Kidney Damage in Long COVID: Studies in Experimental Mice

Abstract

Signs and symptoms involving multiple organ systems which persist for weeks or months to years after the initial SARS-CoV-2 infection (also known as PASC or long COVID) are common complications of individuals with COVID-19. We recently reported pathophysiological changes in various organs post-acute infection of mice with mouse hepatitis virus-1 (MHV-1, a coronavirus) (7 days) and after long-term post-infection (12 months). One of the organs severely affected in this animal model is the kidney, which correlated well with human studies showing kidney injury post-SARS-CoV-2 infection. Our long-term post-infection pathological observation in kidneys includes the development of edema and inflammation of the renal parenchyma, severe acute tubular necrosis, and infiltration of macrophages and lymphocytes, in addition to changes observed in both acute and long-term post-infection, which include tubular epithelial cell degenerative changes, peritubular vessel congestion, proximal and distal tubular necrosis, hemorrhage in the interstitial tissue, and vacuolation of renal tubules. These findings strongly suggest the possible development of renal fibrosis, in particular in the long-term post-infection. Accordingly, we investigated whether the signaling system that is known to initiate the above-mentioned changes in kidneys in other conditions is also activated in long-term post-MHV-1 infection. We found increased TGF-β1, FGF23, NGAL, IL-18, HIF1-α, TLR2, YKL-40, and B2M mRNA levels in long-term post-MHV-1 infection, but not EGFR, TNFR1, BCL3, and WFDC2. However, only neutrophil gelatinase-associated lipocalin (NGAL) increased in acute infection (7 days). Immunoblot studies showed an elevation in protein levels of HIF1-α, TLR-2, and EGFR in long-term post-MHV-1 infection, while KIM-1 and MMP-7 protein levels are increased in acute infection. Treatment with a synthetic peptide, SPIKENET (SPK), which inhibits spike protein binding, reduced NGAL mRNA in acute infection, and decreased TGF-β1, BCL3 mRNA, EGFR, HIF1-α, and TLR-2 protein levels long-term post-MHV-1 infection. These findings suggest that fibrotic events may initiate early in SARS-CoV-2 infection, leading to pronounced kidney fibrosis in long COVID. Targeting these factors therapeutically may prevent acute or long-COVID-associated kidney complications.

Keywords: SARS-CoV-2; fibrosis; inflammation; kidney; long COVID.

Figure 1

Altered mRNA expressions 7-days post-MHV-1 infection (acute). (A) EGFR mRNA level is not significantly changed in infected as well as treatment with SPK. (B) TGFβ mRNA level is not significantly changed in infected as well as treatment with SPK. (C) FGF23 mRNA level is not significantly changed in infected as well as treatment with SPK. (D) NGAL mRNA level is significantly increased in the infected group, while its mRNA level is significantly decreased following SPK treatment. (E–L) mRNA level is not significantly changed in the infected group, while its mRNA level decreased not significantly following SPK treatment. Values are the mean SD of three independent experiments. * = p < 0.05 is statistically significant. ns = nonsignificant.

Figure 2

Altered mRNA expressions 12 months post-MHV-1 infection (chronic). (A) EGFR mRNA level is not significantly changed in infected as well as treatment with SPK. (B) TGF-β1 mRNA level is significantly increased in the infected group, while its mRNA level is significantly decreased following SPK treatment. (C) FGF-23 mRNA level is significantly increased in the infected group, and treatment with SPK leads to substantial elevation. (D) NGAL mRNA level is significantly increased in the infected group, while its mRNA level decreased not significantly following SPK treatment. (E) IL-18 mRNA level is significantly increased in the infected group, while its mRNA level decreased not significantly following SPK treatment. (F) HIF-1α mRNA level is significantly increased in the infected group, and treatment with SPK leads to substantial elevation. (G) TLR-2 mRNA level is significantly increased in the infected group, and treatment with SPK leads to significant elevation. (H) TNF-R1 mRNA level is not significantly changed in infected as well as treatment with SPK. (I) YKL-40 mRNA level is significantly increased in the infected group, while its mRNA level increased not significantly following SPK treatment. (J) BCL3 mRNA level is not significantly increased in the infected group, while its mRNA level is significantly decreased following SPK treatment. (K) B2M mRNA level is significantly increased in the infected group, and treatment with SPK leads to significant elevation. (L) WFDC2 mRNA level is not significantly changed in infected as well as treatment with SPK. Values are the mean SD of three independent experiments. * = p < 0.05 is statistically significant; ** = p < 0.01 is statistically significant; *** = p < 0.001 is statistically significant; **** = p < 0.0001 is statistically significant; ns = nonsignificant.

Figure 3

(A) Representative immunoblots changes in HIF1α, TLR2, and EGFR protein levels. All three protein levels increased 12 months post-infection in kidney cells in mice. Treatment with SPK (5 mg/kg) reduced all three protein levels significantly; HIF-1α and TLR2 levels are reduced below the normal (B,C) and EGFR levels, approximating the control (D). Values are the mean SD of three independent experiments. * p < 0.05 is statistically significant vs. Sham; † p < 0.05 is statistically significant vs. MHV-1.

Figure 4

(A,B) Alteration of the immunofluorescence levels of KIM-1, MMP-7, YKL-40, and EGFR in the kidneys of mice 7 days post-MHV-1 infection. KIM-1 and MMP-7 levels are significantly elevated compared to the normal group (Sham). However, YKL-40 and EGFR expressions are markedly reduced in comparison with the health control (Sham). Values are the mean SD of three independent experiments. Scale bar = 35 mm. * p < 0.05 is statistically significant increase vs. Sham; † p < 0.05 is considered to be statistically significant decrease vs. MHV-1.

Figure 5

(A,B) Alteration of the immunofluorescence levels of KIM-1, MMP-7, YKL-40, and EGFR in the kidneys of mice 12 months post-MHV-1 infection. KIM-1, MMP-7 and YKL-40 levels are significantly reduced compared to the normal (Sham). However, EGFR expressions are increased in comparison with the health control (Sham). Fluorescence intensities for those markers confirmed the immunofluorescence findings. Values are the mean SD of three independent experiments. Scale bar = 35 mm. * p < 0.05 is a statistically significant decrease vs. Sham; † p < 0.05 is considered to be a statistically significant increase vs. MHV-1.