The Role Of Progranulin In Hyperhomocysteinemia-Induced Kidney Injury

ObjectiveHyperhomocysteinemia (hHcys) has been recognized as a critical risk and pathogenic factor in the progression of end stage renal disease (ESRD). Recently, evidence is accumulating that hHcys may directly act on glomerular cells to induce glomerular dysfunction and consequent glomerular sclerosis, leading to ESRD. As we all know, glomerular filtration barrier consists of glomerular endothelial cells, glomerular basement membrane and podocytes. The injury of podocytes may lead to proteinuria, while dysfunction of endothelial cells may increase inflammation response. Glomerular endothelial cells and podocytes are considered as an important role in maintaining renal function and glomerular filtration barrier selective permeability. The mechanisms involved in hHcys-induced kidney injury, especially in podocytes and glomerular endothelial cells,have not been fully understood.Progranulin (PGRN) is an autocrine growth factor, contains seven-and-a-half repeats of a cysteine-rich motif. PGRN is abundantly expressed in epithelial cells, immune cells, neurons, and chondrocytes, and reportedly plays a critical role in many physiological and disease processes such as:cell proliferation, tumorigenesis, insulin resistance, frontotemporal dementia, wound healing and inflammation. Our previous studies have found that PGRN attenuates kidney injury induced by acute kidney injury through the suppression of inflammation response. However, the role of PGRN in hHcys-nduced chronic kidney disease have not elucidated.Wnt/β-catenin signaling has a central role in mediating podocyte dysfunction and proteinuria, which may lead to kidney injury. PGRN loss caused aberrant activation of the Wnt signaling pathway during frontotemporal lobar degeneration. Nothing further is known about the relationship of PGRN with Wnt/β-catenin signaling in kidney injury.We employed this model in vivo and vitro to determine the effect and the mechanism of PGRN in hyperhomocysteinemia-induced kidney injury.Methods and Results1. The expression of PGRN in mice kidney after hHcys injuryEight weeks wild male C57BL/6J mice were divided into two groups randomly. After a 1-week recovery period from uninephrectomy, the mice were maintained on a normal diet or a folate-free (FF) diet to induce hHcys for 10 weeks. Plasma total Hcys was measured by high performance liquid chromatography (HPLC) analysis for model evaluation. The HPLC assay results showed plasma tHcys levels were significantly higher than in normal diet group, suggesting that hHcys mouse model to replicate successfully. Urinary albumin excretion was measured using a mouse albumin enzyme-linked immunosorbent assay (ELISA) quantitation kit. Urine albumin-to-creatinine ratio was significantly increased in hHcys mice. Glomerular morphology changes were observed by periodic acid-schiff stain (PAS) staining and electron microscope, in hHcys mice, showed that varying degrees of enhanced mesangial expansion, glomerular basement membrane uneven thickness, podocyte foot processes fusion, which indicated that the renal function and glomerular were impaired. Plasma PGRN was measured by ELISA. PGRN expression was measured using western blot(WB), and immunohistochemistry(IHC) staining. Coimrnunofluorescence staining for PGRN and renal cells marker were used in the kidney from mice with hHcys. Both of PGRN in serum and renal cortex were reduced. Immunofluorescence microscopy detection displayed PGRN protein decreased in glomerular endothelial cells, podocytes from the mice with hHcys. In vitro, glomerular endothelial cells (GEnC), podocytes (HPC), rat mesangial cells (RMC) and human renal proximal tubular cells (HK-2) were treated by different concentration of L-Hcys. WB analyses showed that PGRN levels were concentration dependently reduced in GEnC and HPC.2. The role of PGRN in mice kidney after hHcys injuryEight weeks wild male C57BL/6J mice and PGRN-deficient mice (B6(Cg)-Grntm1.1AidiJ) mice were divided into four groups randomly. Detection of plasma tHcys levels, Urine albumin-to-creatinine ratio, PAS staining and electron microscope, the results showed that compared with plasma tHcys, urine albumin-to-creatinine ratio was significantly increased and morphology aggravate in PGRN-deficient mice. Chemokines and cytokines in renal cortex were measured with real time RT-PCR. Macrophages(CD68) and neutrophils (Ly6B) expression were observed by immunohistochemistry. PGRN deficiency increased the levels of inflammation responses and macrophages(CD68), neutrophils (Ly6B) accumulation. The expression and the morphology of slit diaphragm protein nephrin and podocin were detected by WB and immunofluorescence, meanwhile WB detected the protein level of occludin (a tight-junction protein). PGRN deficiency exacerbated hHcys-induced nephrin and podocin injury and reduced the expression of occludin. In vitro, glomerular endothelial cells (GEnC) were subjected the treatment of L-Hcys or L-Hcys with recombinant PGRN (rPGRN) pretreatment, immunofluorescence analysis clarified the damaged ZO-1 (a tight-junction protein) protein was recovered in GEnC after pretreatment with rPGRN, accompanied by increased expression of ZO-1. accompanied by increased expression of ZO-1.3. The mechanism of PGRN ameliorated hyperhomocysteinemia-induced glomerular injuryWB analysis showed that FF diet and PGRN deficiency induced activation of p1-catenin in the kidney. In vitro, treatment of GEnC and HPC with L-Hcys induced the activation of β-catenin, moreover rPGRN pretreatment restored the expression of activated (β-catenin to some extent.ConclusionsOur results for the first time found that PGRN was reduced in the renal cortex of hHcys mice model and PGRN levels were concentration dependently reduced in GEnC and HPC. We further showed that PGRN deficiency exacerbated glomerular injury, proinflammatory response, inflammatory cells accumulation, podocytes slit diaphragm protein and tight-junction protein damaged in mice after hHcys. PGRN reversed GEnC dysfunction with Hcys treatment and protected hHcys-induced kidney injury. In this work, we suggested that Wnt/β-catenin is one of potential signaling pathways that links PGRN to hyperhomocysteinemia-nduced glomerular injury. Our study may help illuminate the mechanism of hHcys-induced kidney injury, moreover pharmacological targeting of PGRN may help design new approach to develop therapeutic strategies for treatment of hHcys-induced kidney injury.