High Elmo1 expression aggravates and low Elmo1 expression prevents diabetic nephropathy

Abstract

Human genome-wide association studies have demonstrated that polymorphisms in the engulfment and cell motility protein 1 gene (ELMO1) are strongly associated with susceptibility to diabetic nephropathy. However, proof of causation is lacking. To test whether modest changes in its expression alter the severity of the renal phenotype in diabetic mice, we have generated mice that are type 1 diabetic because they have the Ins2(Akita) gene, and also have genetically graded expression of Elmo1 in all tissues ranging in five steps from ∼30% to ∼200% normal. We here show that the Elmo1 hypermorphs have albuminuria, glomerulosclerosis, and changes in the ultrastructure of the glomerular basement membrane that increase in severity in parallel with the expression of Elmo 1. Progressive changes in renal mRNA expression of transforming growth factor β1 (TGFβ1), endothelin-1, and NAD(P)H oxidase 4 also occur in parallel with Elmo1, as do the plasma levels of cystatin C, lipid peroxides, and TGFβ1, and erythrocyte levels of reduced glutathione. In contrast, Akita type 1 diabetic mice with below-normal Elmo1 expression have reduced expression of these various factors and less severe diabetic complications. Remarkably, the reduced Elmo1 expression in the 30% hypomorphs almost abolishes the pathological features of diabetic nephropathy, although it does not affect the hyperglycemia caused by the Akita mutation. Thus, ELMO1 plays an important role in the development of type 1 diabetic nephropathy, and its inhibition could be a promising option for slowing or preventing progression of the condition to end-stage renal disease.

Keywords: 3′-untranslated region; fibrosis; reactive oxygen species.

Fig. 1.

Generation of Akita diabetic mice having five graded expression levels of Elmo1. (A) The gene-targeting strategy. (a) The target locus, Elmo1, into which an exogenous 3′-untranslated region (3′-UTR) is introduced by homologous recombination. Coding sequences and the endogenous 3′-UTR of the Elmo1 gene are shown as black and yellow columns, respectively. (b) The targeting vector contains a loxP sequence, the c-Fos 3′-UTR (blue), a Neo gene with the MC1 promoter (pMC1), loxP, and the 3′-UTR of the bovine growth hormone gene (bGH 3′-UTR; red). TK, thymidine kinase gene. (c) The resulting locus after homologous recombination; the Elmo1 gene is now in its low-expression form (Elmo1^L) because the stability of its mRNA is now controlled by the destabilizing 3′-UTR of Fos. (d) The resulting locus after Cre-lox P recombination; the Elmo1 gene is now in its high-expression form (Elmo1^H) because the stability of its mRNA is now controlled by the stabilizing 3′-UTR of bGH. (B) In vitro comparison of the effects of 3′-UTRs and their G-rich elements (GREs) on expression of a green fluorescent protein gene. Fluorescence of colonies with the 3′-UTRs and GREs of bGH, Elmo1, and Fos is shown. (C) Mean fluorescence levels of the single cells with the 3′-UTR and GRE of bGH, Elmo1, and Fos from different colonies. *P < 0.05, **P < 0.01 vs. Elmo1. (D) The mRNA levels of Elmo1 in the kidney of the nondiabetic and Akita diabetic mice having five graded expression levels of Elmo1 at age 40 wk. *P < 0.05, **P < 0.01 vs. Elmo1 WT genotype.

Fig. 2.

Systemic parameters affecting diabetic nephropathy in Akita mice with five graded expressions of Elmo1 at age 40 wk. Dotted lines indicate nondiabetic WT levels. *P < 0.05, **P < 0.01 vs. Elmo1 WT. (A) Plasma glucose levels. (B) Plasma insulin levels. (C) Systolic blood pressure (BP) determined with a tail-cuff method. (D) Plasma levels of lipid peroxide (LPO). (E) Erythrocyte levels of reduced glutathione (GSH). (F) Plasma levels of transforming growth factor β1 (TGFβ1).

Fig. 3.

Renal pathology in the Akita diabetic mice having five graded expression levels of Elmo1 at 40 wk of age. Dotted lines indicate nondiabetic WT levels. *P < 0.05, **P < 0.01 vs. Elmo1 WT. (A–E) Periodic acid–Schiff (PAS) staining with hematoxylin of the glomerulus in 40-wk-old male mice. (Scale bar: 50 μm.) (F–J) Transmission electron microscopy of the glomerular basement membrane in 40-wk-old male mice. (Scale bar: 1 μm.) (A and F) L/L Akita mouse. (B and G) L/+ Akita mouse. (C and H) WT Akita mice. (D and I) H/+ Akita mouse. (E and J) H/H Akita mouse. (K) Quantification of PAS-positive mesangial material per total glomerular tuft cross-sectional area. (L) Percentage of open capillary area per glomerular tuft. (M) Thickness of glomerular basement membrane (GBM). (N) Plasma levels of cystatin C. (O) Urinary albumin excretion. (P) Renal expression of Tgfb1. (Q) Renal expression of Edn1. (R) Renal expression of Nox4.