Joseph C.K. Leung

Albumin stimulates interleukin-8 expression in proximal tubular epithelial cells in vitro and in vivo.

Renal tubulointerstitial injury is characterized by inflammatory cell infiltrate; however, the stimuli for leukocyte recruitment are not fully understood. IL-8 is a potent chemokine produced by proximal tubular epithelial cells (PTECs). Whether nephrotic proteins stimulate tubular IL-8 expression remains unknown. Acute exposure of human PTECs to albumin induced IL-8 gene and protein expression time- and dose-dependently. Apical albumin predominantly stimulated basolateral IL-8 secretion. Electrophoretic mobility shift assay demonstrated nuclear translocation of NF-kappaB, and the p65/p50 subunits were activated. NF-kappaB activation and IL-8 secretion were attenuated by the NF-kappaB inhibitors pyrrolidine dithiocarbamate and cell-permeable peptide. Albumin upregulated intracellular reactive oxygen species (ROS) generation, while exogenous H2O2 stimulated NF-kappaB translocation and IL-8 secretion. Albumin-induced ROS generation, NF-kappaB activation, and IL-8 secretion were endocytosis- and PKC-dependent as these downstream events were abrogated by the PI3K inhibitors LY294002 and wortmannin, and the PKC inhibitors GF109203X and staurosporin, respectively. In vivo, IL-8 mRNA expression was localized by in situ hybridization to the proximal tubules in nephrotic kidney tissues. The intensity of IL-8 immunostaining was higher in nephrotic than non-nephrotic subjects. In conclusion, albumin is a strong stimulus for tubular IL-8 expression, which occurs via NF-kappaB-dependent pathways through PKC activation and ROS generation.

Aldehyde dehydrogenase activity in leukemic blasts defines a subgroup of acute myeloid leukemia with adverse prognosis and superior NOD/SCID engrafting potential.

Aldehyde dehydrogenase (ALDH) activity is used to define normal hematopoietic stem cell (HSC), but its link to leukemic stem cells (LSC) in acute myeloid leukemia (AML) is currently unknown. We hypothesize that ALDH activity in AML might be correlated with the presence of LSC. Fifty-eight bone marrow (BM) samples were collected from AML (n=43), acute lymphoblastic leukemia (ALL) (n=8) and normal cases (n=7). In 14 AML cases, a high SSCloALDHbr cell population was identified (ALDH+AML) (median: 14.89%, range: 5.65–48.01%), with the majority of the SSCloALDHbr cells coexpressing CD34+. In another 29 cases, there was undetectable (n=23) or rare (⩽5%) (n=6) SSCloALDHbr population (ALDH−AML). Among other clinicopathologic variables, ALDH+AML was significantly associated with adverse cytogenetic abnormalities. CD34+ BM cells from ALDH+AML engrafted significantly better in NOD/SCID mice (ALDH+AML: injected bone 21.11±9.07%; uninjected bone 1.52±0.75% vs ALDH−AML: injected bone 1.77±1.66% (P=0.05); uninjected bone 0.23±0.23% (P=0.03)) with the engrafting cells showing molecular and cytogenetic aberrations identical to the original clones. Normal BM contained a small SSCloALDHbr population (median: 2.92%, range: 0.92–5.79%), but none of the ALL cases showed this fraction. In conclusion, SSCloALDHbr cells in ALDH+AML might denote primitive LSC and confer an inferior prognosis in patients.

Toll-Like Receptor 4 Promotes Tubular Inflammation in Diabetic Nephropathy

Inflammation contributes to the tubulointerstitial lesions of diabetic nephropathy. Toll-like receptors (TLRs) modulate immune responses and inflammatory diseases, but their role in diabetic nephropathy is not well understood. In this study, we found increased expression of TLR4 but not of TLR2 in the renal tubules of human kidneys with diabetic nephropathy compared with expression of TLR4 and TLR2 in normal kidney and in kidney disease from other causes. The intensity of tubular TLR4 expression correlated directly with interstitial macrophage infiltration and hemoglobin A1c level and inversely with estimated glomerular filtration rate. The tubules also upregulated the endogenous TLR4 ligand high-mobility group box 1 in diabetic nephropathy. In vitro, high glucose induced TLR4 expression via protein kinase C activation in a time- and dose-dependent manner, resulting in upregulation of IL-6 and chemokine (C-C motif) ligand 2 (CCL-2) expression via IκB/NF-κB activation in human proximal tubular epithelial cells. Silencing of TLR4 with small interfering RNA attenuated high glucose-induced IκB/NF-κB activation, inhibited the downstream synthesis of IL-6 and CCL-2, and impaired the ability of conditioned media from high glucose-treated proximal tubule cells to induce transmigration of mononuclear cells. We observed similar effects using a TLR4-neutralizing antibody. Finally, streptozotocin-induced diabetic and uninephrectomized TLR4-deficient mice had significantly less albuminuria, renal dysfunction, renal cortical NF-κB activation, tubular CCL-2 expression, and interstitial macrophage infiltration than wild-type animals. Taken together, these data suggest that a TLR4-mediated pathway may promote tubulointerstitial inflammation in diabetic nephropathy.

Changes of cytokine profiles during peritonitis in patients on continuous ambulatory peritoneal dialysis

Continuous ambulatory peritoneal dialysis (CAPD) has emerged as an important dialysis treatment modality worldwide. One of the major complications is bacterial peritonitis, which may result in subsequent technique failure because of loss of peritoneal clearance or peritoneal fibrosis. Bacterial peritonitis leads to the release of proinflammatory cytokines from resident and infiltrating cells in the peritoneal cavity. We studied 35 patients undergoing CAPD with acute bacterial peritonitis. All patients treated with antibiotics for 2 weeks after the clinical diagnosis of peritonitis had a good recovery. Peritoneal dialysate effluent (PDE) was collected on days 1, 3, 5, 10, 21, and 42 after the start of treatment. Cell populations were monitored by flow cytometry. PDE levels of interleukin-1beta (IL-1), IL-6, transforming growth factor-beta (TGF-beta), and basic fibroblast growth factor (FGF) were measured by enzyme-linked immunosorbent assay. Gene transcription of TGF-beta in macrophages from PDE was measured by quantitative polymerase chain reaction. Bacterial peritonitis was associated with a sharp increase in total cell and neutrophil counts (400-fold) in PDE up to 3 weeks after peritonitis despite clinical remission (P < 0.0001). There was an increased absolute number of macrophages during the first 3 weeks despite the reduced percentage of macrophages among total cells in PDE compared with noninfective PDE. There was a progressive increase in the percentage of mesothelial cells or dead cells in the total cell population in PDE over the entire 6-week period. PDE levels of IL-1, IL-6, TGF-beta, and FGF increased markedly on day 1 before their levels decreased gradually. PDE levels of these cytokines or growth factors were significantly greater than those in noninfective PDE (n = 76) throughout the study period (P < 0.01). Similarly, TGF-beta complementary DNA (cDNA) molecules per macrophage were significantly greater than those of macrophages in noninfective PDE throughout this period (P < 0.01). There was no significant correlation between PDE levels of TGF-beta and TGF-beta cDNA molecules per macrophage, suggesting that peritoneal macrophages are not the only source of TGF-beta in PDE. We conclude there is an active release of proinflammatory cytokines and sclerogenic growth factors through at least 6 weeks despite apparent clinical remission of peritonitis. The peritoneal cytokine networks after peritonitis may potentially affect the physiological properties of the peritoneal membrane.

Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy

BACKGROUND We have previously documented that human mesangial cell (HMC)-derived tumour necrosis factor-alpha (TNF-alpha) is an important mediator involved in the glomerulo-tubular communication in the development of interstitial damage in IgA nephropathy (IgAN). With the strategic position of podocytes, we further examined the function of podocytes in IgAN. METHODS Podocyte markers were examined in renal tissues by immunofluorescence. In vitro experiments were conducted with podocytes cultured with polymeric IgA (pIgA) or conditioned medium prepared from HMC incubated with pIgA (IgA-HMC conditioned medium). RESULTS Glomerular immunostaining for nephrin or ezrin was significantly weaker in patients with IgAN. The immunostaining of IgA and nephrin was distinctly separate with no co-localization. In vitro experiments revealed no effect of pIgA on the expression of these podocyte proteins as IgA from IgAN patients did not bind to podocytes. In contrast, IgA conditioned medium prepared from IgAN patients down-regulated the expression of these podocyte proteins as well as other podocyte markers (podocin and synaptopodin) in cultured podocytes. The mRNA expression of nephrin, erzin, podocin but not synaptopodin correlated with the degree of proteinuria and creatinine clearance. The down-regulation was reproducible in podocytes cultured with TNF-alpha or transforming growth factor-beta (TGF-beta) at concentration comparable to that in the IgA-HMC conditioned medium. The expression of these podocyte proteins was restored partially with a neutralizing antibody against TNF-alpha or TGF-beta and fully with combination of both antibodies. CONCLUSION Our finding suggests podocyte markers are reduced in IgAN. An in vitro study implicates that humoral factors (predominantly TNF-alpha and TGF-beta) released from mesangial cells are likely to alter the glomerular permeability in the event of proteinuria and tubulointerstitial injury in IgAN.

Role for macrophage migration inhibitory factor in acute respiratory distress syndrome

The critical role of macrophage migration inhibitory factor (MIF) in mediating inflammatory lung injury in acute respiratory distress syndrome (ARDS) has been raised recently. The present study has identified enhanced MIF protein expression in alveolar capillary endothelium and infiltrating macrophages in lung tissues from ARDS patients. The possibility that MIF up‐regulates its synthesis in an autocrine fashion in ARDS was tested using cultured endothelial cells stimulated with MIF and a murine model of lipopolysaccharide (LPS)‐induced acute lung injury. MIF induced significant MIF and tumour necrosis factor (TNF)‐α synthesis in cultured endothelial cells and the effect was blocked by neutralizing anti‐MIF antibody. A similar blocking effect was observed when MIF‐stimulated endothelial cells were pretreated with neutralizing anti‐TNF‐α antibody or glucocorticoid, supporting the notion that MIF induced TNF‐α production via an amplifying pro‐inflammatory loop. Treatment with anti‐MIF or glucocorticoid effectively attenuated pulmonary pathology and the synthesis of MIF or TNF‐α in mice with LPS‐induced acute lung injury. Mildly augmented expression of aquaporin 1 (AQP1) was also detected in alveolar capillary endothelium in ARDS. In vitro studies revealed that both MIF and TNF‐α induced a small increase of AQP1 synthesis in cultured endothelial cells. These findings suggest that MIF plays a crucial pathological role leading to alveolar inflammation in ARDS. Anti‐MIF and early glucocorticoid therapy may represent a novel therapeutic approach for reducing alveolar inflammation in ARDS. Copyright

Long-term study of mycophenolate mofetil treatment in IgA nephropathy.

Since the efficacy of mycophenolate mofetil (MMF) to treat immunoglobulin A (IgA) nephropathy is controversial, we extended our original study by following 40 Chinese patients with established IgA nephropathy for 6 years. All patients were maintained on their angiotensin blockade medication and half were randomized to receive MMF for 6 months. After 6 years, 11 patients required dialysis (2 from the MMF and 9 from the control group). Significantly, only 3 treated (as compared to 10 control) patients reached the composite end point of serum creatinine doubling or end-stage renal disease. Linear regression showed the annualized decline in the estimated glomerular filtration rate was significantly less in the MMF-treated group. Urinary protein excretion and the albumin-to-creatinine ratio were lower with MMF treatment during the first 24 months, beyond which there was no difference between groups. Multivariable Cox regression analysis showed that the baseline estimated glomerular filtration rate and proteinuria, and change in the urine albumin-to-creatinine ratio at 1 year to be important predictors of progression to end-stage renal disease. We found that among Chinese patients with IgA nephropathy who had mild histologic lesions and persistent proteinuria despite maximal angiotensin blockade, MMF treatment may result in transient and partial remission of proteinuria in the short-term and renoprotection in the long-term.

THE RENIN-ANGIOTENSIN SYSTEM

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease in developed countries where type 2 diabetes mellitus has reached epidemic proportions. Although the exact pathogenesis of DN is not fully understood and is likely diverse in nature, there are convincing data that the renin-angiotensin system (RAS) is a major mediator of renal injury. Angiotensin II (Ang II), traditionally playing a central role as a mediator of glomerular hemodynamic adaptation and injury, is now recognized to exert proinflammatory action leading to upregulation of chemokines, adhesion molecules, and other fibrogenic growth factors that culminate in a decline of renal function. Hyperglycemia and mechanical stress deriving from glomerular hypertension are the key factors underlying pathogenesis of DN. The common signaling pathways stimulated by high glucose and mechanical insult may act synergistically, thereby accelerating the cell damage. Podocytes are subjected not only to the load of filtered glucose but also to diverse mechanical forces. Both high glucose and mechanical stress may impair the protein systems anchoring the podocyte foot processes in the glomerular basement membrane, therefore blunting resistance of these cells to mechanical forces in addition to the inflammatory insults. Loss of the podocytes is irreversible due to their inability to proliferate and to replenish damaged cells. Podocytes are injured early in the course of DN, which, most likely, underlies further glomerular and renal damage in diabetes. Under normal physiological conditions, podocytes play a specific role in the maintenance of intraglomerular RAS balance with enzymatic activities that predominantly lead to ANG1-7 and ANG1-9 formation, as well as Ang II degradation. ANG1-7 counteracts the proinflammatory actions of Ang II. These enzymatic activities are altered in a nonphysiological environment such as hyperglycemia that mimics diabetic kidney disease. An understanding of the local intraglomerular RAS will provide a novel approach for early stages of DN.

Activation of podocytes by mesangial-derived TNF-α: glomerulo-podocytic communication in IgA nephropathy

We have previously documented that human mesangial cell (HMC)-derived TNF-alpha is an important mediator involved in the glomerulo-tubular communication in the development of interstitial damage in IgA nephropathy (IgAN). With the strategic position of podocytes, we further examined the role of mesangial cells in the activation of podocytes in IgAN. There was no binding of IgA from patients with IgAN to podocytes. Podocytes cultured with IgA from patients with IgAN did not induce the release of growth factors or cytokines. Furthermore, podocytes did not express mRNA of known IgA receptors. In contrast, IgA-conditioned medium (IgA-HMC medium) prepared by culturing HMC with IgA from patients with IgAN for 48 h significantly increased the gene expression and protein synthesis of TNF-alpha by podocytes with a 17-fold concentration above that of IgA-HMC medium. The upregulation of TNF-alpha expression by podocyte was only abolished by a neutralizing antibody against TNF-alpha but not by other antibodies. Exogenous TNF-alpha upregulated the synthesis of TNF-alpha by podocytes in an autocrine fashion. IgA-HMC medium prepared with IgA from patients with IgAN also significantly upregulated the expression of both TNF-alpha receptor 1 and 2 in podocytes. Our in vitro finding suggests podocytes may play a contributory role in the development of interstitial damage in IgAN by amplifying the activation of tubular epithelial cells with enhanced TNF-alpha synthesis after inflammatory changes of HMC.

Polymeric IgA1 from Patients with IgA Nephropathy Upregulates Transforming Growth Factor-β Synthesis and Signal Transduction in Human Mesangial Cells via the Renin-Angiotensin System

The effects of polymeric IgA1 (pIgA1) and monomeric IgA1 (mIgA1) from patients with IgA nephropathy (IgAN) on the renin-angiotensin system (RAS) and TGF-beta synthesis were examined in cultured human mesangial cells (HMC). Both pIgA1 and mIgA1 induced renin gene expression in HMC, in a dose-dependent manner. Similar findings were observed for TGF-beta gene and protein expression. The values measured in HMC incubated with pIgA1 were significantly higher than those in HMC incubated with equivalent amounts of mIgA1. When similar experiments were performed with the addition of either captopril or losartan, there was a significant increase in the renin gene expression by HMC, whereas the synthesis of TGF-beta was markedly reduced. The TGF-beta signal transduction pathways in HMC were studied by measuring the receptor-regulated Smad proteins (Smad 2 and 3) and common-partner Smad proteins (Smad 4). pIgA1 from patients with IgAN upregulated Smad activity in HMC, and the activity observed in HMC that had been preincubated with pIgA1 was readily suppressed with optimal concentrations of captopril or losartan. The effects of pIgA1 on the RAS were further examined in HMC incubated with IgA isolated from 30 patients with IgAN, 30 healthy subjects, and disease control subjects with other diseases. pIgA1 induction of angiotensin II or TGF-beta synthesis in HMC was significantly greater with preparations from patients with IgAN, compared with healthy or disease control subjects. The findings support a pathogenetic role of pIgA1 in IgAN through upregulation of the RAS and TGF-beta, leading to chronic renal failure with renal fibrosis.