1. Volume 79, Issue 9, Pages 987-996 (May 2011)
Low-protein diet supplemented with ketoacids reduces the severity of renal disease in 5/6 nephrectomized rats: a role for KLF15 
Xiang Gao, Lianghu Huang, Fabrizio Grosjean, Vittoria Esposito, Jianxiang Wu, Lili Fu, Huimin Hu, Jiangming Tan, Cijian He, Susan Gray, Mukesh K. Jain, Feng Zheng, Changlin Mei 
Kidney International 
Volume 79, Issue 9, Pages (May 2011)
DOI: /ki
Copyright © 2011 International Society of Nephrology Terms and Conditions

2. Figure 1
General data of rats. Body weight (a) and serum albumin levels (b) of control and 5/6 nephrectomized rats fed with NPD, LPD, or LPD+KA. Data are expressed as means±s.d. *P<0.05, **P<0.01 versus LPD, or LPD+KA; and ##P<0.01 versus LPD; P<0.05 versus control or LPD+KA. LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; NPD, normal protein diet.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

3. Figure 2
Renal function. Proteinuria (a), BUN (b), and Scr (c) levels of control and 5/6 nephrectomized rats fed with NPD, LPD, or LPD+KA. Data are expressed as means±s.d. *P<0.05 versus LPD or LPD+KA; ##P<0.01 versus the other three groups; and P<0.05 versus LPD+KA. BUN, blood urea nitrogen; LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; NPD, normal protein diet; Scr, serum creatinine.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

4. Figure 3
Renal pathology (Masson's trichrome, × 400). Representative of slide from control (a) and 5/6 nephrectomized rats fed with NPD (b), LPD (c), or LPD+KA (d). Increased fibrosis in both glomerulus and tubulointerstitial area, and a prominent inflammatory cell infiltration were seen in tubulointerstitium of NPD-fed 5/6 nephrectomized rats. Semiquantitative measurements of glomerulosclerosis (e) and interstitial fibrosis (f) revealed that dietary protein restriction decreased glomerulosclerosis index and interstitial fibrosis score. *P<0.05 versus control or LPD+KA; and ##P<0.01 versus control, LPD, or LPD+KA. Data are expressed as means±s.d. LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; NPD, normal protein diet.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

5. Figure 4
Renal fibrosis expression. Fibronectin immunostaining (a–f) and mRNA levels of TGF-β and extracellular matrix genes (g–k). Fibronectin staining was barely present in the control kidney (a, × 200). Increased staining was visible in remnant kidney of NPD-fed animal, especially in tubulointerstitium (b). Dietary protein restriction reduced fibronectin staining (c), and the reduction was even more obvious in 5/6 nephrectomized rats fed with LPD+KA (d). Semiquantitative measurements of intensity and area of fibronectin staining showed that kidneys from animals in LPD and LPD+KA had weaker fibronectin staining in glomeruli (e) and tubulointerstitium (f) than kidneys from animals in NPD. *P<0.05, **P<0.01 versus Control or LPD+KA; and ##P<0.01 versus Control, LPD, or LPD+KA. Real-time PCR analysis of mRNA levels of TGF-β (g), fibronectin (h), type I (i), type III (j), and type IV collagen (k) in kidneys from control and 5/6 nephrectomized rats fed with NPD, LPD, and LPD+KA. GAPDH mRNA levels were determined at the same time. Results are expressed as the ratio between each molecule and GAPDH. *P<0.05, **P<0.01 versus control or LPD+KA; and #P<0.05, ##P<0.01 versus control, LPD, or LPD+KA. Data are expressed as means±s.d. GAPDH; glyceraldehyde-3-phosphate dehydrogenase; LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; NPD, normal protein diet; TGF-β, transforming growth factor-β.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

6. Figure 5
Renal inflammatory expression. Monocyte/macrophage immunostaining with an anti-CD68 antibody (a–d) and mRNA levels of TNF-α, MCP-1, CXCL-1, and RANTES (e–h) in kidneys from control and 5/6 nephrectomized rats. CD68-positive cell was rarely seen in control kidney (a, × 400). Increased staining of positive cell was seen in both glomeruli and tubulointerstitial area of kidneys from 5/6 nephrectomized rats fed with NPD (b). The number of CD68-positive cells was decreased in 5/6 nephrectomized rats fed with LPD (c). LPD+KA nearly completely prevented the infiltration of CD68-positive cell (d). mRNA levels of TNF-α (e), MCP-1 (f), CXCL-1 (g), and RANTES (h) were determined by real-time PCR, and results were normalized to GAPDH mRNA levels. **P<0.01 versus Control or LPD+KA; and ##P<0.01 versus control, LPD or LPD+KA. Data are expressed as means±s.d. CXCL-1, chemokine (C-X-C motif) ligand-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; MCP-1, monocyte chemotactic protein-1; NPD, normal protein diet; RANTES, regulated on activation, normal T expressed and secreted; TNF-α, tumor necrosis factor-α.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

7. Figure 6
KLF15 expression. Representative of immunofluorescence staining of KLF15 in renal sections from control (a, magnification × 400), and 5/6 nephrectomized rats on NPD (b), LPD (c), or LPD+KA (d). KLF15-positive cells are abundant in glomeruli of the normal kidney, and the number decreased significantly in 5/6 nephrectomized rats fed with NPD (b, e). Both LPD and LPD+KA diets largely prevented the decrease in KLF15-positive cells (e). mRNA levels of KLF15 were decreased by about 80% after 5/6 nephrectomy (f). LPD and LPD+KA partly prevented the decrease in KLF15 mRNA expression. **P<0.01 versus control or LPD+KA; ##P<0.01 versus control, LPD or LPD+KA; and P<0.05 versus control. Data are expressed as means±s.d. (g) Real-time PCR analysis of KLF15 mRNA levels in kidneys from 30% protein (HPD)- and 6% protein (LPD)-fed normal Sprague–Dawley rats. GAPDH mRNA levels were determined at the same time. **P<0.01 versus HPD. Data are expressed as means±s.d. GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPD, high-protein diet; KLF15, Kruppel-like factor-15; LPD, low-protein diet; LPD+KA, low-protein diet supplemented with ketoacids; NPD, normal protein diet.
Kidney International  , DOI: ( /ki )
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8. Figure 7
KLF15 mRNA expression in mesangial cells was decreased by oxidant, TGF-β1, or TNF-α. H2O2 decreased KLF15 mRNA levels in mesangial cells in a dose-dependent manner (a). TGF-β1 treatment also decreased KLF15 mRNA levels (b). Time-course study showed that a maximal inhibition of KLF15 mRNA by TNF-α occurred 2h after TNF-α treatment (c). TNF-α-mediated inhibition of KLF15 mRNA expression was lost in mesangial cells lacking TNFR1 (d) and mouse embryonic fibroblasts lacking nuclear factor-κB p65 (e). **P<0.01 versus other group; and ##P<0.01 versus control. Data are expressed as means±s.d. H2O2, hydrogen peroxide; KLF15, Kruppel-like factor-15; MC, mesangial cells; MEF, mouse embryonic fibroblast; TGF-β1, transforming growth factor-β1; TNF-α, tumor necrosis factor-α; TNFR1, TNF receptor-1; WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

9. Figure 8
Overexpression of KLF15 decreased fibronectin and type IV collagen mRNA levels. Mesangial cells or HEK293 cells were transiently transfected with a mouse KLF15 complementary DNA (cDNA) or a pcDNA3 plasmid. Cells were collected after 24h of transfection for determining mRNA levels by real-time PCR. Additionally, fibronectin produced by mesangial cells in both the supernatant and the cell layer was measured by enzyme-linked immunosorbent assay. All experiments were repeated at least three times. (a) KLF15, α1 type IV collagen, and fibronectin mRNA levels (corrected by glyceraldehyde-3-phosphate dehydrogenase) in mesangial cells transfected with or without KLF15 cDNA. The levels in control plasmid-transfected cells were arbitrarily defined as one. (b) Fibronectin levels in both supernatant and cell layer were decreased by overexpression of KLF15 in mesangial cells. **P<0.01 versus control. (c) Overexpression of KLF15 in HEK293 cells also decreased mRNA levels of α1 type IV collagen and fibronectin as determined by real-time PCR. **P<0.01 versus control. Data are expressed as means±s.d. KLF, Kruppel-like factor.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

10. Figure 9
Renal pathology (PAS). Representative slides from wild-type (a, × 400; c, × 800) and KLF15−/− uninephrectomized mice (b, × 400; d, × 800). Morphometric analysis further revealed that mesangial area was increased in uninephrectomized KLF15−/− but not in uninephrectomized wild-type mice (e). Data are expressed as means±s.d. PAS, Periodic acid-Schiff; KLF15−/−, Kruppel-like factor-15−/−; WT, wild type.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions