Urinary tract infections are common bacterial infections that affect mankind. They are traditionally diagnosed by routine analysis of urine, urine culture and antibiotic sensitivity profiling to identify the antibiotic regimen to be administered to the patient. The role of the various proteins present in the kidney, and excreted in the urine have always remained in the dark; this has started to gain importance with the recent advancements in the field of mass spectrometric analysis of the urine samples. This study aims to generate the mass spectrometric profile for urinary macromolecules prepared from normal subjects and urinary tract infected subjects (with and without diabetes mellitus).
The urine samples were collected in the presence of the protease inhibitors and transported in cold and the samples were quantified for its protein, urea and creatinine content. The urinary macromolecules were prepared using a 10 kda cutoff filter and the aliquoted samples were lyophilized. The pooled normal (N), non-diabetic UTI (NU), diabetic (D) and diabetic UTI (DU) protein samples were resolved on a 10% SDS polyacrylamide gel and the banding pattern was observed after staining and the expression of Tamm Horsfall protein and transferrin was carried out. The samples were analyzed for the various proteins using MS analysis.
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The protein content in the NU, D, DU samples were considerably higher when compared to the N sample. The number of bands had also increased in the NU, D, DU, while the expression of THP had been considerably reduced.
Key words: Urine, Urinary Tract infections, Diabetes, Mass spectrometric analysis.
Urinary tract infection (UTI) is the most common non-epidemic bacterial infection in humans, posing an immediate danger for the host. Innate, adaptive components as well as the stromal cells including bladder epithelium are involved in UTI prevention and clearance.Â Urine formed in the kidneys, contains metabolic waste products along with glucose, amino acids, inorganic salts and serum proteins.The availability of urine in large volumes through non-invasive procedures, with an appreciable amount of protein content, and stability, urine samples are now preferred for discovery of crucial proteins that could play an important role in disease mechanisms through mass spectrometric analysis and it has gained importance as an attractive biofluid. Recent advancements in the field of clinical proteomics have identified mass spectrometry -based urinary protein and peptide profiling to be the most suitable for clinical application for obtaining information with regard to descriptive urinary proteomics and validation of urinary biomarkers for uro-genital and non-uro-genital diseases.
2. Materials and methods
2.1. Collection of urine sample
The urine samples from urinary tract infected patients and healthy individuals after their consent to participate in the study (Table 1) were collected from The Sri Narayani Hospital and Research Centre and Health Centre, and VIT University, Vellore, after obtaining prior approval for the experimental protocols from the Institutional Human ethical Committee (IRB approval number: 11/06/08/10). The inclusion criteria for the study included UTI patients with positive dipstick analysis for leukocyte and nitrite and with or without Diabetes mellitus and normal individuals while patients who were undergoing treatment for UTI, hepatic disease, history of renal dialysis, indwelling catheters, hypersensitivity to antibiotics, were excluded from the study. The samples collected in the a sterile container were immediately transported to the laboratory.
2.2 Urine analysis
The collected urine sample was analyzed for various parameters such as pH, leucocyte content, nitrite content, urobilinogen, specific gravity, protein, glucose, by multistix (Siemens, Newyork, USA). The amount of protein (Bradford's), Urea (DAM) and creatinine (Jaffe's) present in the sample were quantified.
2.3 Preparation of urinary macromolecule
Urine macromolecules from normal (N), non-diabetic UTI (NU), diabetic (D) and diabetic UTI (DU) protein samples were isolated by ultradiafiltration (10 kD TFF, Pall Biosciences, India) against a buffer containing 100 mM NaCl. The macromolecular fractions were concentrated five to tenfold during this process, and were constantly in the presence of protease inhibitors (Sigma) from the time of collection. Samples were stored at _80_C until assayed. For patient sample comparisons, we added the quantity of macromolecules that would have been contained in the same volume of the original urine as a given quantity of creatinine (i.e., 1 Creat Eq would be the amount of macromolecules contained in the same volume of the original urine as 1 mg of creatinine). Thus, we were adjusting principally for variations in urine dilution between patients, but the samples may contain different quantities of macromolecules from different individuals according to observed variations in this parameter.
2.4 Expression analysis - SDS PAGE and Western blot
Always on Time
Marked to Standard
The urinary macromolecules were separated on a 10% 29.2:0.8 Acrylamide- Bisacrylamide SDS gel at 50V. The gel was then stained with Coommassie Briiliant Blue overstained and then destained to check for the banding pattern. The gels were then probed using in house raised antibodies to check for the expression pattern of Tamm Horsfall protein nad transferrin among the samples N, NU, D, DU using a HRP conjugated secondary antibody and TMB chromogenic system.
Results and discussion
Urine is formed in the kidney by ultrafiltration from the plasma to eliminate waste products such as urea and metabolites. Although the kidney accounts for only 0.5% of total body mass, a large volume of plasma (350-400 ml/100 g tissue/min) flows into the kidney, generating a large amount of ultrafiltrate (150-180 l/day) under normal physiologic conditions [1,2]. Components in the ultrafiltrate such as water, glucose, amino acids, and inorganic salts are selectively reabsorbed, and less than 1% of ultrafiltrate is excreted as urine.
Serum proteins are filtered based on their sizes and charges at the glomeruli . After passing through glomeruli, abundant serum proteins ,for instance, albumin, immunoglobulin light chain,transferrin, vitamin D binding protein, myoglobin, and receptor-associated protein are reabsorbed, mainly by endocytic receptors, megalin, and cubilin in proximal renal tubules [4-8]. Thus, protein concentration in normal donor urine is very low (less than 100 mg/l when urine output is 1.5 l/day), and normal protein excretion is less than 150 mg/day. This is about a factor 1000 less compared with other body fluids such as plasma.
In 24 h, about 900 liters of plasma flows through the kidneys of which 150-180 liters is filtered. However, more than 99% of this primitive urine is reabsorbed. The remainder (the "final" urine) exits the kidney via the ureter into the bladder. Therefore urine may contain information not only from the kidney and the urinary tract but also from more distant organs via plasma obtained by glomerular filtration. In healthy individuals, 70% of the urinary proteome originates from the kidney and the urinary tract, whereas the remaining 30% represents proteins filtered by the glomerulus (3). The urinary proteome analysis might therefore allow the identification of biomarkers of both urogenital and systemic diseases. Urinary proteomics has been conducted by combining various protein concentration and protein separation methods as well as mass spectrometry (MS) technology.
In many studies, two-dimensional gel electrophoresis was employed for protein separation.Through one of these studies, that was conducted by Pieper and coworkers , identified 150 unique proteins using two-dimensional gel electrophoresis and both matrix assisted laser desorption ionization time-of-flight MS and liquid chromatography (LC)-tandem mass spectrometry (MS/MS or MS2). However, one-dimensional and two-dimensional chromatographic approaches have been used in several recent studies, resulting in further protein identification. 295 unique proteins from the exosome fraction using one-dimensional gel electrophoresis and LC-MS/MS was reported earlier by Pisitkun and co-workers. Sun and colleagues  identified 226 unique proteins using onedimensional gel electrophoresis plus LC-MS/MS and multidimensional liquid chromatography (LC/LC)-MS/MS.
Wang and coworkers  applied concanavalin A affinity purification for the enrichment of N-glycoprotein in urine and identified 225 proteins using one-dimensional gel electrophoresis plus LC-MS/MS and LC/LC-MS/MS. Recently, Castagna and colleagues  exploited beads coated with a hexametric peptide ligand library for urinary protein concentration and equalization, and identified 383 unique gene products by LCMS/MS using a linear ion trap-Fourier transform (LTQ-FT)instrument. These researchers combined their set of urinary proteins with others derived from the literature to yield a total of about 800 proteins.
In our study, a pooled normal urine sample was prepared from the age and sex matched healthy subjects after ultrafiltration of the urine collected (N). The macromolecules from urine were also prepared from the Diabetic (D), Non diabetic UTI (NU) and Diabetic UTI (DU). The protein content was found to be increased in the NU, D and DU. The values of protein estimation, urea and creatinine values are represented in Table 1.
Protein Estimation (mg/ml)
Pooled normal (N)
Non-diabetic UTI (NU)
0.835 Â± 0.04
0.724 Â± 0.02
Diabetic UTI (DU)
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Table 1: Panel showing the Urine demographics
Figure 1: Gel picture showing the various bands in the urinary macromolecule
Figure 2: Blot picture showing the expression of THP in the various urinary macromolecules. The expression of THP was found to be reduced in the case of non-diabetic UTI, when compared to normal. In the case of diabetic UTI, the expression of THP was found to be downregulated.
In table 1, It can be seen that protein concentration in non diabetic UTI (NU) is higher than that of pooled normal (N) urine sample and the protein concentration is very low in diabetic sample (D) when compared to the pooled normal (N).The diabetic UTI (DU) sample has slightly less protein concentration than the pooled normal (N) urine sample.
In the SDS-PAGE analysis (Figure 1) the lane 1 is the molecular marker with respect to which the expressions of the proteins were studied, where expression of BSA was found to be the greatest in the non diabetic UTI (NU) sample while the lowest was found to be in diabetic sample (D). Both pooled normal (N) and diabetic UTI showed nearly equal expression but much lower than that of the non diabetic UTI (DU). We further observe that the first band in the lane 1 can be Tamm horsfall protein as its molecular weight is approximately 90KDa, so we observed the expression of Tamm horsfall protein (THP) in all the lanes, the expression was found to be the greatest in pooled normal (N) and diabetic UTI (DU). The expression of THP was found to be very faint (nearly no expression) in non diabetic UTI (NU) and diabetic (D) urine sample.
From the results of western blotting (Fig. 2) the THP expressions were studied. It was seen that the pooled normal urine (N) and the diabetic urine (D) had prominently high expressions of THP, which correlated with the protein quantification results. The expression of THP was found to be considerably low in non diabetic UTI (NU) and there was no expression of THP in the case of in diabetic UTI (DU). From this we can conclude that the level of THP has been reduced during UTI and the reduction can be because of the binding of the THP to the bacteria and eliminating it through the urine.
Fig. 3 represents the blot depicting the expression of Transferrin. Transferrin is a protein, which was found to be down-regulated during UTI, as the iron present in them are scavenged by the bacteria for their growth. In this study we found that the levels of transferrin were down regulated in the case of diabetic, non-diabetic UTI and in diabetic UTI, when compared to the pooled normal. Thus it indicated that the protein was degraded by the
bacterial toxins such a malleobactin, to scavenge iron for their growth.
Figure 3: Blot picture showing the expression of Transferrin in the various urinary macromolecules. The expression of Transferrin was found to be reduced in the case of diabetic, non-diabetic UTI, and diabetic UTI when compared to normal. In the case of diabetic UTI, the expression of THP was found to be downregulated.
Through this study we were able to find out that the levels of THP and Transferrin, two important proteins in the renal milieu were down regulated during UTI.