Successful application of extracorporeal membrane oxygenation due to pulmonary hemorrhage secondary to granulomatosis with polyangiitis.

Extracorporeal membrane oxygenation (ECMO) is increasingly applied in adults with acute refractory respiratory failure that is deemed reversible. Bleeding is the most frequent complication during ECMO support. Severe pre-existing bleeding has been considered a contraindication to ECMO application. Nevertheless, there are cases of successful ECMO application in patients with multiple trauma and hemorrhagic shock or head trauma and intracranial hemorrhage. ECMO has proved to be life-saving in several cases of life-threatening respiratory failure associated with pulmonary hemorrhage of various causes, including granulomatosis with polyangiitis (Wegener's disease). We successfully applied ECMO in a 65-year-old woman with acute life-threatening respiratory failure due to diffuse massive pulmonary hemorrhage secondary to granulomatosis with polyangiitis, manifested as severe pulmonary-renal syndrome. ECMO sustained life and allowed disease control, together with plasmapheresis, cyclophosphamide, corticoids, and renal replacement therapy. The patient was successfully weaned from ECMO, extubated, and discharged home. She remains alive on dialysis at 17 months follow-up.

Urinary podocyte loss is a more specific marker of ongoing glomerular damage than proteinuria.

Podocyte loss contributes to the development of glomerulosclerosis. Although podocyte detachment has been recognized as a new mechanism of podocyte loss in glomerular diseases, its time course and relationship to disease activity are not known. Urinary excretion of viable podocytes was quantified in two models of transient glomerular injury, i.e., rats with puromycin aminonucleoside-induced nephrosis (PAN) and mesangioproliferative nephropathy (anti-Thy 1.1 nephritis model), as well as in a model of continuous glomerular injury, i.e., hypertensive nephropathy (5/6-nephrectomy model), and in aging rats. The number of glomerular Wilm's tumor (WT)-1-positive podocytes and the glomerular expression of cell-cycle proteins in vivo were assessed. Urinary podocyte loss occurred in both primary (PAN) and secondary (anti-Thy 1.1 nephritis) in parallel to the onset of proteinuria. However, subsequently proteinuria persisted despite remission of podocyturia. In continuous glomerular injury, i.e., after 5/6-nephrectomy, podocyturia paralleled the course of proteinuria and of systemic hypertension, whereas no podocyturia became detectable during normal aging (up to 12 mo). Despite podocyte detachment of varying degrees, no decrease in glomerular podocyte counts (i.e., WT-1 positive nuclei) was noted in either disease model. Podocyturia in the PAN and anti-Thy 1.1 nephritis model was preceded by entry of glomerular podocytes into the cell cycle, i.e., cyclin D1, cdc2, and/or proliferating cell nuclear antigen (PCNA) expression. Podocyturia is a widespread phenomenon in glomerular disease and not simply a reflection of proteinuria because it is limited to phases of ongoing glomerular injury. The data suggest that podocyturia may become a more sensitive means to assess the activity of glomerular damage than proteinuria.

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.

Cyclin-dependent kinase 5 is a regulator of podocyte differentiation, proliferation, and morphology.

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cyclin-dependent kinase 5 (CDK5) has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. In this study, we examined the role of CDK5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, CDK5 increased in association with podocyte differentiation. During mouse glomerulogenesis in vivo, CDK5 expression was predominantly detected in podocytes from the capillary loop stage to maturation and persisted in the podocytes of adult glomeruli. In contrast, CDK5 was markedly decreased in the proliferating and dedifferentiated podocytes of mice with anti-glomerular basement membrane nephritis and in human immunodeficiency virus transgenic mice. p35, the activator of CDK5, was also detected in podocytes and the p35/CDK5 complex was active. Cell fractionation studies showed that active p35/CDK5 was mainly localized to the plasma membrane. Specific inhibition of CDK5 in differentiated cultured podocytes, either pharmacologically or with siRNA, induced shape changes, with cellular elongation and loss of process formation compared to the characteristic arborized phenotype. These data suggest a role for CDK5 as a regulator of podocyte differentiation, proliferation, and morphology.

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.

Podocyte proliferation and differentiation in glomerular disease: role of cell-cycle regulatory proteins.

Injury to the podocyte underlies many forms of glomerular disease. In contrast to mesangial and endothelial cells, podocytes do not typically proliferate. Moreover, the lack of proliferation is thought to underlie the development of glomerulosclerosis. Studies have recently shown that the lack of podocyte proliferation is due to an increase in cyclin-dependent kinase inhibitors, which arrest the cell cycle. Current work is aimed at further delineating the mechanisms regulating podocyte proliferation.

DNA damage is a novel response to sublytic complement C5b-9-induced injury in podocytes.

In response to Ab-complement-mediated injury, podocytes can undergo lysis, apoptosis, or, when exposed to sublytic (<5% lysis) amounts of C5b-9, become activated. Following the insertion of sublytic quantities of C5b-9, there is an increase in signaling pathways and growth factor synthesis and release of proteases, oxidants, and other molecules. Despite an increase in DNA synthesis, however, sublytic C5b-9 is associated with a delay in G(2)/M phase progression in podocytes. Here we induced sublytic C5b-9 injury in vitro by exposing cultured rat podocytes or differentiated postmitotic mouse podocytes to Ab and a complement source; we also studied the passive Heymann nephritis model of experimental membranous nephropathy in rats. A major finding was that sublytic C5b-9-induced injury caused an increase in DNA damage in podocytes both in vitro and in vivo. This was associated with an increase in protein levels for p53, the CDK inhibitor p21, growth-arrest DNA damage-45 (GADD45), and the checkpoint kinases-1 and -2. Sublytic C5b-9 increased extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2), and inhibiting ERK-1 and -2 reduced the increase in p21 and GADD45 and augmented the DNA damage response to sublytic C5b-9-induced injury. These results show that sublytic C5b-9 induces DNA damage in vitro and in vivo and may explain why podocyte proliferation is limited following immune-mediated injury.

Mitotic cell cycle proteins increase in podocytes despite lack of proliferation.

BACKGROUND: Podocyte proliferation is an uncommon response to glomerular injury and its lack may underlie the development of glomerulosclerosis. However, whether podocytes have the capacity to enter and finish mitosis and cytokinesis is not known. METHODS: The expression of mitotic cell cycle proteins (phosphorylated Histone 3, Cdc2, cyclin B1 and B2) was examined by immunohistochemistry in kidneys of embryonal mice, transgenic HIV-mice, and rats with experimental membranous nephropathy (passive Heymann nephritis, PHN). Mitotic proteins also were measured by Western blot in glomerular protein from PHN-rats and the activity of mitotic cyclins was quantified by histone kinase assay. RESULTS: Mitotic proteins were increased in embryonal mouse glomeruli during the S- and comma-shaped stages and were absent at the capillary loop stage and in mature rodent glomeruli. There was an increase in podocyte expression of Cdc2, cyclin B1 and B2 and phosphorylated histone 3 in PHN rats, and in HIV transgenic mice. CONCLUSIONS: Podocytes have the ability to increase cell cycle proteins required for mitosis. Without obvious differences in the expression of the major mitotic proteins in PHN- and HIV-nephropathy, a regulatory disturbance in cytokinesis might be responsible for the development of polynucleated cells and a lack of podocyte proliferation in experimental glomerular disease.

Insulin is a potent survival factor in mesangial cells: role of the PI3-kinase/Akt pathway.

BACKGROUND: Elucidating the mechanisms of apoptosis is important for understanding the molecular mechanisms underlying glomerular disease. The phosphatidylinositol 3 kinase (PI3-kinase)/Akt pathway is essential for survival signaling in non-renal cells. However, little is known about the anti-apoptotic effect of insulin and the role of the PI3-kinase/Akt pathway in mesangial cells (MC) apoptosis. METHODS: Apoptosis was induced in wild type, p27Kip1 (p27) -/- and p21Cip1/Waf1 (p21) -/- mouse MC by survival factor withdrawal, actinomycin D, ultraviolet (UV)-B irradiation and cycloheximide in the presence or absence of insulin (1 micromol/L) or insulin-like growth factor-I (IGF-I; 100 ng/mL). The activation and levels of Akt, extracellular signal regulated kinase (ERK) and specific cell cycle proteins were determined by Western blot analysis. RESULTS: Insulin and IGF-I inhibited wild-type MC apoptosis induced by survival factor withdrawal, actinomycin D, ultraviolet-B irradiation and cycloheximide and in p27 -/- MC when apoptosis was induced by survival factor withdrawal. Akt was activated by insulin and IGF-I during apoptosis. Blocking PI3-kinase with LY294002 reduced Akt activation and abrogated the anti-apoptotic effect of insulin. ERK was activated during apoptosis and blocking ERK activation with U0126 or PD98059 partially rescued MC from apoptosis. Moreover, insulin also suppressed ERK activation during apoptosis. Our results also showed that the CDK-inhibitor p21 was increased by insulin and that p21 up-regulation was PI3-kinase/Akt pathway dependent. Furthermore, p21 -/- MC apoptosis induced by survival factor withdrawal was not rescued by insulin in contrast to the wild-type and p27 -/- MC. These data suggest that p21 may have a critical role in the anti-apoptotic effect of insulin. CONCLUSIONS: Insulin is a potent survival factor for MC in response to a number of different apoptotic triggers, and this effect is mediated through the PI3-kinase/Akt pathway. Moreover, ERK and p21 may be involved in anti-apoptotic effect of insulin in MC.

Mechanical stress reduces podocyte proliferation in vitro.

BACKGROUND: Mechanical stretch, a consequence of capillary glomerular hypertension, is thought to be the common final pathway for glomerulosclerosis in systemic hypertension, diabetes, reduced nephron number and focal segmental glomerulosclerosis. However, the effects of stretch on podocyte growth and the mechanisms that underlie this have not been elucidated. METHODS: Mouse podocyte growth (3H-thymidine, MTT-assay, FACS) was measured following the application of mechanical stretch created by vacuum. The expression of specific cell cycle regulatory proteins was examined by RNAse protection assay and Western blot analysis. Control cells were grown under similar conditions, but were not exposed to stretch. RESULTS: Mechanical stretch decreased DNA-synthesis (3H-thymidine incorporation) and cell number (MTT-assay) in podocytes at 24, 48 and 72 hours (P < 0.001 vs. control non-stretched cells), which was not due to apoptosis (Hoechst staining) nor cell detachment. Stretch decreased the mRNA and protein levels of cyclins D1, A and B1 within 24 hours. Stretching cells decreased the activity of Cdk2 (measured by histone H1 kinase assay) at 48 and 72 hours and Cdc2 at 72 hours. In contrast, stretch increased the protein levels of the cyclin dependent kinase inhibitors (CKI) p21Cip/Kip/Waf (p21) and p27Kip1 (p27) within the first 24 hours, and increased the mRNA levels of p57Kip2 (p57) at 72 hours. To examine the role of p21 in inhibiting proliferation induced by stretch, we studied p21-/- podocytes in culture. Stretch did not reduce proliferation in p21-/- podocytes (P> 0.05 vs. non-stretched podocytes; P < 0.001 vs. stretched p21+/+ podocytes). CONCLUSIONS: In contrast to mesangial cells, mechanical stretch decreases the growth of podocytes. This effect is mediated through the regulation of specific cell cycle regulatory proteins. These events may explain the apparent lack of podocyte proliferation in diseases correlated with capillary glomerular hypertension.