Microparticles (MPs) are small (0.1–1.0 µm) vesicles shed from the surface of cells in response to stress. Whether podocytes produce MPs and whether this production reflects glomerular injury are unclear. We examined MP formation in cultured human podocytes (hPODs) and diabetic mice. hPODs were exposed to cyclical stretch, high glucose (HG; 25 mM), angiotensin II, or TGF-β. Urinary podocyte MPs were assessed in three mouse models of diabetic nephropathy: streptozotocin (STZ)-treated, OVE26, and Akita mice. Cyclic stretch and HG increased MP release as assessed by flow cytometry (P<0.01 and P<0.05, respectively, versus controls). Inhibition of Rho-kinase (ROCK) with fasudil blocked HG-induced podocyte MP formation. STZ-treated (8 weeks) mice exhibited increased urinary podocyte MPs compared with age-matched nondiabetic mice. Similarly, 16-week-old OVE26 mice had elevated levels of urinary podocyte MPs compared with wild-type littermates (P<0.01). In 1 week post-STZ–treated and 6- and 12-week-old Akita mice, urinary podocyte MPs increased significantly compared with those MPs in nondiabetic mice, despite normal urinary albumin levels. Our results indicate that podocytes produce MPs that are released into urine. Podocyte-derived MPs are generated by exposure to mechanical stretch and high glucose in vitro and could represent early markers of glomerular injury in diabetic nephropathy.

Mutations in the gene encoding α-actinin-4 (ACTN4), an actin crosslinking protein, are associated with a form of autosomal dominant focal segmental glomerulosclerosis (FSGS). To better study its progression, a transgenic mouse model was developed by expressing murine α-actinin-4 containing a mutation analogous to that affecting a human FSGS family in a podocyte-specific manner using the murine nephrin promoter. Consistent with human ACTN4-associated FSGS, which shows incomplete penetrance, a proportion of the transgenic mice exhibited significant albuminuria (8 of 18), while the overall average systolic BP was elevated in both proteinuric and non-proteinuric ACTN4-mutant mice. Immunofluorescence confirmed podocyte-specific expression of mutant α-actinin-4, and real-time RT-PCR revealed that HA-ACTN4 mRNA levels were higher in proteinuric versus non-proteinuric ACTN4-mutant mice. Only proteinuric mice exhibited histologic features consistent with human ACTN4-associated FSGS, including segmental sclerosis and tuft adhesion of some glomeruli, tubular dilatation, mesangial matrix expansion, as well as regions of podocyte vacuolization and foot process fusion. Consistent with such podocyte damage, proteinuric ACTN4-mutant kidneys exhibited significantly reduced mRNA and protein levels of the slit diaphragm component, nephrin. This newly developed mouse model of human ACTN4-associated FSGS suggests a cause-and-effect relationship between actin cytoskeleton dysregulation by mutant α-actinin-4 and the deterioration of the nephrin-supported slit diaphragm complex. E-mail: ckennedy@uottawa.ca

NADPH oxidase (Nox) enzymes are a significant source of reactive oxygen species, which contribute to glomerular podocyte dysfunction. Although studies have implicated Nox1, -2, and -4 in several glomerulopathies, including diabetic nephropathy, little is known regarding the role of Nox5 in this context. We examined Nox5 expression and regulation in kidney biopsies from diabetic patients, cultured human podocytes, and a novel mouse model. Nox5 expression increased in human diabetic glomeruli compared with nondiabetic glomeruli. Stimulation with angiotensin II upregulated Nox5 expression in human podocyte cultures and increased reactive oxygen species generation. siRNA-mediated Nox5 knockdown inhibited angiotensin II–stimulated production of reactive oxygen species and altered podocyte cytoskeletal dynamics, resulting in an Rac-mediated motile phenotype. Because the Nox5 gene is absent in rodents, we generated transgenic mice expressing human Nox5 in a podocyte-specific manner (Nox5^pod+). Nox5^pod+ mice exhibited early onset albuminuria, podocyte foot process effacement, and elevated systolic BP. Subjecting Nox5^pod+ mice to streptozotocin-induced diabetes further exacerbated these changes. Our data show that renal Nox5 is upregulated in human diabetic nephropathy and may alter filtration barrier function and systolic BP through the production of reactive oxygen species. These findings provide the first evidence that podocyte Nox5 has an important role in impaired renal function and hypertension.

Approaching epidemic levels, diabetic kidney disease (DKD) is now the leading cause of end-stage renal disease (ESRD). Microalbuminuria is an early clinical marker of DKD that results from damage to the glomerular filtration barrier at the level of the highly differentiated glomerular podocyte cells. Injury to these epithelial cells, podocytopathies, includes cellular hypertrophy, foot process effacement, detachment from the glomerular basement membrane, and apoptosis. Here we review the role of a number of recently identified factors that contribute to podocytopathies in DKD. These factors include members of the renin-angiotensin system (RAS), including angiotensin-converting enzyme (ACE) types 1 and 2, prorenin and its receptor, reactive oxygen species (ROS), prostanoids, peroxisome proliferator-activated receptors (PPAR), advanced glycation end-products (AGEs) and their receptors (RAGE), adiponectin, and microRNAs. As the number of therapeutic options that slow, but do not halt, the progression of DKD to ESRD remains limited, a more comprehensive understanding of the signaling events that contribute to this increasingly prevalent disease may identify novel avenues for treatment and prevention.

Since the first demonstration of Nox enzyme expression in the kidney in the early 1990s and the subsequent identification of Nox4, or RENOX, a decade later, it has become apparent that the Nox family of reactive oxygen species (ROS) generating enzymes plays an integral role in the normal physiological function of the kidney. As our knowledge of Nox expression patterns and functions in various structures and specialized cell types within the kidney grows, so does the realization that Nox-derived oxidative stress contributes significantly to a wide variety of renal pathologies through their ability to modify lipids and proteins, damage DNA and activate transcriptional programmes. Diverse studies demonstrate key roles for Nox-derived ROS in kidney fibrosis, particularly in settings of chronic renal disease such as diabetic nephropathy. As the most abundant Nox family member in the kidney, much emphasis has been placed on the role of Nox4 in this setting. However, an ever growing body of work continues to uncover key roles for other Nox family members, not only in diabetic kidney disease, but in a diverse array of renal pathological conditions. The objective of the present review is to highlight the latest novel developments in renal Nox biology with an emphasis not only on diabetic nephropathy but many of the other renal disease contexts where oxidative stress is implicated.

A dynamic cytoskeleton allows podocytes to withstand significant mechanical stress on elevation of intraglomerular capillary pressure (P_gc). However, vasoactive hormones, such as prostaglandin E₂(PGE₂), may challenge the integrity of the actin cytoskeleton, alter podocyte morphology, and compromise glomerular permeability. PGE₂synthesis correlates with the onset of proteinuria and increased P_gcfollowing reduced nephron mass. We investigated the interplay among mechanical stress, cyclooxygenase (COX), E-prostanoid (EP) receptor expression, and the actin cytoskeleton, using an in vitro model of cell stretch. Immortalized mouse podocytes grown on flexible silicone membranes were cyclically stretched (5% elongation, 0.5 Hz) for 2 h. EP₄and COX-2 mRNA increased three- and sevenfold above nonstretched controls, whereas EP₁and COX-1 levels were unchanged. Six hours of stretch resulted in a threefold increase in PGE₂-stimulated cAMP accumulation, a measure of EP₄receptor function, and an increase in COX-2 protein. The stretch-induced effects on COX-2/EP₄expression and EP₄-induced cAMP production were attributable to p38 MAP kinase, as blockade of this pathway, but not of ERK or JNK, abrogated the response. These stretch-induced changes in expression were transcriptionally dependent as they were actinomycin D sensitive. Finally, we investigated the influence of enhanced EP₄signaling on the actin cytoskeleton. Addition of PGE₂resulted in actin filament depolymerization observable only in stretched cells. Our results indicate that key components of the eicosanoid pathway are upregulated by mechanically stimulated p38 MAP kinase in podocytes. Enhanced EP₄receptor signaling may undermine podocyte cytoskeletal dynamics and thereby compromise filtration barrier function under conditions of increased P_gc.