Ablation of Myeloid Cell MRP8 Ameliorates Nephrotoxic Serum-induced Glomerulonephritis by Affecting Macrophage Characterization through Intraglomerular Crosstalk

Toll-like receptor 4 (TLR4) and one of its endogenous ligands myeloid-related protein 8 (MRP8 or S100A8), especially expressed in macrophages, play an important role in diabetic nephropathy and autoimmune disorders. However, detailed mechanisms and consequence of MRP8 expression remain unknown, partly due to embryonic lethality of MRP8 knockout mice. In this study, Myeloid lineage cell-specific MRP8 knockout mice were generated, and nephrotoxic serum-induced glomerulonephritis was developed. Mice with conditional ablation of MRP8 gene in myeloid cells exhibited less severe histological damage, proteinuria and inflammatory changes compared to control mice. Mechanism of MRP8 upregulation was investigated using cultured cells. Co-culture of macrophages with mesangial cells or mesangial cell-conditioned media, but not with proximal tubules, markedly upregulated MRP8 gene expression and inflammatory M1 phenotype in macrophages, which was attenuated in MRP8-deleted bone marrow-derived macrophages. Effects of MRP8 deletion was further studied in the context of macrophage-inducible C-type lectin (Mincle), which is critically involved in maintenance of M1 phenotype of macrophages. MRP8 ablation in myeloid cells suppressed the induction of Mincle expression on macrophages in glomerulonephritis. Thus, we propose that intraglomerular crosstalk between mesangial cells and macrophages plays a role in inflammatory changes in glomerulonephritis, and MRP8-dependent Mincle expression in macrophage may be involved in the process

Osteocrin ameliorates adriamycin nephropathy via p38 mitogen-activated protein kinase inhibition

Natriuretic peptides exert multiple effects by binding to natriuretic peptide receptors (NPRs). Osteocrin (OSTN) binds with high affinity to NPR-C, a clearance receptor for natriuretic peptides, and inhibits degradation of natriuretic peptides and consequently enhances guanylyl cyclase-A (GC-A/NPR1) signaling. However, the roles of OSTN in the kidney have not been well clarified. Adriamycin (ADR) nephropathy in wild-type mice showed albuminuria, glomerular basement membrane changes, increased podocyte injuries, infiltration of macrophages, and p38 mitogen-activated protein kinase (MAPK) activation. All these phenotypes were improved in OSTN- transgenic (Tg) mice and NPR3 knockout (KO) mice, with no further improvement in OSTN-Tg/NPR3 KO double mutant mice, indicating that OSTN works through NPR3. On the contrary, OSTN KO mice increased urinary albumin levels, and pharmacological blockade of p38 MAPK in OSTN KO mice ameliorated ADR nephropathy. In vitro, combination treatment with ANP and OSTN, or FR167653, p38 MAPK inhibitor, reduced Ccl2 and Des mRNA expression in murine podocytes (MPC5). OSTN increased intracellular cyclic guanosine monophosphate (cGMP) in MPC5 through GC-A. We have elucidated that circulating OSTN improves ADR nephropathy by enhancing GC-A signaling and consequently suppressing p38 MAPK activation. These results suggest that OSTN could be a promising therapeutic agent for podocyte injury

Ablation of the N-type calcium channel ameliorates diabetic nephropathy with improved glycemic control and reduced blood pressure

Pharmacological blockade of the N-and L-type calcium channel lessens renal injury in kidney disease patients. The significance of specific blockade of α1 subunit of N-type calcium channel, Ca[v]2.2, in diabetic nephropathy, however, remains to be clarified. To examine functional roles, we mated Ca[v]2.2-/- mice with db/db (diabetic) mice on the C57BLKS background. Ca[v]2.2 was localized in glomeruli including podocytes and in distal tubular cells. Diabetic Ca[v]2.2-/- mice significantly reduced urinary albumin excretion, glomerular hyperfiltration, blood glucose levels, histological deterioration and systolic blood pressure (SBP) with decreased urinary catecholamine compared to diabetic Ca[v]2.2+/+ mice. Interestingly, diabetic heterozygous Ca[v]2.2+/- mice also decreased albuminuria, although they exhibited comparable systolic blood pressure, sympathetic nerve activity and creatinine clearance to diabetic Ca[v]2.2+/+ mice. Consistently, diabetic mice with cilnidipine, an N-/L-type calcium channel blocker, showed a reduction in albuminuria and improvement of glomerular changes compared to diabetic mice with nitrendipine. In cultured podocytes, depolarization-dependent calcium responses were decreased by ω-conotoxin, a Ca[v]2.2-specific inhibitor. Furthermore, reduction of nephrin by transforming growth factor-β (TGF-β) in podocytes was abolished with ω-conotoxin, cilnidipine or mitogen-activated protein kinase kinase inhibitor. In conclusion, Ca[v]2.2 inhibition exerts renoprotective effects against the progression of diabetic nephropathy, partly by protecting podocytes

Increase of Total Nephron Albumin Filtration and Reabsorption in Diabetic Nephropathy

There is a hot debate concerning actual amount of albumin filtered through glomeruli and reabsorbed at proximal tubules in normal kidneys and diabetic conditions. To overcome current technical problems, we generated a drug-inducible megalin knockout mouse line, megalin(lox/lox);Ndrg1-CreER[T2] (or iMegKO), whose protein reabsorption can be shut off anytime by tamoxifen (Tam). After Tam administration, renal megalin protein expression was reduced by 92% compared to wild-type C57BL/6J mice, and renal reabsorption of intravenously-injected retinol binding protein was almost completely abrogated. Urinary albumin excretion increased to 175 μg/day (0.460 mg/mg-creatinine), suggesting that this was the amount of total nephron albumin filtration. Glomerular sieving coefficient of albumin was 1.7 x 10[-5]. By comparing streptozotocin-induced, Tam-treated, diabetic STZ;iMegKO mice with non-STZ;iMegKO mice, we estimated that daily albumin filtration was increased by 1.9-fold, reabsorption was increased by 1.8-fold, and reabsorption efficiency was reduced to 86% by development of diabetes (versus 96% in control). Such abnormalities were well normalized after insulin treatment. Another type 1 diabetic model of Akita;iMegKO mice showed equivalent results. This study reveals actual values and changes of albumin filtration and reabsorption in early diabetic nephropathy, bringing new insights into our understanding of renal albumin dynamics in hyperfiltration status of diabetic nephropath

Natriuretic peptide receptor guanylyl cyclase-A pathway counteracts glomerular injury evoked by aldosterone through p38 mitogen-activated protein kinase inhibition

Guanylyl cyclase-A (GC-A) signaling, a natriuretic peptide receptor, exerts renoprotective effects by stimulating natriuresis and reducing blood pressure. Previously we demonstrated massive albuminuria with hypertension in uninephrectomized, aldosterone-infused, and high salt-fed (ALDO) systemic GC-A KO mice with enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) in podocytes. In the present study, we examined the interaction between p38 MAPK and GC-A signaling. The administration of FR167653, p38 MAPK inhibitor, reduced systolic blood pressure (SBP), urinary albumin excretion, segmental sclerosis, podocyte injury, and apoptosis. To further investigate the local action of natriuretic peptide and p38 MAPK in podocytes, we generated podocyte-specific (pod) GC-A conditional KO (cKO) mice. ALDO pod GC-A cKO mice demonstrated increased urinary albumin excretion with marked mesangial expansion, podocyte injury and apoptosis, but without blood pressure elevation. FR167653 also suppressed urinary albumin excretion without reducing SBP. Finally, we revealed that atrial natriuretic peptide increased phosphorylation of MAPK phosphatase-1 (MKP-1) concomitant with inhibited phosphorylation of p38 MAPK in response to MAPK kinase 3 activation, thereby resulting in decreased mRNA expression of the apoptosis-related gene, Bax, and Bax/Bcl2 ratio in cultured podocytes. These results indicate that natriuretic peptide exerts a renoprotective effect via inhibiting phosphorylation of p38 MAPK in podocytes.</p

シキュウタイ キンイ オヨビ エンイ ニョウサイカン ノ ショウガイ オ ハンエイスル シンキ バイオマーカー ト シテ ノ ニョウチュウ NGAL

京都大学0048新制・課程博士博士(医学)甲第14508号医博第3353号新制||医||974(附属図書館)UT51-2009-D220京都大学大学院医学研究科内科系専攻(主査)教授 稲垣 暢也, 教授 小川 修, 教授 乾 賢一学位規則第4条第1項該当Doctor of Medical ScienceKyoto UniversityDA

Original raw data of each step of the drug screening.

Original raw data of each step of the drug screening supporting the findings of this study.</p

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