1. Volume 86, Issue 6, Pages 1229-1243 (December 2014)
Enhanced glomerular Toll-like receptor 4 expression and signaling in patients with type 2 diabetic nephropathy and microalbuminuria 
Daniela Verzola, Laura Cappuccino, Elena D'Amato, Barbara Villaggio, Fabio Gianiorio, Mariano Mij, Alchiede Simonato, Francesca Viazzi, Gennaro Salvidio, Giacomo Garibotto 
Kidney International 
Volume 86, Issue 6, Pages (December 2014)
DOI: /ki
Copyright © 2014 International Society of Nephrology Terms and Conditions

2. Figure 1
Expression of (a) Toll-like receptor 4 (TLR4) mRNA and (b) protein in microdissected glomeruli (GL) and tubulointerstitium (T) of patients with microalbuminuria (n=12), overt diabetic nephropathy (DN; n=11), and minimal change disease (MCD; n=10), and in normal control kidney (n=13). TLR4 mRNA expression level was determined by real-time PCR. The expression of TLR4 protein was evaluated by immunohistochemical and image analyses. Values are expressed as fold increase ±s.e.m. to the normal kidney. In comparison with normal and disease controls, (a) TLR4 mRNA was markedly overexpressed (∼8- to 10-fold increase) in the GL and T of patients with microalbuminuria and, to a lesser extent (∼4-fold increase), in patients with overt DN. In microalbuminuria, (b) TLR4 protein levels in the GL and T were also upregulated (∼8- and 5-fold increase, respectively). TLR4 protein expression remained high (∼4- and 7-fold increase in the glomerular and tubulointerstitial compartment, respectively) in overt DN. *P<0.05, **P<0.01 vs. controls and MCD.
Kidney International  , DOI: ( /ki )
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3. Figure 2
Immunohistochemistry of Toll-like receptor 4 (TLR4) in microalbuminuria and overt diabetic nephropathy. TLR4 protein was absent or very faintly expressed in (a) the normal kidney and (b) minimal change disease, whereas it was detectable in the glomeruli in microalbuminuria, and it was mainly localized to (c, d) podocytes; in addition, (e) staining for TLR 4 was detected in the tubules. In patients with advanced disease, TLR4 was expressed in (f) the glomeruli and was strongly expressed along (g, h) the proximal tubule brush border. Magnification: × 400: a, b, c, e, g; × 1000: d, f, h. The arrows indicate positive cells.
Kidney International  , DOI: ( /ki )
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4. Figure 3
Phospho-p65 (p-p65) expression in the kidneys of patients with microalbuminuria or overt diabetic nephropathy (DN) as compared with minimal change disease (MCD) and the normal kidney. (a) Normal (n=10) and MCD (n=10) kidneys showed p-p65-positive nuclei in a very small percentage of glomerular cells. This percentage increased significantly in patients with microalbuminuria (n=10) and in those with clinical DN (n=11). Phosphorylated-p65 positivity was observed mainly in podocytes. (b) Double immunostaining for podocyte marker podocin (red) and p-p65 (green) performed on the diabetic glomeruli showed colocalization of the positive staining, as demonstrated by merging (magnification × 400). The arrows indicate p-p65-positive podocytes identified by podocin positivity. (c) Phosphorylated-p65 expression appeared to be nonstatistically different from controls and MCD subjects in tubules of microalbuminuric patients, but it was markedly expressed (∼2.3-fold increase) in the tubular nuclei of patients with overt DN. The degree of positive nuclei was estimated by counting the number of p-p65-positive cells for 100 cells examined in an average of five high-power fields. Data are expressed as mean±s.e.m.; *P<0.05 vs. normal kidney and MCD.
Kidney International  , DOI: ( /ki )
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5. Figure 4
Immunohistochemical analysis of Phospho-p65 (p-p65) in microalbuminuria, overt diabetic nephropathy (DN), minimal change disease (MCD), and the normal kidney. Only few p-p65-positive nuclei were observed in (a) the normal kidney and (b) in MCD, whereas in (c, d) microalbuminuria and (e, f) overt DN p-p65 was markedly expressed in the glomeruli. Phosphorylated p-p65 expression was modestly upregulated in the tubules of microalbuminuric patients (g), but it was markedly expressed in (h) the tubular nuclei of patients with overt DN and (i, j) in tubulointerstitial cell infiltrates. Magnification: × 400: a, b, c, f, g, h, i; × 1000: d, e, j. The arrows indicate positive cells.
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6. Figure 5
Phospho-inhibitor of nuclear factor κB (IκB-α) (p-IκB-α) expression in the kidneys of patients with microalbuminuria (n=9), overt diabetic nephropathy (DN; n=10), and minimal change disease (MCD; n=10), and in normal controls (n=10). (a) P-IκB-α was upregulated both in glomeruli (GL) and in tubuli in microalbuminuria and overt DN. Data are expressed as fold increase±s.e.m. to the normal kidney; *P<0.05, **P<0.01 vs. the normal kidney and MCD. T, tubulointerstitial. (b–g) Immunohistochemical findings. Photos are representative of p-IκB-α expression and pattern. Magnification: × 400: b, c, f, g; × 1000, d, e. Arrows indicate positive cells.
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7. Figure 6
Expression of tumor necrosis factor α (TNF-α) mRNA and protein in patients with microalbuminuria and overt diabetic nephropathy (DN) and in the normal control kidney. (a) TNF-α mRNA was markedly overexpressed in laser capture microdissected glomeruli (GL) and tubulointerstitium of microalbuminuric (n=12) and overt DN (n=11) patients with respect to the normal kidney (n=13). (b) The protein expression of TNF-α in GL and tubuli was very low in normal renal tissue (n=10) (b, c), but it was clearly increased (approximately four- to fivefold) in microalbuminuria (b, d, e) and in overt diabetic nephropathy (b, f, g). Data are expressed as fold increase±s.e.m. to the normal kidney; *P<0.05 vs. the normal kidney. T, tubulointerstitial. Pictures are representative of TNF-α distribution (magnification: × 400: c, e, g; × 1000: d, f). Arrows indicate positive cells.
Kidney International  , DOI: ( /ki )
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8. Figure 7
Expression of TNFR1 mRNA and protein in microalbuminuric and overt diabetic nephropathy (DN) patients and in the normal kidney. (a) TNFR 1 mRNA was upregulated in the tubulointerstitium of microalbuminuric patients (n=12; approximately four- to fivefold increase over controls), whereas in overt disease (n=11) TNFR 1 mRNA was overexpressed both in the glomeruli (GL) and in the tubulointerstitium (approximately fivefold increase over controls). (b) Among groups (n=10 normal kidney; n=10 microalbuminuria; n=10 overt DN), no differences in TNFR1 protein expression were observed. Data are expressed as fold increase±s.e.m. to normal kidney; *P<0.05 vs. normal kidney. T, tubulointerstitial. Pictures are representative of TNFR1 expression. Magnification: × 400: c, d, e.
Kidney International  , DOI: ( /ki )
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9. Figure 8
The expression of IL-6 mRNA and protein. (a) IL-6 mRNA was overexpressed in the glomeruli (GL; ∼5.6-fold increase) in microalbuminuria (n=12) and both in GL and tubulointerstitium (∼8- to 10-fold increase) in overt diabetic nephropathy (DN; n=11), with respect to the normal kidney (n=13). (b) The immunohistochemical analysis showed increased IL-6 signal in the GL of microalbuminuric patients (by approximately threefold over controls) and in the GL (approximately threefold), tubulointerstitium (approximately sixfold) and interstitial cell infiltrates in clinical DN (n=10 normal kidney; n=10 microalbuminuria; n=10 overt DN). Data are expressed as fold increase±s.e.m. to the normal kidney; *P<0.05, **P<0.01. T, tubulointerstitial. c–f are representative of IL-6 expression. Magnification: × 400: c, e; × 1000: d, f. The arrows indicate positive cells.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

10. Figure 9
Expression of CCL2 and CCR2 mRNA and protein. (a) CCL2 mRNA was scarcely detected in the glomeruli (GL) and tubuli of microalbuminuric patients (n=12), whereas it was overexpressed (∼1.5-fold increase) in the tubulointerstitium (T) of patients with overt diabetic nephropathy (DN; n=11), with respect to the normal kidney (n=13). (b) Expression of CCL2 protein by immunohistochemical and image analysis in microalbuminuric (n=9) and overt DN (n=9) patients and in the normal kidney (n=10) biopsies. CCL2 protein expression was not detectable in (c) the normal kidney tissue, but it was observed in (d) the tubule cells and (e) interstitial compartment of patients with overt diabetic disease. (f) CCR2 mRNA was overexpressed in GL both in microalbuminuria (n=12; by ∼2.5- to 3-fold) and in overt disease (n=11; by ∼3-fold) with respect to the normal kidney (n=13). (g) CCR2 protein was significantly upregulated in the GL and tubuli in overt disease (n=10 normal kidney; n=9 microalbuminuria; n=9 overt DN). (h) Normal kidney; (i, l) expression of CCR2 in the GL and (m, n) tubuli of patients with overt DN. Values are expressed as fold increase±s.e.m. to the normal kidney; *P<0.05. Magnification: × 400: c, d, e, h, i, m; × 1000: l, n. Positive glomerular cells and tubuli are indicated by arrows.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions

11. Figure 10
Expression of CCL5 and CCR5 mRNA and protein. (a) CCL5 mRNA was overexpressed in the glomeruli (GL) of microalbuminuric patients (n=12) with respect to overt diabetic nephropathy (DN; n=11) and the normal kidney (n=13). (b) CCL5 protein showed a similar pattern (n=10 normal kidney; n=10 microalbuminuria; n=10 overt DN). (c–e) Images are representative of CCL5 expression. (f) CCR5 mRNA was overexpressed in the GL of microalbuminuric patients (n=12) and in tubulointerstitium (T) of overt DN (n=11), with respect to normal kidney. (g) A similar pattern was observed as for CCR5 protein, which was expressed in the glomerular compartment in microalbuminuria and in tubuli of patients with overt diabetic disease (n=10 normal kidney; n=10 microalbuminuria; n=9 overt DN). (h–J) Images are representative of CCR5 expression. Values are expressed as fold increase±s.e.m. to normal kidney. *P<0.05. Magnification: × 400: c, d, e, h, J; × 1000: i. Glomerular and tubular positivity is indicated by arrows.
Kidney International  , DOI: ( /ki )
Copyright © 2014 International Society of Nephrology Terms and Conditions