Blockade of HMGB1 Attenuates Diabetic Nephropathy in Mice

Abstract

Activation of TLR2 or TLR4 by endogenous ligands such as high mobility group box 1 (HMGB1) may mediate inflammation causing diabetic kidney injury. We determined whether blockade of HMGB1 signaling by: (1) supra-physiological production of endogenous secretory Receptor for Advanced Glycation End-products (esRAGE), a receptor for HMGB1; (2) administration of HMGB1 A Box, a specific competitive antagonist, would inhibit development of streptozotocin induced diabetic nephropathy (DN). Wild-type diabetic mice developed albuminuria, glomerular injuries, interstitial fibrosis and renal inflammation. Using an adeno-associated virus vector, systemic over-expression of esRAGE afforded significant protection from all parameters. No protection was achieved by a control vector which expressed human serum albumin. Administration of A Box was similarly protective against development of DN. To determine the mechanism(s) of protection, we found that whilst deficiency of TLR2, TLR4 or RAGE afforded partial protection from development of DN, over-expression of esRAGE provided additional protection in TLR2^-/-, modest protection against podocyte damage only in TLR4^-/- and no protection in RAGE^-/- diabetic mice, suggesting the protection provided by esRAGE was primarily through interruption of RAGE and TLR4 pathways. We conclude that strategies to block the interaction between HMGB1 and its receptors may be effective in preventing the development of DN.

Figure 1

rAAV-mediated expression of esRAGE in vivo. (a) esRAGE concentration at 10 days post-injection increased in a dose-dependent manner in mice treated with the rAAV-esRAGE vector and was highest in those receiving 5 × 10¹¹ VGC (7.8 ± 0.7 µg/ml) (n = 2 per group). (b) Timecourse for rAAV-esRAGE at 5 × 10¹¹ VGC. The highest expression levels reached at week 6 and remained high at 3 months post-injection (n = 2 per group). (c) Confirmation of esRAGE binding to HMGB1 by Co-immunoprecipitation (Co-IP) and Western blot (WB). esRAGE (51 kDa) and rHMGB1 (31 kDa) complex pulled down with anti-RAGE antibody by Co-IP was detected by either anti-HMGB1 or anti-RAGE antibody by WB. Cropped image of blots exposed for 30 seconds, is shown. Multiple exposures of full-length blots are presented in Supplementary Figure S1. (d) High levels of esRAGE in serum was detected in DN + esRAGE group four weeks after the injection of rAAV-esRAGE (41.4 ± 8.3 µg/ml, n = 6), while the levels of esRAGE in diabetic mice serum treated with rAAV-HSA (n = 2) or no-rAAV (n = 2) were undetectable. (e) esRAGE levels (233.1 ± 31.2 ng/ml) in urine were also detected from these diabetic mice in DN + esRAGE group while esRAGE were undetectable in urine from both diabetic control groups (DN + HSA and DN + No rAAV). Data are presented as mean ± SD.

Figure 2

(a,b) STZ-induced diabetic mice that received rAAV-esRAGE, rAAV-HSA or no virus developed equivalent levels of hyperglycaemia and changes in body weight. (c) A significant increase in albuminuria was detected in diabetic mice as compared to controls (UACR for DN:183.4 ± 50.7 and DN + HSA:184.6 ± 9.7 mg/mmol vs Non-DN:55.0 ± 10.0 mg/mmol). rAAV-esRAGE treated diabetic mice, however, had a significantly lower production of albuminuria (UACR: 117.8 ± 41.8 mg/mmol) compared to both diabetic control groups. Data are presented as mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Glomerular injury was reduced in rAAV-esRAGE treated diabetic mice. Both diabetic control mice (no virus and rAAV-HSA) demonstrated significant glomerular injury, including glomerular hypertrophy (a,b), glomerular hypercellularity (c), glomerular mesangial matrix expansion (d), podocin injury (e,f) and WT1⁺ podocyte injury (g,h) compared to non-diabetic controls. These changes were reduced in rAAV-esRAGE treated diabetic mice. Bars = 50 µm. Data are presented as mean ± SD; **p < 0.01; ***p < 0.001.

Figure 4

(a,b) Significant interstitial collagen accumulation was evident in both diabetic control groups (no virus or rAAV-HSA) versus non-diabetic controls, whilst the deposition was significantly attenuated in rAAV-esRAGE treated diabetic mice. (c–f) Both diabetic control mice (no virus or rAAV-HSA) showed a significant accumulation of CD68⁺ macrophages in both the interstitial (c,e) and glomerular (d,f) versus non-diabetic controls, while rAAV-esRAGE treated diabetic mice showed significantly less macrophage accumulation in both the interstitial and glomerular compartments compared to two control diabetic groups. Bars = 50 µm. Data are presented as mean ± SD; *p < 0.05, ***p < 0.001.

Figure 5

mRNA expression of TLR and RAGE downstream molecules: TNFα (a), CCL2 (b) and CXCL10 (c) were significantly up-regulated in diabetic control groups (no virus or rAAV-HSA) versus non diabetic mice, but were significantly reduced by esRAGE treatment. Results are expressed as a ratio normalised to GAPDH expression. Data are presented as mean ± SD; *p < 0.05, **p < 0.01; ***p < 0.001.

Figure 6

rAAV-esRAGE treated TLR2^−/− diabetic kidney displayed a further protection against the progression of DN. (a,b) TLR2^−/−, TLR4^−/− or RAGE^−/− mice treated with either rAAV-HSA or rAAV-esRAGE exhibited less albuminuria compared to WT DN + HSA group, whilst no further protection was observed in diabetic knockout mice treated with esRAGE compared to those treated with HSA. (c–i) WT diabetic mice treated with rAAV-HSA developed significant renal pathology, including glomerular hypertrophy and hypercellularity, podocyte loss, mesangial expansion, interstitial fibrosis and macrophage accumulation, all of which were attenuated in WT DN + esRAGE mice. TLR2^−/− and TLR4^−/− diabetic mice treated with rAAV-HSA were partially protected against these diabetic kidney injuries (c–i), which is consistent with our previous studies. Treatment with rAAV-esRAGE provided further protection against kidney damage including glomerular hypertrophy (c) and hypercellularity (d), mesangial cells expansion (g), interstitial fibrosis (h) and macrophage accumulation (i) in TLR2^−/− mice, but not in TLR4^−/− diabetic mice (c,d,g–i). Both TLR2^−/− and TLR4^−/− diabetic mice treated with rAAV-esRAGE exhibited further protection against podocyte injury (e). Additional protection against podocin depletion was evident in rAAV-esRAGE TLR4^−/− diabetic mice (f). Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

RAGE^−/− diabetic mice were partially protected against DN. RAGE^−/− diabetic mice received either rAAV-esRAGE or rAAV-HSA treatment were partially protected against DN in terms of glomerular hypertrophy (a), hypercellularity (b), mesangial cells expansion (c), podocin injury (d), podocyte injury (e), interstitial fibrosis (f) and macrophage accumulation (g) compared to WT DN + HSA mice. No further protection was observed in RAGE^−/− DN + esRAGE mice. Data are presented as mean ± SD; *p < 0.05, ***p < 0.001.

Figure 8

Diabetic mice were protected by the administration of HMGB1 A Box. (a) Diabetic mice given saline developed significant albuminuria versus non-diabetic mice (UACR: 165.3 ± 56.3 versus 51.1 ± 9.7 mg/mmol), whilst A Box treated-diabetic mice were protected with significantly less production of albuminuria (110.8 ± 29.6 mg/mmol). (b–h) WT diabetic mice demonstrated significant glomerular injury including glomerular hypertrophy, glomerular hypercellularity, glomerular mesangial matrix expansion, podocin injury, WT1⁺ podocyte injury, interstitial fibrosis and macrophage accumulation compared to non-diabetic controls, but changes were attenuated by A Box treatment. (i) mRNA expression of cytokine (TGF-β and CXCL10), chemokine (CCL2) and pro-fibrotic (fibronectin) genes were significantly up-regulated in saline-treated diabetic kidney versus non-diabetic controls but significantly attenuated in diabetic mice treated with A Box versus saline-treated diabetic mice. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.