Development of late-stage diabetic nephropathy in OVE26 diabetic mice

OVE26 mice are a transgenic model of severe early-onset type 1 diabetes. These mice develop diabetes within the first weeks of life and can survive well over a year with no insulin treatment, and they maintain near normal body weight. To determine whether OVE26 mice provide a valuable model of chronic diabetic nephropathy (DN), OVE26 diabetic mice were compared with their nondiabetic littermates for functional and structural characteristics of DN. OVE26 mice exhibited pronounced polyuria and significant albuminuria by 2 months of age (305 microg/24 h in OVE26 vs. 20 microg/24 h in controls). Albumin excretion rate increased progressively with age and exceeded 15,000 microg/24 h at 9 months of age. The profound loss of albumin led to hypoalbuminemia in some diabetic animals. Albuminuria coincided with an elevation in blood pressure as measured by tail cuff. The glomerular filtration rate (GFR) in OVE26 mice measured using fluorescein isothiocynate inulin clearance demonstrated that GFR increased significantly from 2 to 3 months of age and then decreased significantly from 5 to 9 months. GFR in 9-month-old diabetic mice was significantly lower than that of 9-month-old control mice. The decline in GFR coincided with a significant increase in renal vascular resistance. Structural studies showed an almost twofold increase in kidney weight between 2 and 5 months. Diabetic mice also showed progressively enlarged glomeruli and expanded mesangium with diffuse and nodular expansion of mesangial matrix. Tubulointerstitial fibrosis was also observed in these mice. Glomerular basement membrane was thickened in OVE26 mice. In summary, OVE26 mice demonstrate that most of the characteristics of human DN can be produced by chronic hyperglycemia in a murine model. This model will be useful for improved understanding and treatment of DN.     

Imatinib attenuates diabetic nephropathy in apolipoprotein E-knockout mice

In the diabetic kidney, clinical as well as experimental observations have shown an upregulation of growth factors such as PDGF. These studies, however, were not designed to address whether upregulation of PDGF is merely a manifestation of diabetic renal injury or whether PDGF plays an active role in the pathophysiology of diabetic nephropathy. The objectives of this study were first to assess whether PDGF-dependent pathways are involved in the development of diabetic nephropathy and second to determine the effects of PDGF receptor antagonism on this disorder and associated molecular and cellular processes. This study used the diabetic apolipoprotein E-knockout (apoE-KO) mouse, a recently described model of accelerated diabetic nephropathy. Diabetes was induced by injection of streptozotocin in 6-wk-old apoE-KO mice. Diabetic animals received treatment with a tyrosine kinase inhibitor that inhibits PDGF action, imatinib (STI-571, 10 mg/kg per d orally) or no treatment for 20 wk. Nondiabetic apoE-KO mice served as controls. This model of accelerated renal disease with albuminuria as well as glomerular and tubulointerstitial injury was associated with increased renal expression of PDGF-B, proliferating cells, and alpha-smooth muscle actin-positive cells. Furthermore, there was increased accumulation of type I and type IV collagen as well as macrophage infiltration. Imatinib treatment ameliorated both renal functional and structural parameters of diabetes as well as overexpression of a number of growth factors, collagens, proliferating cells, alpha-smooth muscle actin-positive cells, and macrophage infiltration within the kidney. Tyrosine kinase inhibition with imatinib seems to retard the development of experimental diabetic nephropathy.     

Genetics of diabetic nephropathy: lessons from mice

Although diabetic nephropathy occurs only in a minority of diabetic patients (approximately 30%), it is the major single cause of end-stage renal disease in the United States. Hyperglycemia and hypertension are important factors predisposing patients to nephropathy, however, accumulating evidence points to critical genetic factors that predispose only a subset of diabetic patients to nephropathy. Defining the genes responsible for nephropathy risk in human populations has proven challenging. Comparative genomics using the robust genetic reagents available in the laboratory mouse should provide a complementary approach to defining genes that may predispose to diabetic nephropathy in mice and human beings. In this article we review studies that have started to identify genetic risk factors for diabetic nephropathy in mice and the multiple approaches that may be used to elucidate the genetic pathogenesis of this disorder.     

Increased fibrovascular invasion of subcutaneous polyvinyl alcohol sponges in SPARC-null mice

The expression of SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is elevated in endothelial cells participating in angiogenesis in vitro and in vivo. SPARC acts on endothelial cells to elicit changes in cell shape and to inhibit cell cycle progression. In addition, SPARC binds to and diminishes the mitotic activity of vascular endothelial growth factor. To determine the effect(s) of SPARC on angiogenic responses in vivo, we implanted polyvinyl alcohol sponges subcutaneously into wild-type and SPARC-null mice. On days 12 and 20 following implantation, SPARC-null mice showed increased cellular invasion of the sponges in comparison to wild-type mice. Areas of the sponge with the highest cell density exhibited the highest numbers of vascular profiles in both wild-type and SPARC-null animals. The endothelial component of the vessels was substantiated by immunoreactivity with three different markers specific for endothelial cells. Although sponges from SPARC-null relative to wild-type mice were populated by significantly more cells and blood vessels, an increase in the ratio of vascular to nonvascular cells was not apparent. No differences in the percentage of proliferating cells within the sponge were detected between wild-type and SPARC-null sections. However, elevated levels of vascular endothelial growth factor were associated with sponges from SPARC-null versus wild-type mice. An increase in vascular endothelial growth factor production was also observed in SPARC-null primary dermal fibroblasts relative to those of wild-type cells. In conclusion, we have shown that the fibrovascular invasion of polyvinyl alcohol sponges is enhanced in mice lacking SPARC, and we propose that increased levels of vascular endothelial growth factor account, at least in part, for this response.     

PPARalpha agonist fenofibrate improves diabetic nephropathy in db/db mice

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the ligand-activated nuclear receptor superfamily, and plays an important role in lipid metabolism and glucose homeostasis. The purpose of this study is to determine whether the activation of PPARalpha by fenofbrate would improve diabetes and its renal complications in type II diabetes mellitus. Male C57 BLKS db/db mice and db/m controls at 8 weeks of age were divided to receive either a regular diet chow (db/db, n=8; db/m, n=6) or a diet containing fenofibrate (db/db, n=8; db/m, n=7). Mice were followed for 8 weeks. Fenofibrate treatment dramatically reduced fasting blood glucose (P<0.001) and HbA1c levels (P<0.001), and was associated with decreased food intake (P<0.01) and slightly reduced body weight. Fenofibrate also ameliorated insulin resistance (P<0.001) and reduced plasma insulin levels (P<0.05) in db/db mice. Hypertrophy of pancreatic islets was decreased and insulin content markedly increased (P<0.05) in fenofibrate-treated diabetic animals. In addition, fenofibrate treatment significantly reduced urinary albumin excretion (P<0.001). This was accompanied by dramatically reduced glomerular hypertrophy and mesangial matrix expansion. Furthermore, the addition of fenofibrate to cultured mesangial cells, which possess functional active PPARalpha, decreased type I collagen production. Taken together, the PPARalpha agonist fenofibrate dramatically improves hyperglycemia, insulin resistance, albuminuria, and glomerular lesions in db/db mice. The activation of PPARalpha by fenofibrate in mesangial cells may partially contribute to its renal protection. Thus, fenofibrate may serve as a therapeutic agent for type II diabetes and diabetic nephropathy.     

Accelerated diabetic nephropathy in mice lacking the peroxisome proliferator-activated receptor alpha

Peroxisome proliferator-activated receptor (PPAR)alpha, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at determining the role of PPARalpha in the pathogenesis of diabetic nephropathy using PPARalpha-knockout mice and cultured murine mesangial cells. Diabetes was induced using a low-dose streptozotocin protocol in 8-week-old male 129 SvJ PPARalpha-knockout and wild-type mice. Diabetic PPARalpha-knockout and wild-type mice developed elevated fasting blood glucose (P < 0.001) and HbA1c levels (P < 0.001). Renal functional and histopathological changes in diabetic and nondiabetic PPARalpha-knockout and wild-type mice were evaluated after 16 weeks of hyperglycemia. PPARalpha immunostaining of the cortical tubules of diabetic wild-type mice was elevated by hyperglycemia. In diabetic PPARalpha-knockout mice, renal disease with accompanying albuminuria, glomerular sclerosis, and mesangial area expansion was more severe than in diabetic wild-type mice (P < 0.05) and was accompanied by increased levels of serum free fatty acids and triglycerides (P < 0.01). Furthermore, they exhibited increased renal immunostaining for type IV collagen and osteopontin, which was associated with increased macrophage infiltration and glomerular apoptosis. There were no significant differences in these indexes of renal disease between nondiabetic PPARalpha-knockout and wild-type mice and diabetic PPARalpha wild-type mice. In vitro studies demonstrated that high glucose levels markedly increased the expression of type IV collagen, transforming growth factor-beta1, and the number of leukocytes adherent to cultured mesangial cells. Adherence of leukocytes was inhibited by the PPARalpha agonist fenofibrate. Taken together, PPARalpha deficiency appears to aggravate the severity of diabetic nephropathy through an increase in extracellular matrix formation, inflammation, and circulating free fatty acid and triglyceride concentrations. PPARalpha agonists may serve as useful therapeutic agents for type 1 diabetic nephropathy.     

Endothelial nitric oxide synthase deficiency produces accelerated nephropathy in diabetic mice

Functionally significant polymorphisms in endothelial nitric oxide synthase (eNOS) and reduced vascular eNOS activity have been associated with increased human diabetic nephropathy (DN), but the pathogenic role of eNOS deficiency in the development of DN has not yet been confirmed. This study characterizes the severity of DN in eNOS(-/-) mice that were backcrossed to C57BLKS/J db/db mice. Although the severity of hyperglycemia was similar to C57BLKS/J db/db mice, by 26 wk, eNOS(-/-) C57BLKS/J db/db mice exhibited dramatic albuminuria, arteriolar hyalinosis, increased glomerular basement membrane thickness, mesangial expansion, mesangiolysis, and focal segmental and early nodular glomerulosclerosis. Even more remarkable, eNOS(-/-) C57BLKS db/db exhibited decreases in GFR to levels <50% of that in eNOS(+/+) C57BLKS db/db, as confirmed by increased serum creatinine. In summary, eNOS(-/-) db/db mice provide the most robust model of type II DN that has been described to date and support a role for deficient eNOS-derived NO production in the pathogenesis of DN.     

Preventive effects of syngeneic bone marrow transplantation on diabetic nephropathy in mice

Treatment with autologous bone marrow transplantation (ABMT) can change the natural history of diabetes in patients with new-onset Type 1 diabetes (T1D). Effects of syngeneic bone marrow transplantation (syn-BMT) on diabetic nephropathy were studied in streptozotocin-induced diabetic mice. Diabetic mice received sibling's bone marrow on days 3, 10, 20, or 40 after T1D onset, respectively. Renal pathology, levels of oxidative stress, and the expressions of angiotensinogen (AGT), monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor beta 1 (Tgf-β1) mRNA were investigated. Treatment with syn-BMT when disease was early-onset reduced mesangial area expansion and kidney enlargement; besides, if it is given on day 10, syn-BMT attenuated glomerular hypertrophy. Oxidative stress factors such as catalase (CAT) and superoxide radical anion O₂₋ (O₂₋) were markedly maintained by syn-BMT compared to mice without treatment. In diabetic mice without treatment, renal AGT and MCP-1 mRNA were increased, while they were effectively suppressed by syn-BMT. But it showed no changes or even increment in Tgf-β1 mRNA after syn-BMT. Syn-BMT, if applied when disease was early-onset, ameliorated diabetic renal injury. These preventive effects could be partly via maintaining oxidative stress and expression of AGT and MCP-1 in kidney in streptozotocin-diabetic mice.     

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.     

Intravenous administration of hepatocyte growth factor gene ameliorates diabetic nephropathy in mice

Diabetic nephropathy is characterized by progressive loss of renal function, persistent proteinuria, and relentless accumulation of extracellular matrix leading to glomerulosclerosis and interstitial fibrosis. This study investigated the potential effects of long-term expression of exogenous hepatocyte growth factor (HGF) on normal and diabetic kidneys. Intravenous injection of human HGF gene via naked plasmid vector resulted in abundant HGF protein specifically localized in renal glomeruli, despite an extremely low level of transgene mRNA in the kidney. In uninephrectomized mice made diabetic with streptozotocin, delivery of exogenous HGF gene ameliorated the progression of diabetic nephropathy. HGF attenuated urine albumin and total protein excretion in diabetic mice. Exogenous HGF also mitigated glomerular mesangial expansion, reduced fibronectin and type I collagen deposition, and prevented interstitial myofibroblast activation. In addition, HGF prevented kidney cells from apoptotic death in the glomeruli and tubulointerstitium. Moreover, expression of HGF inhibited renal expression of TGF-beta1 and reduced urine level of TGF-beta1 protein. Therefore, despite the effects of HGF on diabetic nephropathy being controversial, these observations suggest that supplementation of HGF is beneficial in ameliorating diabetic renal insufficiency in mice.     

Overexpression of connective tissue growth factor in podocytes worsens diabetic nephropathy in mice

Connective tissue growth factor (CTGF) is a potent inducer of extracellular matrix accumulation. In diabetic nephropathy, CTGF expression is markedly upregulated both in podocytes and mesangial cells, and this may play an important role in its pathogenesis. We established podocyte-specific CTGF-transgenic mice, which were indistinguishable at baseline from their wild-type littermates. Twelve weeks after streptozotocin-induced diabetes, these transgenic mice showed a more severe proteinuria, mesangial expansion, and a decrease in matrix metalloproteinase-2 activity compared to diabetic wild-type mice. Furthermore, diabetic transgenic mice exhibited less podocin expression and a decreased number of diffusely vacuolated podocytes compared to diabetic wild-type mice. Importantly, induction of diabetes in CTGF-transgenic mice resulted in a further elevation of endogenous CTGF mRNA expression and protein in the glomerular mesangium. Our findings suggest that overexpression of CTGF in podocytes is sufficient to exacerbate proteinuria and mesangial expansion through a functional impairment and loss of podocytes.     

Angiogenesis in diabetic nephropathy

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, plays a key role in both physiologic and pathologic events, including wound healing, cancer, and diabetes. Neovascularization has been implicated in the genesis of diverse diabetic complications such as retinopathy, impaired wound healing, neuropathy, and, most recently, diabetic nephropathy. Diabetic nephropathy is one of the major microvascular-associated complications in diabetes and is the leading cause of end-stage renal disease worldwide. In this review we describe the major factors involved in the pathologic glomerular microvascular alterations in response to hyperglycemia and the possible use of anti-angiogenic therapies for the treatment 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.     

Insight into the genetics of diabetic nephropathy through the study of mice

PURPOSE OF REVIEW: To discuss mouse models of diabetic nephropathy and their use in discovering genetic risk factors predisposing to diabetic nephropathy. RECENT FINDINGS: Despite occurring in only 10-40% of diabetic patients, diabetic nephropathy is the largest single cause of end stage renal disease in the USA. Accumulated evidence points to critical genetic factors that predispose a subset of diabetic patients to nephropathy.Defining the genes that confer risk for nephropathy in human populations has proven challenging. The use of robust genetic reagents available in the laboratory mouse provides a complementary approach to defining genes that predispose to diabetic nephropathy in mice and humans. These findings support the existence of dominant mutations predisposing to diabetic nephropathy in mice as well as substantiating an important role for eNOS in forestalling the development of diabetic nephropathy. SUMMARY: When studied for a sufficient duration of diabetic hyperglycemia, some strains of mice exhibit changes similar to those of human diabetic nephropathy. The unique genetic reagents in mice should help accelerate the identification of genes predisposing to diabetic nephropathy.