OJHAS Vol. 9, Issue 3:
(Jul - Sep, 2010)
Urinary Peptide Levels in Patients with Chronic Renal Failure
Department of Biochemistry
and Genetics, School of Medicine, St Matthews University, Grand Cayman,
British West Indies
Nagaraj M Phani,
Department of Biochemistry,
Kasturba Medical College, Manipal University, Manipal 576104, India,
Department of Biochemistry,
Kasturba Medical College, Manipal University, Manipal 576104, India
Department of Biochemistry,
Kasturba Medical College, Manipal University, Manipal 576104, India.
Dr. Mungli Prakash,
Address For Correspondence
Associate Professor in Biochemistry
St Matthews University, School
Grand Cayman, British West
Prakash M, Phani NM, Kavya R, Supriya M. Urinary peptide levels in
patients with chronic renal failure. Online J Health Allied Scs.
Submitted: Mar 19 2010;
Sep 25, 2010; Published: Oct 15, 2010
Peptide levels in urine are found to be decreased in renal failure.
In the current study urinary peptide levels were determined in chronic
renal failure (CRF) patients. Method: 86 CRF patients and 80
healthy controls were selected for the study. Urinary proteins and peptide
levels were determined by spectrophotometer based Lowry and Bradford
methods. Urinary creatinine levels were determined by clinical chemistry
analyzer. Results: There was significant decrease in urinary
peptide levels in CRF patients and Urinary % peptides were significantly
decreased in CRF patients as compared to healthy controls. Urinary %
peptides correlated negatively with proteinuria. Conclusion:
we have found decrease in urinary peptides and % urinary peptides in
CRF patients and possibly measurement of % urinary peptides may possibly serve
as better indicator in early detection of impairment in renal function.
Key Words: Peptiduria; Proteinuria; Chronic renal failure; Urinary peptides
renal failure (CRF) encompasses a spectrum of different pathophysiologic
processes associated with abnormal kidney function, and a progressive
decline in glomerular filtration.(1) The kidneys attempt to compensate
for renal damage by hyperfiltration within the remaining functional
nephrons and over a time period this hyperfiltration causes further
loss of function. End stage renal disease represents a stage of CRF
where the accumulation of toxins, fluid, and electrolytes normally excreted
by the kidneys results in uremic syndrome.(1)
is common finding in CRF patients and current evidence indicates that
the presence of proteinuria is an early marker of an increased risk
of progressive kidney disease, poor cardiovascular outcome and death.(2-4)
Proteins are too large to pass through the glomeruli into the urine,
but the low molecular weight proteins (less than 1000 kD) are freely
filtered by the glomerulus and this depends upon their size, configuration,
electrical charge.(5) Until recently, it had been believed that the proteins
reaching the renal tubules had been completely reabsorbed.(6) But, recently it had been postulated
that the filtered albumin is taken up by HK-2 cells via a receptor mediated
process and it is degraded by the lysosomal enzymes and the resulting
peptides were exocytosed to the basolateral sides of the cells.(7) The
exact anatomic location of this pathway has not been determined, it
likely takes place in cells distal to the glomerular basement membrane,
most probably in tubular epithelial cells.(8-11) In the current study
we have measured urinary peptides in CRF patients compared them with that
of the healthy individuals to see the difference in excretion of these
six CRF patients admitted to the nephrology ward and 80 healthy controls
were included in the study. The urine sample bottles were stored at
4ºC during the period of collection. Samples were centrifuged at 3000
rpm for 10 min and analyzed immediately after the collection period.
Informed consent was taken from the subjects involved in the study followed
by ethical clearance from the institutional review board.
chemicals such as Bradford reagent and bovine serum albumin (BSA) were
obtained from Sigma Chemicals, St Louis, MO, USA. All other reagents
were of analytical grade.
stock: BSA was dissolved in phosphate buffered saline (PBS). Standard
curves were prepared by dissolving BSA to get the following final concentrations;
for Bradford assay: 2, 4, 6, 8, and 10 μg/mL; for Lowry assay: 50,
100,150, 200, and 250 μg/mL.
standardized the modified Lowry’s assay for determining levels of
total urinary proteins; the reagents were prepared as follows: reagent
A: 2% sodium carbonate, reagent B1: 1% sodium potassium tartarate,reagent
B2: 0.5% CuSO4 in reagent B1, reagent C: 50 mL reagent A + 1 mL reagent
B2, and reagent D: 1N Folin-Ciocalteau reagent.
and peptide levels in urine were measured using a Genesys 10UV spectrophotometer
whereas urine creatinine levels were determined by a Clinical Chemistry
Automated Analyzer (Hitachi 912). Both Lowry and Bradford assays were
performed after diluting the urine samples suitably. Dilutions were
made according to our dilution factors proposed by Prakash et al.(12)
proteins, together with urinary peptides, were measured using the Lowry
assay,(13) whereas urinary proteins were determined using
the Bradford assay.(14)
Urinary peptide levels were determined by subtracting the Bradford’s
value from Lowry’s value of the same urine sample (Lowry value –
Bradford value). All calculations were done using separate calibration
curves prepared for each method.
Lowry estimation, 0.2 mL of the diluted urine sample was taken in two
sets of eppendorf tubes (sets 1 and 2) while 0.2 mL of 145 mM NaCl was
taken in another tube and labeled as reagent blank (RB). To RB and to
set 1, 1 mL of reagent C was added while 1 mL of reagent A was added
to set 2 tubes. The tubes were shaken vigorously and incubated for 10
min at room temperature. Reagent D was added to all the tubes at the
end of 10min and the tubes were vortexed; this step is crucial for color
development. The tubes were incubated at room temperature for 30 min
and the absorbance was read at 600 nm. After correcting for respective
blanks, absorbance values of set 2 samples were subtracted from their
analysis: Statistical analysis was done using statistical package
for social sciences (SPSS) version 16. Independent sample t test was
used to compare mean values and Pearson’s correlation was used to
correlate between the parameters. p<0.05 is considered significant.
depicted in Table 1, there was no significant difference in urine creatinine
levels between healthy controls and CRF patients on conservative management.
There was no significant difference in total urinary proteins (determined
by Lowry assay) per liter of urine between two groups, however there
was significant proteinuria (determined by Bradford’s assay) in CRF
patients on conservative management when compared to healthy controls
(p<0.0001). We have found significant difference in both to total
urinary proteins (Lowry assay) gram of creatinine (p<0.0001) and
urinary proteins (Bradford’s assay) per gram of creatinine (p<0.0001)
between both groups. There was significant difference in urinary peptides
per liter of urine between CRF patients on conservative management compared
to healthy controls (p<0.0001). However, there was no significant
difference in urinary peptide levels expressed per gram of creatinine
between CRF patients on conservative management and healthy controls.
There was a significant decrease in percent urinary peptides in CRF
patients on conservative management compared to healthy controls (p<0.0001).
On applying Pearson’s correlation, percent urinary peptides correlated
negatively with the grams of proteins per gram of creatinine (r2
= 0.426, p<0.01) (Figure 1).
Table 1: Independent sample t test for all the determined biochemical parameters
in both healthy controls and chronic renal failure cases (values expressed
as mean ± standard error of mean).
Healthy Controls (n
Chronic Renal Failure Cases (n = 86)|
|Gm Proteins/L (Lowry’s method)
|Gm Proteins/L (Bradford’s method)
Cr (Lowry’s method)
Cr (Bradford’s method)
Peptides/L (Lowry – Bradford)
– Bradford) x 100 Lowry (% urinary peptides)
*P <0.0001 compared to healthy
Correlation between percent urinary peptides and urinary proteins per
gram of creatinine
have found significant increase in proteinuria and decrease in peptide
excretion in CRF patients indicating increased leakage of intact proteins
through the glomerulus and decreased reabsorption from renal tubules.
Normally renal tubules will reabsorb the proteins filtered by glomerulus
and within the tubules proteins are degraded to peptides and amino acids
which will enter into amino acid pool and some amount of peptides and
amino acids will be secreted by tubules into urine.(7-11,15) Previous
authors have also shown significant decrease in urinary peptides in
renal failure cases in different settings.(12,16) We have found significant
decrease in % urinary peptides in CRF patients which indirectly indicates
decrease in filtration load on glomerulus as there is leakage of intact
protein from it. Previous authors have also shown measurement of % urinary
peptides is better marker for indirectly measuring filtered load on
kidneys.(12,16) In the current study we have found significant
decrease in filtered load on glomerulus with % urinary peptides at 47.32
as compared to 97.12 in healthy controls (table 1). This possibly indicates
leakage of intact protein from glomerulus their by decreasing the filtration
load on it.
We have found significant decrease in urinary peptides along with decrease
in % urinary peptides. This can be probably due to decrease in tubular
reabsorption and degradation of intact protein possibly due to tubular
damage and leakage of lysosomal enzymes into urine. Previous study has
supporting finding for this in which they have shown significant tubular
damage and leakage of lysosomal enzymes into urine in renal failure
cases.(16) The possible leakage and loss of lysosomal enzymes decreases
the capacity of functioning tubules there by decreased reabsorption
of intact proteins escaped in glomerular filtration there by findings
their way into urine. The findings in our study shown similar findings
where by CRF patients shown decrease in urinary peptides and increase
in urinary proteins and also there was negative correlation between
proteinuria and % urinary peptides (Figure 1) supporting the above findings
by other authors.
conclusion, we have found decrease in urinary peptides and % urinary
peptides in CRF patients and possibly measurement of % urinary peptides may
possibly serve as better indicator in early detection of impairment in renal
- Skorecki K, Green
J, Brenner BM. Chronic renal failure. In: Harrisons principles of internal
medicine, Kasper DL, Braunwald E, Fauci AS (Editors), 16th edition,
McGraw-Hill, New York, page 1663-7.
- Gerstein HC, Mann
JF, Yi Q. Albuminuria and risk of cardiovascular events, death
and heart failure in diabetic and nondiabetic individuals. JAMA 2001;286:421-6.
- Mann JF, Gerstein
HC, Pogue J, Bosch J, Yusuf S. Renal insufficiency as a predictor of
cardiovascular outcomes and the impact of ramipril: the HOPE randomized
trial. Ann Intern Med 2001;134:629-36.
- Burton C, Harris
KPG. The role of proteinuria in the progression of chronic renal failure.
Am J Kidney Dis 1996;27:765-75.
- Lemley K. Proteinuria
or peptiduria: where’s the beef? J Lab Clin Med 2005;145:228-230.
- Maack T, Park CH,
Camargo MJ. Renal filtration and metabolism of proteins. In: The Kidney,
physiology and pathophysiology. Seldin DW, Giebish G (editors), New
York, 1985: page 1773-804.
- Gudehithlu KP,
Pegoraro AA, Dunea G, Arruda JAL, Singh A. Degradation of albumin by
the renal proximal tubule cell and the subsequent fate of its fragments.
Kidney Int 2004;65:2113-2122.
- Osicka TM, Pratt
LM, Comper WD. Glomerular capillary wall permeability to albumin and
horseradish peroxidase. Nephrology 1996;2:199-212.
- Osicka TM, Comper
WD. Protein degradation during renal passage in normal kidneys is inhibited
in experimental albuminuria. Clin Sci 1997;93:65-72.
- Osicka TM, Panagiotopoulos
S, Jerums G, Comper WD. Fractional clearance of albumin is influenced
by albumin degradation. Clin Sci 1997;93:557-564.
- Burne MJ, Panagiotopoulos
S, Jerums G, Comper WD. Alterations in renal Degradation of albumin
in early experimental diabetes: a new factor in the mechanism of albuminuria.
Clin Sci 1998;95:67-72.
- Prakash M, Shetty
JK, Dash S, Barik BK, Sarkar A, Prabhu R. Determination of urinary
peptides in patients with proteinuria. Indian J Nephrol
- Lowry OH, Rosenbrough
NJ, Farr AI, Randall RT. Protein measurement with the Folin phenol reagent.
J Biol Chem 1951;193:265-75.
- Bradford MM. A
rapid and sensitive method for the quantitation of microgram quantities
of proteins utilizing the principle of protein dye binding. Anal Biochem
- Russo LM, Bakris
G, Comper W. Renal handling of albumin: a critical review of basic concept
and perspective. Am J Kidney Dis 2002;39:899-919.
- Singh A, Gudehithlu
KP, Le G, Litbarg NO, Khalili V, Vernik J, Hart P, Arruda JAL, Dunea
G. Decreased urinary peptide excretion in patients with renal disease.
Am J Kidney Dis. 2004;44:1031-1038.