Macrophage-mediated glucolipotoxicity via myeloid-related protein 8/toll-like receptor 4 signaling in diabetic nephropathy

Dyslipidemia is an independent risk factor for the development and progression of diabetic nephropathy (DN). In this review, we summarize mouse models with both diabetes and dyslipidemia, and their associated complications. We then discuss molecules potentially involved in deterioration of DN by dyslipidemia. We focus especially upon toll-like receptor 4 (TLR4) and one of its endogenous ligands, myeloid-related protein 8 (MRP8 or S100A8), since we have found that their mRNA levels are commonly increased in glomeruli of type 1 (streptozotocin [STZ]-induced) and type 2 (A-ZIP/F-1 lipoatrophic) diabetic mice. Gene expression of MRP8 and Tlr4 is further upregulated during worsening of STZ-induced DN by a high fat diet (HFD). Moreover, these HFD-induced changes are accompanied by enhanced gene expression of CCAAT element binding protein β and phosphorylation of c-Jun N-terminal kinase in the kidney, which have also been reported in pancreatic β cells under diabetic-hyperlipidemic conditions. Effects of a HFD upon DN are cancelled in Tlr4 knockout mice. Macrophages are the predominant source of MRP8 in glomeruli. In cultured macrophages, combinatorial treatment with high glucose and palmitate amplifies MRP8 expression in a Tlr4-dependent manner, and recombinant MRP8 protein markedly increases gene expression of the inflammatory cytokines interleukin-1β and tumor necrosis factor α. Here, we propose ‘macrophage-mediated glucolipotoxicity’ via activation of MRP8/TLR4 signaling as a novel mechanism of pathophysiology for DN.


Introduction
Since only one-third of patients with type 1 diabetes develop diabetic nephropathy (DN), we should consider the role of factors other than hyperglycemia in the pathophysiology of DN, including genetic, epigenetic, environmental and metabolic aspects. Several reports describe hyperlipidemia or dyslipidemia as an independent risk factor for the progression of DN in type 1 and type 2 diabetes, as well as for atherosclerotic complications [1][2][3][4]. Using type 1 (streptozotocin [STZ]-induced) and type 2 (db/db) diabetic mouse models, we have confirmed that treatment of diabetic mice with a high fat diet (HFD) exacerbates albuminuria and glomerular lesions [5]. Of note, single nucleotide polymorphisms in acetyl-CoA carboxylase b gene, which plays an important role in the regulation of fatty acid metabolism, exhibit a potent association with proteinuria in patients with type 2 diabetes [6,7]. Accordingly, a concept of synergistic toxicity caused by glucose and lipid, described as 'glucolipotoxicity', has emerged in recent years. However, the underlying molecular mechanism is still obscure, especially in renal complication [8]. Here we will discuss diabetic-hyperlipidemic mouse models and glucolipotoxicity in the kidney.
Diabetic-hyperlipidemic mouse models As described above, several clinical and experimental phenomena have highlighted the synergistic effects of hyperglycemia and hyperlipidemia upon the development and progression of diabetic complications including nephropathy. Despite the fact that there are several limitations associated with the difference in hyperlipidemia between rodents and humans, mouse models are still most widely used to study complications caused by diabetes and hyperlipidemia. The reasons include small animal size, short generation time, the ease of induction of diabetes, hyperlipidemia or gene manipulation, and cost effectiveness [9]. Hence, in the last decade diabetic-hyperlipidemic mouse models have been used for genetic modification, pharmacological treatment and/or some particular chow diets that abundantly contain fat and/or cholesterol. In this section, representative mouse models are summarized.
STZ-induced diabetic mice with HFD feeding (STZ ? HFD) A supplemental HFD on STZ-treated diabetic mice increases blood triglyceride and free fatty acid concentrations, at least in part, because of insulin deficiency, suggesting that this model might be useful especially for analyzing pathophysiology by high triglyceride-rich lipoprotein and/or high free fatty acids coexisting with high glucose conditions. In STZ ? HFD mice, there are several reports describing vascular complications such as cardiovascular dysfunction [21], retinopathy [22], neuropathy [23] and nephropathy [5,24].
Treatment of wild-type mice with STZ and HFD synergistically increases albuminuria [5] and expands mesangial area (Fig. 1). Induction of diabetes by STZ causes a marked increase in urine volume and creatinine clearance of normal diet-fed and HFD-fed animals, respectively, suggesting that glomerular hyperfiltration has occurred. On the other hand, HFD treatment reduces urine volume and creatinine clearance in STZ mice ( Fig. 1), suggesting that HFD is not causing more hyperfiltration but is causing nonhemodynamic actions which will be discussed below. Fig. 1 Effects of STZ and/or HFD upon mesangial expansion (a), urine volume (b) and creatinine clearance (c) in wildtype mice. nSTZ-ND non STZnormal diet, nSTZ-HFD non STZ-high fat diet, STZ-ND STZ-normal diet, STZ-HFD STZ-high fat diet. Data are mean ± SEM. n = 4-11. *p \ 0.01, **p \ 0.001. Modified from Kuwabara and others [5] Clin Exp Nephrol (2014) 18:584-592 585 A-ZIP/F-1 lipoatrophic diabetic mice A-ZIP/F-1 mice are a genetic mouse model of lipoatrophic diabetes, characterized by severe insulin resistance, dyslipidemia including hypertriglyceridemia and high free fatty acids, and fatty liver [25,26]. This model is based upon dominant-negative expression of B-ZIP transcription factors of both C/EBP and Jun families under the control of aP2 enhancer/promoter, causing paucity of adipose tissue.
A-ZIP/F-1 mice may serve as a useful tool for studying DN, because they manifest severe nephrotic syndrome and typical histopathological renal lesions which are glomerular hypertrophy, diffuse and pronounced mesangial expansion and accumulation of extracellular matrix [27].
Notably, these renal changes are reversible to some extent by replacement therapy with a fat-derived hormone leptin [27].

Other mouse models
There are a few other diabetic-hyperlipidemic mouse models such as non-obese diabetic mice or Ins2 Akita diabetic mice combined with HFD feeding [28,29], but their renal involvement has not been characterized well. Regardless of the models described above, differences in genetic backgrounds critically affect glucose and lipid metabolism among mouse strains [30]. Furthermore, even similar levels of hyperglycemia cause distinct renal changes among different strains and species. For instance, the DBA/2 strain is highly susceptible to DN, whereas the C57BL/6 strain is relatively resistant [31][32][33]. In addition, since cholesteryl ester transfer protein is inactive in rodents, HDL is the dominant lipoprotein in mice [34]. Apolipoprotein B in rodents also differs from that in humans [35].

Molecules involved in glucolipotoxicity in the kidney and pancreatic b cells
Although glucotoxicity and lipotoxicity were originally proposed as independent concepts, Prentki et al. reported a novel concept of glucolipotoxicity in pancreatic b cells in 1996. They reported that elevated ambient levels of glucose and free fatty acid cause synergistic inhibition of insulin secretion [36]. On the other hand, they reported that increased intracellular glucose-derived metabolites inhibit enzymes for b-oxidation, leading to cytosolic accumulation of lipids [37]. Subsequently, there have been several reports about the molecular mechanism underlying glucolipotoxicity involved in pancreatic b cell dysfunction and insulin resistance [38][39][40]. Furthermore, phenomena of glucolipotoxicity are also observed in DN of humans [1][2][3][4] and rodents [41,42], but their pathophysiology remains largely unknown [8]. Here, we will compare glucolipotoxicity upon pancreatic b cell dysfunction and DN.
c-Jun N-terminal kinase (JNK) JNK plays a pivotal role in ER stress-induced 'unfolded protein response' in innate immune system [43]. It was later revealed that ER stress-induced JNK activation is associated with chronic inflammation or high ambient fatty acid levels in obesity or type 2 diabetes [44,45]. In pancreatic b-cells, high glucose concentrations augment lipotoxicity through JNK activation, at least partly, in an ER stress-dependent manner [46,47]. In our diabetic-hyperlipidemic model [5], treatment with STZ and HFD synergistically increases phosphorylation of IjB and mRNA expression of proinflammatory genes in the kidney, in parallel with phosphorylation of JNK, but not with phosphorylation of other mitogen-activated protein (MAP) kinases such as p38 or extracellular signal-regulated kinase (ERK) (Fig. 2).
CCAAT element binding protein beta (C/EBPb) CCAAT element binding protein beta (C/EBPb) is one of the transcriptional repressors of insulin gene and induced Fig. 2 Western blot analysis for phosphorylation of MAP kinases and IjB in kidney of STZ ? HFD mice. p-/t-p38 phosphorylated/total p38 MAP kinase, p/tERK phosphorylated/total extracellular signalregulated kinase, p/tJNK phosphorylated/total c-Jun N-terminal kinase, pIjB phosphorylated inhibitor of jB. Modified from Kuwabara and others [5] by chronic hyperglycemia [48]. C/EBPb is increased by fatty acids through the Per-Arnt-Sim kinase (PASK) pathway [49] in pancreatic b cells. Since PASK is also induced by high glucose conditions, these mechanisms may possibly exert glucolipotoxic effects. In the kidney, C/EBPb is increased in diabetic rats, but not other C/EBP isoforms [50]. Furthermore, renal upregulation of C/EBPb mRNA in STZ-induced diabetic mice is further enhanced by additional HFD feeding in our experiments [5]. Of note, JNK/AP-1 and C/EBPb pathways may also contribute to glucolipotoxicity-induced renal damage through upregulation of myeloid-related protein 8 (MRP8, also known as S100A8 or calgranulin A), whose gene promoter region contains AP-1 binding site [51,52] and C/EBP motif [53,54], as discussed in the next section.

Fetuin A
Over the last few years, there has been growing evidence for fatty acid-induced lipotoxicity, such as insulin resistance, through toll-like receptor 4 (TLR4) [55][56][57]. However, it is still controversial whether fatty acid stimulates TLR4 directly or indirectly. Recently, fetuin A has been identified as an adopter protein combining fatty acids and TLR4 [58], and its plasma levels are elevated in diabetic humans and mice [59,60]. ER stress induced by high glucose and palmitate increases the expression of fetuin A [60], suggesting that fetuin A could hypothetically participate in glucolipotoxicity upon macrophages.

MRP8/TLR4
MRP8 was originally identified as a cytoplasmic calciumbinding protein in neutrophils and monocytes [61]. MRP8, by making a heterodimer with MRP14 (or S100A9), has become widely recognized as a potent endogenous ligand for TLR4 in various diseases including septic shock and vascular and autoimmune disorders [62][63][64]. To identify candidate disease-modifying molecules in DN, we have performed microarray analysis using isolated glomeruli from two different diabetic models of mice-STZ-induced insulin-dependent diabetic mice and lipoatrophic insulinresistant A-ZIP/F-1 mice. We then focused upon MRP8 and Tlr4, because expression of both genes is commonly increased in these two models [5]. It is noteworthy that diabetic-hyperlipidemic mice such as STZ-HFD mice or A-ZIP/F-1 mice show remarkable upregulation of MRP8 and Tlr4 compared to control non-diabetic mice (Fig. 3). Since macrophages are identified as the major source of MRP8 in the glomeruli of STZ-HFD mice [5], we examined the effects of high glucose and fatty acid on the expression of MRP8 (Fig. 4) and Tlr4 in cultured macrophages. This in vitro study showed that treatment with fatty acid amplifies MRP8 expression only under high ambient glucose conditions. Although Tlr4 is expressed slightly more in high glucose conditions than in low glucose conditions, fatty acid does not alter Tlr4 expression [5]. In addition, synergistic effects with high glucose and fatty acid on macrophages and diabetic kidneys are abrogated by Tlr4 deletion [5] (Fig. 4). Moreover, we have observed that recombinant MRP8 protein markedly increases gene expression of the inflammatory cytokines interleukin-1b and tumor necrosis factor a (TNF-a) in cultured macrophages (submitted) [62]. Similarly, macrophages also play an important role in insulin resistance and b-cell dysfunction through fatty acid-induced TLR4 activation [65,66]. Particularly in the kidney, MRP8 produced by infiltrated macrophages might exert glucolipotoxic effects upon diabetic glomeruli in a paracrine manner, potentially leading to mesangial expansion, podocyte injury, glomerular sclerosis and albuminuria (Fig. 5), because TLR4 is reportedly expressed in healthy or injured glomerular intrinsic cells including mesangial cells [67,68], endothelial cells [67,69] and podocytes [70,71]. Taken together, we propose 'macrophage-mediated glucolipotoxicity' via activation of MRP8/TLR4 signaling as a novel concept for pathophysiology of DN (Fig. 5).
To understand the clinical implication of MRP8 expression in humans, we have carried out immunohistochemical analysis of MRP8 expression in renal biopsy samples from patients with DN, obesity-related glomerulopathy (ORG) and non-obese, non-diabetic controls (which are minor glomerular abnormality [MGA] and minimal change nephrotic syndrome [MCNS]). We have not been able to obtain reliable antibody against TLR4 to date. The rank orders of glomerular and tubulointerstitial MRP8 protein expression levels  There are several subtypes of macrophages including M1 and M2 in tissue injury and repair [72][73][74]. During the course of renal ischemia/reperfusion injury [75] and unilateral ureteral obstruction [76], switch from proinflammatory M1 to anti-inflammatory or profibrotic M2 subtype occurs in macrophages infiltrating the tubulointerstitium.
Here, we have carried out preliminary analysis of M1 and M2 macrophages in glomeruli of STZ ? HFD mice by studying gene expression levels of CD11c (or Itgax) and CD206 (or Mrc1) as markers of M1 and M2 subtypes, respectively [77,78] (Fig. 6). In wild-type mice, treatment with STZ alone does not affect glomerular expression of CD11c and CD206 genes, and addition of HFD to STZ causes a 100 % increase in CD11c and a 30 % increase in CD206, suggesting relative predominance of M1 subtype in diabetic-hyperlipidemic conditions. Furthermore, in Tlr4 KO mice, the stimulatory effects of HFD upon STZ treatment are canceled both for CD11c and CD206 genes, and simple STZ treatment increases CD11c expression by twofold and increases CD206 expression by three-fold, suggesting the presence of M2 predominant status. These results imply that TLR4-mediated signal is partially suppressing M2 subtype in STZ-normal diet mice and enhancing M1 subtype in STZ-HFD mice. These findings are in good agreement with previous reports indicating that treatment of macrophages with MRP8 induces M1 subtype (through TLR4 as lipopolysaccharide does) [61,72,76] and MRP8-expressing macrophages exhibits M1 characteristics by secretion of TNF-a and interleukin-6 [74,79]. Formally, M1/M2 subtype analysis had to be carried out by analyzing isolated macrophages extracted from tissues.
Furthermore, in STZ ? HFD animals, the levels of macrophage infiltration and extracellular matrix accumulation are proportional and progressive, suggesting that M1-M2 switching does not occur spontaneously in this model of DN. In glomeruli of STZ ? HFD mice,[80 % of MRP8 signals co-localize with macrophage marker Mac2 (or Lgals3) [5], whereas collecting duct epithelial cells are the main source of MRP8 expression in unilateral ureteral obstruction [76].
In conclusion, a number of epidemiological and experimental studies have revealed that glucotoxicity and lipotoxicity cause synergistic effects upon the development and progression of DN. Macrophages have emerged as a potential contributor for mediating glucolipotoxicity through activation of MRP8/TLR4 signaling in diabetic glomeruli in our experiments. Although further studies are needed to understand regulation and potential role of MRP8/TLR4 signaling, targeting key molecules involved in this pathway may lead to novel therapeutic strategy to combat DN.
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