Constitutive renal Rel/nuclear factor-κB expression in Lewis polycystic kidney disease rats

Abstract

Author contributions: Schwensen KG conducted the animal study, performed sample collection and histological staining; Ta MHT performed the immunofluorescence, Western blotting, and qPCR experiments, analyzed data and drafted the manuscript; Huso DL and Watnick T developed the Pkd2 knockout mouse model and provided paraffin embedded sections that were utilized for staining; Liuwantara D assisted with data interpretation and provided technical guidance with experimental methods; Rangan GK conceived of the study, conducted the animal study, performed sample collection and histological staining and reviewed and edited the manuscript; all authors read and approved of the final manuscript. Supported by Basic Study(LPK, a genetic ortholog of human nephronopthsis-9) from postnatal weeks 3 to 20. At each timepoint, renal disease progression and the mRNA expression of NF-κB-dependent genes (TNFa and CCL2) were determined. NF-κB was also histologically assessed in human PKD tissue. RESULTS:Progressive kidney enlargement in LPK rats was accompanied by increased renal cell proliferation and interstitial monocyte accumulation (peaking at weeks 3 and 10 respectively), and progressive interstitial fibrosis (with a smooth muscle actin and Sirius Red deposition significantly increased compared to Lewis kidneys from weeks 3 to 6 onwards). Rel/NF-κB proteins (phosphorylated-p105, p65, p50, c-Rel and RelB) were expressed in cystic epithelial cells (CECs) of LPK kidneys as early as postnatal week 3 and sustained until latestage disease at week 20. From weeks 10 to 20, nuclear p65, p50, RelB and cytoplasmic IκBa protein levels, and TNFa and CCL2 expression, were upregulated in LPK compared to Lewis kidneys. NF-κB proteins were consistently expressed in CECs of human PKD. The DNA damage marker γ-H2AX was also identified in the CECs of LPK and human polycystic kidneys. CONCLUSION:Several NF-κB proteins are consistently expressed in CECs in human and experimental PKD. These data suggest that the upregulation of both the canonical and non-canonical pathways of NF-κB signaling may be a constitutive and early pathological feature of cystic renal diseases. Core tip: Until now, there has been limited information regarding the specific nuclear factor (NF)-κB proteins involved in polycystic kidney disease (PKD) and their expression throughout disease progression. Our study demonstrated that a diverse array of NF-κB proteins is expressed in the renal cyst-lining cells of a chronic rodent model of PKD, and that NF-κB expression is constitutive over time. NF-κB was also identified in human PKD, suggesting that NF-κB upregulation is common to renal cystic disease models. Our data suggest that components of both the canonical and non-canonical NF-κB pathway are upregulated in PKD. Future studies should be directed at verifying whether specific NF-κB inhibition can attenuate interstitial inflammation and cyst growth, and slow the decline in renal function in in vivo models of PKD.Ta MHT, Schwensen KG, Liuwantara D, Huso DL, Watnick T, Rangan GK. Con...

Effects of TORC1 Inhibition during the Early and Established Phases of Polycystic Kidney Disease

“…We hypothesised that sirolimus would negatively regulate NF-κB via mTOR in PKD, but found that it did not alter the renal expression of the p65 DNA binding activity, p-p105, or NF-κB-dependent genes TNFα and CCL2 . A previous study in LPK rats demonstrated that renal TNFα and CCL2 are elevated in the late stages of disease (week 10 to 20) [ 22 ], and our current data shows that sirolimus was ineffective in suppressing transcription these genes. Further in vitro studies in cystic renal cells are required to prove a direct linkage between NF-κB and mTOR.…”

“…Three studies were undertaken: (i) Study 1 : To determine the time-dependent changes in the expression of downstream targets for TORC1 (phosphorylated S6 ribosomal protein and 4E-BP1) and TORC2 (phosphorylated Akt) in male Lewis and LPK rats with disease progression, kidney tissue was examined at postnatal weeks 1, 3, 6, 10, 16 and 20 (n = 3 for Lewis and n = 6 for LPK per timepoint) using archival tissue from another experiment [ 22 ]; (ii) Study 2 : To determine the effects of early treatment with sirolimus on renal cystic disease in LPK rats, three week old male rats received either the vehicle or sirolimus (0.2 mg/kg per day by intraperitoneal injection for 5 days per week) from week 3 until week 10 (n = 9 for LPK+sirolimus, n = 11 for LPK+vehicle). Age-matched male Lewis animals received either sirolimus or vehicle (n = 4 per group).…”

Effect of dimethyl fumarate on renal disease progression in a genetic ortholog of nephronophthisis

“…Western blot was done using a previously described method. 36 Blots were probed with NF-jB p105/p50 (1:1000, ab32360, Abcam) and b-actin (1:1000, #4970, Cell Signaling) and visualized on an Odyssey Imaging System (Licor). Densitometry was quantified using ImageJ (v1.47, National Institutes of Health, United States) and normalized using b-actin.…”

Reactive Oxygen Species and Redox Signaling in Chronic Kidney Disease

“…NF-κB consists of a family of transcription factors that play critical roles in inflammation, as well as cell proliferation, differentiation, and survival [110]. Several in vitro studies, as well as experimental animal models and human studies have shown persistent activation of NF-κB and its pathogenic role in mediating chronic inflammation in CKD [111][112][113][114]. This redox-sensitive transcription factor can modulate DNA-binding activity of its multiple homo-and heterodimers, activate or inactivate the inhibitory κB kinase complex, or activate NF-κB by alternative inhibitor κBα phosphorylation [115].…”