Fig 1.
A) Fn14 mRNA expression is upregulated in disease vs vehicle in the NTN model (8% and 32% vs vehicle; p<0.001). B) Immunohistochemistry Score of Fn14 in kidney tissue in NTN model. The IHC staining of kidney shows increase in Fn14 protein consistent with the extent of injury in the NTN model (p<0.005). C) Fn14 IHC reveals progressive increase in Fn14 immunopositivity in glomeruli, vessels and tubular epithelium with disease progression in NTN model. D) Linear regression graph show a positive correlation between Fn14 expression by IHC- staining with the disease severity score by H&E in NTN model (p = 0.0008), R2 = 0.914 E) Fn14 mRNA expression is increased with progression of disease in MRL/lpr mice spontaneous mouse model of Lupus Nephritis. Kidneys from MRL/lpr mice were harvested at 22 weeks of age. Aged matched controls were kidneys from MRL/MPJ mice- comparable strain that does not develop Lupus Nephritis. F) IHC showing Fn14 expression in kidney of MRL/lpr model. Fn14 expression was most prominent in arterioles and glomerulus. G) Strong association of Fn14 with inflammation score in MRL/lpr model. Linear regression plot showing correlation of Fn14 expression with inflammation (p<0.0001) and R2 = 0.63.
Fig 2.
Fn14 concentration is increased in NTN model of kidney injury.
(A) Increased concentration of Fn14 was detected in serum of mice injected with nephrotoxic serum (NTS) (vehicle vs NTS-**p = 0.006, Mann-Whitney test). Serum analysis was performed at the end of the study at 21 days. (B) Rapid increase in Fn14 concentration in urine (closed circles) apparent two days post NTS injection, was accompanied by development of proteinuria (closed triangles), and remained elevated through the duration of the study. (C) Urinary Fn14 strongly correlated with urinary albumin (r = 0.8803; p<0.0001, Spearman’s test) and urinary MCP-1 (r = 0.8978; p<0.0001, Spearman’s test), on day 7 of NTN induction.
Fig 3.
Fn14 is upregulated in Folic Acid induced acute kidney injury.
Mice were systemically dosed with either vehicle or FA. (A) BUN and (B) Urine microalbumin are increased in FA treated mice 24h after dosing. Histology from (C) vehicle control kidney and (D) FA-treated mice show tubular dilatation and attenuation in FA dosed kidneys, indicating acute kidney injury. (E) sFn14 in serum and (F) sFn14 in urine is increased in FA-treated mice 24h after dosing. (G) qRT-PCR shows up-regulation of Fn14 mRNA in FA induced AKI kidneys. IHC from vehicle control kidney (H) and (I) FA-treated mice shows increased Fn14 immuno-reactivity in tubular epithelia of FA dosed mouse kidneys.
Fig 4.
Fn14 and TWEAK expression in DN.
A total of sixty male, 8-week-old ZSF1 obese and lean littermates were used in this study. (A) Urine levels of sFn14 and mRNA expression in kidney from ZSF1 lean (control) and obese (disease) rats (lean vs obese *p<0.0001). (B) Spearman correlation between urine sFn14 and total protein (r = 0.92, p<0.0001) in lean and obese ZSF1 rats (n = 5/group). (C) Plasma concentration of sFn14 (n = 32) and sTWEAK (n = 24) in healthy subjects. (D) Urine levels of sFn14 and sTWEAK in healthy controls (n = 10) and DN patients (n = 26). Data were normalized to urine creatinine, *p<0.0001, Mann-Whitney test. (E) Spearman correlation between urine sFn14, total protein (r = 0.5653, p<0.0005) and MCP-1 (r = 0.5790, p<0.0005), in healthy and DN patients (n = 36). (F) Immunohistochemistry showing Fn14 expression in kidney sections of healthy and DN patients. Fn14 is predominantly expressed in a fine reticular pattern that traces the glomerular mesangium and perivascular smooth muscle in the cortex of normal kidneys (arrows). In diabetic nephropathy, there is marked upregulation of Fn14 expression in the expanded mesangium of affected glomeruli (arrows).