Richard F. Ransom

The podocyte and diabetes mellitus: is the podocyte the key to the origins of diabetic nephropathy?

Purpose of reviewPodocyte injury plays a key role in the development of diabetic nephropathy. This review discusses recent advances in our understanding of mechanisms of podocyte injury in diabetes mellitus and the associated alterations in the function of the glomerular filtration barrier. Recent findingsThe effects of hyperglycemia on critical podocyte parameters including cell–cell interactions, attachment to the glomerular basement membrane, and podocyte apoptosis have been determined in both cell culture and in-vivo models of diabetes mellitus. The podocyte has also been identified as a target of action for insulin and growth hormone, hormones with significant roles in the altered homeostasis of diabetes mellitus. SummaryUnderstanding the cellular and molecular basis for changes in podocyte structure and function in diabetes mellitus may lead to novel diagnostic tools and treatment strategies for diabetic nephropathy.

Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction

Nephrotic syndrome (NS) is characterized by structural changes in the actin‐rich foot processes of glomerular podocytes. We previously identified high concentrations of the small heat shock protein hsp27 within podocytes as well as increased glomerular accumulation and phosphorylation of hsp27 in puromycin aminonucleoside (PAN) ‐induced experimental NS. Here we analyzed murine podocytes stably transfected with hsp27 sense, antisense, and vector control constructs using a newly developed in vitro PAN model system. Cell morphology and the microfilament structure of untreated sense and antisense transfectants were altered compared with controls. Vector cell survival, polymerized actin content, cell area, and hsp27 content increased after 1.25 μg/ml PAN treatment and decreased after 5.0 μg/ml treatment. In contrast, sense cells were unaffected by 1.25 μg/ml PAN treatment whereas antisense cells showed decreases or no changes in all parameters. Treatment of sense cells with 5.0 μ g/ml PAN resulted in increased cell survival and cell area whereas antisense cells underwent significant decreases in all parameters. Hsp27 provided dramatic protection against PAN‐induced microfilament disruption in sense > vector > antisense cells. We conclude that hsp27 is able to regulate both the morphological and actin cytoskeletal response of podocytes in an in vitro model of podocyte injury.—Smoyer, W. E., Ransom, R. F. Hsp27 regulates podocyte cytoskeletal changes in an in vitro model of podocyte process retraction. FASEB J. 16, 315–326 (2002)

Phosphatidylinositol 4-phosphate is a major source of GPCR-stimulated phosphoinositide production

The plasma membrane lipid PI4P is the main source of phosphoinositides generated by GPCR activation. Sourcing phosphoinositides Agonist binding to certain G protein–coupled receptors (GPCRs) stimulates members of the phospholipase C (PLC) family of enzymes to hydrolyze the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2), which results in the generation of the intracellular second messengers DAG and IP3. Noting that certain PLCs also hydrolyze the lipid phosphatidylinositol 4-phosphate (PI4P) at the Golgi, de Rubio et al. performed fluorescence-based imaging to monitor the spatiotemporal regulation of lipid hydrolysis in various cell types stimulated through different GPCRs, as well as the resulting activation of the DAG-dependent kinase PKD. Their findings suggest that PI4P, but not PI4,5P2, at the plasma membrane is the main source of GPCR-stimulated second messenger production. Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P2) to generate the second messengers inositol trisphosphate (IP3) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein–coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation–dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P2-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P2 from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P2. Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P2 pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

Protamine Sulfate and Oxidant Stress Cause Similar Changes in Hsp27 Intracellular Distribution in Cultured Podocytes

Protamine Sulfate and Oxidant Stress Cause Similar Changes in Hsp27 Intracellular Distribution in Cultured Podocytes

Differential Renal Expression of Hsp27 and αB-Crystallin in Rats Following Acute Ischemia † 1837

The mammalian small stress proteins αB-crystallin and hsp27 are structurally related; the latter is induced by a variety of insults including heat and ischemia. Both proteins protect cells from these insults, possibly by molecular chaperoning and/or by stabilization of the cytoskeleton. This study shows the amounts and localization of hsp27 and αB-crystallin in the renal cortex (CO), isolated glomeruli (GL), inner (IM), and outer medulla (OM) at 6 h, 24 h, and 5 days following sham-operation (sham) or severe renal ischemia (60 min complete renal artery occlusion). The amount of hsp27 in sham kidneys was approximately 0.1 ng hsp27/μg total protein in CO and OM, 0.3 ng/μg in GL, and 1 ng/μg in the IM, while the amount ofαB-crystallin was 0.2 ng/μg in CO and GL, 1 ng/μg in OM, and 4 ng/μg in IM as determined by Western blotting. Ischemia led to a 200 to 500% increase in the amount of hsp27 in CO and OM at all times. There was a 200% increase in hsp27 in IM at 6 h, no change at 24 h, and a 50% decrease at five days. The amount of hsp27 in GL decreased by 50% at 5 days. No change in the amount of αB-crystallin was detected after ischemia, while hsp70 was induced to a similar extent and with the same distribution as hsp27. In both sham and ischemic kidneys the non-phosphorylated isoform of hsp27 predominated in OM, IM, and GL, while in CO the majority of hsp27 was mono-phosphorylated. Immuno-fluorescence microscopy of sham kidneys revealed that hsp27 was primarily localized in the smooth muscle cells with weak staining of glomeruli and medullary capillary endothelial cells, while αB-crystallin was found throughout the medulla with weak staining of Bowmans capsule. The distribution of hsp27 and aB-crystallin was unchanged after ischemia. We conclude 1) hsp27 and αB-crystallin are differentially expressed in normal rat kidney and undergo differential regulation in response to ischemia, 2) hsp27 induction is part of a general stress response, and 3) hsp27 may play an important role in the pathogenesis of or recovery from ischemia.