With the goal of identifying optimal platforms for developing better models of diabetic nephropathy in mice, we compared renal effects of streptozotocin (STZ)-induced diabetes among five common inbred mouse strains (C57BL/6, MRL/Mp, BALB/c, DBA/2, and 129/SvEv). We also evaluated the renal consequences of chemical and genetic diabetes on the same genetic background (C57BL/6). There was a hierarchical response of blood glucose level to the STZ regimen among the strains (DBA/2 > C57BL/6 > MRL/MP > 129/SvEv > BALB/c). In all five strains, males demonstrated much more robust hyperglycemia with STZ than females. STZ-induced diabetes was associated with modest levels of albuminuria in all of the strains but was greatest in the DBA/2 strain, which also had the most marked hyperglycemia. Renal structural changes on light microscopy were limited to the development of mesangial expansion, and, while there were some apparent differences among strains in susceptibility to renal pathological changes, there was a significant positive correlation between blood glucose and the degree of mesangial expansion, suggesting that most of the variability in renal pathological abnormalities was because of differences in hyperglycemia. Although the general character of renal involvement was similar between chemical and genetic diabetes, Akita mice developed more marked hyperglycemia, elevated blood pressures, and less variability in renal structural responses. Thus, among the strains and models tested, the DBA/2 genetic background and the Akita (Ins2(+/C96Y)) model may be the most useful platforms for model development.

Previous studies have shown that Akita mice bearing the Ins2(+/C96Y) mutation have significant advantages as a type I diabetes platform for developing models of diabetic nephropathy (DN; Gurley SB, Clare SE, Snow KP, Hu A, Meyer TW, Coffman TM. Am J Physiol Renal Physiol 290: F214-F222, 2006). In view of the critical role for genetic factors in determining susceptibility to DN in humans, we investigated the role of genetic background on kidney injury in Akita mice. To generate a series of inbred Akita mouse lines, we back-crossed the Ins2(C96Y) mutation more than six generations onto the 129/SvEv and DBA/2 backgrounds and compared the extent of hyperglycemia and renal disease with the standard C57BL/6-Ins2(+/C96Y) line. Male mice from all three Akita strains developed marked and equivalent hyperglycemia. However, there were significant differences in the level of albuminuria among the lines with a hierarchy of DBA/2 > 129/SvEv > C57BL/6. Renal and glomerular hypertrophy was seen in all of the lines, but significant increases in mesangial matrix compared with baseline nondiabetic controls were observed only in the 129 and C57BL/6 backgrounds. In F1(DBA/2 x C57BL/6)-Ins2(+/C96Y) mice, the extent of albuminuria was similar to the parental DBA/2-Ins2(+/C96Y) line; they also developed marked hyperfiltration. These studies identify strong effects of genetic background to modify the renal phenotype associated with the Ins2(C96Y) mutation. Identification of these naturally occurring strain differences should prove useful for nephropathy modeling and may be exploited to allow identification of novel susceptibility alleles for albuminuria in diabetes.

Aims The F-actin-binding protein Drebrin inhibits smooth muscle cell (SMC) migration, proliferation and pro-inflammatory signaling. Therefore, we tested the hypothesis that Drebrin constrains atherosclerosis. Methods and results SM22-Cre+/Dbnflox/flox/Ldlr-/- (SMC-Dbn-/-/Ldlr-/-) and control mice (SM22-Cre+/Ldlr-/-, Dbnflox/flox/Ldlr-/-, and Ldlr-/-) were fed a Western diet for 14-20 weeks. Brachiocephalic arteries of SMC-Dbn-/-/Ldlr-/- mice exhibited 1.5- or 1.8-fold greater cross-sectional lesion area than control mice at 14 or 20 wk, respectively. Aortic atherosclerotic lesion surface area was 1.2-fold greater in SMC-Dbn-/-/Ldlr-/- mice. SMC-Dbn-/-/Ldlr-/- lesions comprised necrotic cores that were two-fold greater in size than those of control mice. Consistent with their bigger necrotic core size, lesions in SMC-Dbn-/- arteries also showed more transdifferentiation of SMCs to macrophage-like cells: 1.5- to 2.5-fold greater, assessed with BODIPY or with CD68, respectively. In vitro data were concordant: Dbn-/- SMCs had 1.7-fold higher levels of KLF4 and transdifferentiated to macrophage-like cells more readily than Dbnflox/flox SMCs upon cholesterol loading, as evidenced by greater up-regulation of CD68 and galectin-3. Adenovirally mediated Drebrin rescue produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. During early atherogenesis, SMC-Dbn-/-/Ldlr-/- aortas demonstrated 1.6-fold higher levels of reactive oxygen species than control mouse aortas. The 1.8-fold higher levels of Nox1 in Dbn-/- SMCs was reduced to WT levels with KLF4 silencing. Inhibition of Nox1 chemically or with siRNA produced equivalent levels of macrophage-like transdifferentiation in Dbn-/- and Dbnflox/flox SMCs. Conclusions We conclude that SMC Drebrin limits atherosclerosis by constraining SMC Nox1 activity and SMC transdifferentiation to macrophage-like cells. Translational perspective Drebrin is abundantly expressed in vascular smooth muscle cells (SMCs) and is up-regulated in human atherosclerosis. A hallmark of atherosclerosis is the accumulation of foam cells that secrete pro-inflammatory cytokines and contribute to plaque instability. A large proportion of these foam cells in humans derive from SMCs. We found that SMC Drebrin limits atherosclerosis by reducing SMC transdifferentiation to macrophage-like foam cells in a manner dependent on Nox1 and KLF4. For this reason, strategies aimed at augmenting SMC Drebrin expression in atherosclerotic plaques may limit atherosclerosis progression and enhance plaque stability by bridling SMC-to-foam-cell transdifferentiation.

Reactive oxygen species (ROS) exacerbate atherosclerosis (athero). ROS levels are elevated by specific non-coding, small nucleolar (sno) RNAs encoded within introns of the Rpl13a gene. We therefore tested the hypothesis that these snoRNAs promote athero, using “snoKO” mice deficient in Rpl13a snoRNAs, but not in Rpl13a itself. ROS levels assessed by CellROX Orange were 35% lower in snoKO than snoRNA +/+ aorta frozen sections ( p <0.01). After 14 wk on Western diet, female snoKO/ Apoe -/- unexpectedly showed total cholesterol levels 20% higher than Apoe -/- mice in (1,046 vs 869 mg/dl, p <0.05). Despite this, neointimal lesions in brachiocephalic artery (BCA) cross-sections were 50% smaller in snoKO/ Apoe -/- than in Apoe -/- mice, and lumen size was 45% larger (both p <0.01, n=8/group). Similar data were obtained in males: snoKO/ Apoe -/- mice had 40% smaller BCA lesion areas ( p <0.02, n=8/group). After being stained for cholesteryl ester with BODIPY, for ACTA2 by immunofluorescence and for DNA (Hoechst), BCAs from female snoKO/ Apoe -/- mice (n=9) demonstrated 50% less foam cell-positive and 95% more ACTA2 + area, 40% less necrotic core area, and a 60% lower prevalence of ACTA2 + foam cells ( p <0.05 for each). Thus, Rpl13a snoRNAs promote vascular ROS and athero. Assessed by MitoSOX Red, ROS levels were 25% lower in snoKO than WT M1-polarized bone marrow-derived Mϕs in vitro (n=3, p<0.05). To identify mechanisms linking the Rpl13a snoRNAs to ROS and athero, we performed LC-MS/MS on WT and snoKO aortic SMCs. COX4I2 was expressed 5.7-fold higher in snoKO than WT SMCs by MS/MS, and 2.5-fold higher in snoKO SMCs by immunoblot (n=3/group, p <0.05). As part of mitochondrial complex IV, COX4I2 lowers cellular ROS levels. We used CRISPR/Cas9 to create 293T cells lacking either the RPL13a -snoRNA U34A or the irrelevant snoRNA U25 . With mRNA from these cells we performed reverse transcription at low [dNTP] followed by qPCR (RTL-P) for COX4I2 . Inversely proportional to the degree of mRNA 2’- O -methylation (mediated by snoRNAs), the RTL-P efficiency was 4-fold higher in U34A -knockout than control cells (3 clones/genotype, p <0.01). Thus, COX4I2 mRNA appears to be regulated by RPL13A -snoRNA-guided 2’- O -methylation in a manner that could link Rpl13a snoRNAs, vascular ROS, and athero.