Effects of zinc in podocytes and cortical collecting duct in vitro and Dahl salt-sensitive rats in vivo

Abstract

Zinc is one of the essential divalent cations in the human body and a fundamental microelement involved in the regulation of many cellular and subcellular functions. Experimental studies reported that zinc deficiency is associated with renal damage and could increase blood pressure. It was proposed that zinc dietary supplementation plays a renoprotective role. Our study aimed to investigate the effects of zinc on intracellular signaling in renal cells and explore the correlation between dietary zinc and the progression of salt-induced hypertension. The impact of extracellular zinc concentrations on two different kidney epithelial cell types, podocytes and principal cells of the cortical collecting duct (CCD), was tested. In podocytes, a rise in extracellular zinc promotes TRPC6 channel-mediated calcium entry but not altered intracellular zinc levels. However, we observe the opposite effect in CCD cells with no alteration in calcium levels and steady-state elevation in intracellular zinc. Moreover, prolonged extracellular zinc exposure leads to cytotoxic insults in CCD cells but not in podocytes, characterized by increased cell death and disrupted cytoskeletal organization. Next, we tested if dietary zinc plays a role in the development of hypertension in Dahl salt-sensitive rats. Neither zinc-rich nor deficient diets impact the regular development of salt-sensitive hypertension. These results suggest specialized roles for zinc in renal function, implicating its involvement in proliferation and apoptosis in CCD cells and calcium signaling and cytoskeletal dynamics modulation in podocytes. Further research is required to elucidate the detailed mechanisms of zinc action and its implications in renal health and disease.

Keywords: blood pressure; calcium; kidney; podocyte; zinc.

Figure 1

Zinc induces changes in intracellular calcium levels in podocytes without affecting intracellular zinc levels. Representative fluorescence images of intracellular zinc (A) and calcium (B) changes in human podocytes in response to acute application of ZnCl₂ (100 μM). In podocytes, ZnCl₂ application did not cause changes in intracellular zinc levels (A), but caused changes in intracellular calcium levels (B). The statistical summaries show the changes in intracellular calcium with time and maximum ΔF/F_base ratio before and after application (podocytes control: 1.2 ± 0.4 vs. 2.6 ± 1.1, n ≥ 20, N ≥ 3, Mann-Whitney, p < 0.0001). Additionally, calcium response in podocytes was blunted in 0 Ca²⁺ solution as well as in the presence of SAR7334 (20 μM), a specific TRPC6 inhibitor (C; Control vs. 0 Ca²⁺: 2.6 ± 1.1 vs. 1.6 ± 0.7, p < 0.0001; Control vs. SAR: 2.6 ± 1.1 vs. 1.4 ± 0.6, p < 0.0001; and 0 Ca²⁺ vs. SAR: 1.6 ± 0.7 vs. 1.4 ± 0.6, p = 0.0215; n ≥ 20 cells, N ≥ 3, ANOVA). The scale bar is 100 μm.

Figure 2

Zinc induces changes in intracellular zinc levels in mCCD without affecting intracellular calcium levels. Representative fluorescence images of intracellular zinc (A) and calcium (B) changes in mCCD in response to acute application of ZnCl₂ (100 μM). In mCCD, ZnCl₂ application caused changes in intracellular zinc levels (A) but did not cause changes in intracellular calcium levels (B). The statistical summaries show changes in zinc with time and maximum ΔF/F_base ratio before and after application (mCCD: 0.98 ± 0.03 vs. 3.78 ± 0.95; n ≥ 20 cells, N ≥ 3, t test, p < 0.001). The scale bar is 100 μm for the main images and 30 μm for the zoomed-in sections.

Figure 3

Zinc induces cell death in mCCD, but not in podocytes. Representative images of cell death in mCCD (A) and immortalized human podocytes (B) within 20 h after ZnCl₂ application. Incubation of podocytes with 100 μM of ZnCl₂ (∗∗∗, ###p < 0.001, ANOVA, N = 6) resulted in a higher number of Nuclear Green-marked cells after 20 h compared to the control. H₂O₂ (1 mM) was used as a positive control in both cases. The scale bar is 1 mm.

Figure 4

Impact of zinc on cytoskeletal organization in mCCD cells and podocytes. Immunofluorescence images of mCCD cells (A) and podocytes (B) stained for microtubule (α-Tubulin, top row) and actin network (F-actin, middle row) under control conditions and after treatment with ZnCl₂ (100 μM). In the control group, the microtubule network appears dense and uniformly distributed with fewer gaps and less defined cell boundaries, while the actin filaments form a continuous and intricate network. Upon ZnCl₂ treatment, the microtubule network becomes more perinuclear dense with more defined cell boundaries, and the actin network shows noticeable fragmentation and reduced filamentous length. In the merged images, cyan represents microtubules (α-Tubulin), and red represents actin (F-actin). Quantitative analysis of the total filament length and the length of filaments ≥10 μm showed a significant reduction in filament length upon ZnCl₂ treatment compared to controls, both in mCCD cells (C, Total length: Control 7.4 ± 4.8 vs. Zn 6.9 ± 3.8 μm; Length ≥ 10 μm: Control 16 ± 5.2 vs. Zn 14.6 ± 3.9 μm; n ≥ 3, N ≥ 3, Kolmogorov-Smirnov, p < 0.001) and in podocytes (D, Total length: Control 7 ± 5.2 vs. Zn 6.6 ± 4 μm; Length ≥ 10 μm: Control 16 ± 8.4 vs. Zn 15.6 ± 6.2 μm; n ≥ 3, N ≥ 3, Kolmogorov-Smirnov, p < 0.001). The scale bar is 100 μm.

Figure 5

Zinc supplementary or zinc-deficient diets do not affect salt-induced hypertension in Dahl salt-sensitive (ss) rats. The development of mean MAP in male (A) and female (B) Dahl SS rats fed with zinc-deficient (<5 ppm) or zinc-supplementary (180 ppm) high-salt diets over 28 days. We observed no difference in MAP between the supplementary and deficient diets. Additionally, no changes were detected in diuresis (C, D), urine electrolytes, kidney weight, heart weight, and body weight (E, F) under zinc-deficient or zinc-supplementary high-salt diets within 28 days. These results suggest that zinc supplementation does not have a significant impact on blood pressure and renal function in Dahl SS rat model of salt-sensitive hypertension. 24 h zinc excretion (G), as well as zinc/creatinine ratio (H), were significantly higher in the zinc-supplementary group at Day 28, compared to control and zinc-deficient groups. Light grey, white, and dark grey represent control, zinc-deficient, or zinc-supplementary diets, respectively.