The Mechanism Of Renal Impairment In Patients With Multiplemyeloma Caused By Bence Jones Protein In Vitro

BackgroundMultiple myeloma (MM) is a B cell neoplasm of the bonemarrow with a complex array of clinical manifestations including anemia, bone lesions, hypercalcemia, renal dysfunction, and compromised immune function. It accounts for10%of all hematologic malignancies.Renal impairment is a frequent complication of MM and is associated with significant morbidity and is seen in25to50%of cases.The presence of renal insufficiency in patients with myeloma is of prognostic importance because it is associated with a significant increase in morbidity and mortality. The median survival in patients with myeloma is approximately3years, compared the patients with renal injury only0.91years and renal failure is one of the most common causes of death, second only to infection.The mechanisms of MM-associated kidney injury are complex and multifaceted. Although several factors such as dehydration, infections, hypercalcemia, nephrotoxic drugs, and contrast media may result in decreased glomerular filtration rate, in the majority of patients, renal failure is caused by pathogenic light chains. BJP can cause a variety of pathological changes in the kidneys, including "myeloma kidney", or cast nephropathy; light chain amyloidosis (AL); monoclonal Ig deposition disease (MIDD); Fanconi syndrome.Cast nephropathy (CN) is the most common pattern of renal injury and is seen on30%to50%of renal biopsies. When the concentration of BJP in glomerular ultrafiltrate exceeds the reabsorptive capacity of proximal tubular epithelium, BJP may coprecipitate with Tamm-Horsfall protein (THP) in distal tubules to produce casts. The huge number of BJP blocked the tubules and impaired renal function, resulting in the cast nephropathy. The study found that the formation of tube affected the occurrence and development of cast nephropathy, but the direct toxicity of BJP on renal tubular epithelial cells also played an important role. Matsuura et al found some of the BJP can produce toxic effects on renal tubular epithelial cells, lead to apoptosis in vitro experiments. They confirmed that the BJP has amidase activity, contributed to the pathogenesis of renal impairment in multiple myeloma. However, not all of BJP caused renal injury, may be associated with the BJP amidase activity.The renal impairment in patients with AL-amyloidosis and monoclonal immunoglobulin deposition disease are due to a large number of light chain and their fragments secreted by myeloma cells abnormal deposited in the kidney. The type of light chain is not the same in AL-amyloidosis and monoclonal immunoglobulin deposition disease, the more common λ type light chain occurred in amyloidosis, whereas in monoclonal immunoglobulin deposition disease type κ light chain accounted for about70%. The special structure of the light chain protein can cause a variety of pathological changes.However, the renal toxicity of BJP has not yet completely understood. The amount of BJP secreted from patients with MM and the level of renal impairment were not consistent. The patients with high concentrations of BJP did not occurred damage of kidney, in the contrary, some patients with lower level of BJP display severe renal impairment. It is concluded that not all of the BJP with renal toxicity, there are other mechanisms of renal impairment in patients with multiple myeloma.ObjectiveThis study was purposed to investigate the relationship between the catalysis of Bence Jones protein in the urine of patients with multiple myeloma and toxicity on the renal proximal tubular cells in the vitro study, and to explore the potential mechanism for the toxicity of BJP on renal impairment in patients with multiple myeloma.Methods1The purification of BJPTake24-hour urine of patients with MM, adding261g of solid ammonium sulfate per1000ml, at4℃overnight, remove the supernatant after centrifugation. Precipitate dissolved with PB solution (pH7.0), then used the dialysis method to remove inorganic salts, concentrated. Sephadex G-100gel chromatography was used to separate and purify the protein. The eluent solution is the PB (PH7.0). Collect the protein peaks and concentrate. Desalting fraction of crude separating mixture could be chromatographed by DEAE Sepharose CL-6B equilibrated and eluted with pH7.0,50mM Tris buffer.Collect the protein peaks and identified by12%SDS-PAGE electrophoresis. Concentrated and packed into the-80℃refrigerator.2Culture cells LLC-PK1Anchorage-dependent cells LLC-PK1was collected from renal tubular epithelial of Hampshire pig and cultured with M199culture medium with8%serum in37℃and5%CO2conditions. The cells were passaged every48hours.3The catalytic level of BJP hydrolysis BANPAThe amidase activity of BJP can clearage peptide p-nitroanilides, which are chromogenic substrates for trypsin. Because of N-alpha-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPNA) contain the p-nitroanilides structure, we used to measure the Michaelis-Menten constant (Km) and catalytic constant (kcat) of the amidase activity of BJP. Taken150μl different concentrations of BAPNA preheated to37℃, added50μl BJP(terminal concentration:5mg/ml), control groups were given equivalent volume of Tris buffer(0.05mol/L, PH7.4). Reaction for5minutes at37℃, added the acetic acid to terminate the reaction. The absorption values (A) examined at405nm by Microplate reader, zero setting with a blank group. Calculate absorbance difference ΔA between the experimental group and control group, used the Hanes plot to calculate the value of Km and kcat.4The proliferation inhibition of BJP to LLC-PK1Assay the cell proliferation and cell proliferation inhibition rate to the proliferation by MTT colorimetryCells LLC-PK1were incubated in96well plate. After24h incubation, BJP whose terminal concentration were2.5mg/ml、5mg/ml and10mg/ml were put into M199culture medium that was Serum free for further culture respectively. Noting but PBS were put into control groups which contains3multiple pores respectively. After24h incubation, MTT20μl were put into each pore. Culture medium were sucked out and dimethyl sulfoxide were added after a further culture for4h. Absorbance A570nm of each pore were measured by using the enzyme mark instrument. Inhibitory rate of cell proliferation A value in observational group-A value in control group/A value in observational group was measured.5Flow CytometryLLC-PK1subcultured conventionally in M199culture medium containing8%bovine serum is incubated in6-well plate and cultivated by adherent culture methods for24h. The culture solution was replaced by medium that is Serum free and after24h, they are cocultured with BJP (terminal concentration are2.5mg/ml、5mg/ml and10mg/ml respectively) of patients whose number is5、6、8. Noting but PBS are put into control groups which contains3multiple pores respectively. The cells were collected after24h and PBS was washed twice.5μl Annexin V-FITC and5μl PI solution were put into uniform mixing of1～5×105cells and500μl结合液that was incubated at room temperature without light and analysed by Flow Cytometry.6Statistical analysisT test and two-way ANOVA are adopted and measurement data was expressed by x±s. Spearman analytical method was applied into correlate analysis and SPSS13.0was used for data processing. It was indicated that the difference was significance when P≤0.05.Result1Clinical data of MM patientAccording to the diagnostic criteria and the risk index of the patients,13MM patients had been diagnosed. The patients could be divided into2groups by serum creatinine (Scr):one was renal impairment (n=5)if Scr≥178μmol/L and another was without renal impairment (n=8) if Scr<178μmol/L. There was obvious difference in Scr between two groups after Independent-Sample Test (P=0.044), while there was no obvious difference in other factors such as age, blood urea nitrogen (BUN),light chain and24h protein.2The purification of BJP protein and The SDS-PAGE electrophoresis identificationAfter purification the BJP protein collected form13MM patient was identified by the SDS-PAGE electrophoresis identification and there are8typeκ and5typeλ, light chain. The immuneglobulin light chain molecular weight was about25kD and κlight chain often exist in the shape of monomer in vivo while λ, light chain often exist in the shape of dimmer formed by noncovalent interaction. By means of the SDS-PAGE electrophoresis identification, there was a single band of25kD inκlight chain while there were2bands of25kD and50kD respectively inλ light chain. 3BJP enzyme kinetic parameter3.1Kmand kcat of BJPEnzyme activity was measured by enzyme kinetic index Km which reflect the appetency between enzyme and substrate and catalytic constant kcat that reflect the catalytic reaction speed of substrate. The more efficiently the enzyme catalyze, the higher the kcat was. The results showed that all of the Km value of the BJP were the order of10-4mol/L. There were significant differences of the kcat value among different BJP. No.7and No.10were negative value and nonsense.3.2The optimum pH of the BJP catalytic activityAccording to the outcome of3.1, the kcat value of No.5, No.6, No.8BJP was higher5-40times than others. Taken these three BJPs hydrolyses BAPNA to measure their catalytic activity in the range of PH (5.5,6.0,6.5,7.0,7.4,8.0). The results showed that BJP activity was higher in the PH7.0～8.0and the catalytic activity was the highest when PH is7.4. With the increase and decreased of PH value, the catalytic activity of BJP decreased.4The toxic effects of BJP on LLC-PK1cellsThe inhibition ratio of cell proliferation was detected by MTT experiment.The inhibition ratio of cell proliferation of group with renal impairment respectively were7.4±4.5%,13.3±9.4%,21.1±13.6%, the inhibition ratio of cell proliferation of group without renal impairment respectively were1.9±3.3%,1.3±4.9%,1.7±3.9%.It was found by MTT assay that at the5,10mg/ml concentration, the toxic effects of BJP of the gourp with renal impairment was significant higher than that of the gourp without renal impairment (P<0.05).5The relationship between the catalysis of BJP and toxicityAccording to the toxicity of BJP, all of BJP were divided into toxic and non-toxic group. There was obvious difference in the value of Km and kcat between two groups after Independent-Sample Test (P<0.05). It is concluded that the kcat of toxic group were higher than the non-toxic group, while the Km was lower than the non-toxic group.The inhibition ratio of cell proliferation and the kcat value of B JP analysised by Spearman analytical method to identify their correlation. The results showed that there was a positive correlation between the inhibition ratio of cell proliferation and the kcat value of BJP in different concentration (P<0.01for all). The correlation coefficients are0.837,0.872,0.833respectively.6Analysis of Apoptotic by flow cytometryThe flow cytometry showed that the percentage of the apoptotic and necrosis cells in control group, BJP at2.5,5,10mg/ml concentration group respectively were0.7±0.1%,1.9±0.1%,2.7±0.4%,5.9±0.7%and5.1±0.3%,9.3±3.0%,13.3±2.2%,22.0±6.4%. It was found that at the5,10mg/ml concentration, the poptotic and necrosis cells was significant higher than that of the control gourp (P<0.05).BJP could induce apoptosis and necrosis of the cells when reached a certain concentration and this effect enhanced with concentration of BJP.ConclusionIt is concluded that BJP with amidase activity was one of the pathogenesis in renal impairment in patients with multiple myeloma. could induce apoptosis and necrosis of the cells when reached a certain concentration and this effect enhanced with concentration of BJP.The catalysis of BJP and toxicity to renal tubular epithelial cells had a positive correlation, and toxic effect of BJP on renal tubular epithelial cells was through inhibiting proliferation and inducing apoptosis and necrosis of the cells.