Farhad R. Danesh

Diabetic Nephropathy : Mechanisms of Renal Disease Progression

Diabetic nephropathy is characterized by excessive amassing of extracellular matrix (ECM) with thickening of glomerular and tubular basement membranes and increased amount of mesangial matrix, which ultimately progress to glomerulosclerosis and tubulo-interstitial fibrosis. In view of this outcome, it would mean that all the kidney cellular elements, i.e., glomerular endothelia, mesangial cells, podocytes, and tubular epithelia, are targets of hyperglycemic injury. Conceivably, high glucose activates various pathways via similar mechanisms in different cell types of the kidney except for minor exceptions that are related to the selective expression of a given molecule in a particular renal compartment. To begin with, there is an obligatory excessive channeling of glucose intermediaries into various metabolic pathways with generation of advanced glycation products (AGEs), activation of protein kinase C (PKC), increased expression of transforming growth factor-β (TGF-β), GTP-binding proteins, and generation of reactive oxygen species (ROS). The ROS seem to be the common denominator in various pathways and are central to the pathogenesis of hyperglycemic injury. In addition, there are marked alterations in intraglomerular hemodynamics, i.e., hyperfiltration, and this along with metabolic derangements adversely compounds the hyperglycemia-induced injury. Here, the information compiled under various subtitles of this article is derived from an enormous amount of data summarized in several excellent literature reviews, and thus their further reading is suggested to gain in-depth knowledge of each of the subject matter.

3-Hydroxy-3-methylglutaryl CoA reductase inhibitors prevent high glucose-induced proliferation of mesangial cells via modulation of Rho GTPase/ p21 signaling pathway: Implications for diabetic nephropathy

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, also known as statins, are lipid-lowering agents widely used in the prevention of coronary heart disease. Recent experimental and clinical data, however, indicate that the overall benefits of statin therapy may exceed its cholesterol-lowering properties. We postulate that statins may ameliorate the detrimental effects of high glucose (HG)-induced proliferation of mesangial cells (MCs), a feature of early stages of diabetic nephropathy, by preventing Rho isoprenylation. Rat MCs cultured in HG milieu were treated with and without simvastatin, an HMG-CoA reductase inhibitor. Simvastatin inhibited HG-induced MC proliferation as measured by [3H]thymidine incorporation. This inhibitory effect was reversed with geranylgeranyl pyrophosphate, an isoprenoid intermediate of the cholesterol biosynthetic pathway. At the cell-cycle level, the HG-induced proliferation of MCs was associated with a decrease in cyclin dependent kinase (CDK) inhibitor p21 protein expression accompanied by an increase in CDK4 and CDK2 kinase activities. Simvastatin reversed the down-regulation of p21 protein expression and decreased CDK4 and CDK2 kinase activities. Exposure of MCs to HG was associated with an increase in membrane-associated Ras and Rho GTPase protein expression. Cotreatment of MCs with simvastatin reversed HG-induced Ras and Rho membrane translocation. Immunofluorescence microscopy revealed that the overexpression of the dominant-negative RhoA led to a significant increase in p21 expression. Our data suggest that simvastatin represses the HG-induced Rho GTPase/p21 signaling in glomerular MCs. Thus, this study provides a molecular basis for the use of statins, independently of their cholesterol-lowering effect, in early stages of diabetic nephropathy.

Mitochondrial fission triggered by hyperglycemia is mediated by ROCK1 activation in podocytes and endothelial cells.

Several lines of evidence suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of microvascular complications of diabetes, including diabetic nephropathy. However, the signaling pathways by which hyperglycemia leads to mitochondrial dysfunction are not fully understood. Here we examined the role of Rho-associated coiled coil-containing protein kinase 1 (ROCK1) on mitochondrial dynamics by generating two diabetic mouse models with targeted deletions of ROCK1 and an inducible podocyte-specific knockin mouse expressing a constitutively active (cA) mutant of ROCK1. Our findings suggest that ROCK1 mediates hyperglycemia-induced mitochondrial fission by promoting dynamin-related protein-1 (Drp1) recruitment to the mitochondria. Deletion of ROCK1 in diabetic mice prevented mitochondrial fission, whereas podocyte-specific cA-ROCK1 mice exhibited increased mitochondrial fission. Importantly, we found that ROCK1 triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. These findings provide insights into the unexpected role of ROCK1 in a signaling cascade that regulates mitochondrial dynamics.

Identification of MicroRNA-93 as a Novel Regulator of Vascular Endothelial Growth Factor in Hyperglycemic Conditions

Vascular endothelial growth factor (VEGF) is a dimeric glycoprotein that plays a crucial role in microvascular complications of diabetes, including diabetic nephropathy. However, the precise regulatory mechanisms governing VEGF expression in the diabetic milieu are still poorly understood. Here, we provide evidence that microRNA-93 (miR-93) regulates VEGF expression in experimental models of diabetes both in vitro and in vivo. Comparative microRNA expression profile arrays identified miR-93 as a signature microRNA in hyperglycemic conditions. We identified VEGF-A as a putative target of miR-93 in the kidney with a perfect complementarity between miR-93 and the 3′-untranslated region of vegfa in several species. When cotransfected with a luciferase reporter construct containing the mouse vegfa 3′-untranslated region, expression of miR-93 markedly decreased the luciferase activity. We showed that forced expression of miR-93 in cells abrogated VEGF protein secretion. Conversely, anti-miR-93 inhibitors increased VEGF release. Transfection of miR-93 also prevented the effect of high glucose on VEGF downstream targets. Using transgenic mice containing VEGF-LacZ bicistronic transcripts, we found that inhibition of glomerular miR-93 by peptide-conjugated morpholino oligomers elicited increased expression of VEGF. Our findings also indicate that high glucose decreases miR-93 expression by down-regulating the promoter of the host MCM7 gene. Taken together, our findings provide new insights into the role of miR-93 in VEGF signaling pathway and offer a potentially novel target in preventing the progression of diabetic nephropathy.

MicroRNA-29c is a signature microRNA under high glucose conditions that targets Sprouty homolog 1, and its in vivo knockdown prevents progression of diabetic nephropathy.

Although several recent publications have suggested that microRNAs contribute to the pathogenesis of diabetic nephropathy, the role of miRNAs in vivo still remains poorly understood. Using an integrated in vitro and in vivo comparative miRNA expression array, we identified miR-29c as a signature miRNA in the diabetic environment. We validated our profiling array data by examining miR-29c expression in the kidney glomeruli obtained from db/db mice in vivo and in kidney microvascular endothelial cells and podocytes treated with high glucose in vitro. Functionally, we found that miR-29c induces cell apoptosis and increases extracellular matrix protein accumulation. Indeed, forced expression of miR-29c strongly induced podocyte apoptosis. Conversely, knockdown of miR-29c prevented high glucose-induced cell apoptosis. We also identified Sprouty homolog 1 (Spry1) as a direct target of miR-29c with a nearly perfect complementarity between miR-29c and the 3′-untranslated region (UTR) of mouse Spry1. Expression of miR-29c decreased the luciferase activity of Spry1 when co-transfected with the mouse Spry1 3′-UTR reporter construct. Overexpression of miR-29c decreased the levels of Spry1 protein and promoted activation of Rho kinase. Importantly, knockdown of miR-29c by a specific antisense oligonucleotide significantly reduced albuminuria and kidney mesangial matrix accumulation in the db/db mice model in vivo. These findings identify miR-29c as a novel target in diabetic nephropathy and provide new insights into the role of miR-29c in a previously unrecognized signaling cascade involving Spry1 and Rho kinase activation.

Targeting of RhoA/ROCK Signaling Ameliorates Progression of Diabetic Nephropathy Independent of Glucose Control

OBJECTIVE—RhoA, a small GTPase protein, and its immediate downstream target, Rho kinase (ROCK), control a wide variety of signal transduction pathways. Recent studies have shown that fasudil, a selective ROCK inhibitor, may play a pivotal role in a number of pathological conditions, ranging from cardiovascular diseases to pulmonary hypertension and erectile dysfunction. Considerable evidence suggests that some of the beneficial effects of statins may also stem from their modulatory effects on RhoA/ROCK signaling. In the current study, we hypothesized that pharmacological blockade of the RhoA/ROCK pathway with either fasudil or simvastatin would ameliorate progression of diabetic nephropathy. RESEARCH DESIGN AND METHODS—In two separate experiments, diabetic db/db mice received fasudil (10 mg · kg− · day− i.p.) or simvastatin (40 mg · kg− · day− p.o.) for 16 weeks. Untreated db/db and db/m mice served as controls. RESULTS—The kidney cortices of untreated db/db mice displayed increased ROCK activity compared with db/m mice. The fasudil-treated mice exhibited a significant reduction in ROCK activity, albuminuria, glomerular collagen IV accumulation, and urinary collagen IV excretion compared with untreated db/db mice. Interestingly, blood glucose was unaffected by fasudil administration. Treatment with simvastatin significantly attenuated RhoA activation in the kidney cortices of db/db mice and resulted in a significant reduction of albuminuria and mesangial matrix expansion. CONCLUSIONS—Based on these results, we propose that RhoA/ROCK blockade constitutes a novel approach to the treatment of diabetic nephropathy. Our data also suggest a critical role for RhoA/ROCK activation in the pathogenesis of diabetic nephropathy.

Injectable Multidomain Peptide Nanofiber Hydrogel as a Delivery Agent for Stem Cell Secretome

Peptide hydrogels show immense promise as therapeutic materials. Here we present a rationally designed multidomain peptide that self-assembles into nanofibers approximately 8 nm wide, 2 nm high, and micrometers in length in the presence of Mg(2+). At a concentration of 1% by weight, the peptide forms an extensive nanofibers network that results in a physically cross-linked viscoelastic hydrogel. This hydrogel undergoes shear thinning and then quickly recovers nearly 100% of its elastic modulus when the shearing force is released, making it ideal for use as an injectable material. When placed in the presence of human embryonic stem cells (ESCs), the nanofibrous hydrogel acts like a sponge, soaking up the vast array of growth factors and cytokines released by the ESCs. The peptide hydrogel sponge can then be removed from the presence of the ESCs and placed in a therapeutic environment, where it can subsequently release these components. In vitro experiments demonstrate that release of stem cell secretome from these hydrogels in the presence of glomerular epithelial cells treated with high glucose significantly decreased protein permeability in a model of diabetes-induced kidney injury. Tracking experiments were then performed to determine the fate of the hydrogel upon injection in vivo. Hydrogels labeled with a Gd(3+) MRI contrast agent were injected into the abdominal cavity of mice and found to remain localized over 24 h. This implies that the hydrogel possesses sufficient rigidity to remain localized and release stem cell secretome over time rather than immediately dissolving in the abdominal cavity. Together, the shear thinning and recovery as observed by rheometry as well as secretome absorption and release in vivo demonstrate the potential of the nanofibrous multidomain peptide hydrogel as an injectable delivery agent.

Modulatory effects of HMG-CoA reductase inhibitors in diabetic microangiopathy

3‐Hydroxy‐3‐methyl‐glutaryl CoA (HMG‐CoA) reductase inhibitors or statins are competitive inhibitors of the rate‐limiting enzyme in cholesterol biosynthesis. Several large landmark clinical studies have shown a marked reduction of cardiovascular mortality and morbidity in patients treated with statins. Because of the strong association between serum cholesterol levels and coronary artery disease, investigators initially assumed that the predominant beneficial effects of statins result from their lipid‐lowering properties. However, more recent observations have suggested that the clinical benefits of statins may be in part independent of their cholesterol‐lowering effects. The pleiotropic or cholesterol‐independent effects of statins might result from preventing the production of isoprenoids. Isoprenoids serve as important lipid attachments for the post‐translational modification of a variety of proteins such as small GTP binding proteins implicated in intracellular signaling. The list of different pleiotropic effects of statins is still growing and, among others, includes the modulatory effects of statins on endothelial function, oxidative stress, coagulation, plaque stability, and inflammation. The pleiotropic effects of statins represent an area of great interest in prevention and therapy of cardiovascular and other chronic diseases. An area of particular interest is the potential beneficial effects of statins in diabetes and its micro/macrovascular complications. This review summarizes our current understanding of the pleiotropic effects of statins in diabetes and the modulatory effects of statins in various pathobiological pathways involved in diabetes and its complications.—Danesh, F. R., Kanwar, Y. S. Modulatory effects of HMG‐CoA reductase inhibitors in diabetic microangiopathy. FASEB J. 18, 805–815 (2004)

HMG CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosin regulatory light chain phosphorylation

The beneficial effects of statins are usually assumed to stem from their ability to reduce cholesterol biosynthesis. However, because statins are potent inhibitors of the mevalonate, which governs diverse cell signaling pathways, inhibition of 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A reductase may also result in pleiotropic effects. The present study describes a novel pleiotropic effect of statins on vascular endothelial growth factor (VEGF)‐induced glomerular endothelial cell (GEnC) hyperpermeability. Using live cell imaging with green fluorescent protein‐tagged myosin regulatory light chain (MLC) and correlative biochemical analyses, we investigated 1) VEGF signaling pathway leading to GEnC hyperpermeability and 2) the modulatory effects of statins on VEGF signaling. Our findings indicate that VEGF stimulation elicits a robust increase in GEnC permeability. The signaling pathway that mediates VEGF‐induced GEnC hyperpermeability involves RhoA activation leading to actin cytoskeletal remodeling, MLC diphosphorylation, and enhanced paracellular gap formation. Remarkably, cotreatment of endothelial cells with simvastatin, a hydrophobic statin, reversed VEGF‐induced GEnC hyperpermeability by preventing MLC diphosphorylation, and cytoskeletal remodeling. In summary, this study identifies RhoA and MLC phosphorylation as key mediators of VEGF‐induced endothelial cell hyperpermeability and demonstrates the modulatory effects of statins on VEGF signaling pathway.

New Insights Into Molecular Mechanisms of Diabetic Kidney Disease

Diabetic kidney disease remains a major microvascular complication of diabetes and the most common cause of chronic kidney failure requiring dialysis in the United States. Medical advances over the past century have substantially improved the management of diabetes mellitus and thereby have increased patient survival. However, current standards of care reduce but do not eliminate the risk of diabetic kidney disease, and further studies are warranted to define new strategies for reducing the risk of diabetic kidney disease. In this review, we highlight some of the novel and established molecular mechanisms that contribute to the development of the disease and its outcomes. In particular, we discuss recent advances in our understanding of the molecular mechanisms implicated in the pathogenesis and progression of diabetic kidney disease, with special emphasis on the mitochondrial oxidative stress and microRNA targets. Additionally, candidate genes associated with susceptibility to diabetic kidney disease and alterations in various cytokines, chemokines, and growth factors are addressed briefly.