Protective role of growth hormone against hyperhomocysteinemia-induced glomerular injury

Abstract

The present study investigated the protective role of growth hormone (GH) against hyperhomocysteinemia (hHcys)-induced activations of reactive oxygen species/hypoxia-inducible factor (HIF)-1α, epithelial-mesenchymal transition (EMT), and consequent glomerular injury. A hHcys model was induced by folate free diet in mice. The urine protein excretion significantly increased while plasma GH levels dramatically decreased in hHcys. Real-time reverse transcription polymerase chain reaction showed that GH receptor (GHR) level increased in the cortex of hHcys mice, which mainly occurred in podocytes as shown by confocal microscopy. Recombinant mouse growth hormone (rmGH) treatment (0.02 mg/kg, once a day for 6 weeks) significantly restored the plasma GH, inhibited GHR upregulation and attenuated proteinuria. Correspondingly, rmGH treatment also blocked hHcys-induced decrease in the expression of podocin, a podocyte slit diaphragm molecule, and inhibited the increases in the expression of desmin, a podocyte injury marker. It was also demonstrated that in hHcys the expression of epithelial markers, p-cadherin and ZO-1, decreased, while the expression of mesenchymal markers, antifibroblast-specific protein 1 (FSP-1) and α-SMA, increased in podocytes, which together suggest the activation of EMT in podocytes. Nicotinamide adenine dinucleotide phosphate oxidase (Nox)-dependent superoxide anion (O2 (.-)) and hypoxia-inducible factor-1α (HIF-1α) level in the hHcys mice cortex was markedly enhanced. These hHcys-induced EMT enhancement and Nox-dependent O2 (.-)/HIF-1α activation were significantly attenuated by rmGH treatment. HIF-1α level increased in Hcys-treated cultured podocytes, which were blocked by rmGH treatment. Meanwhile, homocysteine (Hcys)-induced EMT in cultured podocytes was significantly reversed by HIF-1α siRNA. All these results support the view that GH ameliorates hHcys-induced glomerular injury by reducing Nox-dependent O2 (.-)/HIF-1α signal pathway and EMT.

Fig. 1

Plasma GH level, mRNA and protein level of GHRs in glomeruli of mice on normal diet or FF diet with or without GH treatment. A. ELISA data of GH level in mice plasma (n=6/group). B. Real time PCR analysis of GHR mRNA expression. The data are presented as the fold changes compared with mice on the normal diet (n=7/group). C. Representative confocal fluorescent images for GHR and desmin in glomeruli from different mice. D. Summarized co-localization coefficient data showing co-localization levels of desmin and GHR in different groups of mice (n=4/group). * P<0.05 vs. mice on the normal group, # P<0.05 vs. vehicle-treated mice on hHcys group.

Fig. 2

Urinary protein, albumin excretion and morphological features of glomeruli in C57BL/6J mice on the normal diet or folate free diet with or without GH treatment. A. Urinary total protein excretion of mice on the normal diet or folate free diet with or without GH treatment (n=8–11/group). B. Urinary albumin excretion (n=9–11/group). C. Photomicrographs show typical glomerular structure (original magnification, 400×) (n=6 /group). D. Summarized data of glomerular damage index (GDI) by semiquantitation of scores in four different groups of mice. For each kidney section, 50 glomeruli were randomly chosen for calculation of GDI. * P<0.05 vs. mice on the normal group, # P<0.05 vs. vehicle-treated mice on hHcys group.

Fig. 3

Effects of GH on hHcys-induced changes in mRNA and protein expression of podocin and desmin in glomeruli from mice on the normal diet or FF diet. A. Real time PCR analysis of podocin mRNA level in the glomeruli of different groups of mice (n=6–7/group). B. Western blot analysis of podocin protein expression (n= 6/group). C. Summarized data of podocin protein density. D. Typical images of podocin staining in glomeruli (n=4/group). E. Real time PCR analysis of desmin mRNA expression in the glomeruli of different groups of mice (n=5–7/group). F. Western blot analysis of desmin protein expression (n=5/group). G. Summarized data of desmin protein density. H. Typical images of desmin staining in glomeruli (n=4/group). * P<0.05 vs. mice on the normal group, # P<0.05 vs. vehicle-treated mice on hHcys group.

Fig. 4

Immunofluorescent staining of EMT markers in glomeruli from mice on the normal diet or FF diet with or without GH treatment. A. Typical confocal microscopic results of Podocin vs. ZO-1 or P-cadherin staining in glomeruli from mice on the normal diet or FF diet with or without GH treatment (n=4 /group). B. Typical confocal microscopic results of podocin vs. FSP-1 or α-SMA staining in glomeruli from mice, respectively (n=4/group).

Fig. 5

GH inhibited hHcys-induced podocytes EMT in glomeruli from different groups of mice. A. Real time PCR analysis of ZO-1 and P-cadherin mRNA expression in glomeruli of different groups of mice (n=6–7/group). B. Western blot analysis of ZO-1 and P-cadherin protein expression in different groups of mice (n=5–7/group). C. Summarized data of ZO-1 and P-cadherin density. D. Real time PCR analysis of α-SMA and FSP-1 mRNA expression in glomeruli of different groups of mice (n=6–7/group) E. Western blot analysis of α-SMA and FSP-1 protein expression in different groups of mice. F. Relative quantitation of protein expression for Western blot analysis from different groups of mice (n=6/group). * P<0.05 vs. mice on the normal group, # P<0.05 vs. vehicle-treated mice on hHcys group.

Fig. 6

GH reduced hHcys-induced Nox-dependent O₂^·− and HIF-1α level in renal cortex tissue. A. Representative ESR spectra traces for Nox-dependent O₂^·− production in the cortex from mice on a normal diet or FF diet with or without GH treatment. B. Summarized data show fold changes in Nox-dependent O₂^·− production, which are normalized to vehicle-treated mice on the normal diet under different conditions (n=6/group). O₂^·− was measured by ESR with a trapping substance for SOD-sensitive O₂^·−. C. Western blot analysis of HIF-1α level in different groups of mice. D. Relative quantitation of protein expression for Western blot analysis from different groups of mice (n=6/group). * P<0.05 vs. mice on the normal group, # P<0.05 vs. vehicle-treated mice on hHcys group.

Fig. 7

Effect of rmGH on hHcy-induced increases in HIF-1α production in podocytes and HIF-1siRNA blocked hHcy-induced podocytes EMT. A. Western blot analysis of HIF-1α expression in different groups of mice. B. Relative quantitation of protein expression for Western blot analysis from different groups of cell (n=5/group). C. Western blot analysis of P-cadherin expression in different groups of cells. D. Summarized data of P-cadherin protein density. (n=5/group). E. Representative images show the expression of P-cadherin and FSP-1 as epithelial marker and mesenchymal marker in different groups (n=5/group).* P<0.05 vs. podocyte on the normal group, # P<0.05 vs. vehicle-treated podocyte on hHcys group.