Table 1.
TLR-related signalling: Genes that deviate from levels in kidneys from Group1 BW mice&.
Table 2.
Correlations and significances.
Figure 1.
Renal expression of DNaseI, TLR and Clec4e in (NZBxNZW)F1 mice grouped according to glomerular location of EDS.
The mice were sorted into 3 main groups according to kidney morphology (A); pre-nephritic mice (Group 1, no EDS (n = 6); mice with mesangial EDS deposits (Group 2 (n = 12)), or mice with EDS in GBM (Group 3 (n = 5)). Magnification ×40 k. In B, mRNA expression levels for renal DNaseI, TLR7–9 and for the Clec4e receptor with corresponding expression of proteins in kidneys of group 1–3 mice are demonstrated. DNaseI gene expression levels correlate inversely with TLR7–9 and Clec4e receptor expression levels, both with respect to transcription and translation of the genes in Group 3 of BW mice. For statistics, see Table 2. Inserts in the DNaseI immunofluorescence panels represent western blots of the DNaseI. An unpaired t-test was performed (*: p<0,05; ***: p<0,0001).
Figure 2.
Silencing of renal DNaseI correlates with chromatin deposition in GBM and severe proteinuria, while exposure of chromatin correlates significantly with activation of TLR7–9 and Clec4e.
Reduced expression of renal DNaseI correlates inversely with chromatin-IgG deposits in GBM (A) and with severe proteinuria (B). TLR7–9 activation is not significantly correlated with silenced renal DNaseI (C), while a consequence of reduced DNaseI enzyme activity, i.e. exposure of chromatin in e.g. GBM demonstrate a strong and highly significant correlation with TLR7–9 activation (D). ). Similarly, the Clec4e receptor activation is not significantly correlated with silenced renal DNaseI (E), while exposure of secondary necrotic chromatin demonstrate a strong and highly significant positive correlation with Clec4e activation (F).
Figure 3.
Expression of the MMP2 and MMP9 in kidneys of BW mice.
Data demonstrate an insignificant increase in renal MMP2, but not in MMP9 mRNA levels in Group 3 mice compared with mice from Group 1 and 2 (A), and a corresponding increase in MMP2 protein (western blot, B) or in activated MMP2 (zymography, C). Serum concentrations of MMP2 and MMP9 were stable in all stages of the disease as demonstrated by quantitative ELISA for MMP2 and MMP9 (Figure 3D). In agreement with the hypothesis that MMP2 gene expression is linked to activation of TLRs, TLR8 correlates significantly with expression of MMP2 (r = 0,63, p<0,001, E).
Table 3.
Basic clinical, serological and histological data.
Figure 4.
Expression patterns of DNaseI, TLR7–9 in kidneys from patients with lupus nephritis.
A profound silencing of DNaseI gene expression in kidneys with membranoproliferative lupus nephritis (ISN/RPS class IV, A) compared with DNaseI mRNA levels in control kidneys. Western blot analyses of renal proteins demonstrated a single band corresponding to the MW of human DNaseI (B). Low band intensities were solely found in the kidney samples that demonstrated considerably reduced DNaseI mRNA levels (LN 1–3, A and B). Similarly, immunohistochemistry analyses of kidney sections demonstrated strong staining intensity of the DNaseI protein in normal control kidneys (C) and in ISN/RPS class II kidneys (C). In ISN/RPS class IV kidneys, DNaseI staining was barely detectable (C), in agreement with the low DNaseI mRNA levels in these patients. Control analyses in other renal diseases (like diabetic nephrosclerosis or membranoproliferative glomerulonephritis type 2) demonstrated DNaseI staining intensity similar to that in normal kidneys, and kidneys with ISN/RPS class II lupus nephritis (C). The qPCR analyses revealed that TLR7 and TLR 8 were up-regulated in class IV nephritis, although without reaching statistical significance (D). The TLR 9 and Clec4e (data not shown) mRNA levels did not differ at all from the control group. In harmony with this, immunohistochemical staining of kidneys with class IV lupus nephritis revealed increased staining intensity of TLR8 (E). An unpaired t-test is performed (*p<0,05).
Figure 5.
Expression of MMP2 and MMP9 in kidneys from patients with human ISN/RPS class IV lupus nephritis.
The MMP2 and MMP9 mRNA levels were significantly up-regulated in class IV lupus nephritis compared with control kidneys (A). The high MMP2 and MMP9 mRNA levels were also reflected by increased protein levels as demonstrated by western blots (B) and by immunohistochemistry (C). Taken together, MMP2 and MMP9 mRNA and protein levels are highly expressed in human class IV lupus nephritis. An unpaired t-test is performed (*p<0,05).
Figure 6.
Principal component analysis (PCA) of murine (A) and human (B,C) parameters included in this study.
PCA biplots aim to optimally display variances and not correlations. The angles between the various biplot axes serve as good indicators of the correlations among the variables (shown as arrows). Similarly, the position of the samples of individual mice (shown as the signs 1, 2 and 3 for Group 1–3 mice) relative to the arrows, provide good indications as to which variable(s) have had the largest effect on disease progression. The result of the biplots demonstrates that groups emerging from this analysis perfectly correlated with the groups of BW mice as defined in Figure 1, defined as pre-nephritic BW mice (Group 1), BW mice with deposits of EDS in the mesangial matrix (Group 2) or with deposits in the GBM (Group 3). In B, a similar biplot has been generated for the human data, where the 3 patients with low DNaseI expression levels are included. In C, all 4 patients with lupus nephritis class IV are included, i.e. also the patient with normal renal DNaseI. As is demonstrated, the vector for DNaseI differs in B and C, thus demonstrating the impact of DNaseI levels on the biplot. For all biplots, the most striking observations are that DNaseI vector points away from the diseased individuals (to the left in the biplots), while MMP2, MMP9, TLRS and EDS associated with GBM are clustered and points at the most severely diseased murine and human individuals, in harmony with the statistical analyses demonstrated in Table 2.