The most common type of renal injury in patients with multiple myeloma is “myeloma kidney,” a predominantly tubulointerstitial kidney disease that is characterized by cast formation and marked interstitial fibrosis. We previously demonstrated that excessive endocytosis of myeloma light chains (LCs) induces inflammatory cytokines in human proximal tubule epithelial cells (PTECs). In the present studies, we examined whether LCs directly induce epithelial-mesenchymal transition (EMT) in human renal PTECs. Human LCs were purified from the urine of myeloma patients with modest renal insufficiency. We found that LCs induce marked cellular morphologic alterations in PTECs, accompanied with increased mRNA levels for the extracellular matrix components, collagen IV and fibronectin. Using semiquantative immunoblotting and RT-PCR analyses in human PTECs exposed to 25 μM LCs for up to 72 hours, we observed that the expression of E-cadherin decreases after 24 hours, whereas the expression of α-SMA increased, consistent with the acquisition of mesenchymal characteristics. In contrast, exposure to human serum albumin (160 μM) had no effect on the expression of EMT-related molecules, and its effect was not different from the cells without protein absorption. Human cytokine antibody arrays showed that human PTECs exposed to LCs have increased expressions of profibrotic IL-1, IL-6, IL-8, IL-1ra, MCP-1/4, M-CSF, and TIMP-1/2, as well as EGF, VEGF, TNF-α/β, sTNF-R, and TGF-β. However, the induction of EMT by LCs was not blocked by a neutralizing TGF-β1 antibody, suggesting that this action was TGF-β1 independent. In contrast, LC-induced EMT was markedly attenuated by silencing the p38 MAPK gene using a siRNA. The use of 500 ng/mL bone morphogenetic protein 7 (BMP-7) or 10−8 M pituitary adenylate cyclase-activating polypeptide (PACAP) induced the formation of cell aggregates and the reacquisition of E-cadherin expression and renal proximal tubule epithelial morphology within the confluent cell monolayer during LC treatment. These findings demonstrate that LCs are a direct stimulus for EMT in human renal PTECs. LC-induced EMT involves multiple cytokines and is modulated by p38 MAPK but appears to be independent of the action of TGF-β. LC-induced EMT may be an important mechanism for kidney involvement in myeloma and may be reversible upon administration of exogenous BMP-7 or PACAP.

Migration of human proximal tubule cells (HKC-5) was stimulated by epidermal growth factor (EGF), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1). Integrin signaling via phosphorylation of focal adhesion kinase (FAK) appears to play a central role in cell migration. Once stimulated, FAK undergoes autophosphorylation at tyrosine (Y) 397, followed by phosphorylation of several sites including Y576/Y577 which increases FAK's kinase activity, as well as at Y407, Y861, and Y925. EGF, HGF, and IGF-1 stimulate FAK phosphorylation in various cells. We showed that endothelin stimulated phosphorylation of Y397 in fibroblasts but not HKC-5 cells. After EGF stimulation, HKC-5 cells showed no change in tyrosine phosphorylation at FAK Y397, 407, 576, 861, or 925. Similarly, HGF and IGF-1 did not stimulate the phosphorylation of FAK Y397 in HKC-5 cells. Further, after inhibition of FAK expression by siRNA, cell migration was similar to cells treated with non-target siRNA and responded to EGF with increased migration. Thus, in proximal tubule cells, stimulation of cell migration by growth factors was independent of augmented FAK tyrosine phosphorylation.

Infections are second only to cardiovascular disease as the leading cause of morbidity and mortality among hemodialysis patients. In the United States, there is a high incidence and prevalence of tunneled dialysis catheters. The incidence and optimal treatment of catheter-associated bacteremia (CAB) have not been clearly defined. We retrospectively analyzed the outcomes of all causes of bacteremia in 62 patients with tunneled catheter access in our hemodialysis program through the period from May 1, 2000 to May 1, 2004. We compared outcomes in patients whose catheters were promptly removed (within 72 hours) vs. those who received continued antibiotic without early catheter removal (catheter salvage attempted). Catheter-associated infections were defined as positive blood cultures without another identifiable source. Data recorded for each patient included the number of catheter days, the identity of the infecting organisms, methods of treatment (catheter removal within 3 days or attempted salvage with antibiotics alone), complications and outcomes. Successful treatment was defined as non-recurrence with the same organism for at least two months post treatment. All patients with CAB were treated with at least a 14-day course of intravenous antibiotics regardless of whether the catheter was exchanged. During the 48-month study period, 62 patients developed 146 episodes of bacteremia (21.5 episodes per 1000 catheter-days). Forty of 62 patients (65%) had fever and shaking chills while on HD. 116 of 148 infections (78%) were caused by gram-positive cocci, 22 of 148 (15%) were caused by gram-negative rods, 8 (5%) gram-positive rods and two episodes (1%) were yeast infections. Attempted catheter salvage with antibiotics alone was successful in 29 of 76 episodes (38.1%) vs. 59 of 70 episodes (84.2%) who underwent catheter exchange in addition to antibiotic therapy (p < .0001, Fisher's exact test). Complications occurred in 20 of 62 patients (33.8%) with CAB. These included 9 patients with infective endocarditis, osteomyelitis in 2 patients, splenic abscess and death in 1 patient, and acute MI in 9 patients. We conclude that serious complications are common in HD patients with CAB. Attempted salvage with antibiotic therapy without catheter removal is unlikely to eradicate CAB. We conclude that prompt removal of catheter is associated with better outcome. The best treatment of CAB is prompt catheter removal.

Proximal tubule cell migration is likely a critical early event in recovery from acute renal injury with spreading and migration of cells to cover denuded basement membrane. Growth factors (GFs), including epidermal (EGF), hepatocyte (HGF) and insulin-like growth factor-1 (IGF-1), may play an important role in repair. We have previously shown that each stimulates migration in immortalized human proximal renal tubule cells under control conditions. We now investigated the effect of GFs on migration of these cells after oxidant stress. The cells were cultured in serum- and growth factor-free medium. Confluent cultures were exposed to H2O2 for 1 hour, washed, then scraped with a pipette tip. GFs were added and the area of migration was measured over the next 6 hours. After oxidant stress with 750 μM peroxide, migration decreased to 60% of control (uninjured, no GFs) values. After EGF and HGF migration returned nearly to control values while IGF-1 actually stimulated migration to 131 ± 12 (SEM) % of control values. Adding EGF or HGF to IGF-1 further stimulated migration to 163 ± 16% and 166 ± 13% of control values respectively, representing an increase of ˜ 2.7× over peroxide injured cells (with no GFs). EGF and HGF were not additive. With more severe injury (1.2-1.5 mM H2O2), there was modest stimulation by HGF and IGF-1. There was still some additivity of EGF and HGF with IGF-1, averaging ˜ 1.5× peroxide alone, but values remained depressed compared to uninjured cells not treated with GFs. In summary, the GFs examined stimulated human proximal tubule cell migration after oxidant stress, with IGF-1 being the most potent, especially in combination with EGF or HGF. The stimulation was most pronounced with mild injury. We conclude that the stimulation of migration by growth factors may contribute to their beneficial effect after acute injury.

Background. Both insulin‐like growth factor‐1 (IGF‐1) and epidermal growth factor (EGF) stimulate proliferation of various renal tubule epithelial cells in culture including proximal tubule cells. In some epithelial cells, the effects of EGF and IGF‐1 are additive or synergistic. The effects of EGF and IGF‐1 in cultured tubule epithelial cells following injury are limited. Methods. Immortalized human proximal tubules cultured in serum‐free defined medium were exposed to 0.3–1.5 mM peroxide for 1 h then washed and growth factors were added. ATP was measured by chemiluminescence, proliferation by [3H]thymidine uptake, and receptor expression by flow cytometry. Results. Immediately after 1.5 mM peroxide exposure, ATP levels were depressed to as low as ∼ 15% of normal but had recovered to near normal levels by 4 h. Proliferation was depressed in a dose‐dependent manner by peroxide. At the lowest doses of peroxide both EGF (20 ng/mL) and IGF‐1 (390 ng/mL) stimulated proliferation. As the concentration of peroxide increased, EGF lost its ability to stimulate proliferation and in fact antagonized IGF‐1 which when added alone remained effective at stimulating proliferation even at the highest levels of peroxide exposure. EGF and peroxide depressed EGF receptor expression but there were no changes in IGF‐1 receptor expression with any maneuver. Conclusion. The effects of EGF to antagonize IGF‐1 are distal to IGF‐1 receptor expression. The effects of these growth factors under control conditions do no translate to effects after injury.

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BACKGROUND:In proteinuric states increased cytokine production through endocytosis of filtered proteins by proximal tubule cells (PTCs) has been proposed as a major mechanism mediating tubulointerstitial injury and progressive kidney disease. We studied the effects of six different light chains (LCs) on the production of cytokines in cultured human PTCs.METHODS:LCs were isolated and purified from the urine of patients with myeloma and human PTCs were exposed to either LC or human serum albumin (HSA) for up to 24 hours. LC endocytosis was monitored by immunocytochemistry. Cytokines were determined by enzyme-linked immunosorbent assay (ELISA) in the supernatants and activation of nuclear factor-kappa B (NF-kappaB) was detected by electrophoretic mobility shift assays (EMSA) and immunocytochemistry.RESULTS:Endocytosis of LCs induced the release of interleukins (IL) IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1); however, there was considerable variability among the six different LCs. In contrast, HSA had no effect on cytokine production even at very high concentrations. Removal of LC-containing media resulted in cessation of IL-6 release. LC-induced cytokine release was associated with nuclear translocation of NF-kappaB subunits p50 and p65, as demonstrated by both EMSA and immunocytochemistry. Inhibitors of NF-kappaB, aspirin and pyrrolidineditiocarbamate (PDTC) markedly suppressed LC-induced cytokine production.CONCLUSION:LC endocytosis leads to production of inflammatory cytokines through activation of NF-kappaB. This may be an important mechanism of chronic tubulointerstitial inflammation process commonly seen in multiple myeloma. These findings also point out a potential role by filterable low-molecular-weight proteins, like LCs, in PTC injury during all proteinuric diseases.

We evaluated the effect of eight species of light chains on cultured human kidney proximal tubule cell proliferation. Exposure to light chains for 48 hours caused dose-dependent inhibition in tritium (3H)-thymidine incorporation by simian virus 40 immortalized human proximal tubule cells, although the effect was variable among different species of light chains. We studied cytotoxic effects of selected toxic light chains in further detail. Two of these light chains caused significant DNA degradation. A λ-light chain caused lactate dehydrogenase release from exposed cells at 48 hours, but not at 24 hours. Cytomorphological and electron microscopic examination of cells exposed to light chains for 24 hours showed condensed nuclei, cell detachment, paucity of mitotic activity, and apoptosis, and at 48 hours of exposure, changes consistent with necrosis. Apoptosis assay by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling method showed a sixfold increase in the number of apoptotic cells exposed to the same λ-light chain for 24 hours. Rhodamine-phalloidin staining showed variable but significant disruptions in the actin cytoskeleton. These studies show that some myeloma light chains are toxic to cultured human proximal tubule cells and induce cytoskeletal injury and DNA damage consistent with apoptosis followed by secondary necrosis. Direct proximal tubule cell toxicity may be an important mechanism of renal involvement in multiple myeloma.