Jharna Saha

Reduction in podocyte density as a pathologic feature in early diabetic nephropathy in rodents: Prevention by lipoic acid treatment

BackgroundA reduction in the number of podocytes and podocyte density has been documented in the kidneys of patients with diabetes mellitus. Additional studies have shown that podocyte injury and loss occurs in both diabetic animals and humans. However, most studies in animals have examined relatively long-term changes in podocyte number and density and have not examined effects early after initiation of diabetes. We hypothesized that streptozotocin diabetes in rats and mice would result in an early reduction in podocyte density and that this reduction would be prevented by antioxidants.MethodsThe number of podocytes per glomerular section and the podocyte density in glomeruli from rats and mice with streptozotocin (STZ)-diabetes mellitus was determined at several time points based on detection of the glomerular podocyte specific antigens, WT-1 and GLEPP1. The effect of insulin administration or treatment with the antioxidant, α-lipoic acid, on podocyte number was assessed.ResultsExperimental diabetes resulted in a rapid decline in apparent podocyte number and podocyte density. A significant reduction in podocytes/glomerular cross-section was found in STZ diabetes in rats at 2 weeks (14%), 6 weeks (18%) and 8 weeks (34%) following STZ injection. Similar declines in apparent podocyte number were found in STZ diabetes in C57BL/6 mice at 2 weeks, but not at 3 days after injection. Treatment with α-lipoic acid substantially prevented podocyte loss in diabetic rats but treatment with insulin had only a modest effect.ConclusionSTZ diabetes results in reduction in apparent podocyte number and in podocyte density within 2 weeks after onset of hyperglycemia. Prevention of these effects with antioxidant therapy suggests that this early reduction in podocyte density is due in part to increased levels of reactive oxygen species as well as hyperglycemia.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed and cofilin was de-phosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiologic steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Rosiglitazone reduces renal and plasma markers of oxidative injury and reverses urinary metabolite abnormalities in the amelioration of diabetic nephropathy

Recent studies suggest that thiazolidinediones ameliorate diabetic nephropathy (DN) independently of their effect on hyperglycemia. In the current study, we confirm and extend these findings by showing that rosiglitazone treatment prevented the development of DN and reversed multiple markers of oxidative injury in DBA/2J mice made diabetic by low-dose streptozotocin. These diabetic mice developed a 14.2-fold increase in albuminuria and a 53% expansion of renal glomerular extracellular matrix after 12 wk of diabetes. These changes were largely abrogated by administration of rosiglitazone beginning 2 wk after the completion of streptozotocin injections. Rosiglitazone had no effect on glycemic control. Rosiglitazone had similar effects on insulin-treated diabetic mice after 24 wk of diabetes. Podocyte loss and glomerular fibronectin accumulation, other markers of early DN, were prevented by rosiglitazone in both 12- and 24-wk diabetic models. Surprisingly, glomerular GLUT1 levels did not increase and nephrin levels did not decrease in the diabetic animals; neither changed with rosiglitazone. Plasma and kidney markers of protein oxidation and lipid peroxidation were significantly elevated in the 24-wk diabetic animals despite insulin treatment and were reduced to near-normal levels by rosiglitazone. Finally, urinary metabolites were markedly altered by diabetes. Of 1,988 metabolite features identified by electrospray ionization time of flight mass spectrometry, levels of 56 were altered more than twofold in the urine of diabetic mice. Of these, 21 were returned to normal by rosiglitazone. Thus rosiglitazone has direct effects on the renal glomerulus to reduce reactive oxygen species accumulation to prevent type 1 diabetic mice from development of DN.

Podocyte-specific overexpression of GLUT1 surprisingly reduces mesangial matrix expansion in diabetic nephropathy in mice.

Increased expression of the facilitative glucose transporter, GLUT1, leads to glomerulopathy that resembles diabetic nephropathy, whereas prevention of enhanced GLUT1 expression retards nephropathy. While many of the GLUT1-mediated effects are likely due to mesangial cell effects, we hypothesized that increased GLUT1 expression in podocytes also contributes to the progression of diabetic nephropathy. Therefore, we generated two podocyte-specific GLUT1 transgenic mouse lines (driven by a podocin promoter) on a db/m C57BLKS background. Progeny of the two founders were used to generate diabetic db/db and control db/m littermate mice. Immunoblots of glomerular lysates showed that transgenic mice had a 3.5-fold (line 1) and 2.1-fold (line 2) increase in GLUT1 content compared with wild-type mice. Both lines showed similar increases in fasting blood glucose and body weights at 24 wk of age compared with wild-type mice. Mesangial index (percent PAS-positive material in the mesangial tuft) increased 88% (line 1) and 75% (line 2) in the wild-type diabetic mice but only 48% (line 1) and 39% (line 2) in the diabetic transgenic mice (P < 0.05, transgenic vs. wild-type mice). This reduction in mesangial expansion was accompanied by a reduction in fibronectin accumulation, and vascular endothelial growth factor (VEGF) levels increased only half as much in the transgenic diabetic mice as in wild-type diabetic mice. Levels of nephrin, neph1, CD2AP, podocin, and GLUT4 were not significantly different in transgenic compared with wild-type mice. Taken together, increased podocyte GLUT1 expression in diabetic mice does not contribute to early diabetic nephropathy; surprisingly, it protects against mesangial expansion and fibronectin accumulation possibly by blunting podocyte VEGF increases.

Podocyte-specific GLUT4-deficient mice have fewer and larger podocytes and are protected from diabetic nephropathy.

Podocytes are a major component of the glomerular filtration barrier, and their ability to sense insulin is essential to prevent proteinuria. Here we identify the insulin downstream effector GLUT4 as a key modulator of podocyte function in diabetic nephropathy (DN). Mice with a podocyte-specific deletion of GLUT4 (G4 KO) did not develop albuminuria despite having larger and fewer podocytes than wild-type (WT) mice. Glomeruli from G4 KO mice were protected from diabetes-induced hypertrophy, mesangial expansion, and albuminuria and failed to activate the mammalian target of rapamycin (mTOR) pathway. In order to investigate whether the protection observed in G4 KO mice was due to the failure to activate mTOR, we used three independent in vivo experiments. G4 KO mice did not develop lipopolysaccharide-induced albuminuria, which requires mTOR activation. On the contrary, G4 KO mice as well as WT mice treated with the mTOR inhibitor rapamycin developed worse adriamycin-induced nephropathy than WT mice, consistent with the fact that adriamycin toxicity is augmented by mTOR inhibition. In summary, GLUT4 deficiency in podocytes affects podocyte nutrient sensing, results in fewer and larger cells, and protects mice from the development of DN. This is the first evidence that podocyte hypertrophy concomitant with podocytopenia may be associated with protection from proteinuria.

Targeted Lipidomic and Transcriptomic Analysis Identifies Dysregulated Renal Ceramide Metabolism in a Mouse Model of Diabetic Kidney Disease.

Both type 1 and type 2 diabetes are associated with altered lipid metabolism, which might in part contribute to debilitating complications such as diabetic kidney disease (DKD). Ceramides are bioactive sphingolipids that have been implicated in a variety of diseases as they can regulate cellular responses to stress and invoke a myriad of downstream signaling responses. To investigate a potential role of altered ceramide metabolism in DKD, we utilized a highly sensitive and specific mass spectrometry (MS) method to quantitatively measure species in plasma and kidney cortex from the C57BLKS db/db mouse model of DKD and littermate controls. Long-chain ceramides (C14:0, C16:0, C18:0, C20:0) and a glucosylceramide (Glu-Cer C18:0) were increased in diabetic mouse plasma, while long-chain (C14:0, C16:0, C18:0) and very-long-chain (C24:0, C24:1) ceramides and a glucosylceramide (Glu-Cer C16:0) were decreased in diabetic mouse kidney tissue. Kidney and plasma ceramide levels correlated to functional and histopathological features of DKD. Transcriptomic analysis of mouse kidney tissue revealed expression changes indicative of decreased ceramide synthesis (Degs2, Smpd2) and increased conversion to sphingosine (Acer2) and downstream sphingosine-1-phosphate signaling. Correlation analysis identified a negative relationship between plasma and kidney tissue levels of ceramide C16:0 and ceramide C24:1. Overall, the findings suggest a previously unrecognized role for ceramide metabolism in DKD.

Maintenance of GLUT4 expression in smooth muscle prevents hypertension‐induced changes in vascular reactivity

Previous studies have shown that expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice and that total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity in hypertension. To demonstrate that the effect of GLUT4 overexpression on vascular responses is dependent on vascular smooth muscle GLUT4 rather than on some systemic effect we developed and tested smooth‐muscle‐specific GLUT4 transgenic mice (SMG4). When made hypertensive with angiotensin II, both wild‐type and SMG4 mice exhibited similarly increased systolic blood pressure. Responsiveness to phenylephrine, serotonin, and prostaglandin F2α was significantly increased in endothelium‐intact aortic rings from hypertensive wild‐type mice but not in aortae of SMG4 mice. Inhibition of Rho‐kinase equally reduced serotonin‐stimulated contractility in aortae of hypertensive wild‐type and SMG4‐mice. In addition, acetylcholine‐stimulated relaxation was significantly decreased in aortic rings of hypertensive wild‐type mice, but not in rings of SMG4 mice. Inhibition of either prostacylin receptors or cyclooxygenase‐2 reduced relaxation in rings of hypertensive SMG4 mice. Inhibition of cyclooxygenase‐2 had no effect on relaxation in rings of hypertensive wild‐type mice. Cyclooxygenase‐2 protein expression was decreased in hypertensive wild‐type aortae but not in hypertensive SMG4 aortae compared to nonhypertensive controls. Our results demonstrate that smooth muscle expression of GLUT4 exerts a major effect on smooth muscle contractile responses and endothelium‐dependent vasorelaxation and that normal expression of GLUT4 in vascular smooth muscle is required for appropriate smooth muscle and endothelial responses.