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. 2020 Mar 31;10(1):5719.
doi: 10.1038/s41598-020-62016-9.

Podocyte-targeted Heme Oxygenase (HO)-1 overexpression exacerbates age-related pathology in the rat kidney

Elpida Poulaki  1 , Maria G Detsika  2 , Eythimia Fourtziala  1 , Elias A Lianos  3 , Hariklia Gakiopoulou  1
Affiliations

Podocyte-targeted Heme Oxygenase (HO)-1 overexpression exacerbates age-related pathology in the rat kidney

Elpida Poulaki et al. Sci Rep. .

Abstract

Although Heme Oxygenase-1 (HO-1) induction in various forms of kidney injury is protective, its role in age-related renal pathology is unknown. In the ageing kidney there is nephron loss and lesions of focal glomerulosclerosis, interstitial fibrosis, tubular atrophy and arteriolosclerosis. Underlying mechanisms include podocyte (visceral glomerular epithelial cell/GEC) injury. To assess whether HO-1 can attenuate ageing - related lesions, rats with GEC-targeted HO-1 overexpression (GECHO-1 rats) were generated using a Sleeping Beauty (SB) transposon system and extent of lesions over a 12-month period were assessed and compared to those in age-matched wild-type (WT) controls. GECHO-1 rats older than 6 months developed albuminuria that was detectable at 6 months and became significantly higher compared to age-matched WT controls at 12 months. In GECHO-1 rats, lesions of focal segmental and global glomerulosclerosis as well as tubulointerstitial lesions were prominent while podocytes were edematous with areas of foot process effacement and glomerular basement membrane thickening and wrinkling. GECHO-1 rats also developed hemoglobinuria and hemosiderinuria associated with marked tubular hemosiderin deposition and HO-1 induction, while there was depletion of splenic iron stores. Kidney injury was of sufficient magnitude to increase serum lactate dehydrogenase (LDH) and was oxidative in nature as shown by increased expression of 8-hydroxydeoxyguanosine (8-OHdg, a byproduct of oxidative DNA damage) in podocytes and tubular epithelial cells. These observations highlight a detrimental effect of podocyte-targeted HO-1 overexpression on ageing-related renal pathology and point to increased renal iron deposition as a putative underlying mechanism.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Changes in urine albumin excretion, serum creatinine and serum urea levels in GECHO-1 and WT rats with time. Albuminuria, expressed as Ualb/Ucr (mg albumin/mg creatinine) (a), Serum Creatinine (mg/dL) (b) and Serum Urea (mg/dL) (c) in 2-, 4-, 6-, and 12-month old GECHO-1 rats and WT controls. Values are expressed as mean ± SEM. ****p < 0.0001 12 month old GECHO-1 vs 12 month old WT, ####p < 0.0001 12 month old GECHO-1 vs 2 month old GECHO-1, **p < 0,01 12 month old GECHO-1 vs 12 month old WT.
Figure 2
Figure 2
Glomerular and non-glomerular injury in aging GECHO-1 rats. Glomerular and tubulointerstitial lesions in 12-month old rats with GEC-targeted HO-1 overexpression (GECHO-1, a,b,c,d) and age-matched WT control rats (e,f,g,h). In glomeruli of GECHO-1 rats, focal or global segmental sclerosing lesions were more extensive (a vs e, arrows: segmental sclerosis; arrowhead: global sclerosis, PAS stain x100), as were cystic dilatation of tubular lumens (b vs f, HE stain x100), interstitial inflammatory infiltrates [stars in c vs g, PAS stain x200 (c) and HE stain x100 (g)], presence of proteinaceous casts (c vs g), and thickening of the Bowman’s capsule [d vs h, HE stain x400 (d); PAS stain x400 (h)]. Hyalinosis lesions were also present in sclerosed glomeruli of GECHO-1 rats (d, arrow). HE: hematoxylin/eosin. PAS: periodic acid Schiff.
Figure 3
Figure 3
Electron microscopy of 12-month old GECHO-1 rat glomeruli compared to age-matched WT. (a) Electron microscope image (magnification: x5600) of glomeruli in a 12-month old WT rat showing well-preserved foot processes and glomerular basement membrane. (b,c) Electron microscope images (magnification: x4400) of glomeruli in a 12-month old GECHO-1 rat with segmental glomerular lesion, hyalinosis (white arrow in b), wrinkled basal lamina (black arrow in b), extensive effacement of foot processes and abundant vacuoles within epithelial cells (star in b) and GEC swelling (stars in c).
Figure 4
Figure 4
ΗΟ-1 expression and hemosiderin deposition in aging GECHO-1 rat kidney tissue. (a) prominent HO-1 protein immunolocalization in epithelial cells of the proximal tubule arising from the urinary pole of the glomerulus and in epithelial cells of surrounding tubules (x600). HO-1 is also expressed in podocytes (black arrow), as expected from targeted HO-1 overexpression achieved in these cells. (b) HO-1 protein expression in an albuminuric WT rat (less prominent and extensive in tubules) (x400). Hemosiderin staining (Prussian blue, x400) in kidney sections of 12-month old GECHO-1 (c) and WT rat (d).
Figure 5
Figure 5
Changes in HO-1 expression in aging GECHO-1 and WT rats. Immunolocalization of HO-1 protein HO-1 expression in cortical sections of 2-, 6-, and 12-month old GECHO-1 rats and in age-matched WT controls, magnification x400. Age-dependent increase in HO-1 expression in GECHO-1 rats (a,b,c) compared to WT controls (d,e,f).
Figure 6
Figure 6
Hemosiderin deposition in kidney and spleen sections of aging GECHO-1 and WT rats. Hemosiderin deposition (Prussian blue stain x200) in renal cortical sections of 2-, 6-, and 12-month old GECHO-1 rats and age-matched WT controls as well as in spleen sections of 12-month old GECHO-1 rats and age-matched WT controls. Fine hemosiderin granules in cytoplasm of tubular epithelial cells became apparent as early as 2 months in GECHO-1 rats (a). Coarse granules of hemosiderin deposition in tubular epithelial cells and in lumen of distal tubules (c, black arrow) of 6- and 12-month old GECHO-1 rats (b,c). Deposition most prominent at 12 months. No detectable hemosiderin in cortical section of 2-month old WT rat (e). Detectable but sparsely distributed hemosiderin in cortical sections of 6- and 12-month old WT rats (f,g, arrows). Markedly reduced hemosiderin deposition in spleen sections of 12-month-old GECHO-1 rats (d) compared to age-matched WT controls (h).
Figure 7
Figure 7
Presence of oxidative stress in aging GECHO-1 rats. Immunolocalization of 8-Ohdg (a marker of oxidative RNA and DNA damage by reactive oxygen and nitrogen species) in GECHO-1 12-month old rat. Prominent 8-OHdG immunolocalization detected in podocytes (b, arrow) and in tubular epithelial cells of an albuminuric GECHO-1 rat (b,c) compared to a 12-month old WT (a), magnification x400.
Figure 8
Figure 8
Changes in serum lactate dehydrogenase (LDH) and iron (Fe) levels in GECHO-1 and WT rats. Serum LDH (IU/L) and Fe (μg/dL) in 2-, 4-, 6-, and 12-month old WT and GECHO-1 rats. Values are expressed as mean ± SEM. (a): Significant increase in LDH at 12-month old GECHO-1 rats compared to WT controls. (b): No significant changes in serum iron. ****p < 0.0001 12 month old GECHO-1 vs 12 month old WT.
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