Effect Of Low Protein Diet Supplemented With α-Ketoacids On Renal Fibrosis And Its Mechanisms

Chronic kidney disease (CKD) is one of the most common kidney diseases and may cause end stage renal disease without effective therapeutic interventions. Renal fibrosis is the result of extracellular matrix accumulation and a hallmark of progressive CKD. Multiple factors, including oxidative stress, chronic inflammation and transforming growth factorβ(TGF-β), are all implicated in the development and progression of renal fibrosis, but the exact mechanisms of CKD remain unknown. Kruppel-like factor 15 (KLF15) is a zinc finger-containing transcription factor and plays an important role in regulation of differentiation, proliferation and apoptosis. In 2007, Fisch S et al reported that KLF15 may play an important role in heart fibrosis, and further study indicated that the levels of type I collagen transcription and the activity of connective tissue growth factor (CTGF) promoter were decreased by KLF15. As heart fibroses share similar mechanisms, KLF15 may also be an important factor in renal fibrosis.Dietary protein restriction is a major therapy in CKD. Lowering dietary protein intake to slow down the progression of renal failure would decrease the metabolic burden, especially the levels of urea nitrogen. Since malnutrition is a concern for long-term protein restriction, ketoacida, a nitrogen free substitution for the essential amino acids, have been prescribed together with low protein diet to CKD patients. Ketoacids are equally efficient as their essential amino acids counterpart in protein synthesis. Furthermore, they reduce accumulation of nitrogen waste products by decreasing exogenous nitrogen supply and enhancing reutilization of stored nitrogen. However, it is still unclear whether low protein diet supplemented with ketoacids is better than low protein diet alone in renal protection of CKD patients, and the mechanisms by which they decrease the progression of CKD are unknown. Knowing that oxidative stress and inflammation are implicated in the development of renal fibrosis, we hypothesized that low protein diet supplemented with ketoacids might offer a better protective effect than low protein diet alone. We further evaluated the effect of diet treatment on the level of KLF15 expression and explored the mechanisms in the progression of renal fibrosis in a CKD rat model.Forty-five 5/6 nephrectomy SD rats were equally randomized into three groups:NPD group (22% protein); LPD group (6% protein), and LPD+KA group (5% protein+1% ketoacids), all for 24 weeks. Sham operated rats (n=12) were used as control. Blood and urine samples were collected for biochemical test. Remnant or control kidneys were removed, embedded and sliced in sections, and stained with Masson's, type I collagen and fibronectin immunohistochemical staining as well as electron microscope study. Total RNA was extracted from renal tissue and expressions of fibrosis-related genes including TGF-β1, typeⅠ, type III, type IV collagen and fibronection were examined by real-time PCR. BUN, Proteinuria, decreased renal function, glomerular sclerosis and tubulointerstitial fibrosis were observed in the remnant kidneys of NPD group. Protein restriction ameliorated above changes, and the effect was more obvious in LPD+KA group (P<0.05) than LPD group. Lower body weight and serum albumin level were found in LPD group, indicating protein malnutrition (P<0.05). Since protein malnutrition has been reported to cause oxidative stress and inflammation, which are implicated in the development and progression of renal fibrosis, it seems that low protein diet supplemented with ketoacids plays a more protective role than low protein diet alone in protecting remnant kidney function against progressive injury. The effect may be mediated by ameliorating malnutrition, thus protecting the remnant kidney against oxidative stress and inflammation injury.Levels of oxidative stress and inflammation were further measured to convince the role that they played in protecting CKD renal function by low protein diet supplemented with ketoacids. The results of MDA, oxybolt and nitrotyrosine immunohistochemical staining showed that renal lipid and protein oxidative products, and chronic inflammation as characterized by extensive macrophage infiltration and increased mRNA expression of cytokine and chemokines (TNF-a, MCP-1, CXCL-1 and RANTES) were substantially reduced in LPD and LPD+KA groups, and the reduction in LPD+KA group was more pronounced than that in LPD group (P<0.05). These findings indicate that low protein diet supplemented with ketoacids played a better role in blocking the progression of renal pathology of CKD by mediating oxidative stress and inflammation injury.Then, we studied the role of KLF15 in renal fibrosis and further explored the underlying mechanism. Immunofluorescence revealed that large numbers of KLF15 positive cells were present in both glomeruli and interstitial of the control group. The levels of KLF15 mRNA and the number of KLF15 positive cells were drastically decreased in remnant kidney of 5/6 nephrectomized rats. While restriction of protein intake partially restored the levels of KLF15, the effect was more pronounced in LPD+KA group than that in the groups using dietary protein alone (P<0.05).It was found that in vitro mRNA levels of KLF15 in rat mesangial cells (RMCs) were decreased by TGF-β1, TNF-a, as well as H2O2 (P<0.05) Since the suppressive effect of TNF-a on KLF15 mRNA expression was largely lost in cells lacking TNF receptor 1 (mesangial cells from TNF receptor 1 knock-out mice TNFR1-/-) or p65 subunit of NFκB (embryonic fibroblasts from NFκB p65 deficient mice RelA-/-), both TNF receptor 1 and NFκB are likely required for TNF-a mediated inhibition of KLF15 expression. For further study of KLF15, RMCs and human embryo kidney cells (HEK293) were transfected with a KLF15 cDNA expression vector, and total RNA, cellular protein and culture supernatant were collected for Real-time PCR or ELISA study. It was found that overexpression of KLF15 was associated with a significant decrease in type IV collagen and fibronection expression (P<0.05), suggesting that KLF15 may also have a role in extracellular matrix regulation in renal fibrosis, and the improvement of renal disease by dietary treatment was associated with a partial restoration of KLF15 expression in remnant kidney.In conclusion, the results of our study indicate that low protein diet supplemented with ketoacids plays a more renal protective role than low protein diet alone in the 5/6 nephrectomy rat model. The effect may be mediated by ketoacids through ameliorating oxidative stress and inflammation injury in remnant kidney tissue. The expression of KLF15 could be affected by oxidative stress, inflammation and multiple factors, and it may also play a role in extracellular matrix regulation in renal fibrosis in CKD. The improvement of renal disease by dietary treatment was associated with a partial restoration of KLF15 expression in remnant kidney.