Deletion of the Formin, Drf1, is Protective Against Renal Damage in a Murine Model of Diabetes
Authors: Manigrasso Michaele Beth , Rosario Rosa , Ramasamy Ravichandran , D'Agati Vivette , Schmidt Ann Marie 
Published: November 2023

Our studies have shown the cytoplasmic domain of the receptor for advanced glycation endproducts (RAGE) binds to the formin molecule, diaphanous‐1 (mDia1). mDia1 is a member of the formin family of intracellular molecules involved in cellular migration which act as effectors of Rho GTPase signaling. In RAGE‐expressing cells devoid of Drf1 (gene encoding mDia1), incubation with RAGE ligands failed to generate reactive oxygen species or activate key signaling cellular stress pathways. Here, we sought to determine if mDia1 plays key roles in a murine model of diabetic nephropathy (DN). Our preliminary data reveal that mDia1 expression is increased in human and murine diabetic podocytes and parietal epithelial cells in a diffuse and global pattern compared to age‐matched controls. Furthermore, expression patterns of mDia1 are highly analogous to those of RAGE. While the role of RAGE has been shown to play a key role in the development of DN, the potential contribution of mDia1 has yet to be elucidated. Therefore, we tested the hypothesis that mDia1 contributes to the development and progression of diabetic renal disease in a murine model.

Male wild‐type and Drf1‐null (WT, Drf1KO; all in the C57BL/6 background) mice were rendered diabetic at 6 weeks of age with streptozotocin and sacrificed after 3 or 6 months of diabetes. Kidneys were harvested and processed for histology and gene expression and stained with periodic‐acid Schiff (PAS) and semi‐quantitative scoring was used to determine the degree of mesangial sclerosis by average findings in >100 glomeruli/mouse (scale 0–3+; 0=absent, 1=mild, 2=moderate, 3=severe). Glomerular basement membrane (GBM) thickness and podocyte foot process effacement (FPE) were measured by ultrastructural analysis (>8 glomeruli/mouse). Gene expression of inflammatory markers (transforming growth factor beta; Tgfb, interleukin 6; Il6, and monocyte chemoattractant protein 1; Ccl2) were assessed by quantitative real time PCR using RNA prepared from whole kidney cortex and normalized to beta‐actin.

This preliminary study suggests that deletion of Drf1 in mice results in a substantial protection against indices of DN by reducing podocyte effacement, inflammation and fibrosis in type 1 DM.

Histological and microscopic observations show that mDiaKO mice have a reduced GBM, less podocyte FPE and a decrease in mesangial sclerosis compared to WT mice after 6 months of diabetes.

Genotype

WT
Drf1KO

ND (n=5)
DM (n=5)
ND (n=5)
DM (n=5)

GBM (nm)
217.3±14.1
288.5±17.46^
228.3±16.5
246.0±23.2*

FPE [%]
9.6±3.6
29.0±6.8^
9.8±2.3*
13.0±4.7*

MS
0.4±0.4
2.0±0.4^
0.0±0.0*
1.3±0.6*#

p<0.05 vs. WT ND,

p<0.05 vs WT DM,

p<0.05 vs Drf1KO ND. Values shown are Mean±SEM.

Ratio of target gene fold‐change normalized to beta‐actin. Gene expression of inflammatory markers in diabetes is downregulated with deletion of mDia1 compared to WT after both 3 and 6 months.

Condition

3 months
6 months

WTND
Drf1KOND
WTDM
Drf1KODM
WTND
Drf1KOND
WTDM
Drf1KODM

n=3
n=3
n=3
n=3
n=3
n=3
n=5
n=3

Tgfb
1
1.02
2.67^#
1.09*
1
0.52ab
7.32ac
0.82abc

Il6
1
0.96
1.49^#
1.21.^*#
1
0.52ab
2.13ac
0.73abc

Ccl2
1
1.01
1.43^#
1.17^*#
1
0.75ab
1.82ac
1.12abc

p<0.05 vs. WT ND 3 months,

p<0.05 vs WT DM 3 months,

p<0.05 vs Drf1KO ND 3 months,

p<0.05 vs WT ND 6 months,

p<0.05 vs WT DM 6 months,

p<0.05 vs Drf1KO ND 6 months. Values shown are Mean±SEM.