Activation of Notch3 in Renal Tubular Cells Leads to Progressive Cystic Kidney Disease

Background: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. Methods: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. Results: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. Conclusions: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions.     

Redefining the Role of ADAM17 in Renal Proximal Tubular Cells and Its Implications in an Obese Mouse Model of Pre-Diabetes

Acute and chronic kidney lesions induce an increase in A Disintegrin And Metalloproteinase domain 17 (ADAM17) that cleaves several transmembrane proteins related to inflammatory and fibrotic pathways. Our group has demonstrated that renal ADAM17 is upregulated in diabetic mice and its inhibition decreases renal inflammation and fibrosis. The purpose of the present study was to analyze how Adam17 deletion in proximal tubules affects different renal structures in an obese mice model. Tubular Adam17 knockout male mice and their controls were fed a high-fat diet (HFD) for 22 weeks. Glucose tolerance, urinary albumin-to-creatinine ratio, renal histology, and pro-inflammatory and pro-fibrotic markers were evaluated. Results showed that wild-type mice fed an HFD became obese with glucose intolerance and renal histological alterations mimicking a pre-diabetic condition; consequently, greater glomerular size and mesangial expansion were observed. Adam17 tubular deletion improved glucose tolerance and protected animals against glomerular injury and prevented podocyte loss in HFD mice. In addition, HFD mice showed more glomerular macrophages and collagen accumulation, which was prevented by Adam17 deletion. Galectin-3 expression increased in the proximal tubules and glomeruli of HFD mice and ameliorated with Adam17 deletion. In conclusion, Adam17 in proximal tubules influences glucose tolerance and participates in the kidney injury in an obese pre-diabetic murine model. The role of ADAM17 in the tubule impacts on glomerular inflammation and fibrosis.     

Sirtuin 7 Deficiency Reduces Inflammation and Tubular Damage Induced by an Episode of Acute Kidney Injury

Acute kidney injury (AKI) is a public health problem worldwide. Sirtuins are a family of seven NAD+-dependent deacylases, Overexpression of Sirtuin 1, 3, and 5 protect against AKI. However, the role of Sirtuin 7 (Sirt7) in AKI is not known. Here, we analyzed how Sirt7 deficient mice (KO-Sirt7) were affected by AKI. As expected, wild-type and Sirt7 heterozygotes mice that underwent renal ischemia/reperfusion (IR) exhibited the characteristic hallmarks of AKI: renal dysfunction, tubular damage, albuminuria, increased oxidative stress, and renal inflammation. In contrast, the KO-Sirt7+IR mice were protected from AKI, exhibiting lesser albuminuria and reduction in urinary biomarkers of tubular damage, despite similar renal dysfunction. The renoprotection in the Sirt7-KO+IR group was associated with reduced kidney weight, minor expression of inflammatory cytokines and less renal infiltration of inflammatory cells. This anti-inflammatory effect was related to diminished p65 expression and in its active phosphorylation, as well as by a reduction in p65 nuclear translocation. Sirt7 deficient mice are protected from AKI, suggesting that this histone deacetylase promotes tubular damage and renal inflammation. Therefore, our findings indicate that Sirt7 inhibitors may be an attractive therapeutic target to reduce NFκB signaling.     

Both Specific Endothelial and Proximal Tubular Adam17 Deletion Protect against Diabetic Nephropathy

ADAM17 is a disintegrin and metalloproteinase capable of cleaving the ectodomains of a diverse variety of molecules including TNF-α, TGF-α, L-selectin, and ACE2. We have previously demonstrated that renal ADAM17 is upregulated in diabetic mice. The role of endothelial (eAdam17) and proximal tubular (tAdam17) Adam17 deletion in renal histology, modulation of the renin angiotensin system (RAS), renal inflammation, and fibrosis was studied in a mouse model of type 1 Diabetes Mellitus. Moreover, the effect of Adam17 deletion in an in vitro 3D cell culture from human proximal tubular cells under high glucose conditions was evaluated. eAdam17 deletion attenuates renal fibrosis and inflammation, whereas tAdam17 deletion decreases podocyte loss, attenuates the RAS, and decreases macrophage infiltration, α-SMA and collagen accumulation. The 3D in vitro cell culture reinforced the findings obtained in tAdam17KO mice with decreased fibrosis in the Adam17 knockout spheroids. In conclusion, Adam17 deletion either in the endothelial or the tubular cells mitigates kidney injury in the diabetic mice by targeting different pathways. The manipulation of Adam17 should be considered as a therapeutic strategy for treating DN.     

Tubular IKKβ Deletion Alleviates Acute Ischemic Kidney Injury and Facilitates Tissue Regeneration

Acute kidney injury (AKI) is a common renal injury leading to relevant morbidity and mortality worldwide. Most of the clinical cases of AKI are caused by ischemia reperfusion (I/R) injury with renal ischemia injury followed by reperfusion injury and activation of the innate immune response converging to NF-ĸB pathway induction. Despite the clear role of NF-ĸB in inflammation, it has recently been acknowledged that NF-ĸB may impact other cell functions. To identify NF-ĸB function with respect to metabolism, vascular function and oxidative stress after I/R injury and to decipher in detail the underlying mechanism, we generated a transgenic mouse model with targeted deletion of IKKβ along the tubule and applied I/R injury followed by its analysis after 2 and 14 days after I/R injury. Tubular IKKβ deletion ameliorated renal function and reduced tissue damage. RNAseq data together with immunohistochemical, biochemical and morphometric analysis demonstrated an ameliorated vascular organization and mRNA expression profile for increased angiogenesis in mice with tubular IKKβ deletion at 2 days after I/R injury. RNAseq and protein analysis indicate an ameliorated metabolism, oxidative species handling and timely-adapted cell proliferation and apoptosis as well as reduced fibrosis in mice with tubular IKKβ deletion at 14 days after I/R injury. In conclusion, mice with tubular IKKβ deletion upon I/R injury display improved renal function and reduced tissue damage and fibrosis in association with improved vascularization, metabolism, reactive species disposal and fine-tuned cell proliferation.     

Scattered Tubular Cells Markers in Macula Densa of Normal Human Adult Kidney

Background: The scattered tubular cells (STCs) are a population of resident progenitor tubular cells with expansion, self-renewal and epithelial differentiation abilities. Although these cells are localized within the proximal (PTs) and distal (DTs) tubules in a normal adult kidney, their presence has never been demonstrated in human macula densa (MD). The purpose of the present study is to describe the presence of STCs in MD using specific markers such as prominin-1 (CD133), cytokeratin 7 (KRT7) and vimentin (VIM). Methods: We analyzed two sets of three consecutive serial sections for each sample. The first sections of each set were immunostained for nNOS to identify MD, the second sections were immune-stained for CD133 (specific STCs marker) while the third sections were analyzed for KRT7 (another STCs specific marker) and VIM (that stains the basal pole of the STCs) in the first and second sets, respectively, in order to study the co-expression of KRT7 and VIM with the CD133 marker. Results: CD133 was localized in some MD cells and in the adjacent DT cells. Moreover, CD133 was detected in the parietal epithelial cells of Bowman’s capsule and in some proximal tubules (PT). KRT7-positive cells were identified in MD and adjacent DT cells, while KRT7 positivity was mostly confined in both DT and collecting ducts (CD) in the other areas of the renal parenchyma. CD133 and KRT7 were co-expressed in some MD and adjacent DT cells. Some of the latter cells were positive both for CD133 and VIM. CD133 was always localized in the apical part of the cells, whereas the VIM expression was evident only in the cellular basal pole. Although some cells of MD expressed VIM or CD133, none of them co-expressed VIM and CD133. Conclusions: The presence of STCs was demonstrated in human adult MD, suggesting that this structure has expansion, self-renewal and epithelial differentiation abilities, similar to all other parts of renal tubules.     

Rhabdomyolysis-Induced AKI Was Ameliorated in NLRP3 KO Mice via Alleviation of Mitochondrial Lipid Peroxidation in Renal Tubular Cells

Introduction: A recent study showed that early renal tubular injury is ameliorated in Nod-like receptor pyrin domain-containing protein 3 (NLRP3) KO mice with rhabdomyolysis-induced acute kidney injury (RIAKI). However, the precise mechanism has not been determined. Therefore, we investigated the role of NLRP3 in renal tubular cells in RIAKI. Methods: Glycerol-mediated RIAKI was induced in NLRP3 KO and wild-type (WT) mice. The mice were euthanized 24 h after glycerol injection, and both kidneys and plasma were collected. HKC-8 cells were treated with ferrous myoglobin to mimic a rhabdomyolytic environment. Results: Glycerol injection led to increase serum creatinine, aspartate aminotransferase (AST), and renal kidney injury molecule-1 (KIM-1) level; renal tubular necrosis; and apoptosis. Renal injury was attenuated in NLRP3 KO mice, while muscle damage and renal neutrophil recruitment did not differ between NLRP3 KO mice and WT mice. Following glycerin injection, increases in cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), and a decrease in the glutathione peroxidase 4 (GPX-4) level were observed in the kidneys of mice with RIAKI, and these changes were alleviated in the kidneys of NLRP3 KO mice. NLRP3 was upregulated, and cell viability was suppressed in HKC-8 cells treated with ferrous myoglobin. Myoglobin-induced apoptosis and lipid peroxidation were significantly decreased in siNLRP3-treated HKC-8 cells compared to ferrous myoglobin-treated HKC-8 cells. Myoglobin reduced the mitochondrial membrane potential and increased mitochondrial fission and reactive oxygen species (ROS) and lipid peroxidation levels, which were restored to normal levels in NLRP3-depleted HKC-8 cells. Conclusions: NLRP3 depletion ameliorated renal tubular injury in a murine glycerol-induced acute kidney injury (AKI) model. A lack of NLRP3 improved tubular cell viability via attenuation of myoglobin-induced mitochondrial injury and lipid peroxidation, which might be the critical factor in protecting the kidney.     

Sphingosine 1-Phosphate Receptor 5 (S1P5) Knockout Ameliorates Adenine-Induced Nephropathy

S1P and its receptors have been reported to play important roles in the development of renal fibrosis. Although S1P₅ has barely been investigated so far, there are indications that it can influence inflammatory and fibrotic processes. Here, we report the role of S1P₅ in renal inflammation and fibrosis. Male S1P₅ knockout mice and wild-type mice on a C57BL/6J background were fed with an adenine-rich diet for 7 days or 14 days to induce tubulointerstitial fibrosis. The kidneys of untreated mice served as respective controls. Kidney damage, fibrosis, and inflammation in kidney tissues were analyzed by real-time PCR, Western blot, and histological staining. Renal function was assessed by plasma creatinine ELISA. The S1P₅ knockout mice had better renal function and showed less kidney damage, less proinflammatory cytokine release, and less fibrosis after 7 days and 14 days of an adenine-rich diet compared to wild-type mice. S1P₅ knockout ameliorates tubular damage and tubulointerstitial fibrosis in a model of adenine-induced nephropathy in mice. Thus, targeting S1P₅ might be a promising goal for the pharmacological treatment of kidney diseases.     

Effects of Suramin on Polycystic Kidney Disease in a Mouse Model of Polycystin-1 Deficiency

The aberrant activation of the purinergic signaling pathway has been shown to promote cyst growth and fluid secretion in autosomal dominant polycystic kidney disease (ADPKD). Suramin is an anti-parasitic drug that has strong anti-purinergic properties. Whether suramin could have a therapeutic effect on ADPKD has not been fully investigated. We examined the effect of suramin on cyst progression in a Pkd1 microRNAs transgenic mouse model that presented stable Pkd1 knockdown and moderate disease progression. The Pkd1-deficient mice were treated with suramin (60 mg/kg) by intraperitoneal injection twice a week from postnatal days 35 to 90. Kidney-to-body weight ratios, cyst indices, and blood urea nitrogen (BUN) levels were measured. Cell proliferation and macrophage infiltration were determined by immunohistochemistry. The suramin-treated group had significantly lower renal cyst densities, cell proliferation, and macrophage infiltration compared with saline-treated controls. Suramin significantly inhibited ERK phosphorylation and the expression of Il1b, Il6, Nlrp3, Tgfb, Fn1, P2rx7, and P2ry2 mRNAs in the kidneys. However, BUN levels remained high despite the reduction in cyst growth. Furthermore, plasma cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) levels were significantly higher in the suramin-treated group compared with the control group. Periodic acid-Schiff staining revealed degenerative changes and epithelial cell vacuolation in the non-cystic renal tubules, which indicated phospholipidosis following suramin treatment. These results suggest that suramin may reduce renal cyst growth and inflammation, but the associated tubular cell injuries could limit its therapeutic potential. Other purinergic receptor antagonists with less nephrotoxicity may deserve further investigation for the treatment of ADPKD.     

Empagliflozin Ameliorates Free Fatty Acid Induced-Lipotoxicity in Renal Proximal Tubular Cells via the PPARγ/CD36 Pathway in Obese Mice

High serum levels of free fatty acids (FFAs) could contribute to obesity-induced nephropathy. CD36, a class B scavenger receptor, is a major receptor mediating FFA uptake in renal proximal tubular cells. Empagliflozin, a new anti-diabetic agent, is a specific inhibitor of sodium-glucose co-transporter 2 channels presented on renal proximal tubular cells and inhibits glucose reabsorption. In addition, empagliflozin has shown renoprotective effects. However, the mechanism through which empagliflozin regulates CD36 expression and attenuates FFA-induced lipotoxicity remains unclear. Herein, we aimed to elucidate the crosstalk between empagliflozin and CD36 in FFA-induced renal injury. C57BL/6 mice fed a high-fat diet (HFD) and palmitic acid-treated HK-2 renal tubular cells were used for in vivo and in vitro assessments. Empagliflozin attenuated HFD-induced body weight gain, insulin resistance, and inflammation in mice. In HFD-fed mice, CD36 was upregulated in the tubular area of the kidney, whereas empagliflozin attenuated CD36 expression. Furthermore, empagliflozin downregulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ. Treatment with a PPARγ inhibitor (GW9662) did not further decrease PPARγ expression, whereas a PPARγ antagonist reversed this effect; this suggested that empagliflozin may, at least partly, decrease CD36 by modulating PPARγ. In conclusion, empagliflozin can ameliorate FFA-induced renal tubular injury via the PPARγ/CD36 pathway.     

The Therapeutic Potential of Zinc-Alpha2-Glycoprotein (AZGP1) in Fibrotic Kidney Disease

Chronic kidney disease (CKD) is characterized by a long-term loss of kidney function and, in most cases, by progressive fibrosis. Zinc-alpha2-glycoprotein (AZGP1) is a secreted protein, which is expressed in many different tissues and has been associated with a variety of functions. In a previous study, we have shown in cell culture and in AZGP1 deficient mice that AZGP1 has protective anti-fibrotic effects. In the present study, we tested the therapeutic potential of an experimental increase in AZGP1 using two different strategies. (1) C57Bl/6J mice were treated systemically with recombinant AZGP1, and (2) a transgenic mouse strain was generated to overexpress AZGP1 conditionally in proximal tubular cells. Mice underwent unilateral uretic obstruction as a pro-fibrotic kidney stress model, and kidneys were examined after 14 days. Recombinant AZGP1 treatment was accompanied by better preservation of tubular integrity, reduced collagen deposition, and lower expression of injury and fibrosis markers. Weaker but similar tendencies were observed in transgenic AZGP1 overexpressing mice. Higher AZGP1 levels led to a significant reduction in stress-induced accumulation of tubular lipid droplets, which was paralleled by improved expression of key players in lipid metabolism and fatty acid oxidation. Together these data show beneficial effects of elevated AZGP1 levels in fibrotic kidney disease and highlight a novel link to tubular cell lipid metabolism, which might open up new opportunities for CKD treatment.     

Kidney Response to Chemotherapy-Induced Heart Failure: mRNA Analysis in Normotensive and Ren-2 Transgenic Hypertensive Rats

The aim of the present study was to perform kidney messenger ribonucleic acid (mRNA) analysis in normotensive, Hannover Sprague–Dawley (HanSD) rats and hypertensive, Ren-2 renin transgenic rats (TGR) after doxorubicin-induced heart failure (HF) with specific focus on genes that are implicated in the pathophysiology of HF-associated cardiorenal syndrome. We found that in both strains renin and angiotensin-converting enzyme mRNA expressions were upregulated indicating that the vasoconstrictor axis of the renin–angiotensin system was activated. We found that pre-proendothelin-1, endothelin-converting enzyme type 1 and endothelin type A receptor mRNA expressions were upregulated in HanSD rats, but not in TGR, suggesting the activation of endothelin system in HanSD rats, but not in TGR. We found that mRNA expression of cytochrome P-450 subfamily 2C23 was downregulated in TGR and not in HanSD rats, suggesting the deficiency in the intrarenal cytochrome P450-dependent pathway of arachidonic acid metabolism in TGR. These results should be the basis for future studies evaluating the pathophysiology of cardiorenal syndrome secondary to chemotherapy-induced HF in order to potentially develop new therapeutic approaches.     

Modulation of Tubular pH by Acetazolamide in a Ca2+ Transport Deficient Mice Facilitates Calcium Nephrolithiasis

Proximal tubular (PT) acidosis, which alkalinizes the urinary filtrate, together with Ca²⁺ supersaturation in PT can induce luminal calcium phosphate (CaP) crystal formation. While such CaP crystals are known to act as a nidus for CaP/calcium oxalate (CaOx) mixed stone formation, the regulation of PT luminal Ca²⁺ concentration ([Ca²⁺]) under elevated pH and/or high [Ca²⁺] conditions are unknown. Since we found that transient receptor potential canonical 3 (TRPC3) knockout (KO; -/-) mice could produce mild hypercalciuria with CaP urine crystals, we alkalinized the tubular pH in TRPC3-/- mice by oral acetazolamide (0.08%) to develop mixed urinary crystals akin to clinical signs of calcium nephrolithiasis (CaNL). Our ratiometric (λ340/380) intracellular [Ca²⁺] measurements reveal that such alkalization not only upsurges Ca²⁺ influx into PT cells, but the mode of Ca²⁺ entry switches from receptor-operated to store-operated pathway. Electrophysiological experiments show enhanced bicarbonate related current activity in treated PT cells which may determine the stone-forming phenotypes (CaP or CaP/CaOx). Moreover, such alkalization promotes reactive oxygen species generation, and upregulation of calcification, inflammation, fibrosis, and apoptosis in PT cells, which were exacerbated in absence of TRPC3. Altogether, the pH-induced alteration of the Ca²⁺ signaling signature in PT cells from TRPC3 ablated mice exacerbated the pathophysiology of mixed urinary stone formation, which may aid in uncovering the downstream mechanism of CaNL.     

Kidney Injury Caused by Preeclamptic Pregnancy Recovers Postpartum in a Transgenic Rat Model

Preeclampsia (PE) is characterized by the onset of hypertension (≥140/90 mmHg) and presence of proteinuria (>300 mg/L/24 h urine) or other maternal organ dysfunctions. During human PE, renal injuries have been observed. Some studies suggest that women with PE diagnosis have an increased risk to develop renal diseases later in life. However, in human studies PE as a single cause of this development cannot be investigated. Here, we aimed to investigate the effect of PE on postpartum renal damage in an established transgenic PE rat model. Female rats harboring the human-angiotensinogen gene develop a preeclamptic phenotype after mating with male rats harboring the human-renin gene, but are normotensive before and after pregnancy. During pregnancy PE rats developed mild tubular and glomerular changes assessed by histologic analysis, increased gene expression of renal damage markers such as kidney injury marker 1 and connective-tissue growth factor, and albuminuria compared to female wild-type rats (WT). However, four weeks postpartum, most PE-related renal pathologies were absent, including albuminuria and elevated biomarker expression. Only mild enlargement of the glomerular tuft could be detected. Overall, the glomerular and tubular function were affected during pregnancy in the transgenic PE rat. However, almost all these pathologies observed during PE recovered postpartum.     

Lysophosphatidic Acid Is a Proinflammatory Stimulus of Renal Tubular Epithelial Cells

Chronic kidney disease (CKD) refers to a spectrum of diseases defined by renal fibrosis, permanent alterations in kidney structure, and low glomerular-filtration rate. Prolonged epithelial-tubular damage involves a series of changes that eventually lead to CKD, highlighting the importance of tubular epithelial cells in this process. Lysophosphatidic acid (LPA) is a bioactive lipid that signals mainly through its six cognate LPA receptors and is implicated in several chronic inflammatory pathological conditions. In this report, we have stimulated human proximal tubular epithelial cells (HKC-8) with LPA and 175 other possibly pathological stimuli, and simultaneously detected the levels of 27 intracellular phosphoproteins and 32 extracellular secreted molecules with multiplex ELISA. This quantification revealed a large amount of information concerning the signaling and the physiology of HKC-8 cells that can be extrapolated to other proximal tubular epithelial cells. LPA responses clustered with pro-inflammatory stimuli such as TNF and IL-1, promoting the phosphorylation of important inflammatory signaling hubs, including CREB1, ERK1, JUN, IκΒα, and MEK1, as well as the secretion of inflammatory factors of clinical relevance, including CCL2, CCL3, CXCL10, ICAM1, IL-6, and IL-8, most of them shown for the first time in proximal tubular epithelial cells. The identified LPA-induced signal-transduction pathways, which were pharmacologically validated, and the secretion of the inflammatory factors offer novel insights into the possible role of LPA in CKD pathogenesis.     

TMAO Suppresses Megalin Expression and Albumin Uptake in Human Proximal Tubular Cells Via PI3K and ERK Signaling

Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.     

Effect of Lowering Asymmetric Dimethylarginine (ADMA) on Vascular Pathology in Atherosclerotic ApoE-Deficient Mice with Reduced Renal Mass

The purpose of the work was to study the impact of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH1), on atherosclerosis in subtotally nephrectomized (SNX) ApoE-deficient mice. Male DDAH1 transgenic mice (TG, n = 39) and C57Bl/6J wild-type littermates (WT, n = 27) with or without the deletion of the ApoE gene underwent SNX at the age of eight weeks. Animals were sacrificed at 12 months of age, and blood chemistry, as well as the extent of atherosclerosis within the entire aorta were analyzed. Sham treated (no renal mass reduction) ApoE-competent DDAH1 transgenic and wild-type littermates (n = 11) served as a control group. Overexpression of DDAH1 was associated with significantly lower ADMA levels in all treatment groups. Surprisingly, SNX mice did not exhibit higher ADMA levels compared to sham treated control mice. Furthermore, the degree of atherosclerosis in ApoE-deficient mice with SNX was similar in mice with or without overexpression of DDAH1. Overexpression of the ADMA degrading enzyme, DDAH1, did not ameliorate atherosclerosis in ApoE-deficient SNX mice. Furthermore, SNX in mice had no impact on ADMA levels, suggesting a minor role of this molecule in chronic kidney disease (CKD) in this mouse model.