UNX Mice Research Articles

#2584 Mapping Progression of DKD in ReninAAV UNx db/db mice utilizing time-series RNA sequencing

Abstract Background and Aims The Renin adeno-associated virus (AAV) uninephrectomized (UNx) db/db mouse model of diabetic kidney disease (DKD) recapitulates hallmarks of DKD with hypertension, including albuminuria and glomerulosclerosis. Despite previous characterization of the model, gaps persist in our understanding regarding temporal patterns of gene expression and pathophysiology in the glomeruli. This longitudinal study characterizes isolated glomeruli from ReninAAV UNx db/db mice during progression of DKD with hypertension. Method Female db/db (BKS.Cg-Dock7m +/+ Leprdb/J) received a single i.v. dose of LacZAAV (control) or ReninAAV. Untreated db/m mice (BKS.Cg-Dock7m +/+ Leprdb/J) served as healthy controls. One week after AAV injection, ReninAAV mice underwent UNx, recovered for three weeks, and were subsequently randomized and stratified into 4 groups according to fed blood glucose and body weight. ReninAAV UNx mice were terminated at 4, 8, 12 and 16 weeks (w4-w16) after UNx. Control groups were terminated at 16 weeks. Endpoints included urine and plasma biochemistry, kidney histology (i.e. Collagen 1a1 and Glomerulosclerosis) and RNA sequencing of glomeruli isolated by laser capture microdissection. Results ReninAAV UNx db/db mice showed progressive kidney fibrosis (Col1a1%-area) and albuminuria (uACR) compared to LacZAAV db/db and db/m mice across all timepoints investigated. Furthermore, a significant increase in glomerulosclerosis was observed from w12 and onwards compared to LacZAAV and db/m mice. When compared to LacZAAV mice, more than 1,700 glomerular differentially regulated genes and more than 250 uniquely regulated genes were found at each timepoint, including markers of fibrosis, inflammation and glomerular injury. Several glomerular drug targets exhibited time-specific regulated in ReninAAV UNx db/db mice (e.g. Slc5a2, Egfr, Agtr1a). Regulated genes were segmented into nine clusters with distinct temporal patterns. Among these, a continuous increase in expression of genes associated with extracellular matrix (ECM), small molecule transport and temporal stimulation of metabolic pathways. Finally, a correlation analysis revealed strong associations between ECM and ECM-related pathways vs. glomerulosclerosis and albuminuria. Conclusion The ReninAAV UNx db/db mouse model of hypertensive DKD demonstrates a progressive increase in fibrosis and albuminuria paralleled by dynamic changes in glomerular gene expression signatures. Notably, the identification of temporal patterns of gene expression and pathway regulation provides novel insights into DKD pathophysiology and progression. This underscores the ReninAAV UNx db/db mouse as an applicable model of human DKD in preclinical target and drug discovery.

#5799 COMPENSATORY HYPERTROPHY OF THE KIDNEY CONTRIBUTES TO LOSS OF KLOTHO KIDNEY THROUGH PAKT SIGNALING

Abstract Background and Aims Chronic kidney disease (CKD) represents a significant public health burden worldwide, mainly driven by the increase of the incidence of associated risk factors like diabetes. Decreased renal Klotho expression is an early feature of CKD, driving an increase on circulating FGF23 and phosphate. These alterations in mineral metabolism have a direct negative impact on the progression of renal dysfunction and can cause bone alterations, vascular calcification and heart failure. In parallel, during the early stages of CKD, kidneys activate molecular mechanisms of hypertrophy in an attempt to counteract the loss of renal function. The PI3K/Akt/mTOR pathway, activated by growing factors like insulin-like growing factor-1 (IGF-1) participates in compensatory renal growth. The aim of the present study is to investigate the possible role of PI3K/Akt activation on renal Klotho levels during renal compensatory hypertrophy. Method We generated a mice model of kidney compensatory hypertrophy (UNX mice) in which kidney klotho levels and circulating mineral metabolism parameters were analyzed. Furthermore, some of the mice were treated with inhibitors of the PI3K/Akt/mTOR pathway. In vitro, proximal tubular epithelial human cells (PTEC) were stimulated with IGF-1 in order to activate the PI3K/Akt pathway, and the effects on Klotho expression were determined. Results UNX mice present with an activation of the PI3K/Akt/mTOR pathway in kidney, which correlates with the loss on renal Klotho expression. Moreover, UNX mice showed an increase on both, plasma phosphate and FGF23, and a decrease in the fractional excretion of phosphate (% FEPi). Renal function, estimated by BUN plasma levels, was unaltered. Pharmacological inhibition of the pathway restored Klotho and decreased FGF23 levels, normalizing renal phosphate excretion and blood levels. In vitro, IGF-1 stimulated PI3K/Akt activation in PTEC and induced a decline on Klotho expression, which was restored with PI3K/AKT inhibitors. Conclusion The overactivation of PI3K/Akt/mTOR pathway in renal hypertrophy modulates Klotho expression and has direct effects on FGF23 and phosphate. Our findings constitute an important breakthrough in the research of new therapeutic targets in order to maintain renal Klotho levels, and it may be useful in the treatment of kidney disease patients.

Impaired renal reserve contributes to preeclampsia via the kynurenine and soluble fms-like tyrosine kinase 1 pathway.

To understand how kidney donation leads to an increased risk of preeclampsia, we studied pregnant outbred mice with prior uninephrectomy and compared them with sham-operated littermates carrying both kidneys. During pregnancy, uninephrectomized (UNx) mice failed to achieve a physiological increase in the glomerular filtration rate and during late gestation developed hypertension, albuminuria, glomerular endothelial damage, and excess placental production of soluble fms–like tyrosine kinase 1 (sFLT1), an antiangiogenic protein implicated in the pathogenesis of preeclampsia. Maternal hypertension in UNx mice was associated with low plasma volumes, an increased rate of fetal resorption, impaired spiral artery remodeling, and placental ischemia. To evaluate potential mechanisms, we studied plasma metabolite changes using mass spectrometry and noted that l-kynurenine, a metabolite of l-tryptophan, was upregulated approximately 3-fold during pregnancy when compared with prepregnant concentrations in the same animals, consistent with prior reports suggesting a protective role for l-kynurenine in placental health. However, UNx mice failed to show upregulation of l-kynurenine during pregnancy; furthermore, when UNx mice were fed l-kynurenine in drinking water throughout pregnancy, their preeclampsia-like state was rescued, including a reversal of placental ischemia and normalization of sFLT1 levels. In aggregate, we provide a mechanistic basis for how impaired renal reserve and the resulting failure to upregulate l-kynurenine during pregnancy can lead to impaired placentation, placental hypoperfusion, an antiangiogenic state, and subsequent preeclampsia.

Prostanoid receptors in hyperfiltration-mediated glomerular injury: Novel agonists and antagonists reveal opposing roles for EP2 and EP4 receptors.

Increased fluid-flow shear stress (FFSS) contributes to hyperfiltration-induced podocyte and glomerular injury resulting in progression of chronic kidney disease (CKD). We reported that increased FFSS in vitro and in vivo upregulates PGE2 receptor EP2 (but not EP4 expression), COX2-PGE2 -EP2 axis, and EP2-linked Akt-GSK3β-β-catenin signaling pathway in podocytes. To understand and use the disparities between PGE2 receptors, specific agonists, and antagonists of EP2 and EP4 were used to assess phosphorylation of Akt, GSK3β and β-catenin in podocytes using Western blotting, glomerular filtration barrier function using in vitro albumin permeability (Palb ) assay, and mitigation of hyperfiltration-induced injury in unilaterally nephrectomized (UNX) mice at 1 and 6 months. Results show an increase in Palb by PGE2 , EP2 agonist (EP2AGO ) and EP4 antagonist (EP4ANT ), but not by EP2 antagonist (EP2ANT ) or EP4 agonist (EP4AGO ). Pretreatment with EP2ANT blocked the effect of PGE2 or EP2AGO on Palb . Modulation of EP2 and EP4 also induced opposite effects on phosphorylation of Akt and β-Catenin. Individual agonists or antagonists of EP2 or EP4 did not induce significant improvement in albuminuria in UNX mice. However, treatment with a combination EP2ANT + EP4AGO for 1 or 6 months caused a robust decrease in albuminuria. EP2ANT + EP4AGO combination did not impact adaptive hypertrophy or increased serum creatinine. Observed differences between expression of EP2 and EP4 on the glomerular barrier highlight these receptors as potential targets for intervention. Safe and effective mitigating effect of EP2ANT + EP4AGO presents a novel opportunity to delay the progression of hyperfiltration-associated CKD as seen in transplant donors.

Uninephrectomy and class II PI3K-C2β inactivation synergistically protect against obesity, insulin resistance and liver steatosis in mice

Uninephrectomy (UNx) in living kidney donors for transplantation is now routine clinical practice. While chronic kidney disease, due to bilateral kidney dysfunction, is associated with insulin resistance, liver steatosis, and type 2 diabetes, the metabolic impact of UNx remains unclear. To better understand the crosstalk between the kidney and insulin target tissues, we studied the metabolic consequences of UNx and the potential involvement of class II PI3K-C2β, the inactivation of which has been reported to result in insulin sensitization. Mice underwent UNx or sham operation followed by either normal chow or high-fat diet (HFD). Seventeen weeks post-UNx, mice showed improved glucose tolerance, insulin sensitivity, and decreased HFD-induced liver steatosis. This was associated with an enhanced serum FGF21 and insulin-stimulated Akt signaling in the liver and muscle of both lean and obese mice. Remarkably, the combination of UNx and PI3K-C2β inactivation protected against HFD-induced obesity and further potentiated the metabolic improvement observed in WT UNx mice correlating with a synergistic increase in metabolic tissues of (1) insulin-stimulated Akt signaling (2) FGFR1 and βKlotho expression. We demonstrated a potential beneficial effect of kidney donation and more effectively with PI3K-C2β inactivation to protect against metabolic disorders through a mutual insulin/FGF21 sensitization.

Transcription Factor β-Catenin Plays a Key Role in Fluid Flow Shear Stress-Mediated Glomerular Injury in Solitary Kidney.

Increased fluid flow shear stress (FFSS) in solitary kidney alters podocyte function in vivo. FFSS-treated cultured podocytes show upregulated AKT-GSK3β-β-catenin signaling. The present study was undertaken to confirm (i) the activation of β-catenin signaling in podocytes in vivo using unilaterally nephrectomized (UNX) TOPGAL mice with the β-galactosidase reporter gene for β-catenin activation, (ii) β-catenin translocation in FFSS-treated mouse podocytes, and (iii) β-catenin signaling using publicly available data from UNX mice. The UNX of TOPGAL mice resulted in glomerular hypertrophy and increased the mesangial matrix consistent with hemodynamic adaptation. Uninephrectomized TOPGAL mice showed an increased β-galactosidase expression at 4 weeks but not at 12 weeks, as assessed using immunofluorescence microscopy (p < 0.001 at 4 weeks; p = 0.16 at 12 weeks) and X-gal staining (p = 0.008 at 4 weeks; p = 0.65 at 12 weeks). Immunofluorescence microscopy showed a significant increase in phospho-β-catenin (Ser552, p = 0.005) at 4 weeks but not at 12 weeks (p = 0.935) following UNX, and the levels of phospho-β-catenin (Ser675) did not change. In vitro FFSS caused a sustained increase in the nuclear translocation of phospho-β-catenin (Ser552) but not phospho-β-catenin (Ser675) in podocytes. The bioinformatic analysis of the GEO dataset, #GSE53996, also identified β-catenin as a key upstream regulator. We conclude that transcription factor β-catenin mediates FFSS-induced podocyte (glomerular) injury in solitary kidney.

Calcification of the cavernosal bodies may be responsible for development of erectile dysfunction in uremic apolipoprotein E deficient (apoE−/−) mice

Introduction and objectiveErectile dysfunction's physiopathology in uremia is complex and multifactorial, involving a combination of classical risk factors and specific uremia-related risk factors such as increased oxidative stress, endothelial dysfunction and inflammation. The aim of the study is to investigate the effect of chronic kidney disease (CKD) on vascular calcification and endothelial function of cavernosal bodies in apolipoprotein E deficient (apoE−/−) mice, a well known model of erectile dysfunction. Materials and methodsEight-week-old male apoE−/− mice were randomly assigned to the following 3 groups: (i) subtotally nephrectomised (SNX apoE−/−, 12 mice), (ii) uninephrectomised (UNX apoE−/−, 11 mice) or (iii) sham operated (sham-op apoE−/−, 15 mice). At 16 weeks after surgery, aortas and penile erectile tissues were harvested for histological studies to assess atherosclerosis, vascular calcification, nitrotyrosine staining, total collagen content and macrophage staining. ResultsAt sacrifice, SNX and UNX mice had significantly higher serum urea, total cholesterol, and triglyceride concentrations than sham-op controls. Atherosclerotic lesions in thoracic aorta were significantly larger in uremic apoE−/− mice than in controls. There were no atheromatous lesions in cavernosal bodies or penile artery observed in any group. However, SNX and UNX animals showed a significant increase in calcification score, collagen content and nitrotyrosine staining in cavernosal bodies when compared with controls. The degree of macrophage infiltration was comparable between the 3 groups. ConclusionIn conclusion, even mild renal dysfunction, i.e., after uninephrectomy increases calcification score and aggravates endothelial function of cavernosal bodies in apoE−/− mice and this effect might be linked to increased oxidative stress in penile endothelium.

494-P: Whole-Kidney 3D Imaging and Image Analysis Enables Detailed Quantification of Diabetic Nephropathy Features in the Uninephrectomized db/db Mouse Model

Diabetic nephropathy (DN) is a serious complication of diabetes and the leading cause of end-stage renal disease. To reproducibly test novel therapies, there is a need for precise quantification of DN pathology in rodent models. We compared the accelerated development of DN in male and female uninephrectomized db/db mice using advanced imaging techniques. Unilateral nephrectomy (UNx) was performed in 7-8 weeks old male and female db/db mice. Sham-operated db/+ mice served as controls. Kidneys were preserved for histology and stereology. In a separate study, UNx was performed in 18 weeks old male db/db mice. These mice and control db/+ mice were injected with lectin-594 prior to termination to visualize glomerular morphology by 3D light sheet microscopy. All mice were terminated at 24 weeks of age. Male and female db/db UNx mice showed comparable increases in albuminuria. Total glomerular volume was markedly higher in male and female db/db UNx mice compared to db/+ controls (mean males; 4.42 vs. 2.71 mm3; p&amp;lt;0.001; females; 5.04 vs. 2.69 mm3; p&amp;lt;0.001). Tubulointerstitial collagen III immuno-staining was significantly increased only in female db/db UNx mice compared to db/+ mice. In contrast, total glomerular collagen IV levels were increased in male db/db UNx mice compared to db/+ controls (mean 1.30 vs. 0.80 mm3; p&amp;lt;0.01), and female db/db UNx mice displayed increased glomerular collagen IV compared to male db/db UNx mice (mean 1.80 vs. 1.30 mm3; p&amp;lt;0.05). 3D imaging at 24 weeks following UNx at 18 weeks of age confirmed expansion in glomerular volume (median db/+ 96.000 vs. db/db 145.000 µm3). Moreover, a marked increase was seen in cortical lectin-594 staining, indicating glomerular leakage in db/db UNx mice. In conclusion, male and female db/db UNx mice displayed comparable features of DN and both constitute valid models for DN. The use of whole-kidney 3D imaging provides a novel avenue for assessment of individual glomerular volume and permeability. Disclosure L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. F.E. Sembach: Consultant; Self; Novo Nordisk A/S. Employee; Self; Gubra. U. Roostalu: Employee; Self; Gubra. T. Johansen: Employee; Self; Gubra. J.L. Skytte: Employee; Self; Gubra. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S. Stock/Shareholder; Self; Gubra. Stock/Shareholder; Spouse/Partner; Novo Nordisk A/S. K. Fosgerau: Stock/Shareholder; Self; Gubra. N. Vrang: Board Member; Self; Gubra. Stock/Shareholder; Self; Gubra. J. Jelsing: Stock/Shareholder; Self; Gubra. J. Hecksher-Sørensen: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. T.X. Pedersen: Employee; Self; Gubra.

Pathological and Transcriptome Changes in the ReninAAV db/ db uNx Model of Advanced Diabetic Kidney Disease Exhibit Features of Human Disease.

The ReninAAV db/db uNx model of diabetic kidney disease (DKD) exhibits hallmarks of advanced human disease, including progressive elevations in albuminuria and serum creatinine, loss of glomerular filtration rate, and pathological changes. Microarray analysis of renal transcriptome changes were more similar to human DKD when compared to db/db eNOS-/- model. The model responds to treatment with arterial pressure lowering (lisinopril) or glycemic control (rosiglitazone) at early stages of disease. We hypothesized the ReninAAV db/db uNx model with advanced disease would have residual disease after treatment with lisinopril, rosiglitazone, or combination of both. To test this, ReninAAV db/db uNx mice with advanced disease were treated with lisinopril, rosiglitazone, or combination of both for 10 weeks. All treatment groups showed significant lowering of urinary albumin to creatinine ratio compared to baseline; however, only combination group exhibited lowering of serum creatinine. Treatment improved renal pathological scores compared to baseline values with residual disease evident in all treatment groups when compared to db/m controls. Gene expression analysis by TaqMan supported pathological changes with increased fibrotic and inflammatory markers. The results further validate this model of DKD in which residual disease is present when treated with agents to lower arterial pressure and glycemic control.

Unbiased Stereological Quantification of Key Features of Diabetic Nephropathy

Diabetic nephropathy (DN) is a serious long-term complication of diabetes. There is no curative treatment of DN, and the lack of knowledge about mechanisms leading to DN hampers the development of efficient therapies. To facilitate drug development, the ability to quantify key histopathological changes in animal models of human DN is essential. In order to develop a more precise determination of translatable nephrotic changes, we used novel and refined image analyses and stereological quantification of key features of DN in a model approach combining genetic diabetes (db/db mice) with uninephrectomy (UNx). UNx or sham-operation was performed in db/db or db/+ male and female mice (n=8-16 mice per group) and the study was terminated 10 or 16 weeks after surgery. Diabetes development was similar in sham and UNx db/db mice. Compared to sham-operation, UNx increased average kidney weight in db/db females both after 10 (43%) and 16 weeks (48%). Blood urea nitrogen (BUN) was increased in UNx vs. sham-operated animals 4 weeks post-surgery, but not at termination. Notably, kidney fibrosis was increased in UNx and sham db/db vs. sham db/+ females (16 weeks; mg total collagen; 12.7 ± 1.3 and 10.7 ± 1.8, respectively, vs. 5.7 ± 0.8, p&amp;lt;0.05). Urinary albumin was progressively increased in all db/db groups with significantly lower variance in UNx db/db females than sham (F-test, p=0.02), permitting smaller group sizes in pre-clinical studies. Unbiased stereological quantification is ongoing to establish volume of glomeruli and number of podocytes. Glomerular collagen is quantified by double-fluorescence imaging concomitantly detecting podocin and collagen. In conclusion, renal hypertrophy and raised plasma BUN suggest increased pressure on the remaining kidney in UNx mice, leading to fibrosis and decreased kidney function. The thorough quantification of translatable features of DN in the db/db UNx model may provide refinement of this model enabling improved pharmacological testing of compounds targeting DN. Disclosure T.X. Pedersen: Employee; Self; Gubra. T.T. Johansen: None. L.N. Fink: Employee; Self; Gubra. Stock/Shareholder; Self; Novo Nordisk A/S. N. Vrang: Board Member; Self; Gubra. Employee; Self; Gubra. Stock/Shareholder; Self; Gubra. T. Secher: Employee; Self; Gubra. Employee; Spouse/Partner; Novo Nordisk A/S.

In vivo kinetics of the uremic toxin p-cresyl sulfate in mice with variable renal function.

Uremic toxins such as p-cresyl sulfate (PCS) are associated with increased mortality for chronic kidney disease (CKD) patients, but in vivo PCS toxicity studies are limited due to the lack of a standard animal model. To establish such a model, we measured the pharmacokinetics of PCS in mice with variable renal function. Male Balb/c mice subjected to 5/6 nephrectomy (CRF), unilateral nephrectomy (UNX), or no surgery (controls) were given PCS (po, 50 mg/kg). Blood samples were collected over time and plasma PCS concentrations were measured. Over 4 h, PCS was significantly higher in the plasma of CRF mice (63.28 ± 2.76 mg/L), compared to UNX mice (3.11 ± 0.64 mg/L) and controls (0.39 ± 0.12 mg/L). The PCS half-life was greatest in CRF mice (12.07 ± 0.12 h), compared to 0.79 ± 0.04 h in UNX mice and 0.48 ± 0.02 h in control mice. However, the potential presence of additional uremic toxins along with PCS in CRF mice and rapid PCS clearance in control mice suggest that the UNX mouse would be a better PCS model to study toxicity.

Darbepoetin Alpha Reduces Oxidative Stress and Chronic Inflammation in Atherosclerotic Lesions of Apo E Deficient Mice in Experimental Renal Failure

BackgroundCardiovascular morbidity and mortality is very important in patients with chronic renal failure. This occurs even in mild impairment of renal function and may be related to oxidative stress and chronic inflammation. The nephrectomized apo E knockout mouse is an accepted model for evaluating atherosclerosis in renal dysfunction. Erythropoietin derivates showed anti-oxidative and anti-inflammatory effects. Therefore, this study evaluates the effects of Darbepoetin on markers of oxidative stress and chronic inflammation in atherosclerotic lesions in apo E knockout mice with renal dysfunction.MethodsApo E knockout mice underwent unilateral (Unx, n = 20) or subtotal (Snx, n = 26) nephrectomy or sham operation (Sham, n = 16). Mice of each group were either treated with Darbepoetin or saline solution, a part of Snx mice received a tenfold higher dose of Darbepoetin. The aortic plaques were measured and morphologically characterized. Additional immunhistochemical analyses were performed on tissue samples taken from the heart and the aorta.ResultsBoth Unx and Snx mice showed increased expression of markers of oxidative stress and chronic inflammation. While aortic plaque size was not different, Snx mice showed advanced plaque stages when compared to Unx mice. Darbepoetin treatment elevated hematocrit and lowered Nitrotyrosin as one marker of oxidative stress, inflammation in heart and aorta, plaque stage and in the high dose even plaque cholesterol content. In contrast, there was no influence of Darbepoetin on aortic plaque size; high dose Darbepoetin treatment resulted in elevated renal serum parameters.ConclusionDarbepoetin showed some protective cardiovascular effects irrespective of renal function, i.e. it improved plaque structure and reduced some signs of oxidative stress and chronic inflammation without affecting plaque size. Nevertheless, the dose dependent adverse effects must be considered as high Darbepoetin treatment elevated serum urea. Elevation of hematocrit might be a favorable effect in anemic Snx animals but a thrombogenic risk in Sham animals.

Periaortic adipose tissue-specific activation of the renin-angiotensin system contributes to atherosclerosis development in uninephrectomized apoE−/− mice

Chronic kidney disease (CKD) is an independent risk factor for the development of cardiovascular disease. The perivascular adipose tissue is closely implicated in the development of atherosclerosis; however, the contribution to CKD-associated atherogenesis remains undefined. Eight-week-old apoE-deficient mice were uninephrectomized and fed a high-cholesterol diet starting at 12 wk of age. The atherosclerotic lesion area in the thoracic aorta was comparable in 16-wk-old uninephrectomized (UNX) mice and sham control mice; however, the lesion area was markedly exaggerated in 20-wk-old UNX mice compared with the control (54%, P < 0.05). While the accumulation of monocytes/macrophages and the mRNA expression levels of inflammatory cytokines/chemokines in the thoracic periaortic adipose tissue (PAT) did not differ between the two groups, angiotensinogen (AGT) mRNA expression and the angiotensin II (ANG II) concentration in the PAT were significantly higher in 16-wk-old UNX mice than in the control (1.9- and 1.5-fold increases vs. control, respectively; P < 0.05). ANG II concentrations in both the plasma and epididymal white adipose tissue (WAT) were comparable between the two groups, suggesting that PAT-specific activation of the renin-angiotensin system (RAS) is primarily involved in CKD-associated atherogenesis. The homeostasis model assessment-insulin resistance (HOMA-IR) index and plasma insulin level after glucose loading were significantly elevated in 16-wk-old UNX mice. In vitro stimulation of preadipocytes with insulin exaggerated the AGT mRNA expression along with increased mRNA expression of PPARγ. These findings suggest that PAT-specific RAS activation probably primarily contributes in accelerating atherosclerotic development in UNX mice and could thus represent a therapeutic target for preventing CKD-associated atherogenesis.

ER stress and a chronic inflammatory response are induced in an Angiotensin II/DOCA salt model of chronic kidney disease.

We have developed a C57BL/6 mouse model of Chronic Kidney Disease (CKD). The mice underwent a uninephrectomy (Unx) or a sham operation. Unx mice received 1% NaCl in the drinking water and a 50 mg 21‐day release deoxycorticosterone (DOCA) pellet with Angiotensin II (AngII) infusion (1.5 ng Ang II/minute/g). Mice were sacrificed at day 21. This model produced CKD characterized by hypertensive proteinuria. We hypothesized that hypertension‐induced proteinuria results in endoplasmic reticulum (ER) stress as microarray studies in patients with established proteinuria showed upregulation of unfolded protein response (UPR) genes. In the model, electron microscopy showed disruption of the podocyte filtration barrier. Light microscopy revealed an increase in PAS‐positive intratubular protein casts. RT‐PCR analysis showed increased ER stress markers: GRP78, spliced XBP1, CHOP and TDAG51. TUNEL staining showed increased apoptosis in the tubular epithelium of Unx animals. An increase in T‐cell and macrophage infiltration confirmed an inflammatory response to CKD accompanied by renal interstitial fibrosis as shown by de novo α‐smooth muscle actin and collagen staining. This response may be due to ER stress induced pro‐inflammatory mediators such as NFκB. Future studies will assess whether the therapeutic inhibition of ER stress could alter the progression of chronic inflammation in CKD. Funding, CIHR OSO‐115895.

Abstract 340: Phenotypic Conversion of Perivascular Adipose Tissue in Uninephrectomized Apoe-Deficient Mice: Possible Role in the Early Stage of Atherosclerosis in Chronic Kidney Disease

BACKGROUND: Chronic kidney disease (CKD) is now recognized to be an independent risk factor for cardiovascular disease. Recently, perivascular adipose tissue (PVAT) has been shown to play a crucial role in the pathogenesis of atherosclerosis; however, the role of PVAT in the CKD-associated proatherogenic actions remains undefined. METHOD AND RESULT: Eight-week-old apoE deficient mice underwent uninephrectomy (UNX) or sham operation and were fed a western diet starting at 12 wks of age. UNX mice at the age of 20 wks showed a significant exaggeration of atherosclerotic lesion area (53%, P&lt;0.05). Hemodynamic data, lipid profile, and plasma creatinine concentrations did not differ between the two groups. To elucidate the underlying mechanisms of minor renal insufficiency-induced atherosclerosis, we first examined the number of circulating inflammatory monocytes and the mRNA expression levels of VCAM-1 and ICAM-1 in the vasculature at 16 wks of age. However, there was no difference between the two groups, suggesting that uninephrectomy did not effect on either western diet-induced mobilization of bone marrow-derived monocytes or endothelial activation in the early stage of atherosclerosis. We therefore focused on the PVAT surrounding the thoracic aorta, which showed multilocular fat droplet and high expression levels of brown adipocyte-specific genes. The fat pad weight and cross-sectional area of PVAT including the infiltration of CD45+ cells did not differ between the two groups. While mRNA expression levels of adipocyte differentiation marker genes (PPARγ, FABP4, and adiponectin) were equivalent between the two groups, brown adipocyte-specific marker genes (UCP-1, Elovl3) showed a significantly lower expression in UNX mice (P&lt;0.01). In contrast, white adipocyte-specific marker gene Tcf21 was markedly up-regulated (101%, P&lt;0.05), accompanied by the increased expression of angiotensinogen gene (85%, P&lt;0.05). The concentration of serum angiotensin II did not differ between the two groups. CONCLUSION: Phenotypic conversion of perivascular brown to white adipose tissue along with the depot-specific local activation of renin-angiotensin system is a possible mechanism underlying the early stage of atherosclerosis in CKD.

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 μg/kg) or paricalcitol (0.1 μg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 μm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-β1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-β expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).

Antiatherogenic Effects of Angiotensin Receptor Antagonism in Mild Renal Dysfunction

Angiotensin II (Ang II) increases atherosclerotic cardiovascular disease. Renal damage that is characterized by activation of Ang II markedly potentiates the risk for atherosclerosis, even in the setting of subtle renal impairment. Therefore, whether antagonism of Ang II actions can modify atherosclerosis in a model of mild renal impairment was examined. Apolipoprotein E-deficient spontaneously hyperlipidemic mice underwent uninephrectomy (UNx) or sham operation (sham) followed by treatment with Ang II receptor antagonist losartan or hydralazine for 12 wk. While UNx did not increase the serum creatinine levels, BP and lipids were higher in UNx mice than in age-matched sham controls with intact kidneys. UNx caused a dramatic increase in the extent and the number of atherosclerotic lesions together with greater macrophage-positive area and more disruption in the elastin component of the extracellular matrix versus sham. Ang II antagonism dramatically decreased the UNx-induced acceleration in atherosclerosis in association with decreased macrophage content, linked to decreased macrophage migration in vitro with losartan but not with hydralazine. Aortae of mice treated with Ang II antagonism had fewer elastin breaks together with less immunostaining for the powerful elastolytic enzyme cathepsin S. None of these benefits was observed in the hydralazine-treated mice despite equivalent reduction in BP. These findings support an important role for endogenous Ang II in accelerated atherosclerosis in renal dysfunction and offer a therapeutic intervention with particular benefit in this setting through mechanisms that include reduced vascular macrophage infiltration and preservation of the elastin component of extracellular matrix.

Chronic renal failure accelerates atherogenesis in apolipoprotein E-deficient mice.

Cardiovascular mortality is 10 to 20 times increased in patients with chronic renal failure (CRF). Risk factors for atherosclerosis are abundant in patients with CRF. However, the pathogenesis of cardiovascular disease in CRF remains to be elucidated. The effect of CRF on the development of atherosclerosis in apolipoprotein E-deficient male mice was examined. Seven-week-old mice underwent 5/6 nephrectomy (CRF, n = 28), unilateral nephrectomy (UNX, n = 24), or no surgery (n = 23). Twenty-two weeks later, CRF mice showed increased aortic plaque area fraction (0.266 +/- 0.033 versus 0.045 +/- 0.006; P < 0.001), aortic cholesterol content (535 +/- 62 versus 100 +/- 9 nmol/cm(2) intimal surface area; P < 0.001), and aortic root plaque area (205,296 +/- 22,098 versus 143,662 +/- 13,302 micro m(2); P < 0.05) as compared with no-surgery mice; UNX mice showed intermediate values. The plaques from uremic mice contained CD11b-positive macrophages and showed strong staining for nitrotyrosine. Systolic BP and plasma homocysteine concentrations were similar in uremic and nonuremic mice. Plasma urea and cholesterol concentrations were elevated 2.6-fold (P < 0.001) and 1.5-fold (P < 0.001) in CRF compared with no-surgery mice. Both variables correlated with aortic plaque area fraction (r(2) = 0.5, P < 0.001 and r(2) = 0.3, P < 0.001, respectively) and with each other (r(2) = 0.5, P < 0.001). On multiple linear regression analysis, only plasma urea was a significant predictor of aortic plaque area fraction. In conclusion, the present findings suggest that uremia markedly accelerates atherogenesis in apolipoprotein E-deficient mice. This effect could not be fully explained by changes in BP, plasma homocysteine levels, or total plasma cholesterol concentrations. Thus, the CRF apolipoprotein E-deficient mouse is a new model for studying the pathogenesis of accelerated atherosclerosis in uremia.