Increased cellular senescence and vascular rarefaction exacerbate the progression of kidney fibrosis in aged mice following transient ischemic injury

Abstract

Recent findings indicate that elderly patients with acute kidney injury (AKI) have an increased incidence of progression to chronic kidney disease (CKD) due to incomplete recovery from an acute insult. In the current study, a co-morbid model of AKI was developed to better mimic the patient population and to investigate whether age exacerbates the fibrosis and inflammation that develop in the sequelae of progressive kidney disease following acute injury. Young (8-10 weeks) and aged (46-49 weeks) C57BL/6 mice were subjected to 30 min bilateral renal ischemia-reperfusion (I/R) to induce AKI. The aged animals have greater mortality and prolonged elevation of plasma creatinine correlating with less tubular epithelial cell proliferation compared to the young. Six weeks post-reperfusion, interstitial fibrosis is greater in aged kidneys based on picrosirius red staining and immunolocalization of cellular fibronectin, collagen III and collagen IV. Aged kidneys 6 weeks post-reperfusion also express higher levels of p53 and p21 compared to the young, correlating with greater increases in senescence associated (SA) β-galactosidase, a known marker of cellular senescence. A higher influx of F4/80(+) macrophages and CD4(+) T lymphocytes is measured and is accompanied by increases in mRNA of monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-α (TNF-α). Importantly, microvascular density is significantly less, correlating with an increase in nitro-tyrosine, a marker of oxidative stress. Collectively, these data demonstrate that prolonged acute injury in the aged animals results in an accelerated progression of kidney disease in a chronic state.

Figure 1. Aged mice have greater mortality and prolonged duration of AKI.

A. Percent survival is greater in young mice 6 weeks post-reperfusion (n = 14–15 at day 0). Plasma creatinine (B) and BUN (C) levels were measured at indicated times post-reperfusion (n = 4–10/group) with corresponding shams that are equivalent between young and aged regardless of timepoint. *p≤0.05 indicates a significant difference from the respective sham. **p≤0.05 indicates a significant difference from the young I/R at the same timepoint.

Figure 2. Histopathological injury and apoptosis are similar between young and aged kidneys whereas proliferation differs.

A. Representative micrographs of H&E stained sections at 24 hrs post-reperfusion. Four features of injury examined: intratubular protein casts (arrow, PC), exfoliated cells (*, EC), loss of tubular structure (LTS), and vascular congestion (^∧, VC). B. Injury score at 24 hrs (n = 8/group) post-reperfusion is similar between young and aged. C. The percent apoptotic cells is similar between the two groups as well (n = 3–4/group). D. The percent proliferation is significantly lower in the aged mice (n = 4–7/group). *p≤0.05 indicates a significant difference from young at same timepoint. No significant differences found (p≤0.05) in B and C. Bar = 100 microns.

Figure 3. Fibrosis is greater in aged kidneys 6 weeks post-reperfusion.

Representative micrographs of picrosirius red stained sections at 6 weeks post-reperfusion in the aged (A) and young (C) cortex as well as the aged (B) and young (D) outer medulla. The aged (E, F) and young (G) normal kidney have no interstitial fibrosis. Quantitation of percent picrosirius red staining (G) indicates significantly greater amounts in both the cortex and outer medulla of aged mice 6 weeks post-reperfusion. *p≤0.05 indicates a significant difference from respective normal. **p≤0.05 indicates a significant difference from young I/R. Bar = 100 microns.

Figure 4. Extracellular matrix proteins are greater in aged kidneys compared to young at 6 weeks post-reperfusion.

Representative micrographs of cellular fibronectin (A) and collagen III (B) immunostained sections of the outer medulla of young and aged kidneys. Quantitation of pixels per field indicates significantly increased matrix deposition in the aged kidneys 6 weeks post-reperfusion. (n = 4–5/group). There are equivalent low levels in young and age matched normals. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. Bar = 100 microns.

Figure 5. Expression of p53 and β-galactosidase is greater in aged kidneys at 6 weeks post-reperfusion.

Equivalent low levels are expressed in young and age matched normals. A. p53 expression levels measured by ELISA 6 weeks post-reperfusion (n = 4–6/group). B. Quantitation of percent β-galactosidase positive tubules (n = 4–5/group). *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. C. Representative micrographs of frozen sections of β-galactosidase staining in the outer stripe at 6 weeks post-reperfusion. Some tubules that are β-galactosidase positive are adjacent to fibrotic areas (arrow). Bar = 100 microns. D. Quantification of Western blots for p21 normalized to GAPDH in kidney tissue (n = 3/group) with a representative blot. *p≤0.05 indicates a significant difference between young and aged ischemia.

Figure 6. Inflammation in the cortex and outer medulla 6 weeks post-reperfusion is greater in aged kidneys.

Representative micrographs of F4/80 (A) and CD4 (B) immunostained sections of the outer medulla of young and aged kidneys. Quantitation of pixels per field indicate significantly increased numbers of cells in the aged kidneys 6 weeks post-reperfusion (n = 4–5/group). There are equivalent low levels in young and age matched normals. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. Bar = 100 microns. C. Gene expression of MCP-1 and TNF-α indicate increased mRNA levels in the aged kidneys 6 weeks post-reperfusion (n = 7 for young and 3 for aged mice). *p≤0.05 indicates a significant difference between young and aged ischemia.

Figure 7. Discrete areas of leukocyte infiltrates surrounding the vasculature are only present in aged animals 6 weeks post-reperfusion.

Representative H&E micrograph (A) showing a blood vessel surrounded by leukocytes. It is not the same vessel depicted in subsequent figures. Representative micrographs of serial sections indicate that CD4⁺ T lymphocytes (B) and CD19⁺ B lymphocytes (C) are the predominant immune cells in the area surrounding the blood vessel. Some CD8⁺ T lymphocytes also are present in lower numbers (D); however, F4/80⁺ macrophages do not co-localize with these leukocytes (E). *denotes the lumen of the blood vessel. Hatch marks indicate peripheral margin of the vascular sheath of denoted blood vessel. Bar = 100 microns.

Figure 8. Microvascular loss and oxidative stress are greater in the aged kidney 6 weeks post-reperfusion.

A. Representative micrographs of CD31-immunostained outer medulla from young and aged mice at 6 weeks post-reperfusion. The enlarged view (area indicated by box) highlights CD31⁺ endothelial cells lining the vasculature. Quantitation of CD31 immunostaining is shown in the graph. There is no difference between young and aged normal; however, vascular loss is greater in the aged kidney 6 weeks post-reperfusion. *p≤0.05 indicates a significant difference vs respective normal. **p≤0.05 indicates a significant difference between young and aged ischemia. B. Oxidative stress measured by nitro-tyrosine staining is greater in the outer medulla of aged kidneys 6 weeks post-reperfusion as compared to the young. Images were recorded with identical exposure settings. Bar = 100 microns.