Mechanical stretch induces podocyte hypertrophy in vitro

BACKGROUND: Increased intraglomerular pressure is a final pathway toward glomerulosclerosis in systemic hypertension, diabetes, and focal segmental glomerulosclerosis (FSGS). Increased intraglomerular pressure causes stress-tension, or stretch, on resident glomerular cells. However, the effects of stretch on podocyte growth, and the mechanisms that underlie this, have not been elucidated. METHODS: To test the hypothesis that stretch alters podocyte growth, cultured mouse podocytes were exposed to cyclic mechanical stretch created by vacuum; control cells were grown under similar conditions, but not exposed to stretch. Proliferation (cell cycle phases) and hypertrophy (forward light scatter) were measured in stretched and control podocytes by flow cytometry. The role of the cyclin-dependent kinase (CDK) inhibitors, p21 and p27, was examined by stretching podocytes isolated from p21 and p27 knockout (-/-) mice, and the role of specific signaling pathways was assessed by Western blot analysis and blocking studies. RESULTS: Our results showed that stretch reduced cell cycle progression in wild-type and single p27-/- podocytes and induced hypertrophy in these cells in all phases of the cell cycle at 24, 48, and 72 hours. In contrast, stretch did not induce hypertrophy in single p21-/- and double p21/p27-/- podocytes. Stretch-induced hypertrophy required cell cycle entry, and was prevented by specifically blocking extracellular signal-regulated kinase 1/2 (Erk1/2) or Akt. Although stretch increased p38 activation, inhibition of this pathway had no effect on hypertrophy. CONCLUSION: Mechanical stretch induces hypertrophy in podocytes in vitro in all phases of the cell cycle. This effect is cell cycle dependent, and requires p21, Erk1/2, and Akt. Stretch may play a role in podocyte injury when intraglomerular pressure is increased.     

Differential expression of d-type cyclins in podocytes in vitro and in vivo

The proliferative response of podocytes to injury determines the histological phenotype. Moreover, an apparent lack of podocyte proliferation may underlie the development of glomerulosclerosis. Podocyte proliferation is closely linked with its state of differentiation. However, the mechanisms regulating these processes are not fully elucidated. Because D-type cyclins have been shown to be important in the regulation of proliferation and differentiation, we examined their expression in podocytes in vitro and in vivo. The glomerular expression of cyclins D1 and D3 was examined in vitro in cultured immortalized podocytes by immunostaining and Western blot analysis, and in embryonic mice and rats, the passive Heymann nephritis model of experimental membranous nephropathy in rats, and human immunodeficiency virus (HIV)-transgenic mice. Kidneys from cyclin D1 knockout mice were also examined. Cyclin D1 was abundant in cultured proliferating podocytes, but not in quiescent differentiated podocytes. In contrast, cyclin D3 was abundant in differentiated, but not proliferating podocytes. Cyclin D1 was expressed in embryonic mouse and rat glomeruli during the S- and comma-shaped stages, and was absent in podocytes at the capillary loop stage and in mature rodent glomeruli. Cyclin D1 protein increased after injury in passive Heymann nephritis rats and in HIV-transgenic mice. Cyclin D3 was constitutively and specifically expressed in podocytes in normal rodent glomeruli, and decreases during dedifferentiation and proliferation in HIV-transgenic mice. Kidneys from cyclin D1-/- mice were normal with the podocytes expressing specific differentiation markers. Cyclin D1 is not necessary for the terminal differentiation of podocytes, and expression coincides with cell-cycle entry. In contrast, cyclin D3 expression coincides with podocyte differentiation and quiescence.     

Emergency medicine and environmental activism

Clearfell, burn and sow silviculture is promoted as being the most scientific method of harvesting wet sclerophyll forests in Tasmania. However, it fails to take into account wider social and global environmental issues such as climate change and the carbon cycle. This paper describes an emergency physician's role in the continuing debate surrounding the management of Tasmania's old-growth forests. Like evidence-based medicine, the use of science in this context has its limitations, and such comparisons are discussed.     

Amelioration of diabetic nephropathy in SPARC-null mice

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular protein that inhibits mesangial cell proliferation and also affects production of extracellular matrix (ECM) by regulating transforming growth factor-beta1 (TGF-beta1) and type I collagen in mesangial cells. This study is an investigation of the role of SPARC in streptozotocin (STZ)-induced diabetic nephropathy (DN) of 6-mo duration in wild type (WT) and SPARC-null mice. SPARC expression was evaluated by immunohistochemistry (IHC) and by in situ hybridization (ISH). Deposition of type I and IV collagen and laminin was evaluated by IHC, and TGF-beta 1 mRNA was assessed by ISH. Renal function studies revealed no significant difference in BUN between diabetic SPARC-null mice and diabetic WT mice, whereas a significant increase in albumin excretion was detected in diabetic WT relative to diabetic SPARC-null mice. Diabetic WT animals exhibited increased levels of SPARC mRNA and protein in glomerular epithelial cells and in interstitial cells, in comparison with nondiabetic WT mice. Neither SPARC mRNA nor protein was detected in SPARC-null mice. Morphometry revealed a significant increase in the percentage of the glomerular tufts occupied by ECM in diabetic WT compared with nondiabetic WT mice, although there was no difference in the mean glomerular tuft area among groups. In contrast, diabetic SPARC-null mice did not show a significant difference in the percentage of the glomerular tufts occupied by ECM relative to nondiabetic null mice. Tubulointerstitial fibrosis was ameliorated in diabetic SPARC-null mice compared with diabetic WT animals. Further characterization of diabetic SPARC-null mice revealed diminished glomerular deposition of type IV collagen and laminin, and diminished interstitial deposition of type I and type IV collagen correlated with decreases in TGF-beta 1 mRNA compared with WT diabetic mice. These observations suggest that SPARC contributes to glomerulosclerosis and tubulointerstitial damage in response to hyperglycemia through increasing TGF-beta 1 expression in this model of chronic DN.     

Complement (C5b-9) induces DNA synthesis in rat mesangial cells in vitro

BACKGROUND: The membrane attack complex C5b-9 causes injury in many forms of immune-mediated glomerular diseases characterized by mesangial cell (MC) proliferation and inhibiting C5b-9 decreases MC proliferation in vivo. Membrane insertion of sublytic quantities of the membrane attack complex of complement (C5b-9) is a potent stimulus for cell activation and the production of a variety of cytokines, growth factors, oxidants, matrix components, and other nephritogenic molecules. In vivo, a common response of MC to C5b-9--mediated injury is cell proliferation, an event closely linked to matrix expansion and sclerosis. In this study, we tested the hypothesis that C5b-9 might also serve as a mitogenic stimulus for MCs. METHODS: Rat MCs in vitro were exposed anti-Thy1 antibody and 2% normal PVG serum (a complement source) to induce sublytic C5b-9 attack and DNA synthesis and cell number were measured. Control MCs were exposed to antibody and C6-deficient PVG serum. RESULTS: Sublytic C5b-9--induced injury to MCs is sufficient to induce DNA synthesis. Furthermore, C5b-9 augmented DNA synthesis induced by platelet-derived growth factor (PDGF) and 5% fetal calf serum. C5b-9--induced DNA synthesis was reduced by inhibiting reactive oxygen species (ROS) with superoxide dismutase and catalase, but not by neutralizing the mitogenic growth factors PDGF and basic fibroblast growth factor (bFGF). CONCLUSIONS: This study demonstrates that C5b-9 may directly increase DNA synthesis in cultured MCs, which are mediated in part by the release of ROS, and that C5b-9 also augments DNA synthesis induced in MCs by other known mitogens.     

Clusterin is up-regulated in glomerular mesangial cells in complement-mediated injury

BACKGROUND: Clusterin is a soluble complement regulatory protein that binds to C5b-7 and inhibits generation of membrane attack complex, C5b-9. Glomerular deposition of clusterin has been observed in human and experimental membranous nephropathy in association with C5b-9 and immune deposits. However, it is controversial as to whether clusterin observed in glomeruli is synthesized by the resident glomerular cells or is derived from the circulation. We examined whether clusterin is expressed by resident glomerular cells exposed to complement-mediated injury. METHODS: In vitro, cultured mesangial cells were exposed to antithymocyte serum immunoglobulin G and 5% normal rat serum as a complement source. In vivo, we induced anti-Thy1 nephritis in rats and examined the kidneys on days 8 and 29. RESULTS: We observed increased expression of clusterin in cultured rat glomerular mesangial cells stimulated by sublytic complement attack. We also demonstrated that in comparison with control rats, both a marked increase in clusterin mRNA in the glomeruli and marked deposition of clusterin protein in the mesangial area occurred in the OX-7-treated rats on day 8 in association with C5b-9 deposition and on day 29. CONCLUSION: Clusterin was induced in glomerular mesangial cells during the course of immune-mediated injuries. This up-regulation of clusterin may play a critical role in protecting mesangial cells from complement attack.     

Glomerulonephritis

The differential diagnosis of glomerulonephritis without systemic disease includes poststreptococcal glomerulonephritis, IgA nephropathy, rapidly progressive glomerulonephritis (RPGN), and membranoproliferative glomerulonephritis (MPGN). Glomerular inflammation is probably induced directly by a nephritogenic streptococcal protein in poststreptococcal glomerulonephritis, and by mesangial deposition of abnormally glycosylated IgA1-containing immune aggregates in IgA nephropathy. In crescentic RPGN the role of cellular rather than humoral immune mechanisms is now becoming clear. Many patients with MPGN have chronic hepatitis C infection. There is no effective disease-specific therapy for poststreptococcal glomerulonephritis or IgA nephropathy. RPGN benefits from high-dose steroids and cytotoxic drug therapy with the addition of plasma exchange in disease induced by antibody to glomerular basement membrane. Antiviral therapies reduce the severity of MPGN due to hepatitis C virus. However, various new therapies directed at specific cytokines, growth factors, fibrin deposition, and other mediators of injury are being developed, as well as more specific and less toxic forms of immunotherapy.     

Factors influencing ventilatory muscle recruitment in patients with chronic airflow obstruction

Patients with chronic airflow obstruction (CAO) frequently develop abnormal thoraco-abdominal excursion, but the patterns described are inconsistent and the factors that relate to their development remain unknown. We studied 45 stable patients with FEV1 ranging from 0.36 to 2.1 L. A pattern of ventilatory muscle recruitment (VMR) was established by simultaneously measuring gastric (Pg) and pleural (Ppl) pressures and rib cage (Vrc) and abdominal (Vab) volume displacement with inductance plethysmography. From these tracings, Pg-Ppl plots were constructed and the delta Pg/delta Ppl values were calculated. The delta Pg/delta Ppl was validated in 15 patients with simultaneous analysis of Vab-Pg plots. Pearson's test and multiple regression analyses were used to correlate delta Pg/delta Ppl to factors thought to influence respiratory muscle function such as age, sex, nutritional status (weight/height, albumin), hyperinflation, airflow obstruction, and arterial blood gases. We found a direct correlation between a more positive delta Pg/delta Ppl value and increasing hyperinflation (r = 0.69, p less than 0.0001), increasing airflow obstruction (r = -0.55, p less than 0.001), and decreasing diaphragmatic strength (r2 = 0.32, p less than 0.001). We also found that expiratory Ppl became more positive with decreasing FEV1 (r2 = 0.33, p less than 0.001). This change in VMR was independent of age, sex, nutritional status, and arterial blood gas determinations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expanding community health nursing roles to meet health-care needs of frail elderly. An adult learning model

The increased training needs for community health nurses (CHNs) working with frail elderly offer a variety of challenges in staff development. Santa Clara County addressed these challenges with an innovative model, and geriatric assessment skills building program presented to 40 CHNs. The model uses a team teaching approach, a preceptorship and adult learning theory, making it readily adaptable to a variety of community health settings.