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Table of Contents
Abstract
Introduction
Results
Discussion
Methods
Animal Experiments
Pressure-Volume (PV) Loop Analysis
Renal Function Measurement
Histology And Immunohistochemistry
RNA Extraction And Real Time RT-PCR
Western Blotting
Cell Culture And Induction Of Insulin Resistance
Tandem Mass Tag (TMT)-Mass Spectrometry (MS) Processing And Analysis
NephroSeq Analysis
Statistics
Funding
Duality Of Interest
Author Contributions
Effects Of Global Hmmr Gene Deletion On Body Weight, Systolic, Diastolic And Pulse Blood Pressures On Chow And HF Diet.
Competing Interest Statement
Competing Interest Statement
Abstract
Obesity-related glomerulopathy (ORG) contributes to diabetic nephropathy and kidney cancer, leading to chronic/end-stage kidney disease. To date, treatments for ORG are limited because of incomplete understanding of the disease pathogenesis. Here, we identified a novel role for hyaluronan (HA) and its membrane receptors, CD44 and RHAMM in obesity-associated renal inflammation, fibrosis, tubular injury, and kidney dysfunction. Pharmacological and genetic ablation of HA, CD44 or RHAMM reversed these renal disorders induced by high fat diet feeding in mice in vivo . Increased HA content, and CD44 and RHAMM expression damaged the kidney via activation of TGF-β1/Smad2/3, P38/JNK MAPK and ROCK/ERK pathways. We further established a link between renal insulin resistance and ECM remodelling using human kidney cells in vitro , shedding mechanistic insight into the role of HA, CD44 and RHAMM in the pathogenesis of ORG. Furthermore, in human kidney biopsies gene expression of CD44 and RHAMM was increased in chronic kidney disease and diabetic nephropathy, and their levels were correlated with markers of kidney (dys)function (GFR, serum creatinine, proteinuria). Our findings provide evidence for HA-CD44/RHAMM as a potential therapeutic target in ORG and consequent prevention of chronic kidney disease.
Introduction
The incidence of obesity-related glomerulopathy (ORG) is increasing at an alarming rate worldwide in conjunction with the epidemic of obesity. ORG is associated with poor health outcomes including chronic kidney disease (CKD), end-stage renal disease, and increased mortality. Current treatments for ORG (e. g. weight loss and RAAS inhibitors) are limited and attenuate over time because of incomplete understanding of the disease pathogenesis. Therefore, understanding the mechanisms of ORG and identifying new early intervention strategies would have major clinical and socioeconomic benefits. Extracellular matrix (ECM) remodelling is implicated in the pathogenesis of several metabolic disorders such as cancer[ 1 ], type 2 diabetes[ 2 ], atherosclerosis[ 3 ], obesity[ 4 ] and insulin resistance[ 5 ]. Hyaluronan (HA), a non-sulfated glycosaminoglycan formed linearly by repeating units of glucuronic acid and acetylglucosamine and one of the major ECM constituents, participates in tissue repair and disease progress[ 6 ]. In the healthy kidney, it is mainly found in the interstitium of the inner medulla, where it regulates fluid balance, with limited presence in the outer zone of the outer medulla and virtually void of HA in the cortex[ 7 ]. During pathophysiological conditions of kidney-related diseases such as acute kidney injury (AKI)[ 8 ], diabetic nephropathy[ 9 ], lupus nephritis[ 10 ], chronic cyclosporine nephropathy[ 11 ] and IgA nephropathy[ 12 ], HA accumulation has been associated with renal inflammation and interstitial fibrosis. CD44 is a major cell surface receptor for HA. The HA-CD44 interaction is important in regulating cell proliferation, aggregation, adhesion, angiogenesis and immune cell migration and activation[ 13 , 14 ]. Moreover, recent evidence suggests a pivotal role of CD44 in metabolic regulation[ 15 ], such as skeletal muscle insulin resistance[ 5 ] and obesity[ 16 ]. In the kidney, CD44 is mostly localized on the basolateral membranes of collecting ducts in the inner stripes of outer medulla, the thin descending limb of Henle’s loop and macula densa cells[ 17 ]. CD44 expression in kidney tissue was positively correlated with renal injury, inflammation, and fibrosis in mice[ 18 ]. Genetic deletion of CD44 reduces the number of glomerular lesions in crescentic glomerulonephritis[ 19 ]. Oligosaccharides ameliorated AKI by alleviating CD44-mediated immune responses and inflammation in renal tubular cells[ 20 ]. The interaction between activated memory T cells and HA/CD44 in the glomerular endothelial glycocalyx is critically important in the glomerular homing of T cells in mouse models of lupus nephritis[ 21 ], therefore HA/CD44 plays an important role in mediating pathogenic mechanisms in lupus nephritis[ 10 ]. Upregulation of HA/CD44 contributes to interstitial fibrosis in chronic cyclosporin A (CsA)-induced renal injury and dysfunction[ 11 ]. Upon HA binding, CD44 interacts with different proteins and recruits intracellular signalling molecules to activate NF-κB,[ 22 ] PKC[ 23 ], PI3K/Akt[ 24 ], Src-ERK[ 25 ] and MAPK-signalling pathways[ 26 ], subsequently inducing inflammatory processes and regulating cell adhesion, proliferation, migration, and tumour cell invasion. Receptor for HA mediated motility (RHAMM) is another important HA receptor described by Hardwick et al[ 27 ]. RHAMM is poorly expressed in most homeostatic adult tissues. However, RHAMM mRNA and protein expression are strongly, but transiently, increased in response to injury[ 28 ]. RHAMM differs from CD44 in structure, but they seem to perform similar or synergistic functions in many diseases[ 29 ]. Research indicates that CD44-mediated cellular processes including inflammation, wound healing, tumour formation, and cell migration may require RHAMM surface expression[ 30 ]. In contrast to the many studies on CD44 and kidney diseases[ 19 , 20 ], there are only a few studies on RHAMM, related to renal cell carcinomas[ 31 ]. RHAMM participates in inflammation, angiogenesis[ 32 , 33 ] and tissue response to injury[ 34 ], all of which are important factors in the pathogenesis of kidney disease. We have previously showed that HA accumulation contributed to obesity-associated skeletal muscle insulin resistance, and that reduction of HA or genetic deletion of CD44 improved muscle insulin resistance in obese mice[ 5 , 35 ]. However, the role of HA and CD44 in regulating kidney function in the setting of obesity or ORG has not been studied. Moreover, the involvement of RHAMM in this process is unknown. In this study, we utilised both genetic and pharmacological approaches to reduce the HA-CD44/RHAMM pathway in mice for renal metabolic and functional phenotyping, together with human kidney cell lines and patient biopsies. Our results reveal a novel role of the HA-CD44/RHAMM pathway in the pathogenesis of ORG, which has the potential for developing new treatment strategies for ORG and its associated kidney diseases.
Results
PEGPH20 treatment attenuated obesity-induced renal HA accumulation, tubular damage, renal dysfunction and fibrosis
We have previously shown that in diet-induced obese mice that administering PEGPH20, human recombinant PEGylated hyaluronidase PH-20, at 1mg/kg body weight reduced muscle HA to levels seen in chow-fed mice, decreased fat mass and improved muscle insulin resistance[ 35 ]. Here we assessed its role in ORG. We discovered that kidney cortex HA ( Fig. 1a and 1b ) and outer medulla HA ( Fig. 1a and 1c ) were significantly increased in the HF diet group and these increments were partially rescued by PEGPH20 treatment. Furthermore, PAS staining showed a significant increase of the glomerular area in the HF diet-fed mice relative to chow-fed controls. However, the glomerular areas were not different between vehicle group and PEGPH20 group on HF diet ( Fig. 1a and 1d ). Moreover, the tubular damage, including tubular epithelial cell vacuolar deformation/hypertrophy, tubular dilation, loss of brush border and cell lysis mainly occurred in the cortex and outer stripe of the outer medulla in HF diet mice ( Fig. 1a ). The tubular injury score of HF diet group was significantly higher than that in the chow-fed mice. PEGPH20 treatment partially prevented the tubular damage progression ( Fig. 1e ). In addition, serum creatinine concentration, a marker of renal dysfunction was increased by HF diet feeding in mice but decreased by PEGPH20 treatment ( Fig. 1f ). Renal fibrosis, assessed by collagen deposition and protein expression of α-SMA was increased in HF-fed mice relative to chow-fed controls, which was partially blocked by PEGPH20 treatment ( Fig. 1a , 1g and 1h ). These results demonstrate that HF diet feeding contributed to renal dysfunction, including HA accumulation, tubular injury, elevated serum creatinine levels and fibrosis, which were partially prevented by PEGPH20 treatment.
PEGPH20 blocked the activation of TGF-β1/Smad2/3, P38/JNK MAPK and HA/CD44 pathways and prevented obesity-induced inflammation
We further investigated the mechanisms by which PEGPH20 ameliorated ORG. TGF-β1/Smad2/3 signalling was increased by HF diet feeding in mice, and this was abolished by PEGPH20 treatment ( Fig. 1i-k ). Moreover, the P38/JNK MAPK phosphorylation levels were increased in the HF diet-fed mice, which was blocked in mice treated with PEGPH20 ( Fig. 2a-c ). HA is a major component of the renal ECM and it binds to membrane receptor CD44 and RHAMM regulating physiological and pathological activities. We further investigated whether the renal protective effect of PEGPH20 was dependent upon CD44 and RHAMM. HF diet increased CD44 and RHAMM expression and PEGPH20 treatment reduced CD44 expression ( Fig. 2a and 2d ) in the kidney of HF-fed mice but did not affect RHAMM expression ( Fig. 2a and 2e ). Furthermore, HF diet feeding resulted in an increase, which was abolished by PEGPH20 in ROCK2 expression and ERK/Akt phosphorylation levels ( Fig. 2a and 2f -h), which are important intracellular signalling molecules of the HA-CD44 signalling[ 15 ]. Furthermore, mRNA expression of the pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were increased by HF diet and expression of the anti-inflammatory cytokine IL-10 was decreased by HF diet ( Fig 2i-l ). These changes were all reversed by PEGPH20 treatment. Collectively, these results suggest that PEGPH20 treatment ameliorated obesity-induced renal injury by suppressing the activation of TGF-β1/Smad2/3, P38/JNK MAPK and HA/CD44 pathways and subsequent inflammation cascade.
Global Cd44 gene deletion attenuated obesity-induced HA accumulation, tubular damage, renal dysfunction and fibrosis
To elucidate whether the CD44 receptor mediated renal injury in ORG, Cd44 +/+ and Cd44 -/- mice were fed with HF diet for 16 weeks. Cd44 -/- mice also received either vehicle or PEGPH20 once every 3 days for 28 days. We have previously reported that CD44 contributes to HA-mediated muscle insulin resistance and that the metabolic beneficial effects of PEGPH20 are CD44 dependent[ 5 ]. Here, we show that global deletion of Cd44 gene in HF-fed mice significantly decreased renal cortex and outer medulla HA accumulation ( Fig 3a-c ). Furthermore, PEGPH20 treatment in Cd44 -/- mice caused a further reduction in HA accumulation in the outer medulla but not in the cortex. Global deletion of Cd44 gene in HF-fed mice significantly decreased the tubular injury scores ( Fig. 3a and 3e ), serum creatinine levels ( Fig. 3f ), collagen deposition ( Fig. 3a and 3g ) and α-SMA expression ( Fig. 3a and 3h ) without affecting the glomerular areas ( Fig. 3a and 3d ). PEGPH20 treatment caused a further reduction in serum creatinine level in HF-fed Cd44 -/- mice ( Fig. 3f ). Moreover, the protein expression of TGF-β1 and phosphorylation of Smad2/3 were reduced in HF-fed Cd44 -/- mice relative to HF-fed Cd44 +/+ mice and further decreased after PEGPH20 treatment ( Fig. 3i-k ). These results demonstrate that Cd44 deletion reduced obesity induced kidney injury, which was potentiated by also reducing HA levels. Global Cd44 gene deletion blocked the activation of P38/JNK MAPK and HA/CD44 pathways and prevented obesity-induced inflammation. Global deletion of Cd44 gene significantly lowered the P38/JNK MAPK phosphorylation levels and this was more pronounced in Cd44 -/- mice also treated with PEGPH20 ( Fig. 4a-c ). Furthermore, deletion of the Cd44 gene significantly reduced the protein expression of CD44 ( Fig. 4a and 4d ) but had no effect on the protein expression of RHAMM ( Fig. 4a and 4e ) in the kidney. ROCK2 and ERK/Akt phosphorylation levels were downregulated in HF-fed Cd44 -/- mice when compared with HF-fed Cd44 +/+ mice, and PEGPH20 had no further effects ( Fig. 4a and 4f -h). mRNA levels of IL-1β, TNF-α and IL-6 were markedly decreased, and IL-10 mRNA level was increased in the HF-fed Cd44 -/- mice in comparison to HF-fed Cd44 +/+ mice. PEGPH20 treatment of Cd44 -/- mice further reduced IL-1β, TNF-α and IL-6 mRNA levels and increased IL-10 mRNA level compared to vehicle treated Cd44 -/- mice ( Fig. 4i-l ). This suggests that CD44 deletion suppresses the activation of TGF-β1/Smad2/3, P38/JNK MAPK and HA/CD44 pathways and subsequent inflammation cascade which attenuates obesity-induced renal injury but this is augmented through HA enzymatic degradation with PEGPH20.
Global Hmmr gene deletion reduces obesity-induced HA accumulation, glomerular area expansion, tubular damage, renal dysfunction and fibrosis
The role of RHAMM in renal injury of ORG was explored using Hmmr -null mice ( Hmmr -/- ) and their wildtype littermate controls ( Hmmr +/+ ) fed with an HF or chow diet for 16 weeks. Hmmr -/- and Hmmr +/+ mice body weights were not different on either the chow or HF diet (Supplemental Fig. 2a and 2b ). Systolic and diastolic blood pressures were lower in chow-fed Hmmr -/- mice compared to chow-fed Hmmr +/+ mice, but were similar between genotypes on HF diet (Supplemental Fig. 2c and 2d ). In the renal cortex and outer medulla, a similar level of HA was observed in chow-fed Hmmr +/+ and Hmmr -/- mice. However, HF diet significantly increased HA accumulation in Hmmr +/+ mice, but was significantly reduced in the Hmmr -/- mice ( Fig. 5a-c ). Hmmr gene deletion significantly attenuated HF diet-induced increases in glomerular area, tubular injury score, serum creatinine level, collagen deposition, α-SMA expression, TGF-β1 expression and phosphorylation of Smad2/3 ( Fig. 5a and 5d -k).
Global Hmmr gene deletion blocked the activation of P38/JNK MAPK and HA/CD44 pathways and prevented obesity-induced inflammation
HF-fed Hmmr +/+ mice significantly increased their renal P38/JNK MAPK phosphorylation ( Fig. 6a-c ), CD44 and RHAMM protein expression ( Fig. 6a , 6d , 6e). Additionally, ROCK2 expression ( Fig. 6a , 6f) and the levels of both Akt and ERK phosphorylation ( Fig. 6a , 6g-h) were significantly increased. These were all significantly reduced in the HF-fed Hmmr -/- mice. The cytokines IL-1β, TNF-α and IL-6 mRNA levels were significantly increased in the HF- Hmmr +/+ mice whilst IL-10 was decreased. These cytokine alterations were also all reversed in the HF-fed Hmmr -/- mice. These results suggest that Hmmr gene deletion alleviates obesity-induced kidney injury possibly by inhibiting the activation of TGF-β1/Smad2/3, P38/JNK MAPK and HA/CD44 pathways and subsequent inflammation. In addition, we speculated that the renal protective effect of Hmmr gene deletion was accompanied by reduction of CD44.
CD44 and RHAMM were upregulated in insulin resistant human kidney cells
We next investigated which cell types in the kidney are responsible for obesity-associated renal injury using conditionally immortalized human kidney cells[ 36 ]. Obesogenic and insulin resistant conditions were induced by incubating cells with high glucose, high insulin and inflammatory cytokines (i.e. TNF-α and IL-6)[ 37 ]. Proteomic analysis revealed that CD44 protein expression was increased in insulin resistant podocytes, proximal tubular (PT) cells and mesangial cells, but not glomerular endothelial cells (GEnC) ( Fig. 7a ). Interestingly, RHAMM expression was only increased in insulin resistant podocytes ( Fig. 7b ). HA remodelling was observed in insulin resistant podocytes and PT cells as intracellular HA binding protein (HABP) was decreased in insulin resistant PT cells whereas cell surface hyaluronidase expression was increased in insulin resistant podocytes ( Fig. 7c-d ). Moreover, insulin resistance caused a considerable collagen deposition in podocytes, PT and GEnC cells as evidenced by increased expression of various collagen isoforms, but not in the mesangial cells ( Fig. 7e-k ). Consistently, the protein expression of collagen receptor integrins was also increased by insulin resistance, mostly in PT cells and to a less extend in podocytes and mesangial cells, but not in the GEnC cells ( Fig. 7l-p ). Intriguingly, collagen remodelling (e.g. glycosylation and crosslinking) was induced by insulin resistance mainly in the GEnC cells as evidenced by increased expression of collagen modifying enzymes ( Fig. 7q-t ). These results exhibit human relevance of the mouse work and provide insight into the role of different cell types of the kidney in obesity and insulin resistance-associated renal injury.
Increased CD44 and RHAMM expression correlated with a decline in kidney function in humans
Using publically available transcriptomic datasets from the Nephroseq repository, we found that gene expression of CD44 and RHAMM was increased in kidney biopsies of patients with CKD and diabetic nephropathy when compared to those from healthy living donors ( Fig. 8a-f ). Functionally, CD44 expression was negatively correlated with GFR, but positively correlated with serum creatinine levels in patients with diabetic nephropathy ( Fig. 8g-h ). Likewise, RHAMM expression was positively correlated with proteinuria in patients with diabetic nephropathy ( Fig. 8i ).
Discussion
In the present study, we showed that HF diet feeding in mice led to renal HA accumulation, renal inflammation and fibrosis, and consequent renal injury. Reduction of renal HA by human recombinant hyaluronidase prevented these HF diet-induced renal disorders. Moreover, genetic deletion of CD44 or RHAMM, the two membrane receptors for HA also provided renal protective effects in HF diet-induced obese mice. These results demonstrate a novel role for HA and its receptors in ORG in mice and we further showed that the HA-CD44/RHAMM pathway may cause renal damage by activating the TGF-β1/Smad2/3, P38/JNK MAPK, and ROCK2/ERK/Akt pathways ( Fig. 9 ). ORG, a risk factor for the development of CKD is characterised by glomerular hypertrophy, mesangial matrix expansion, and focal segmental glomerulosclerosis[ 38 ]. Another cardinal feature of ORG is the accumulation of lipid vacuoles in podocytes, mesangial cells, and PT cells with structural and functional changes, suggesting that abnormal lipid metabolism and lipotoxicity may be the major cause of renal dysfunction and activation of proinflammatory processes[ 39 ]. These histologic changes are consistent with what we have observed in the present study. Our study showed a significant increase of the glomerular area in the HF diet-fed mice. Furthermore, the tubular damage, including tubular epithelial cell vacuolar deformation/hypertrophy, tubular dilation, loss of brush border and cell lysis also occurred in mice after 16 weeks of HF diet feeding with an increased serum creatinine concentration. These results demonstrate that HF diet feeding in mice reassembles pathologies of human ORG, therefore representing a valuable experimental model. Upon kidney injury, profibrotic factors can be secreted by injured tubular epithelia and infiltrated inflammatory cells to promote fibroblast differentiation into myofibroblasts and ECM production, leading to renal fibrosis. Tubulointerstitial fibrosis was observed in obesity-related kidney disease[ 40 ]. Renal fibrosis is characterized by pathological deposition of the ECM, which disrupts normal kidney architecture and damages renal function[ 41 ]. The ECM HA was shown to be induced in the initial response phase of kidney injury and remodelled during disease progression of AKI, CKD, and diabetic nephropathy[ 42 ], suggesting a pathological role of HA in kidney injury. Here we also showed increased HA accumulation in ORG in mice. Fibroblast differentiation into myofibroblasts is regulated by classical TGF-β1-dependent Smad-signalling pathway, yet TGF-β1-mediated fibroblast differentiation was dependent upon HA and CD44[ 43 ]. HA production by HA synthase-2 facilitated TGF-β1-dependent fibroblast differentiation via promoting CD44 interaction with epidermal growth factor receptor (EGFR) within membrane-bound lipid rafts[ 43 ]. Our results showing that pharmacological reduction of HA by PEGPH20 or genetic deletion of CD44 ameliorated renal fibrosis (i.e. decreased HA and collagen deposition) and reduced fibroblast differentiation (i.e. decreased α-SMA and TGF-β1/Smad2/3 signalling) in HF-fed mice were consistent with these previous findings[ 43 ]. In addition, CD44 has been demonstrated to be associated with the pathogenesis of crescentic glomerulonephritis[ 19 ], AKI[ 20 ], lupus nephritis[ 10 , 21 ] and CsA-induced renal injury[ 11 ]. CD44 was found to be upregulated and located in dilated tubules in the kidneys of a rat model of AKI[ 44 ]. Moreover, increased CD44 in glomerular parietal epithelial cells in aged mice contributed to glomerular hypertrophy and lower podocyte density accompanied by segmental and global glomerulosclerosis[ 45 ]. In line with these, our results showed that HF-diet feeding for 16 weeks increased renal CD44 protein expression and genetic deletion of CD44 in mice ameliorated obesity-induced tubular injury and renal dysfunction, providing evidence for a crucial role of CD44 in ORG. The biological function of RHAMM is complex, and its extracellular and intracellular functions differ markedly. RHAMM plays a vital role in inflammation, angiogenesis[ 32 , 33 ] and a variety of tissue repair processes[ 34 , 46 ]. RHAMM also interacts with ERK1/2 to regulate tumour metastasis[ 47 ] and is necessary for CD44-mediated skin wound healing[ 48 ]. RHAMM is also an important target for the development of therapies via HA signalling in the tumour microenvironment[ 49 ]. RHAMM leads to progressive fibrosis and is correlated with increased severity of systemic sclerosis. Function-blocking peptides against RHAMM reduced skin fibrosis (dermal thickness and collagen production, deposition and organization), profibrotic gene expression (Tgfb1, c-Myc, Col1a1, Col3a1) and also increased the expression of the antifibrotic adipokines perilipin and adiponectin as well as prevented TGF-β1-induced myofibroblast differentiation in a bleomycin-induced mouse model of systemic sclerosis[ 50 ]. TGF-β1 was found to stimulate RHAMM expression[ 51 ]. However, the role of RHAMM in ORG has not been studied. In the present study, we observed that HF-diet feeding in mice increased RHAMM protein expression and global Hmmr deletion in HF-fed mice significantly reduced renal fibrosis, decreased glomerular areas, improved tubular injury and renal dysfunction. Our data demonstrated a vital role of RHAMM in ORG for the first time. The formation of a triple complex between HA, CD44 and RHAMM on the cell surface during tumorigenesis had been reported[ 52 ]. The role of RHAMM was to enhance CD44 surface localization, stabilize the HA-CD44 interaction and contribute to the activation of ERK1/2 signalling[ 53 ]. It has been reported that cell surface RHAMM associates with several integral protein and non-protein tyrosine kinase receptors including TGF Receptor-1[ 32 ], CD44[ 48 ] and CD44-EGFR complexes[ 43 , 54 ], to regulate HA deposition and CD44 and RHAMM protein expression. In our study, HF diet-induced increases in CD44 protein expression was abolished in HF-fed Hmmr knockout mice. We speculate that beneficial action of Hmmr deletion was associated with reduced cell surface HA-CD44-RHAMM complexes, which however, remains to be further explored. Notably, PEGPH20 treatment or genetic deletion of CD44 had no effect on the glomerular area in HF-fed mice despite improved renal fibrosis and function. However, HF diet-induced increases in glomerular area was reversed by global Hmmr gene deletion. PEGPH20 treatment or genetic deletion of CD44 did not affect RHAMM expression. But HF diet-induced increase in CD44 protein expression in Hmmr +/+ mice was abolished by global Hmmr gene deletion. These results suggest that reversal of glomerular area in obese mice may require the reduction of both CD44 and RHAMM proteins. While CD44 expression was increased in insulin-resistant podocytes, PT cells and mesangial cells, RHAMM was only increased in insulin-resistant podocytes. Therefore, CD44 and RHAMM may have distinct and cell-specific roles in regulating kidney function. Obesity is a chronic low-grade inflammatory condition with up-regulation of proinflammatory cytokines (e.g. TNF-α, IL-6, IL-1β, MCP-1) and free fatty acids in the circulation, and activation of inflammatory pathways (e.g. P38/JNK MAPK pathways), which contribute to kidney hypertrophy and dysfunction. Our results support this concept, where renal fibrosis and dysfunction was associated with elevated inflammation (i.e. increased expression of pro-inflammatory cytokines, decreased expression of anti-inflammatory cytokines, and decreased phosphorylation of P38/JNK), and vice versa in the mouse models that were tested in the present study. The MAPK signalling pathway is a positive regulator of genotoxic stress that can subsequently induce cell apoptosis[ 55 ]. Consistent results were reported by Amos et al., who found that inhibition of p38/JNK activation reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis[ 56 ]. There is mounting evidence that early ECM remodelling is associated with obesity-associated insulin resistance. Previous studies by us and others have shown this to be the case in skeletal muscle, liver, and adipose tissue where insulin resistance is associated with increased deposition of ECM components (e.g. collagens and HA) and their interaction with membrane bound receptors such as integrins and CD44 in obese mice[ 5 , 57 – 60 ]. The kidney also responds to the hormone insulin and reduced insulin action in podocytes leads to impaired glomerular and renal function[ 61 ]. In addition, insulin resistance prevails in patients with CKD and contributes to the progression of renal disease[ 62 ]. The relationship between insulin resistance and CKD also extends to ORG. Patients with ORG exhibited approximately 45% podocyte loss and insulin resistance specifically in podocytes triggers podocyte morphology changes and apoptosis[ 63 ]. Here we showed that in vitro obesogenic and insulin-resistant human kidney cells also underwent ECM remodelling, evidenced by changes in HA catabolism, increased deposition of collagens, changes in collagen modification, as well as increased expression of membrane ECM receptors including integrins, CD44 and RHAMM. Remarkably, these remodifications are cell type specific. While HA remodelling, collagen deposition and ECM receptor upregulation primarily occurred in podocytes and PT cells, collagen modification and crosslinking occurred in GEnC cells. These results support our concept that ECM-receptor activation contributes to insulin resistance[ 5 , 57 ], as podocytes and PT cells are the main cell types responsive to insulin in the kidney[ 64 ]. However, the importance and relative contribution of each cell type to the pathological changes in ORG is unknown and remains to be elucidated. In conclusion, this is the first study to demonstrate that HA and its receptors, CD44 and RHAMM mediate obesity-induced renal inflammation, fibrosis, and dysfunction, possibly via activation of TGF-β1/Smad2/3, P38/JNK MAPK and ROCK/ERK pathways. We further provided evidence that pharmacological and genetic ablation of these molecules in mice reversed adverse renal effects of obesity, therefore an intervention targeting the HA-CD44/RHAMM pathway may represent a novel therapeutic strategy against the progression of obesity-induced kidney injury. Mechanistic work in insulin-resistant human kidney cells in vitro illustrated an association between renal ECM remodelling and insulin resistance. Further studies to elucidate the causal relationship between renal ECM remodelling, kidney insulin resistance, and subsequent obesity-related kidney disease are warranted. Lastly, we showed clinical relevance of CD44 and RHAMM in human CKD and diabetic nephropathy, highlighting their implications and therapeutic potential in wider human kidney diseases.
Methods
Animal experiments
Animal work was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986, the ARRIVE guidelines and the University of Dundee Welfare and Ethical Use of Animals Committee. All Mice were housed in an air-conditioned room at 22±2°C with a 12-h light/12-h dark cycle and had free access to water and food. All mice used in these experiments were male and were fed either a high fat (HF) diet (60% calories as fat, SDS #824054) or maintained on a chow diet (13% calories as fat, DBM; #D/811004) starting at 6 weeks of age for 16 weeks. All mice were studied at 22 weeks of age. Only male mice were studied due to their robust response to HF diet-induced obesity and kidney morphological changes, therefore the current study may limit its clinical relevance to the male gender. Cd44- null mice ( Cd44 -/- ) were obtained on a C57BL/6 background from Jackson Laboratory (Maine; stock no.005085). Hmmr -null mice ( Hmmr -/- ) [RHAMM knock-out] on the C57BL/6 background were a kind gift from Dr Eva Ann Turley (London Health Sciences Centre, London, Canada; The University of Western Ontario, London, ON, Canada) and generated as previously described[ 65 ]. For the reduction of HA content, mice received injections of either vehicle (10mmol/L histidine, 130mmol/L NaCl at pH6.5) or PEGylated recombinant human hyaluronidase PH20 (PEGPH20; Provided under a Material Transfer Agreement with Halozyme Therapeutics, San Diego, CA) at 1 mg/kg through the tail vein, once every 3 days for 28 days. Three different experimental setups were used to examine the role of HA and its receptors in obesity-associated kidney injury, where mice were divided into 4 groups respectively. Study 1: (1) Normal “Chow” diet-fed C57BL/6 mice (Chow); (2) HF diet-fed C57BL/6 mice (HF); (3) HF diet-fed mice receiving vehicle injections (HF Vehicle); (4) HF diet-fed mice receiving PEGPH20 injections (HF PEGPH20). Study 2: (1) HF diet-fed CD44 wildtype littermate control mice (HF Cd44 +/+ ); (2) HF diet-fed Cd44 -null mice (HF Cd44 -/- ); (3) HF diet-fed Cd44 -null mice with vehicle injections (HF Cd44 -/- Vehicle); (4) HF diet-fed Cd44 -null mice with PEGPH20 injections (HF Cd44 -/- PEGPH20). Study 3: (1) Chow diet-fed RHAMM wildtype littermate control mice (Chow Hmmr +/+ ); (2) Chow diet-fed Hmmr -null mice (Chow Hmmr -/- ); (3) HF diet-fed wildtype control mice (HF Hmmr +/+ ); (4) HF diet-fed Hmmr -null mice (HF Hmmr -/- ). At the time of sacrifice, blood and kidney tissue samples were collected. For each mouse, one kidney was snap-frozen at -80°C for gene expression and protein analysis and the other was fixed in 10% formalin for histology.
Pressure-Volume (PV) loop analysis
Pressure volume loop is considered a gold standard for the measurement of cardiac function. Mice were anaesthetized using 2% isoflurane (volume/volume). Admittance catheter (1.2F, Transonic) coupled to ADV500 data acquisition system (Transonic) visualised by LabChart (ADInstruments) sensors, was introduced into the aorta via the carotid artery to measure arterial pressure. During the procedure body temperature of mice was monitored by a rectal thermometer probe. The blood pressure data obtained from the experimental mice were analysed using Lab Chart Pro 8 software (ADInstruments).
Renal function measurement
Serum creatinine level was determined using a Creatinine Assay kit (#ab65340, Abcam). Creatinine concentration was calculated as: Creatitine concentration = (amount of Creatinine nmol × Creatinine molecular weight)/ sample volume µl × sample dilution factor.
Histology and immunohistochemistry
Paraffin-embedded mouse kidney sections (5-μm thickness) were prepared by a routine procedure (3 min in histoclear, three times; 3 min in 100% ethanol, three times; 1 min in 95% ethanol; 1min in 80% ethanol; and 5 min in distilled water). Sections were stained with Periodic acid-Schiff (PAS) staining, using the PAS Stain Kit (#ab150680; Abcam), and Sirius red staining, using Direct Red 80 (#365548; Sigma) and Picric acid (#P6744; Sigma). Immunohistochemical staining of α-SMA was performed using anti-α-SMA(#D4K9N, Cell signaling, 1:200) as previously described[ 5 ], HRP-linked Anti-rabbit (#7074S, Cell signaling, 1:1000) and DAB Substrate kit (#D3939, Sigma). HA was assessed as previously described[ 5 ], using a biotinylated HA-binding protein (#AMS.HKD-BC41, amsbio, 1:200). All staining procedures and image analysis were carried out in a blinded manner. Images were captured using camera mounted on an AxioVision microscope (Zeiss Axioscope, Germany). Quantification of Sirius red, α-SMA and HA staining were assessed in the cortex and outer medullar area using Image J Software. For PAS staining, the glomerular areas were determined by randomly analysing 6 glomeruli in the outer cortex in each kidney section. The kidney cortex and outer medulla were examined for the tubular epithelial cell vacuolar deformation/hypertrophy, tubular dilation, loss of brush border and cell lysis. The degree of tubular damage was calculated using the scoring system described by Haut et al[ 66 ].
RNA extraction and real time RT-PCR
Total RNA was isolated from mouse kidneys using TRIzol reagent (#20130301, Ambion) and reversely transcribed into cDNA using Superscript II 10000U (#2409783, Invitrogen). The mRNA expression levels were determined by real-time quantitative RT-PCR using an Applied Biosystems (QuantStudio 7 Flex, Thermofisher, USA). The sequences of specific primers are listed in Supplemental Table 1. Data were normalized to the 18S expression and quantified using the 2 -ΔΔCT method.
Western blotting
Kidney homogenates were prepared in 1 × Lysis buffer containing 92mg/ml Sucrose, 0.1% β-Mercaptaethanol, 1mM Na 3 VO 4 , 1mM Benzamidine, and 0.1M PMSF using 0.5mm zirconium oxide beads (#E-1626, ZROB05; Next Advance). The supernatant was collected after centrifugation at 9000 rpm for 5 min. The protein concentration was measured using a Bradford Reagent (#B6916; Sigma). Protein samples (40μg) were run on a 10% SDS-page gel (at 150 V) and transferred to nitrocellulose membranes (#10600002, Lytiva Amersham TM ). The following primary antibodies were used to detect respective proteins by incubation overnight at 4°C in 5% BSA: CD44 (#E7k2y, Cell signaling, 1:1000), RHAMM (#E7S4Y, Cell signaling, 1:1000), TGF-β1 (#ab179695, Abcam, 1:1000), Phospho-Smad2 (Ser 465/467 )/Smad3 (Ser 423/425 ) (#8828S, Cell signaling, 1:1000), Smad2/3 (#3102S, Cell signaling, 1:1000), Phospho-p38 MAPK (Thr 180 /Tyr 182 ) (#9211, Cell signaling, 1:1000), p38 MAPK (#9212, Cell signaling, 1:1000), Phospho-SAPK/JNK (Thr 183 /Tyr 185 ) (#9251, Cell signaling, 1:1000), SAPK/JNK (#9252, Cell signaling, 1:1000), Phospho-Akt (Thr 308 ) (#4056, Cell signaling, 1:1000), Akt (#9272, Cell signaling, 1:1000), Phospho-p44/42 MAPK (Erk1/2) (Thr 202 /Tyr 204 ) (#4370, Cell signaling, 1:1000), p44/42 MAPK (Erk1/2) (#4695, Cell signaling, 1:1000), ROCK2 (#8236, Cell signaling, 1:1000), GAPDH (#D16H11, Cell signaling, 1:1000), and Tubulin (#ab6046, Abcam, 1:1000). The membranes were then washed six times with TBST containing 0.1% Tween-20 and incubated in either HRP-goat-anti-sheep (#XDP1015101, RD SYSTEMS) or HRP-goat-anti-rabbit (#D11103-05, LI-COR) secondary antibodies for 1 h at room temperature. Densitometric quantification was performed on scanned images using the ImageJ studio.
Cell culture and induction of insulin resistance
Conditionally immortalized human podocytes were maintained in RPMI-1640 containing L-glutamine and NaHCO 3 , supplemented with 10% FBS (Sigma Aldrich, Gillingham, UK). Proximal tubular (PT) cells were cultured in DMEM-HAM F-12 (Lonza) containing 36ng/ml hydrocortisone (Sigma) 10ng/ml EGF (Sigma), 40pg/ml Tri-iodothyronione (Sigma) and 10% Fetal bovine Serum (FBS). Glomerular Endothelial Cells (GEnC) were maintained in Endothelial Cell Growth Medium-2, containing microvascular SingleQuots Supplement Pack in 5% FBS (Lonza). Cells were studied after 12-14 days of differentiation at 37°C and were free of Mycoplasma infection. To induce obesogenic and insulin resistant condition, cells were cultured in the presence of 100nmol/l insulin (Tocris, Bristol, UK), 25mmol/l glucose (Sigma), 1ng/ml TNF-α and 1ng/ml IL-6 (R&D systems, Abingdon, UK). D-Mannitol (Sigma) was used as a control for osmotic pressure[ 37 ].
Tandem Mass Tag (TMT)-Mass Spectrometry (MS) processing and analysis
Total cell protein was extracted in RIPA lysis buffer (ThermoFisher) and aliquots of each sample were digested with trypsin (2.5µg per 100µg protein; 37°C, overnight), labelled with Tandem Mass Tag (TMT) ten plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, LE11 5RG, UK). Labelled samples were pooled and 50μg was desalted using a SepPak cartridge (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in 20 mM ammonium hydroxide, pH 10, prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). The sample was loaded onto an XBridge BEH C18 Column (130Å, 3.5 µm, 2.1 mm X 150 mm, Waters, UK) and peptides eluted with an increasing gradient (0-95%) of 20 mM Ammonium Hydroxide in acetonitrile, pH 10, over 60 minutes. The resulting fractions were evaporated to dryness and resuspended in 1% formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system and spectra were acquired using an Orbitrap Fusion Lumos mass spectrometer controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS-MS3 workflow. Raw data files for the Total proteome analyses were processed and quantified using Proteome Discoverer software v2.1 (Thermo Scientific) and searched against the UniProt human database (September 2018: 152,927 entries) using the SEQUEST algorithm. The reverse database search option was enabled, and all data was filtered to satisfy false discovery rate (FDR) of 5%. The data output from the Proteome Discoverer 2.1 analysis was further handled, processed and analysed using Microsoft Office Excel, GraphPad Prism and R. Normalization and differential analysis were performed in R in the same manner as RNA-seq data.
NephroSeq Analysis
CD44 and RHAMM gene expression was analysed by Nephroseq v5 ( https://nephroseq.org/ ) using datasets of Nakagawa CKD Kidney[ 67 ] and Ju CKD Glomeruli and Tubulointerstitium[ 68 ]. Correlation of CD44 and RHAMM gene expression with GFR, serum creatinine level and proteinuria were analysed in datasets of Ju CKD Tubulointerstitium and Schmid Diabetes Tubulointerstitium[ 69 ]. Pearson correlations were performed for statistical analysis with p measuring statistical significance, r measuring the linear correlation between two variables and r 2 measuring how close the data to the fitted regression line.
Statistics
All data except Nephroseq analyses were expressed as mean ± SEM and analysed using Prism GraphPad Software version 9. The unpaired, two-tailed Student’s t test was used to identify statistical significance between two groups. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. The data were considered statistically significant at p < 0.05.
Funding
This work was supported by Diabetes UK 15/0005256 and 22/0006477 (LK), British Heart Foundation PG/18/56/33935 (LK), Natural Science Foundation of Jilin Province YDZJ202301ZYTS175 (BQ), BQ and XW were supported by China Scholarship Council. This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking (JU) under grant agreement No 115974. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA and JDRF. Any dissemination of results reflects only the author’s view; the JU is not responsible for any use that may be made of the information it contains. The Medical Research Council also funded this project (Senior Clinical Fellowship to RJMC MR/K010492/1).
Duality of Interest
No potential conflicts of interest relevant to this article were reported.
Author Contributions
BQ and LK contributed to conceptualization and experimental design, researched data, contributed to discussion and data interpretation, wrote the manuscript. VM, XW, AB, CM AL, KH, and CH researched data, contributed to discussion and data interpretation, reviewed and edited the manuscript. WJ, MB and RJMC contributed to discussion and data interpretation, reviewed and edited the manuscript. All authors approved the final version of this manuscript. The authors received no editorial assistance. LK is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Effects of global Hmmr gene deletion on Body weight, Systolic, diastolic and pulse blood pressures on chow and HF diet.
Competing Interest Statement
Competing Interest Statement
The authors have declared no competing interest.
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