Abstract
The kidneys play a vital role in the excretion of metabolic
waste products such as urea, creatinine and uric acid,
regulation of extracellular fluid volume, serum osmolality and
electrolyte concentrations, removal of acids, alkalies as well
as the production of hormones like erythropoietin and
1,25 dihydroxyvitamin D and Renin Assessment of renal function
is important in the management of patients with kidney disease
or pathologies affecting renal function. Tests of renal
function have utility in identifying the presence of renal
disease, monitoring the response of kidneys to treatment, and
determining the progression of renal disease.
Key words: glomerular filtration rate, BUN, creatinine,
cystatin C, proteinuria
Generally, for estimation of serum creatinine, blood urea,
blood urea nitrogen (BUN), serum protein, serum uric acid,
serum calcium, serum phosphorous, HbA1c and blood glucose
levels, a random blood sample suffices. Fasting state is
needed for fasting blood glucose and serum lipid levels.
However, the effect of recent high protein ingestion may
increase creatinine and urea levels to a significant extent.
Also hydration status can have a significant effect on BUN
measurement.
Collection of midstream urine for urine analysis is required
as this sample is less likely to be contaminated by epithelial
cells and commensal bacteria.
For timed urine collections such as for the 24-hour urine
creatinine clearance, it is essential that urine be collected
accurately over the required period as under or over
collection will affect final results.
Procedures
a) Urine Analysis
The best specimen for urine analysis is freshly voided
midstream urine. Midstream urine is utilized as it is less
likely to be contaminated by commensal bacteria and epithelial
cells.
Urine analysis involves assessment of urine characteristics to
aid in diagnosis and consists of physical observation,
chemical and microscopic analysis. Physical observation
involves assessing color and clarity. The normal color of
urine is straw colored in the presence of dehydration urine is
a darker color. Red urine may indicate hematuria or porphyria
or represent the dietary intake of food like beets. Cloudy
urine may be seen in the presence of pyuria due to urinary
tract infection. Specific gravity is an indicator of renal
concentrating ability may be measured using refractometry or
chemically by use of urine dipstick. The physiologic range for
specific gravity is 1.003 to 1.030 and is increased with
concentrated urine and decreased with dilute urine. A fixed
specific gravity of 1010 indicates chronic renal disease.
b) Urine dipstick
Dipstick uses dry chemistry methods to detect for the presence
of protein, glucose, blood, ketones, bilirubin, urobilinogen,
nitrite, and leukocyte esterase. These may be performed as a
point-of-care test near a patient. The color changes following
interaction of the urine with the chemical reagents
impregnated on the paper of the dipstick are compared to the
color chart guide to interpret the results.
Protein will not be detectable in normal urine specimens.
Bilirubin is not detected in normal urine. Glucose is not
detected in healthy patients but may be seen in diabetes
mellitus, pregnancy, and renal glycosuria when the renal
threshold of 180 mg/dl is decreased. The presence of ascorbic
acid (vitamin C) and some antibiotics may affect results.
Blood may be present after renal tract injury or infection,
with ascorbic acid causing a falsely negative result. Urine
dipstick detects the globin portion of hemoglobin, and thus
cannot detect the difference between the presence of myoglobin
or hemoglobin in urine. Additionally, both intact red blood
cells (RBC) and hemoglobinuria are detected. In normal urine
RBC per high-power field is between 0 to 3 and white blood
cells (WBC) between 0 and 5. Ketones are present in fasting,
severe vomiting, or diabetic ketoacidosis. Urine dipstick only
detects acetoacetate and acetone, not the ketone
beta-hydroxybutyrate. Bilirubin is detected in the presence of
conjugated hyperbilirubinemia, urobilinogen may normally be
present but is absent in conjugated hyperbilirubinemia and
increased in the presence of prehepatic jaundice and
hemolysis. Nitrite and leucocyte esterase are indicators of
urinary tract infection. Some bacteria, for example,
Enterobacteriaceae,
convert nitrates to nitrites.
The microscopic analysis involves wet-prep analysis of urine
to assess in the presence of cells, casts, and crystals as
well as micro-organisms. Red blood casts usually denote
glomerulonephritis while white blood cell casts are consistent
with pyelonephritis. Presence of WBC and WBC casts indicates
infection; red blood cells indicate renal injury; RBC casts
indicate tubular damage or glomerulonephritis. Hyaline casts
consist of protein and may occur in glomerular disease.
Crystals may also be identified in urine and are indicative of
the following conditions:
Triple phosphate crystals are rectangular are
seen in pseudogout.
Uric acid crystals are needle-shaped and are associated
with gout.
Oxalate crystals are envelope shaped and are present in
ethylene glycol poisoning or primary and secondary
hyperoxaluria.
Cysteine crystals are hexagonal and observed in
cystinuria.
Albuminuria and Proteinuria
Albuminuria refers to the presence of albumin in urine - 30 to
300 mg per day. Microalbuminuria is a term to describe a
moderate increase in the level of urine albumin. It occurs
when the kidney leaks small amounts of albumin into the urine,
in other words, when an abnormally high permeability for
albumin in the glomerulus of the kidney occurs. Normally, the
kidneys filter albumin, so if albumin is found in the urine,
then it is a marker of kidney disease. The term
microalbuminuria is now discouraged by Kidney Disease
Improving Global Outcomes and has been replaced by moderately
increased albuminuria. . Albuminuria is used as a marker for
detection of incipient nephropathy in diabetics; it is an
independent marker for the cardiovascular disease since it
connoted increased endothelial permeability and is also a
marker of chronic renal impairment. Urine albumin may be
measured in 24-hour urine collections or early morning/random
specimens as an albumin/creatinine ratio. Presence of
albuminuria on two occasions with the exclusion of a urinary
infection indicates glomerular dysfunction. The presence of
albuminuria for 3 or more months is indicative of chronic
kidney disease. Frank proteinuria is defined as greater than
300 mg per day of protein. Normal urine protein up to 150 mg
per day is formed by 30% albumin; 30% globulins and 40% Tamm
Horsfall protein. Increased amounts of protein in urine may be
due to:
Glomerular proteinuria: Seen in
glomerulonephritis or nephrotic syndrome
Tubular proteinuria: Seen in interstitial nephritis
Overflow proteinuria: Seen in multiple myeloma-Bence Jones
protein, myoglobinuria, urinary tract inflammation or tumor
Urine protein may be measured using either a 24-hour urine
collection or random urine protein: creatinine ratio (early
morning sample preferred and more representative of the
24-hour sample).
The KDIGO classification defines 3 stages of albuminuria:
A1: Less than 30 mg/g creatinine
A2: 30 to 300 mg/g creatinine
A3: Greater than 300 mg/g creatinine
When the urine protein excretion exceeds 3.5 g/day and
associated with edema, hypoalbuminemia, and
hypercholesterolemia is called Nephrotic Syndrome.
Blood Urea and Blood Urea Nitrogen (BUN)
Urea or BUN is a nitrogen-containing compound formed in the
liver as the end product or protein metabolism and urea cycle.
About 85% of urea is eliminated via kidneys; the rest is
excreted via the gastrointestinal tract. Blood urea is
increased in conditions where renal clearance decreased (in
acute and chronic renal failure/impairment). Urea may also
increase in other conditions not related to renal diseases
such as upper GI bleeding, dehydration, catabolic states, and
high protein diets. Urea may be decreased in starvation,
low-protein diet, and severe liver disease. Serum creatinine
is a more accurate assessment of renal function than urea;
however, urea is increased earlier in renal disease.
Creatinine
The most commonly used endogenous marker for assessment of
glomerular function is Creatinine. The calculated clearance of
creatinine is used to provide an indicator of GFR. This
involves the collection of urine over a 24-hour period.
Creatinine clearance is then calculated using the equation:
C = (U x V) / P
C = clearance, U = urinary concentration, V = urinary flow
rate (volume/time ie ml/min), and P = plasma concentration
Creatinine clearance should be corrected for body surface
area. Improper or incomplete urine collection is one of
the major issues affecting the accuracy of this test, hence
timed collection is advantageous. Furthermore, due to tubular
secretion, creatinine overestimates GFR by around 10% to 20%.
Creatinine is the by-product of creatine phosphate in muscle,
and it is produced at a constant rate by the body. For the
most part, creatinine is cleared from the blood entirely by
the kidney. Decreased clearance by kidney results in an
increased blood creatinine. The amount of creatinine produced
per day depends on muscle bulk, and thus, there is a
difference in creatinine ranges between males and females with
lower creatinine values in children and those with decreased
muscle bulk. Diet also influences creatinine values.
Creatinine can change as much as 30% after ingestion of red
meat. As GFR increases in pregnancy lower creatinine values
are found in pregnancy. Additionally, serum creatinine is a
later indicator of renal impairment-renal function is
decreased by 50% before a rise in serum creatinine is
observed.
Serum creatinine is also utilized in GFR estimating equations
such as the Modified Diet in Renal Disease (MDRD) and the
CKD-EPI equation. These eGFR equations are superior to serum
creatinine alone since they include race, age, and gender
variables. GFR is classified into the following stages based
on the kidney disease.
Improving Global Outcomes (KDIGO) stages of chronic kidney
disease (CKD):
Stage 1 GFR greater than 90 ml/min/1.73 m
Stage 2 GFR between 60 to 89 ml/min/1.73 m
Stage 3a GFR 45 to 59 ml/min/1.73 m
Stage 3b GFR 30 to 44 ml/min/1.73 m
Stage 4 GFR of 15 to 29 ml/min/1.73 m
Stage 5-GFR less than 15 ml/min/1.73 m (end-stage renal
disease)
These provide an easier estimation of GFR without collection
of urine or use of exogenous materials. However, as they
utilize serum creatinine, they are also affected by the issues
around serum creatinine measurement, hence the correction for
race, gender, and age.
The ratio of Blood Urea to Creatinine can be useful to
differentiate Prerenal from Renal causes when the Blood Urea
is increased. The normal ratio is 20:1. In Pre-renal disease
the ratio is close to 50:1, while in intrinsic renal disease
it is closer to 10:1.
Cystatin C
Cystatin C is a low-molecular-weight protein which functions
as a protease inhibitor produced by all nucleated cells in the
body. It is formed at a constant rate and freely filtered by
the kidneys. Serum levels of cystatin C are inversely
correlated with the glomerular filtration rate (GFR). In other
words, high values indicate low GFRs, while lower values
indicate higher GFRs, similar to creatinine. The renal
handling of cystatin C differs from creatinine. While both are
freely filtered by glomeruli, once cystatin C is filtered, it
is reabsorbed and metabolized by proximal renal tubules,
unlike creatinine. Thus, under normal conditions, cystatin C
does not enter the final excreted urine to any significant
degree. Cystatin C is measured in serum and urine. The
advantages of cystatin C over creatinine are that it is not
affected by age, muscle bulk, or diet, and various reports
have indicated that it is a more reliable marker of GFR than
creatinine particularly in early renal impairment. Cystatin C
has also be incorporated into eGFR equations such as the
combined creatinine-cystatin KDIGO CKD-EPI equation.
Cystatin C concentration may be affected by the presence of
cancer, thyroid disease, and smoking.
Tests of Tubular Function
The renal tubules play an important role in reabsorption of
electrolytes, water, and maintaining acid-base balance.
Electrolytes, sodium, potassium, chloride, magnesium,
phosphate can be measured in urine as well as glucose.
Measurement of urine osmolality allows for assessment of
concentrating ability of urine tubules. A urinary osmolality
greater than 750 mOsmol/Kg H2O implies a normal concentrating
ability of tubules. A water deprivation test can be used to
exclude nephrogenic diabetes insipidus. Also in distal renal
tubular acidosis (dRTA), an ammonium chloride test can be used
to confirm the diagnosis of distal RTA with failure to acidify
the urine to a pH less than 5.3. In Fanconis syndrome, there
is aminoaciduria, glycosuria, and phosphaturia and bicarbonate
wasting (proximal RTA).
Assessment of Renal Function
There are a number of clinical laboratory tests that are
useful in investigating and evaluating kidney function.
Clinically, the most practical tests to assess renal function
are to get an estimate of the glomerular filtration rate (GFR)
and to check for proteinuria (albuminuria).
The best overall indicator of the glomerular function is the
glomerular filtration rate (GFR). The normal GFR for an adult
male is 90 to 120 mL per minute. A GFR of less than 15 ml per
minute is considered to be end-stage renal failure requiring
renal replacement therapy, e.g., dialysis. The presence of a
normal GFR does not exclude the presence of renal disease
which may be evidenced by the presence of
albuminuria/proteinuria or imaging.
Interfering Factors
a) Creatinine
Preanalytical issues such as high-protein intake and increased
muscle bulk may lead to elevated creatinine levels not
representative of actual renal function in an individual.
Likewise, serum creatinine as a marker of renal function is
often unreliable in the those with decreased muscle bulk such
as the elderly, amputees and is individuals affected by
muscular dystrophy. Creatinine is commonly measured on
automated analyzers using either a colorimetric reaction known
as the Jaffe reaction or an enzymatic assay. The Jaffe
reaction involves the formation of an alkaline picrate. It is
subject to negative (for example, bilirubin) and positive
interferences (for example, ketones and proteins). Various
modifications to the Jaffe reaction have been made to overcome
some of these issues.
b) BUN
Serum urea/BUN concentrations may also be raised in the
presence of a high-protein diet or with patients using oral
corticosteroids.
c) Urine Albumin and Protein
Urine albumin or protein may be increased in the presence of
conditions not related to renal disease, for example, posture
and exercise (called Orthostatic Proteinuria) and in febrile
illnesses. Furthermore, in the presence of a urinary tract
infection, urine protein levels may be raised without the
presence of any intrinsic renal pathology.
Reference
Praful B. Godkar and Darshan P. Godkar: Textbook of Medical
Laboratory Technology. 2003; Published by Bhalani Publishing
House, New Delhi.