Rat Podocytes

Acute Glucose Fluctuation Promotes Autophagy and IL-1β and TNF-α Generation in Rat Podocytes

Diabetic nephropathy (DN) is a serious diabetes-related kidney disease in which acute glucose fluctuation (AGF) is one risk factor that can lead to greater kidney damage than steady-state hyperglycemia. But the exact mechanisms are not clear. Hu's team is trying to find out how AGF affects the progression of DN. They used rat podocytes cultured under 72-hour AGF to monitor cell proliferation, apoptosis, oxidative stress and autophagy.

CCK-8 showed a reduced viability of cells in high‑glucose group (HG) compared with normal glucose group (NG), and also in AGF group compared with both NG and HG group (Fig. 1B). Rates of apoptosis were not significantly different between groups (Fig. 1C and D). Autophagy, a major component of podocytes, was measured by western blot analysis of autophagy biomarkers (LC3II/I and Beclin-1). AGF treatment significantly elevated LC3II/I and Beclin-1, while the NG and HG group showed a decrease (Fig. 2A and B). Confocal microscopy demonstrated a stronger presence of yellow and red puncta in AGF compared with HG (Fig. 2C and D). In order to test whether AGF influences inflammation, mRNA and protein concentrations of TNF-α and interleukin-1β (IL-1β) were measured in the treated rat podocytes. Both cytokines were higher in the HG and AGF groups than in the NG group, and both were substantially higher in the AGF group than in the HG group (Fig. 3). This indicates that AGF promotes inflammatory response in rat podocytes.

Fig. 1. Effect of acute blood fluctuation on rat podocyte proliferation and apoptosis (Hu Z, Fang W, et al., 2021).

Fig. 2. AGF induces autophagy in rat podocytes (Hu Z, Fang W, et al., 2021).

Fig. 3. AGF promotes pro-inflammatory cytokine generation (Hu Z, Fang W, et al., 2021).

Rac1 and RhoA Activity Influences Rat Podocyte Permeability and Migration

Podocyte foot processes maintain glomerular permeability and are regulated by insulin through the PKGIα signaling pathway. Disruption of this mechanism in diabetes impacts the actin cytoskeleton, which depends on Rho GTPases. Rachubik et al. used Rho GTPases inhibitors and siRNA in cultured cells and Zucker rats to explored PKGIα-Rac1-RhoA's role in podocyte cytoskeletal organization.

Insulin significantly increased glomerular albumin permeability (P_alb). This increase was reduced by Rac1 inhibitors EHT1864 and NSC23766 by 32% and 57%, respectively, (Fig. 4A). Conversely, RhoA inhibitors (Ripasudil acid and Y27632) increased P_alb by 240% and didn't affect P_alb with insulin. Similar trends were observed in podocyte layer albumin permeability (Fig. 4B). Insulin increased albumin flux by 197%, reduced in the presence of Rac1 inhibitors. RhoA inhibitors also elevated permeability through the podocyte monolayer. To investigate how small GTPases influence cytoskeleton dynamics and podocyte migration, they utilized NSC23766 and Y27632 inhibitors. Quantitative analysis showed that insulin increased F-actin near the plasma membrane (Fig. 5). They hypothesized that this insulin-driven cytoskeletal reorganization is Rac1-dependent, as preincubation with the Rac1 inhibitor NSC23666 lessened the effect. RhoA inhibition also mimicked insulin's effect (Fig. 5). Wound-healing assay revealed insulin boosted podocyte migration, blocked by NSC23766, but not by Y27632, indicating insulin's role as a key Rac1-dependent actin remodeling and migration regulator.

Fig. 4. Rho family GTPases regulate glomerular filtration barrier permeability (Rachubik R, Szrejder M, et al., 2022).

Fig. 5. Insulin redistributes F-actin to the cell periphery via Rac1 signaling pathway (Rachubik R, Szrejder M, et al., 2022).