus alleviating the symptoms of renal fibrosis in 5/6Nx rats, which revealed BAE was a potential compound to relieve renal fibrosis effect.

Assessing the effects of tempol on renal fibrosis, inflammation, and oxidative stress in a high-salt diet combined with 5/6 nephrectomy rat model: utilizing oxidized albumin as a biomarker.

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of chronic kidney disease (CKD), prompting the exploration of antioxidants as a potential therapeutic avenue for mitigating disease progression. This study aims to investigate the beneficial impact of Tempol on the progression of CKD in a rat model utilizing oxidized albumin as a biomarker. METHODS: After four weeks of treatment, metabolic parameters, including body weight, left ventricle residual weight, kidney weight, urine volume, and water and food intake, were measured. Systolic blood pressure, urinary protein, oxidized albumin level, serum creatinine (Scr), blood urea nitrogen (BUN), 8-OHdG, TGF-ß1, and micro-albumin were also assessed. Renal fibrosis was evaluated through histological and biochemical assays. P65-NF-κB was quantified using an immunofluorescence test, while Smad3, P65-NF-κB, and Collagen I were measured using western blot. TNF-α, IL-6, MCP-1, TGF-ß1, Smad3, and P65-NF-κB were analyzed by RT-qPCR. RESULTS: Rats in the high-salt diet group exhibited impaired renal function, characterized by elevated levels of blood urea nitrogen, serum creatinine, 8-OHdG, urine albumin, and tubulointerstitial damage, along with reduced body weight. However, these effects were significantly ameliorated by Tempol administration. In the high-salt diet group, blood pressure, urinary protein, and oxidized albumin levels were notably higher compared to the normal diet group, but Tempol administration in the treatment group reversed these effects. Rats in the high-salt diet group also displayed increased levels of proinflammatory factors (TNF-α, IL-6, MCP1) and profibrotic factors (NF-κB activation, Collagen I), elevated expression of NADPH oxidation-related subunits (P65), and activation of the TGF-ß1/Smad3 signaling pathway. Tempol treatment inhibited NF-κB-mediated inflammation and TGF-ß1/Smad3-induced renal fibrosis signaling pathway activation. CONCLUSION: These findings suggest that Tempol may hold therapeutic potential for preventing and treating rats undergoing 5/6 nephrectomy. Further research is warranted to elucidate the mechanisms underlying Tempol's protective effects and its potential clinical applications. Besides, there is a discernible positive relationship between oxidized albumin and other biomarkers, such as 8-OHG, urinary protein levels, mALB, Scr, BUN, and TGF-ß1 in a High-salt diet combined with 5/6 nephrectomy rat model. These findings suggest the potential utility of oxidized albumin as a sensitive indicator for oxidative stress assessment.

C-Phycoerythrin Prevents Chronic Kidney Disease-Induced Systemic Arterial Hypertension, Avoiding Oxidative Stress and Vascular Dysfunction in Remanent Functional Kidney.

Chronic kidney disease (CKD) is a burden in low- and middle-income countries, and a late diagnosis with systemic arterial hypertension (SAH) is the major complication of CKD. C-phycoerythrin (CPE) is a bioactive compound derived from Phormidium persicinum that presents anti-inflammatory and antioxidant effects in vitro and nephroprotective effects in vivo. In the current study, we determine the antihypertensive effect of CPE in a 5/6 nephrectomy-induced CKD model using twenty normotensives male Wistar rats, grouped into four groups (n = 5): sham; sham + CPE; 5/6 nephrectomy (NFx); and NFx + CPE. Treatment started a week post-surgery and continued for five weeks, with weekly hemodynamic evaluations. Following treatment, renal function, oxidative stress, and the expression of vascular dysfunction markers were assessed. The renal function analysis revealed CKD hyperfiltration, and the hemodynamic evaluation showed that SAH developed at the third week. AT1R upregulation and AT2R downregulation together with Mas1/p-Akt/p-eNOS axis were also observed. CPE treatment mitigated renal damage, preserved renal function, and prevented SAH with the modulation of the vasodilative AT1R, AT2R, and Mas1/pAKT/peNOS axis. This result reveals that CPE prevented CKD progression to SAH by avoiding oxidative stress and vascular dysfunction in the kidneys.

Comparison of the surgical resection and infarct 5/6 nephrectomy rat models of chronic kidney disease.

The 5/6 nephrectomy rat remnant kidney model is commonly used to study chronic kidney disease (CKD). This model requires the removal of one whole kidney and two-thirds of the other kidney. The two most common ways of producing the remnant kidney are surgical resection of poles, known as the polectomy model, or ligation of superior and inferior segmental renal arteries, resulting in pole infarction. These models have much in common, but also major phenotypic differences, and thus respectively model unique aspects of human CKD. The purpose of this review is to summarize phenotypic similarities and differences between these two models and their relation to human CKD while emphasizing their vascular phenotype. In this article, we review studies that have evaluated arterial blood pressure, the renin-angiotensin-aldosterone-system, autoregulation, nitric oxide, single-nephron physiology, angiogenic and antiangiogenic factors, and capillary rarefaction in these two models. In terms of phenotypic similarities, both models spontaneously develop hallmarks of human CKD including uremia, fibrosis, capillary rarefaction, and progressive renal function decline. They both undergo whole organ hypertrophy, hyperfiltration of functional nephrons, reduced renal expression of vascular endothelial growth factor, increased renal expression of antiangiogenic thrombospondin-1, impaired renal autoregulation, and abnormal vascular nitric oxide physiology. In terms of key phenotypic differences, the infarction model develops rapid-onset, moderate to severe systemic hypertension and the polectomy model develops early normotension followed by mild to moderate hypertension. Rats subjected to the infarction model have a markedly more active renin-angiotensin-aldosterone system. Comparison of these two models facilitates understanding of how they can be used for studying CKD pathophysiology.

Altered vitamin K biodistribution and metabolism in experimental and human chronic kidney disease.

Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low-density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy.

Lactobacillus rhamnosus L34 attenuates chronic kidney disease progression in a 5/6 nephrectomy mouse model through the excretion of anti-inflammatory molecules.

BACKGROUND: Although pathogenic gut microbiota causes gut leakage, increases translocation of uremic toxins into circulation and accelerates CKD progression, the local strain of Lactobacillus rhamnosus L34 might attenuate gut leakage. We explored the effects of L34 on kidney fibrosis and levels of gut-derived uremic toxins (GDUTs) in 5/6 nephrectomy (5/6Nx) mice. METHODS: At 6 weeks post-5/6Nx in mice, either L34 (1 × 106 CFU) or phosphate buffer solution (as 5/6Nx control) was fed daily for 14 weeks. In vitro, the effects of L34-conditioned media with or without indoxyl sulfate (a representative GDUT) on inflammation and cell integrity (transepithelial electrical resistance; TEER) were assessed in Caco-2 (enterocytes). In parallel, the effects on proinflammatory cytokines and collagen expression were assessed in HK2 proximal tubular cells. RESULTS: At 20 weeks post-5/6Nx, L34-treated mice showed significantly fewer renal injuries, as evaluated by (i) kidney fibrosis area (P < 0.01) with lower serum creatinine and proteinuria, (ii) GDUT including trimethylamine-N-oxide (TMAO) (P = 0.02) and indoxyl sulfate (P < 0.01) and (iii) endotoxin (P = 0.03) and serum TNF-α (P = 0.01) than 5/6Nx controls. Fecal microbiome analysis revealed an increased proportion of Bacteroidetes in 5/6Nx controls. After incubation with indoxyl sulfate, Caco-2 enterocytes had higher interleukin-8 and nuclear factor κB expression and lower TEER values, and HK2 cells demonstrated higher gene expression of TNF-α, IL-6 and collagen (types III and IV). These indoxyl sulfate-activated parameters were attenuated with L34-conditioned media, indicating the protective role of L34 in enterocyte integrity and renal fibrogenesis. CONCLUSION: L34 attenuated uremia-induced systemic inflammation by reducing GDUTs and gut leakage that provided renoprotective effects in CKD.

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

BACKGROUND: The 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. METHODS: 5/6 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. RESULTS: 5/6 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. CONCLUSION: Our 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.

Regulation of SARS CoV-2 host factors in the kidney and heart in rats with 5/6 nephrectomy-effects of salt, ARB, DPP4 inhibitor and SGLT2 blocker.

BACKGROUND: Host factors such as angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine-subtype-2 (TMPRSS2) are important factors for SARS-CoV-2 infection. Clinical and pre-clinical studies demonstrated that RAAS-blocking agents can be safely used during a SARS-CoV-2 infection but it is unknown if DPP-4 inhibitors or SGLT2-blockers may promote COVID-19 by increasing the host viral entry enzymes ACE2 and TMPRSS2. METHODS: We investigated telmisartan, linagliptin and empagliflozin induced effects on renal and cardiac expression of ACE2, TMPRSS2 and key enzymes involved in RAAS (REN, AGTR2, AGT) under high-salt conditions in a non-diabetic experimental 5/6 nephrectomy (5/6 Nx) model. In the present study, the gene expression of Ace2, Tmprss2, Ren, Agtr2 and Agt was assessed with qRT-PCR and the protein expression of ACE2 and TMPRSS2 with immunohistochemistry in the following experimental groups: Sham + normal diet (ND) + placebo (PBO); 5/6Nx + ND + PBO; 5/6Nx + high salt-diet (HSD) + PBO; 5/6Nx + HSD + telmisartan; 5/6Nx + HSD + linagliptin; 5/6Nx + HSD + empagliflozin. RESULTS: In the kidney, the expression of Ace2 was not altered on mRNA level under disease and treatment conditions. The renal TMPRSS2 levels (mRNA and protein) were not affected, whereas the cardiac level was significantly increased in 5/6Nx rats. Intriguingly, the elevated TMPRSS2 protein expression in the heart was significantly normalized after treatment with telmisartan, linagliptin and empagliflozin. CONCLUSIONS: Our study indicated that there is no upregulation regarding host factors potentially promoting SARS-CoV-2 virus entry into host cells when the SGLT2-blocker empagliflozin, telmisartan and the DPP4-inhibitor blocker linagliptin are used. The results obtained in a preclinical, experimental non-diabetic kidney failure model need confirmation in ongoing interventional clinical trials.

Uremia-Induced Gut Barrier Defect in 5/6 Nephrectomized Mice Is Worsened by Candida Administration through a Synergy of Uremic Toxin, Lipopolysaccharide, and (1➔3)-ß-D-Glucan, but Is Attenuated by Lacticaseibacillus rhamnosus L34.

A chronic kidney disease (CKD) causes uremic toxin accumulation and gut dysbiosis, which further induces gut leakage and worsening CKD. Lipopolysaccharide (LPS) of Gram-negative bacteria and (1➔3)-ß-D-glucan (BG) of fungi are the two most abundant gut microbial molecules. Due to limited data on the impact of intestinal fungi in CKD mouse models, the influences of gut fungi and Lacticaseibacillus rhamnosus L34 (L34) on CKD were investigated using oral C. albicans-administered 5/6 nephrectomy (5/6Nx) mice. At 16 weeks post-5/6Nx, Candida-5/6Nx mice demonstrated an increase in proteinuria, serum BG, serum cytokines (tumor necrotic factor-α; TNF-α and interleukin-6), alanine transaminase (ALT), and level of fecal dysbiosis (Proteobacteria on fecal microbiome) when compared to non-Candida-5/6Nx. However, serum creatinine, renal fibrosis, or gut barrier defect (FITC-dextran assay and endotoxemia) remained comparable between Candida- versus non-Candida-5/6Nx. The probiotics L34 attenuated several parameters in Candida-5/6Nx mice, including fecal dysbiosis (Proteobacteria and Bacteroides), gut leakage (fluorescein isothiocyanate (FITC)-dextran), gut-derived uremic toxin (trimethylamine-N-oxide; TMAO) and indoxyl sulfate; IS), cytokines, and ALT. In vitro, IS combined with LPS with or without BG enhanced the injury on Caco-2 enterocytes (transepithelial electrical resistance and FITC-dextran permeability) and bone marrow-derived macrophages (supernatant cytokines (TNF-α and interleukin-1 ß; IL-1ß) and inflammatory genes (TNF-α, IL-1ß, aryl hydrocarbon receptor, and nuclear factor-κB)), compared with non-IS activation. These injuries were attenuated by the probiotics condition media. In conclusion, Candida administration worsens kidney damage in 5/6Nx mice through systemic inflammation, partly from gut dysbiosis-induced uremic toxins, which were attenuated by the probiotics. The additive effects on cell injury from uremic toxin (IS) and microbial molecules (LPS and BG) on enterocytes and macrophages might be an important underlying mechanism.

Positron emission tomography imaging of renal mitochondria is a powerful tool in the study of acute and progressive kidney disease models.

Mitochondrial dysfunction plays a critical role in the pathogenesis of kidney diseases via ATP depletion and reactive oxygen species overproduction. Nonetheless, few studies have reported the renal mitochondrial status clinical settings, partly due to a paucity of methodologies. Recently, a positron emission tomography probe, 18F-BCPP-BF, was developed to non-invasively visualize and quantitate the renal mitochondrial status in vivo. Here, 18F-BCPP-BF positron emission tomography was applied to three mechanistic kidney disease models in rats: kidney ischemia-reperfusion, 5/6 nephrectomy and anti-glomerular basement membrane glomerulonephritis. In rats with ischemia-reperfusion, a slight decrease in the kidney uptake of 18F-BCPP-BF was accompanied by morphological abnormality of the mitochondria in the proximal tubular cells after three hours of reperfusion, when the kidney function was slightly declined. In 5/6 nephrectomy and rats with anti-glomerular basement membrane glomerulonephritis, the kidney uptake of 18F-BCPP-BF cumulatively decreased with impairment of the kidney function, which was accompanied by a reduction of mitochondrial protein and a pathological tubulointerstitial exacerbation rather than glomerular injury. The 18F-BCPP-BF uptake in the injured kidney was suggested to represent the volume of healthy tubular epithelial cells with normally functioning mitochondria. Thus, this positron emission tomography probe can be a powerful tool for studying the pathophysiological meanings of the mitochondrial status in kidney disease.

CSE/H2S system alleviates uremic accelerated atherosclerosis by regulating TGF-ß/Smad3 pathway in 5/6 nephrectomy ApoE-/- mice.

BACKGROUND: Hydrogen sulfide (H2S) has been shown to inhibit the atherosclerosis development and progression. It is produced by cystathionine γ-lyase (CSE) in the cardiovascular system. In our previous study, it has been shown that CSE/H2S system plays a significant role in the changes of uremic accelerated atherosclerosis (UAAS), but the mechanism is not known clearly. METHODS: In this study, we explored the antagonism of CSE/H2S system in UAAS and identified its possible signaling molecules in ApoE-/- mice with 5/6 nephrectomy and fed with atherogenic diet. Mice were divided into sham operation group (sham group), UAAS group, sodium hydrosulfide group (UAAS+NaHS group) and propargylglycine group (UAAS+PPG group). Serum creatinine, urea nitrogen, lipid levels and lesion size of atherosclerotic plaque in the aortic roots were analyzed. Meanwhile, the expression of CSE, TGF-ß and phosphorylation of Smad3 were detected. RESULTS: Compared with sham group, the aortic root of ApoE-/- mice in the UAAS group developed early atherosclerosis, the levels of total cholesterol, triglyceride, low-density lipoprotein-cholesterol, serum creatinine and urea nitrogen were also higher than that in the sham group. NaHS administration can inhibit the development of atherosclerosis, but PPG administration can accelerate the atherosclerosis development. Meanwhile, the protein expression levels of CSE and TGF-ß and phosphorylation of Smad3 significantly decreased in the UAAS mice. Treatment of UAAS mice with NaHS inhibited TGF-ß protein expression and Smad3 phosphorylation decrease, but PPG treatment had the opposite effect. CONCLUSIONS: The CSE/H2S system is of great importance for treating atherosclerosis in patients with chronic kidney disease, and it may protect the vascular from atherosclerosis through the TGF-ß/Smad pathway.

Temporal Alterations in Mitochondrial ß-Oxidation and Oxidative Stress Aggravate Chronic Kidney Disease Development in 5/6 Nephrectomy Induced Renal Damage.

Five-sixths nephrectomy (5/6Nx) model is widely used for studying the mechanisms involved in chronic kidney disease (CKD) progression, a kidney pathology that has increased dramatically in recent years. Mitochondrial impairment is a key mechanism that aggravates CKD progression; however, the information on mitochondrial bioenergetics and redox alterations along a time course in a 5/6Nx model is still limited and in some cases contradictory. Therefore, we performed for the first time a time-course study of mitochondrial alterations by high-resolution respirometry in the 5/6Nx model. Our results show a decrease in mitochondrial ß-oxidation at early times, as well as a permanent impairment in adenosine triphosphate (ATP) production in CI-linked respiration, a permanent oxidative state in mitochondria and decoupling of these organelles. These pathological alterations are linked to the early decrease in complex I and ATP synthase activities and to the further decrease in complex III activity. Therefore, our results may suggest that mitochondrial bioenergetics impairment is an early event in renal damage, whose persistence in time aggravates CKD development in the 5/6Nx model.

Tissue, urine and blood metabolite signatures of chronic kidney disease in the 5/6 nephrectomy rat model.

INTRODUCTION: Progressive chronic kidney disease (CKD) is an important cause of morbidity and mortality. It has a long asymptomatic phase, where routine blood tests cannot identify early functional losses, and therefore identifying common mechanisms across the many etiologies is an important goal. OBJECTIVES: Our aim was to characterize serum, urine and tissue (kidney, lung, heart, spleen and liver) metabolomics changes in a rat model of CKD. METHODS: A total of 17 male Wistar rats underwent 5/6 nephrectomy, whilst 13 rats underwent sham operation. Urine samples were collected weekly, for 6 weeks; blood was collected at weeks 0, 3 and 6; and tissue samples were collected at week 6. Samples were analyzed on a nuclear magnetic resonance spectroscopy platform with multivariate and univariate data analysis. RESULTS: Changes in several metabolites were statistically significant. Allantoin was affected in all compartments. Renal asparagine, creatine, hippurate and trimethylamine were significantly different; in other tissues creatine, dimethylamine, dimethylglycine, trigonelline and trimethylamine were significant. Benzoate, citrate, dimethylglycine, fumarate, guanidinoacetate, malate, myo-inositol and oxoglutarate were altered in urine or serum. CONCLUSION: Although the metabolic picture is complex, we suggest oxidative stress, the gut-kidney axis, acid-base balance, and energy metabolism as promising areas for future investigation.

Dietary methionine restriction modulates renal response and attenuates kidney injury in mice.

Methionine restriction (MR) extends the lifespan across several species, such as rodents, fruit flies, roundworms, and yeast. MR studies have been conducted on various rodent organs, such as liver, adipose tissue, heart, bones, and skeletal muscle, to elucidate its benefits to the healthspan; however, studies of the direct effect of MR on kidneys are lacking. To investigate the renal effects of MR, we used young and aged unilateral nephrectomized and 5/6 nephrectomized (5/6Nx) mice. Our studies indicated that MR mice experienced polydipsia and polyuria compared with control-fed counterparts. Urine albumin, creatinine, albumin-to-creatinine ratio, sulfur amino acids, and electrolytes were reduced in MR mice. Kidneys of MR mice up-regulated genes that are involved in ion transport, such as Aqp2, Scnn1a, and Slc6a19, which indicated a response to maintain osmotic balance. In addition, we identified renoprotective biomarkers that are affected by MR, such as clusterin and cystatin C. Of importance, MR attenuated kidney injury in 5/6Nx mice by down-regulating inflammation and fibrosis mechanisms. Thus, our studies in mice show the important role of kidneys during MR in maintaining osmotic homeostasis. Moreover, our studies also show that the MR diet delays the progression of kidney disease.-Cooke, D., Ouattara, A., Ables, G. P. Dietary methionine restriction modulates renal response and attenuates kidney injury in mice.

Impact of chronic kidney dysfunction on serum Sulfatides and its metabolic pathway in mice.

Serum sulfatides are critical glycosphingolipids that are present in lipoproteins and exert anticoagulant effects. A previous study reported decreased levels of serum sulfatides in hemodialysis patients and suggested an association with cardiovascular disease. However, the mechanism of changes in serum sulfatides in chronic kidney dysfunction has not been well investigated. The current study examined whether a chronic kidney disease (CKD) state could decrease serum sulfatide levels using 5/6 nephrectomy (5/6NCKD) mice, an established CKD murine model, and studied the mechanisms contributing to diminished sulfatides. 5/6NCKD mice and sham operation control mice were sacrificed at the 4th or 12th postoperative week (POW) for measurement of serum sulfatide levels. Hepatic sulfatide content, which is the origin of serum sulfatides, and the expression of sulfatide metabolic enzymes in liver tissue were assessed as well. The 5/6NCKD mice developed CKD and showed increased serum creatinine and indoxyl sulfate. The serum levels and hepatic amounts of sulfatides were significantly decreased in 5/6NCKD mice at both 4 and 12 POW, while the degradative enzymes of sulfatides arylsulfatase A and galactosylceramidase were significantly increased. In a Hepa1-6 murine liver cell line, indoxyl sulfate addition caused intracellular levels of sulfatides to decrease and degradative enzymes of sulfatides to increase in a manner comparable to the changes in 5/6NCKD mice liver tissue. In conclusion, chronic kidney dysfunction causes degradation of sulfatides in the liver to decrease serum sulfatide levels. One explanation of these results is that indoxyl sulfate, a uremic toxin, accelerates the degradation of sulfatides in liver tissue.

An optimized 5/6 nephrectomy mouse model based on unilateral kidney ligation and its application in renal fibrosis research.

5/6 Nephrectomy (PNx) on rat and mouse mimics renal failure after loss of kidney function in human, and it has been widely used in CKD researches. However, existing methods for PNx model construction present high mortality of animals after modeling due to hemorrhage and infection in or after surgery. Here, we report a novel and highly efficient PNx modeling method to simulate conventional 5/6 nephrectomy, which significantly reduced the mortality of animals and simplified the modeling procedures. In this novel modeling method, we directly ligated the upper and lower poles of left kidney after removal the right kidney 1 week later (l-PNx), which leads to necrosis of ligated upper and lower poles of the kidney and mimics the conventional 5/6 nephrectomy (c-PNx). After modeling 4 and 12 weeks, the serum creatinine, BUN and proteinuria levels were strongly increased in both c-PNx and l-PNx model. Importantly, compared with the c-PNx, l-PNx model present more severe renal fibrosis estimated by Masson staining, IHC and western blotting. The results showed that the protein levels of α-SMA were significantly increased in the kidney of c-PNx and l-PNx models, but more increase was found in l-PNx model. It is noteworthy that, compared with c-PNx model, the survival rate of l-PNx model was markedly increased. In summary, we established a novel and efficient 5/6 nephrectomy model, which can mimic conventional 5/6 nephrectomy to construct a renal fibrosis and renal failure mouse model, that is conducive to mechanism and treatment researches of CKD.

Diet-induced hyperhomocysteinemia impairs vasodilation in 5/6-nephrectomized rats.

Plasma homocysteine is elevated in patients with impaired renal function, and markedly so at end-stage renal disease. As chronic kidney disease and hyperhomocysteinemia are also independent risk factors for cardiovascular disease, the latter is hypothesized to accelerate vascular abnormalities following renal failure. This study aimed to investigate the combined effect of impaired renal function and hyperhomocysteinemia on vascular function. We show that in 5/6-nephrectomized rats, a model of chronic kidney disease, a methionine-rich diet for 8 weeks induces moderate hyperhomocysteinemia, exacerbates hypertension, and attenuates the vascular response to acetylcholine, sodium nitroprusside, 8-bromo-cGMP, and isoprenaline. However, plasma nitrate/nitrite and total NOS activity in the thoracic aorta were not affected. Collectively, the data imply that hyperhomocysteinemia and end-stage renal disease synergistically impair endothelium-dependent and endothelium-independent vasodilatation by blocking the cGMP/protein kinase G and/or cAMP/protein kinase A pathways. 5/6-Nephrectomized rat with hyperhomocysteinemia induced by a methionine-rich diet would be a useful model for elucidating the pathogenesis of vascular impairment in patients with end-stage renal disease.

Metabonomic Profiling Reveals Difference in Altered Metabolic Pathways Between Chronic Kidney Disease and High-Fat-Induced Insulin Resistance in Rats.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is closely correlated with the development of insulin resistance (IR). Until now, the underlying molecular mechanisms remain to be elucidated. This study aimed to identify metabolites and molecular pathways unique to CKD-induced IR. METHODS: Ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS) analysis coupled with orthogonal partial least square discriminant analysis (OPLS-DA) were performed to profile metabolites in the serum, liver, and muscle tissues and to analyze molecular pathways in relation to CKD- and high fat diet (HFD)-induced IR in the rats. RESULTS: At 18 weeks after the 5/6 Nx operation, CKD induction was demonstrated by renal histology and biochemical tests. Furthermore, both CKD-induced IR and HFD-induced IR rats showed significantly greater levels of fasting insulin and homeostasis model assessment of insulin resistance (HOMA-IR). In the UPLC-MS in combination with OPLS-DA analysis, we identified 101, 59, and 41 differential metabolites in the serum, liver, and muscle, which were associated with the CKD-induced IR, while 58, 38, and 17 differential metabolites in the serum, liver, and muscle were revealed in the HFD-induced rats compared to controls. Moreover, compared to HFD-induced IR rats, those with CKD-induced IR exhibited abnormal pathways primarily in the tryptophan metabolism, arginine metabolism, and trimethylamine oxide metabolite. Interestingly, altered metabolites in the CKD-induced IR and HFD-induced IR displayed an opposite direction. CONCLUSION: Alterations in metabolites and relevant pathways were significantly different between the CKD- and HFD-induced IR rats. These findings may offer important information regarding the pathogenesis specific to IR caused by the decline in the renal function.

Combined Inhibition of Soluble Epoxide Hydrolase and Renin-Angiotensin System Exhibits Superior Renoprotection to Renin-Angiotensin System Blockade in 5/6 Nephrectomized Ren-2 Transgenic Hypertensive Rats with Established Chronic Kidney Disease.

BACKGROUND/AIMS: We found recently that increasing renal epoxyeicosatrienoic acids (EETs) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, shows renoprotective actions and retards the progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). This prompted us to examine if additional protection is provided when sEH inhibitor is added to the standard renin-angiotensin system (RAS) blockade, specifically in rats with established CKD. METHODS: For RAS blockade, an angiotensin-converting enzyme inhibitor along with an angiotensin II type receptor blocker was used. RAS blockade was compared to sEH inhibition added to the RAS blockade. Treatments were initiated 6 weeks after 5/6 NX in TGR and the follow-up period was 60 weeks. RESULTS: Combined RAS and sEH blockade exhibited additional positive impact on the rat survival rate, further reduced albuminuria, further reduced glomerular and tubulointerstitial injury, and attenuated the decline in creatinine clearance when compared to 5/6 NX TGR subjected to RAS blockade alone. These additional beneficial actions were associated with normalization of the intrarenal EETs deficient and a further reduction of urinary angiotensinogen excretion. CONCLUSION: This study provides evidence that addition of pharmacological inhibition of sEH to RAS blockade in 5/6 NX TGR enhances renoprotection and retards progression of CKD, notably, when applied at an advanced stage.