Macrophages in mouse type 2 diabetic nephropathy: correlation with diabetic state and progressive renal injury

BACKGROUND: Macrophage-mediated renal injury has been implicated in progressive forms of glomerulonephritis; however, a role for macrophages in type 2 diabetic nephropathy, the major cause of end-stage renal failure, has not been established. Therefore, we examined whether macrophages may promote the progression of type 2 diabetic nephropathy in db/db mice. METHODS: The incidence of renal injury was examined in db/db mice with varying blood sugar and lipid levels at 8 months of age. The association of renal injury with the accumulation of kidney macrophages was analyzed in normal db/+ and diabetic db/db mice at 2, 4, 6, and 8 months of age. RESULTS: In db/db mice, albuminuria and increased plasma creatinine correlated with elevated blood glucose and hemoglobin A1c (HbA1c) levels but not with obesity or hyperlipidemia. Progressive diabetic nephropathy in db/db mice was associated with increased kidney macrophages. Macrophage accumulation and macrophage activation in db/db mice correlated with hyperglycemia, HbA1c levels, albuminuria, elevated plasma creatinine, glomerular and tubular damage, renal fibrosis, and kidney expression of macrophage chemokines [monocyte chemoattractant protein-1 (MCP-1), osteopontin, migration inhibitory factor (MIF), monocyte-colony-stimulating factor (M-CSF)]. The accrual and activation of glomerular macrophages also correlated with increased glomerular IgG and C3 deposition, which was itself dependent on hyperglycemia. CONCLUSION: Kidney macrophage accumulation is associated with the progression of type 2 diabetic nephropathy in db/db mice. Macrophage accumulation and activation in diabetic db/db kidneys is associated with prolonged hyperglycemia, glomerular immune complex deposition, and increased kidney chemokine production, and raises the possibility of specific therapies for targeting macrophage-mediated injury in diabetic nephropathy.     

Macrophages in streptozotocin-induced diabetic nephropathy: potential role in renal fibrosis.

BACKGROUND: Renal fibrosis is central to the progression of diabetic nephropathy; however, the mechanisms responsible for fibroblast and matrix accumulation in this disease are only partially understood. Macrophages accumulate in diabetic kidneys, but it is unknown whether macrophages contribute to renal fibrosis. Therefore, we examined whether macrophage accumulation is associated with the progression of renal injury and fibrosis in type 1 diabetic nephropathy and whether macrophages exposed to the diabetic milieu could promote fibroblast proliferation. METHODS: Kidney macrophages, renal injury and fibrosis were analysed in diabetic C57BL/6J mice at 2, 8, 12 and 18 weeks after streptozotocin injection. Isolated rat bone marrow macrophages were stimulated with diabetic rat serum or carboxymethyllysine (CML)-bovine serum albumin (BSA) to determine whether macrophage-conditioned medium could promote the proliferation of rat renal (NRK-49F) fibroblasts. RESULTS: Progressive injury and fibrosis in diabetic nephropathy was associated with increased numbers of kidney macrophages. Macrophage accumulation in diabetic mice correlated with hyperglycaemia (blood glucose, HbA1c levels), renal injury (albuminuria, plasma creatinine), histological damage and renal fibrosis (myofibroblasts, collagen IV). Culture supernatant derived from bone marrow macrophages incubated with diabetic rat serum or CML-BSA induced proliferation of fibroblasts, which was inhibited by pre-treating fibroblasts with interleukin-1 (IL-1) receptor antagonist or the platelet-derived growth factor (PDGF) receptor kinase inhibitor, STI-571. CONCLUSION: Kidney macrophage accumulation is associated with the progression of renal injury and fibrosis in streptozotocin-induced mouse diabetic nephropathy. Elements of the diabetic milieu can stimulate macrophages to promote fibroblast proliferation via IL-1- and PDGF-dependent pathways which may enhance renal fibrosis.     

巨噬细胞Bruton酪氨酸激酶基因特异性敲除减轻糖尿病小鼠肾脏损害的作用及机制

目的探讨巨噬细胞Bruton酪氨酸激酶(Btk)基因特异性敲除对糖尿病小鼠肾脏损害的作用及机制。方法选用巨噬细胞Btk基因特异性敲除(Btk-/-)小鼠和C57BL/6N小鼠链脲菌素(STZ,50 mg/kg)造模成功后随机分为正常组、糖尿病组、Btk-/-组和Btk-/-糖尿病组。12周后测定小鼠一般指标,观察肾组织病理学改变,免疫荧光检测肾组织巨噬细胞标志物CD68表达,免疫组化检测足细胞标志物WT1和Nephrin的表达,Western印迹检测细胞外基质纤维连接蛋白(FN)、IV型胶原(IV-Col)及转化生长因子β1(TGF-β1),巨噬细胞激活标志物诱导型一氧化氮合酶(iNOS)、磷酸化(p)-Btk,炎性因子白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)和丝裂原活化蛋白激酶(MAPK)信号通路p-p38MAPK、p-JNK、p-ERK以及核因κB(NF-κB)通路p-p65、p-IκB蛋白水平变化;实时荧光定量PCR检测炎性因子IL-1β、TNF-α、单核细胞趋化蛋白1(MCP-1)mRNA表达。结果与糖尿病组相比,Btk-/-糖尿病组尿白蛋白明显减少,肾组织损伤明显减轻,肾脏巨噬细胞CD68表达明显减少,足细胞标志物WT1及Nephrin表达明显增加,细胞外基质FN、IV-Col及TGF-β1表达明显减少,炎性因子IL-1β、TNF-α及MCP-1表达明显降低,p-JNK、p-ERK、p-p38MAPK及p-p65、p-IκB表达明显下调(均P<0.05)。结论巨噬细胞Btk特异性敲除可能通过MAPK、NF-κB通路降低炎性因子的表达从而对糖尿病小鼠肾脏起到保护作用。     

IL-18 contributes to renal damage after ischemia-reperfusion

IL-18 is a proinflammatory cytokine produced by macrophages and other cell types present in the kidney during ischemia-reperfusion injury (IRI), but its role in this injury is unknown. Here, compared with wild-type mice, IL-18(-/-) mice subjected to kidney IRI demonstrated better kidney function, less tubular damage, reduced accumulation of neutrophils and macrophages, and decreased expression of proinflammatory molecules that are downstream of IL-18. For determination of the relative contributions of leukocytes and parenchymal cells to IL-18 production and subsequent kidney damage during IRI, bone marrow-chimeric mice were generated. Wild-type mice engrafted with IL-18(-/-) hemopoietic cells showed less kidney dysfunction and tubular damage than IL-18(-/-) mice engrafted with wild-type bone marrow. In vitro, macrophages produced IL-18 mRNA and protein in response to ischemia. These data suggest bone marrow-derived cells are the key contributors to IL-18-mediated effects of renal IRI. Finally, similar to IL-18(-/-) mice, pretreatment of wild-type mice with IL-18-binding protein was renoprotective in this model of IRI. In conclusion, IL-18, derived primarily from cells of bone marrow origin, contributes to the renal damage observed during IRI. IL-18-binding protein may have potential as a renoprotective therapy.     

Monocyte chemoattractant protein-1 promotes the development of diabetic renal injury in streptozotocin-treated mice

Diabetic nephropathy involves a renal inflammatory response induced by the diabetic milieu. Macrophages accumulate in diabetic kidneys in association with the local upregulation of monocyte chemoattractant protein-1 (MCP-1); however, the contribution of macrophages to renal injury and the importance of MCP-1 to their accrual are unclear. Therefore, we examined the progression of streptozotocin (STZ)-induced diabetic nephropathy in mice deficient in MCP-1 in order to explore the role of MCP-1-mediated macrophage accumulation in the development of diabetic kidney damage. Renal pathology was examined at 2, 8, 12 and 18 weeks after STZ treatment in MCP-1 intact (+/+) and deficient (-/-) mice with equivalent blood glucose and hemoglobin A1c levels. In MCP-1(+/+) mice, the development of diabetic nephropathy was associated with increased kidney MCP-1 production, which occurred mostly in tubules, consistent with our in vitro finding that elements of the diabetic milieu (high glucose and advanced glycation end products) directly stimulate tubular MCP-1 secretion. Diabetes of 18 weeks resulted in albuminuria and elevated plasma creatinine in MCP-1(+/+) mice, but these aspects of renal injury were largely suppressed in MCP-1(-/-) mice. Protection from nephropathy in diabetic MCP-1(-/-) mice was associated with marked reductions in glomerular and interstitial macrophage accumulation, histological damage and renal fibrosis. Diabetic MCP-1(-/-) mice also had a smaller proportion of kidney macrophages expressing markers of activation (inducible nitric oxide synthase or sialoadhesin) compared to diabetic MCP-1(+/+) mice. In conclusion, our study demonstrates that MCP-1-mediated macrophage accumulation and activation plays a critical role in the development of STZ-induced mouse diabetic nephropathy.     

Intercellular adhesion molecule-1 deficiency is protective against nephropathy in type 2 diabetic db/db mice

Diabetic nephropathy is a leading cause of end-stage renal failure and is a growing concern given the increasing incidence of type 2 diabetes. Diabetic nephropathy is associated with progressive kidney macrophage accumulation and experimental studies suggest that intercellular adhesion molecule (ICAM)-1 facilitates kidney macrophage recruitment during type 1 diabetes. To ascertain the importance of ICAM-1 in promoting type 2 diabetic nephropathy, the development of renal injury in ICAM-1 intact and deficient db/db mice with equivalent hyperglycemia and obesity between ages 2 and 8 mo was examined and compared with results with normal db/+ mice. Increases in albuminuria (11-fold), glomerular leukocytes (10-fold), and interstitial leukocytes (three-fold) consisting of predominantly CD68+ macrophages were identified at 8 mo in diabetic db/db mice compared with nondiabetic db/+ mice. In comparison to db/db mice, ICAM-1-deficient db/db mice had marked reductions in albuminuria at 6 mo (77% downward arrow) and 8 mo (85% downward arrow). There was also a significant decrease in glomerular (63% downward arrow) and interstitial (83% downward arrow) leukocytes in ICAM-1-deficient db/db mice, which were associated with reduced glomerular hypertrophy and hypercellularity and tubular damage. The development of renal fibrosis (expression of TGF-beta1, collagen IV, and interstitial alpha-smooth muscle actin) was also strikingly attenuated in the ICAM-1-deficient db/db mice. Additional in vitro studies showed that macrophage activation by high glucose or advanced glycation end products could promote ICAM-1 expression on tubular cells and macrophage production of active TGF-beta1. Thus, ICAM-1 appears to be a critical promoter of nephropathy in mouse type 2 diabetes by facilitating kidney macrophage recruitment.     

Cholecystokinin metabolism in man and dogs.

We have developed a sensitive, specific and reproducible radioimmunoassay for cholecystokinin (CCK) with which basal levels of CCK of between 400-800 pg/ml have been measured in normal man, in patients with diabetes and with duodenal ulcer disease, and in normal dogs. After a meal, circulating levels of CCK rose to 1000-1200 pg/ml in human subjects. Release of CCK was more rapid in diabetic and duodenal ulcer patients than in normal subjects, but elevated postprandial levels persisted much longer in normal subjects. Patients with the Zollinger-Ellison syndrome had elevated values of cholecystokinin which rose after a meal. Lack of correlation between elevated basal levels of gastrin and CCK in patients with the Zollinger-Ellison syndrome suggest that the hypercholecystokininemia may be absolute. The disappearance half-time of exogenous CCK was about 21/2 minutes in normal subjects as well as in diabetic and duodenal ulcer patients. Studies in dogs demonstrated no uptake of basal levels of cholecystokinin by the kidney; on infusion of exogenous CCK-33, the kidney extracted 43% of the total CCK presented and 56% of the integrated CCK. We conclude that: 1) circulating basal and postprandial levels of CCK may be measured in a reproducible fashion; 2) postprandial release of CCK is more rapid in diabetic and duodenal ulcer patients than in normal man; 3) the disappearance half-time of exogenous CCK in man and dogs is about 21/2 minutes; 4) the kidney is a major site for uptake of CCK.     

Deletion of MK2 signalling in vivo inhibits small Hsp phosphorylation but not diabetic nephropathy.

It is supposed that some stress-induced heat shock proteins (Hsps) are regulated through e.g. stimulation of the p38MAPK/MK(MAPKAP)-2 signalling pathway. It has been postulated from in vitro experiments that phosphorylation of Hsp25(rodents)/Hsp27(human), the major phosphorylation substrate of MK2, is responsible for mesangial contractility and glomerular hyperfiltration in the diabetic kidney. To verify this hypothesis in vivo we studied the renal function of nondiabetic and streptozotocin (STZ)-induced, diabetic MK2(-/-) mice in comparison to wild-type (WT) control mice. Following 8 weeks of hyperglycaemia, light microscopy showed increased glomerulosclerosis and tubulointerstitial renal fibrosis in both diabetic study groups. Protein analysis demonstrated that Hsp25 phosphorylation is stimulated upon high-glucose condition but inhibited in the diabetic MK2(-/-) mice. However, we found the kidney-body weight ratio significantly increased in diabetic WT and MK2(-/-) mice. No difference regarding the increased expression of the extracellular matrix proteins and TGF-beta1 between both diabetic study groups was observed. Importantly, diabetic MK2(-/-) mice showed no protection against renal hyperfiltration in the diabetic state and the development of diabetic albuminuria. Although activation of p38MAPK has been previously shown in diabetes mellitus, our results indicate that blockade of the downstream MK2/Hsp25 signalling pathway does not interfere with the development of early diabetic nephropathy.     

Macrophage scavenger receptor-a-deficient mice are resistant against diabetic nephropathy through amelioration of microinflammation

Microinflammation is a common major mechanism in the pathogenesis of diabetic vascular complications, including diabetic nephropathy. Macrophage scavenger receptor-A (SR-A) is a multifunctional receptor expressed on macrophages. This study aimed to determine the role of SR-A in diabetic nephropathy using SR-A-deficient (SR-A(-/-)) mice. Diabetes was induced in SR-A(-/-) and wild-type (SR-A(+/+)) mice by streptozotocin injection. Diabetic SR-A(+/+) mice presented characteristic features of diabetic nephropathy: albuminuria, glomerular hypertrophy, mesangial matrix expansion, and overexpression of transforming growth factor-beta at 6 months after induction of diabetes. These changes were markedly diminished in diabetic SR-A(-/-) mice, without differences in blood glucose and blood pressure levels. Interestingly, macrophage infiltration in the kidneys was dramatically decreased in diabetic SR-A(-/-) mice compared with diabetic SR-A(+/+) mice. DNA microarray revealed that proinflammatory genes were overexpressed in renal cortex of diabetic SR-A(+/+) mice and suppressed in diabetic SR-A(-/-) mice. Moreover, anti-SR-A antibody blocked the attachment of monocytes to type IV collagen substratum but not to endothelial cells. Our results suggest that SR-A promotes macrophage migration into diabetic kidneys by accelerating the attachment to renal extracellular matrices. SR-A may be a key molecule for the inflammatory process in pathogenesis of diabetic nephropathy and a novel therapeutic target for diabetic vascular complications.     

Deletion of protein kinase C-beta isoform in vivo reduces renal hypertrophy but not albuminuria in the streptozotocin-induced diabetic mouse model

The protein kinase C (PKC)-beta isoform has been implicated to play a pivotal role in the development of diabetic kidney disease. We tested this hypothesis by inducing diabetic nephropathy in PKC-beta-deficient (PKC-beta(-/-)) mice. We studied nondiabetic and streptozotocin-induced diabetic PKC-beta(-/-) mice compared with appropriate 129/SV wild-type mice. After 8 weeks of diabetes, the high-glucose-induced renal and glomerular hypertrophy, as well as the increased expression of extracellular matrix proteins such as collagen and fibronectin, was reduced in PKC-beta(-/-) mice. Furthermore, the high-glucose-induced expression of the profibrotic cytokine transforming growth factor (TGF)-beta1 and connective tissue growth factor were significantly diminished in the diabetic PKC-beta(-/-) mice compared with diabetic wild-type mice, suggesting a role of the PKC-beta isoform in the regulation of renal hypertrophy. Notably, increased urinary albumin-to-creatinine ratio persisted in the diabetic PKC-beta(-/-) mice. The loss of the basement membrane proteoglycan perlecan and the podocyte protein nephrin in the diabetic state was not prevented in the PKC-beta(-/-) mice as previously demonstrated in the nonalbuminuric diabetic PKC-alpha(-/-) mice. In summary, the differential effects of PKC-beta deficiency on diabetes-induced renal hypertrophy and albuminuria suggest that PKC-beta contributes to high-glucose-induced TGF-beta1 expression and renal fibrosis, whereas perlecan, as well as nephrin, expression and albuminuria is regulated by other signaling pathways.     

Diminished loss of proteoglycans and lack of albuminuria in protein kinase C-alpha-deficient diabetic mice

Activation of protein kinase C (PKC) isoforms has been implicated in the pathogenesis of diabetic nephropathy. We showed earlier that PKC-alpha is activated in the kidneys of hyperglycemic animals. We now used PKC-alpha(-/-) mice to test the hypothesis that this PKC isoform mediates streptozotocin-induced diabetic nephropathy. We observed that renal and glomerular hypertrophy was similar in diabetic wild-type and PKC-alpha(-/-) mice. However, the development of albuminuria was almost absent in the diabetic PKC-alpha(-/-) mice. The hyperglycemia-induced downregulation of the negatively charged basement membrane heparan sulfate proteoglycan perlecan was completely prevented in the PKC-alpha(-/-) mice, compared with controls. We then asked whether transforming growth factor-beta1 (TGF-beta1) and/or vascular endothelial growth factor (VEGF) is implicated in the PKC-alpha-mediated changes in the basement membrane. The hyperglycemia-induced expression of VEGF165 and its receptor VEGF receptor II (flk-1) was ameliorated in PKC-alpha(-/-) mice, whereas expression of TGF-beta1 was not affected by the lack of PKC-alpha. Our findings indicate that two important features of diabetic nephropathy-glomerular hypertrophy and albuminuria-are differentially regulated. The glucose-induced albuminuria seems to be mediated by PKC-alpha via downregulation of proteoglycans in the basement membrane and regulation of VEGF expression. Therefore, PKC-alpha is a possible therapeutic target for the prevention of diabetic albuminuria.     

Intrarenal dopamine inhibits progression of diabetic nephropathy

The kidney has a local intrarenal dopaminergic system, and in the kidney, dopamine modulates renal hemodynamics, inhibits salt and fluid reabsorption, antagonizes the renin-angiotensin system, and inhibits oxidative stress. The current study examined the effects of alterations in the intrarenal dopaminergic system on kidney structure and function in models of type 1 diabetes. We studied catechol-O-methyl-transferase (COMT)(-/-) mice, which have increased renal dopamine production due to decreased dopamine metabolism, and renal transplantation was used to determine whether the effects seen with COMT deficiency were kidney-specific. To determine the effects of selective inhibition of intrarenal dopamine production, we used mice with proximal tubule deletion of aromatic amino acid decarboxylase (ptAADC(-/-)). Compared with wild-type diabetic mice, COMT(-/-) mice had decreased hyperfiltration, decreased macula densa cyclooxygenase-2 expression, decreased albuminuria, decreased glomerulopathy, and inhibition of expression of markers of inflammation, oxidative stress, and fibrosis. These differences were also seen in diabetic mice with a transplanted kidney from COMT(-/-) mice. In contrast, diabetic ptAADC(-/-) mice had increased nephropathy. Our study demonstrates an important role of the intrarenal dopaminergic system to modulate the development and progression of diabetic kidney injury and indicate that the decreased renal dopamine production may have important consequences in the underlying pathogenesis of diabetic nephropathy.     

Intercellular adhesion molecule-1-deficient mice are resistant against renal injury after induction of diabetes

Diabetic nephropathy is a leading cause of end-stage renal failure. Several mechanisms, including activation of protein kinase C, advanced glycation end products, and overexpression of transforming growth factor (TGF)-beta, are believed to be involved in the pathogenesis of diabetic nephropathy. However, the significance of inflammatory processes in the pathogenesis of diabetic microvascular complications is poorly understood. Accumulation of macrophages and overexpression of leukocyte adhesion molecules and chemokines are prominent in diabetic human kidney tissues. We previously demonstrated that intercellular adhesion molecule (ICAM)-1 mediates macrophage infiltration into the diabetic kidney. In the present study, to investigate the role of ICAM-1 in diabetic nephropathy, we induced diabetes in ICAM-1-deficient (ICAM-1(-/-)) mice and ICAM-1(+/+) mice with streptozotocin and examined the renal pathology over a period of 6 months. The infiltration of macrophages was markedly suppressed in diabetic ICAM-1(-/-) mice compared with that of ICAM-1(+/+) mice. Urinary albumin excretion, glomerular hypertrophy, and mesangial matrix expansion were significantly lower in diabetic ICAM-1(-/-) mice than in diabetic ICAM-1(+/+) mice. Moreover, expressions of TGF-beta and type IV collagen in glomeruli were also suppressed in diabetic ICAM-1(-/-) mice. These results suggest that ICAM-1 is critically involved in the pathogenesis of diabetic nephropathy.     

Overexpression of thioredoxin1 in transgenic mice suppresses development of diabetic nephropathy.

BACKGROUND: Oxidative stress has been suggested to play an important role in the pathogenesis of diabetic nephropathy. In the present study, the effects of thioredoxin1 (TRX1) overexpression, a small protein with antioxidant property, on the development of diabetic nephropathy in streptozotocin-induced diabetic animals were investigated using TRX1 transgenic mice (TRX1-Tg). METHODS: Eight-week-old male TRX1-Tg and wild-type mice littermates (WT) mice were treated either with streptozotocin (200 mg/kg) or vehicle alone. After 24 weeks of treatment, diabetic nephropathy and oxidative stress were assessed in these four groups of mice, by biochemical analyses of blood and urine, as well as by histological analyses of the kidneys. RESULTS: Haemoglobin A1c (HbA1c) levels of diabetic TRX1-Tg were not significantly different from those of the diabetic WT. Nevertheless, an augmented urinary albumin excretion observed in diabetic WT was significantly diminished in diabetic TRX1-Tg. Histological study revealed that pathological changes such as mesangial matrix expansion and tubular injury were significantly prevented in diabetic TRX1-Tg accompanied by a reduced tendency of expression of transforming growth factor-beta as compared with diabetic WT. In parallel, urinary excretion of 8-hydroxy-2'-deoxyguanosine and acrolein adduct and the immunostaining intensities of these markers in the kidney were significantly higher in diabetic WT compared with non-diabetic mice. The markers were significantly suppressed in diabetic TRX1-Tg, an indication of systemic and renal oxidative stress attenuation by TRX1 overexpression. CONCLUSION: These findings indicated the significant role of oxidative stress in the development of diabetic nephropathy and a potential inhibition of progression of nephropathy by TRX1.     

Distinct macrophage phenotypes contribute to kidney injury and repair

The ischemically injured kidney undergoes tubular cell necrosis and apoptosis, accompanied by an interstitial inflammatory cell infiltrate. In this study, we show that iNos-positive proinflammatory (M1) macrophages are recruited into the kidney in the first 48 hours after ischemia/reperfusion injury, whereas arginase 1- and mannose receptor-positive, noninflammatory (M2) macrophages predominate at later time points. Furthermore, depletion of macrophages before ischemia/reperfusion diminishes kidney injury, whereas depletion at 3 to 5 days after injury slows tubular cell proliferation and repair. Infusion of Ifnγ-stimulated, bone marrow-derived macrophages into macrophage-depleted mice at the time of kidney reperfusion restored injury to the level seen without macrophage depletion, suggesting that proinflammatory macrophages worsen kidney damage. In contrast, the appearance of macrophages with the M2 phenotype correlated with the proliferative phase of kidney repair. In vitro studies showed that IFNγ-stimulated, proinflammatory macrophages begin to express markers of M2 macrophages when cocultured with renal tubular cells. Moreover, IL-4-stimulated macrophages with an M2 phenotype, but not IFNγ-stimulated proinflammatory macrophages, promoted renal tubular cell proliferation. Finally, tracking fluorescently labeled, IFNγ-stimulated macrophages that were injected after injury showed that inflammatory macrophages can switch to an M2 phenotype in the kidney at the onset of kidney repair. Taken together, these studies show that macrophages undergo a switch from a proinflammatory to a trophic phenotype that supports the transition from tubule injury to tubule repair.     

The lack of cyclin kinase inhibitor p27(Kip1) ameliorates progression of diabetic nephropathy

Cyclin kinase inhibitor p27(Kip(1)) (p27) has been shown to be upregulated in glomeruli of diabetic animals and mesangial cells cultured under high glucose. This study was an investigation of the role of p27 in the progression of diabetic nephropathy. Mice deficient in p27 (p27 -/-) and wild-type mice (p27 +/+) were studied 12 wk after diabetes induction by streptozotocin. Blood glucose and BP were comparable between diabetic p27 +/+ and p27 -/- mice. The kidney weight to body weight ratio and glomerular volume increased in diabetic p27 +/+ mice. In contrast, these parameters did not change in diabetic p27 -/- mice. Similarly, albuminuria developed in diabetic p27 +/+ mice but not in diabetic p27 -/- mice. The mesangial expansion was significantly milder in diabetic p27 -/- mice than that in diabetic p27 +/+ mice. These changes were associated with a similar increase in glomerular TGF-beta expression in diabetic p27 +/+ and p27 -/- mice. However, glomerular protein expression of fibronectin, a target of TGF-beta, increased only in diabetic p27 +/+ mice. In mesangial cells cultured from p27 +/+ mice, exposure to high glucose caused significant increases in total protein content and [(3)H]-leucine incorporation. On the other hand, high glucose caused a significant reduction in these parameters in cells from p27 -/- mice. Phosphorylation of 4E-BP1, the translation inhibitor, increased after exposure to high glucose in p27 +/+ cells. In p27 -/- cells, the level of phosphorylated 4E-BP1 was higher than that in control p27 +/+ cells and decreased under high glucose conditions. In conclusion, renal hypertrophy, glomerular hypertrophy, and albuminuria did not develop, and mesangial expansion was milder in diabetic p27 -/- mice despite glomerular TGF-beta upregulation. These results suggest that controlling p27 function may ameliorate diabetic nephropathy.     

Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in mice

Angiotensin-converting enzyme 2 (ACE2) degrades angiotensin II to angiotensin-(1-7) and is expressed in podocytes. Here we overexpressed ACE2 in podocytes in experimental diabetic nephropathy using transgenic methods where a nephrin promoter drove the expression of human ACE2. Glomeruli from these mice had significantly increased mRNA, protein, and activity of ACE2 compared to wild-type mice. Male mice were treated with streptozotocin to induce diabetes. After 16 weeks, there was no significant difference in plasma glucose levels between wild-type and transgenic diabetic mice. Urinary albumin was significantly increased in wild-type diabetic mice at 4 weeks, whereas albuminuria in transgenic diabetic mice did not differ from wild-type nondiabetic mice. However, this effect was transient and by 16 weeks both transgenic and nontransgenic diabetic mice had similar rates of proteinuria. Compared to wild-type diabetic mice, transgenic diabetic mice had an attenuated increase in mesangial area, decreased glomerular area, and a blunted decrease in nephrin expression. Podocyte numbers decreased in wild-type diabetic mice at 16 weeks, but were unaffected in transgenic diabetic mice. At 8 weeks, kidney cortical expression of transforming growth factor-β1 was significantly inhibited in transgenic diabetic mice as compared to wild-type diabetic mice. Thus, the podocyte-specific overexpression of human ACE2 transiently attenuates the development of diabetic nephropathy.     

RAGE control of diabetic nephropathy in a mouse model: effects of RAGE gene disruption and administration of low-molecular weight heparin

Diabetic nephropathy is a major microvascular complication in long-standing diabetic patients who eventually undergo renal dialysis or transplantation. To prevent development of this disease and to improve advanced kidney injury, effective therapies directed toward the key molecular target are required. In this study, we examined whether inhibition of the receptor for advanced glycation end products (RAGE) could attenuate changes in the diabetic kidney. Here, we show that inactivation of the RAGE gene in a mouse model of diabetic nephropathy results in significant suppression of kidney changes, including kidney enlargement, increased glomerular cell number, mesangial expansion, advanced glomerulosclerosis, increased albuminuria, and increased serum creatinine compared with wild-type diabetic mice. The degree of kidney injury was proportional to RAGE gene dosage. Furthermore, we show that low-molecular weight heparin (LMWH) can bind RAGE at a mean equilibrium dissociation constant (K(d)) value of approximately 17 nmol/l and act as an antagonist to RAGE. LMWH treatment of mice significantly prevented albuminuria and increased glomerular cell number, mesangial expansion, and glomerulosclerosis in a dose-dependent manner; it also significantly improved the indexes of advanced-stage diabetic nephropathy. This study provides insight into the pathological role of RAGE in both early- and advanced-phase diabetic nephropathy and suggests that RAGE antagonists will be a useful remedy in the treatment of diabetic nephropathy.     

GM-CSF Promotes Macrophage Alternative Activation after Renal Ischemia/Reperfusion Injury

After kidney ischemia/reperfusion (I/R) injury, monocytes home to the kidney and differentiate into activated macrophages. Whereas proinflammatory macrophages contribute to the initial kidney damage, an alternatively activated phenotype can promote normal renal repair. The microenvironment of the kidney during the repair phase mediates the transition of macrophage activation from a proinflammatory to a reparative phenotype. In this study, we show that macrophages isolated from murine kidneys during the tubular repair phase after I/R exhibit an alternative activation gene profile that differs from the canonical alternative activation induced by IL-4–stimulated STAT6 signaling. This unique activation profile can be reproduced in vitro by stimulation of bone marrow-derived macrophages with conditioned media from serum-starved mouse proximal tubule cells. Secreted tubular factors were found to activate macrophage STAT3 and STAT5 but not STAT6, leading to induction of the unique alternative activation pattern. Using STAT3-deficient bone marrow-derived macrophages and pharmacologic inhibition of STAT5, we found that tubular cell-mediated macrophage alternative activation is regulated by STAT5 activation. Both in vitro and after renal I/R, tubular cells expressed GM-CSF, a known STAT5 activator, and this pathway was required for in vitro alternative activation of macrophages by tubular cells. Furthermore, administration of a neutralizing antibody against GM-CSF after renal I/R attenuated kidney macrophage alternative activation and suppressed tubular proliferation. Taken together, these data show that tubular cells can instruct macrophage activation by secreting GM-CSF, leading to a unique macrophage reparative phenotype that supports tubular proliferation after sterile ischemic injury.