Renal Function Tests – Indication with normal values

Dr Jayadeep
Email  : jayadeepbp@gmail.com 

Functions of kidney

• Excretion of Metabolic Waste Products, Foreign Chemicals, Drugs, and Hormone Metabolites
• Regulation of water – electrolyte balances, body fluid osmolality
• Regulation of arterial pressure
• Regulation of acid-base balance
• Regulation of Erythrocyte Production
• Regulation of 1,25–Dihydroxyvitamin D3 Production
• Gluconeogenesis 

OBJECTIVES OF RFT

• To detect possible renal damage and assessment of its severity
• To diagnose renal disease
• To observe the progress of renal disease 

Gives information about

• Renal blood flow
• Glomerular filtration
• Renal tubular function
• Urinary out flow unhindered by any obstruction 

4 groups of tests

• Urine analysis
• Concentration & dilution tests
• Blood chemistry
• Renal clearance tests 

[1] URINE ANALYSIS

• Under physical, chemical, Bacteriological and Microscopic examination 

PHYSICAL EXAMINATION
Assess 24 hour urinery out put [volume], Appearance, Colour, Turbidity, pH, Specific gravity, osmolality etc

Volume

• Is a measure of glomerular filtration and tubular reabsorption
• Normal urine volume varies from 500 – 2,500 ml
  • L/24 hr – typical in health
• Temperate climates: output of 800-2500 ml urine per day is usual.
• Dependent upon subject’s activity, hydration status, diet and body size.
• Sudden changes in volume of urine can indicate problems with ability to concentrate urine
• Children: ca 1.5 ml/Kg of b.w./1 hour!

Oliguria

• Urine volume less than 400 ml/24 hours or < 1 ml/kg
• In hypotension or hypovolaemia & intrinsic renal pathology 

Polyuria – urine output of > 2 litres/24 hours

• Disturbance in the tubular concentrating capacity or ADH failure [ diabetes insipidus]
• Increased osmotic load (diabetes mellitus)
• Excessive water intake (physiological response)
• Drug-induced (outdated tetracycline, lithium)
• Deficiency of vasopressin
• Associated with nocturia

Anuria < 100 mL
Total anuria is usually due to obstruction in the urinary tract.

Colour –appearance

• Normally Amber light coloured
• Very clear urine with high frequency of urination indicates it’s less likely to be a bacterial problem
• Deep yellow -Concentrated urine, Jaundice
• Red urine- Haematuria, Haemoglobinuria Myoglobinuria, Porphyria, Beet root ingestion, Drugs like rifampicin, pyridium
• Cloudy -Infection
• Milky – Chyluria Pyuria Phosphaturia
• Dark on standing –Porphyria, Alkaptonuria
• Turbidity-seen in Infection, Nephrotic syndrome, proteinuria

Osmolality

• Measures urine concentrating ability
• Normal – 400-900 mOsm/Kg H20
• Can reach Max – 1200 mOsm/Kg H20
• Useful for determining whether ionic imbalances exist in subject
• Depends on # of particles, not size or charge
• Largely due to ADH
• Prior to collection, fluid intake restricted
• First void submitted for evaluation
• Measuring using the fact of freezing point depression
• Increased -dehydration, DM, hyperglycemia, hypernatremia
• Decreased -overhydration, hyponatremia, Diabetes insipidus

Urinary pH

• Normally acidic
• Normal range – 4.5-8
• Diagnostic significance- when it is studied serially in response to acid load in suspected renal tubular acidosis
• Iimportant when studying metabolism of various nutrients e.g. glucose during exercise

Specific gravity

• A measure of density of urine measured with density of water
• With a refractometer or urinometer
• Gives rough estimate of osmolarity
• Normal  -1.003 – 1.030 ; Average – 1.018
• The higher the number = the more concentrated urine
• A fixed specific gravity of 1.010 is characteristic of chronic renal insufficiency
• Iincreased -Lack of fluids, Increased ADH, Glomerular disorders
• Falsely high-when Glucose, dye or protein in urine

Decreased -Dilute urine, Decreased ADH [diabetes insipidus ], primary tubular disorders

b. CHEMICAL TESTS
To assess the permeability of glomerular membrane

To see the presence of proteinGlucose, RBCs, Hemoglobin, Ketone bodies, Nitrites, bilirubin

Using Dipstick tests -paper strips impregnated with appropriate reagents & indicator dyes
Modern dipsticks with multiplied zones -For Protein, hemoglobin, glucose, urobilinogen, nitrite, leukocytes, specific gravity, and pH etc

Testys for Protein

• A 24-hour urine collection and measurement of protein is the most accurate
• Normally small amount of protein is excreted in the urine which may not exceed 150 mg/24 hours
• Screening tests Dipstix test and acid precipitation test 

Dipstix test

• A paper strip impregnated with bromophenol blue dye which changes to blue in the presence of protein at a suitable pH (pH 3)
• As the strip has a yellow background the colour change is observed as green
• The intensity of green is proportional to concentration of protein in urine
• Disadvantages – colour change is pH-dependent and a highly alkaline urine can induce it
• The test has to be done on fresh urine
• It has a low sensitivity for other proteins such as globulins and Bence-Jones protein.
• The lower limit of detection ranges from 50-100 mg/dl.

Acid precipitation test

• A more sensitive but less specific test
• Eight drops of sulphosalicylic acid are added to 2 ml of urine
• A precipitate forms in the presence of protein
• Light chains and low-molecular-weight proteins are detected by this technique.
• False positive results occur with penicillin, PAS, etc 

Mild proteinuria – chronic interstitial disease, febrile illness and congestive cardiac failure

Small amount – severe urinary tract infection or obvious haematuria

Large amount (3 g/day or more) – glomerular disease.

Urine protein/urine creatinine ratio

• When 24 hours’ collection of urine is difficult or impractical as in children or patients with urinary fistulae, urine protein/urine creatinine ratio can be calculated in spot urine sample.
• Due to diurnal variation the best sample is obtained at mid morning
• A value of < 0.3 is considered normal, 0.3 – 3.0 is abnormal, and > 3 indicates massive proteinuria

Microalbuminuria

• Conventional methods cannot detect urinary albumin excretion of 20 to 200 mcg/min, referred to as microalbuminuria
• It is a particularly useful test for detecting incipient diabetic nephropathy
• The urine sample is collected under standard conditions after rest of 2 hours, overnight (8 hours) or early morning.
• A very specific and rapid method is the radio-immunoassay technique
• UAE can however increase with exercise, hypertension, cardiac failure, urinary tract infection and after drinking large amounts of fluid
• Bence-Jones proteins -are light chains excreted by patients suffering from monoclonal gammopathies. It is not detected by dipstix and is best identified by immunoelectrophoresis of urine 

Glucose

• Using Benedict’s test
• Dipstix are specific
• Normally –ve
• Positive urine glucose– Increased blood glucose,Low renal threshold,Other tubular diseases
• False +ve– Ascorbic acid
• Renal glycosuria is not infrequent in the elderly where renal threshold for glucose is lowered below normal of 180 mg/dl or in inherited tubular defects (e.g. Fanconi’s syndrome) 

Blood

• Very sensitive; 2 or more cells can produce result
• Sometimes TOO sensitive, giving false positives
• Can’t distinguish between blood and free Hb, so usually double-check with microscope
• Nitrites are positive when UTI with gram +ve bacteria

C. Bacteriological examination

• By proper & aseptic collections of mid stream specimen of urine
• The presence of any bacteria in suprapubic aspirate should be considered indicative of bacterial infection.Urine cultures should follow
• Bacterial counts of more than 105/ml indicate significant bacteriuria
• Llower counts cannot be ignored in suprapubic specimen, patients on treatment with antibiotics
• Immunosuppressed individuals and symptomatic infection

d. Urine Microscopy

• The genitalia should be cleaned with soap and water and a mid-stream specimen should be asked for.
• If a ‘clean catch’ specimen cannot be obtained, it is better to do a suprapubic aspiration
• Microscopic examination should be done immediately as delay facilitates bacterial growth and disintegration of cellular components of sediments
• Both uncentrifuged and centrifuged samples should be examined
• Should check for- Red cells, pus cells, epithelial cells, crystals, urinery casts

WBC cells -0-1 HPF

• Presence of more than 5 wbcs/hpf suggest infection, pyelonephritis, inflammation of GUT
• Should be complemented with urine cultures 

RBC cells [0-1 hpf]

• Large number of RBCs with renal diseases, lower urinary tract disease, exercise
• Dysmorphic appearance of RBCs in glomerular aetiology 

Epithelial cells [0-2 HPF] : Increased in bladder inflammation, tubular injury etc

Casts

• Cylindrical bodies formed by coagulation of Tomm-Horsfall glycoprotein within the tubules
• Hallmark of renal parenchymal disease
• The material contained within the tubular lumen at the time of cast formation gets entrapped within the cast
• Often seen normally after exercise
• Hyaline casts-non specific ;seen in normal urine
• Granular casts-in pyelonephritis
• Red cell casts –in a/c glomerulonephritis
• WBC casts– in Proliferative GMN,Interstitial nephritis
• Waxy – Advanced renal failure
• Fatty– Nephrotic syndrome,Fabry’s disease
• Mixed– Proliferative GMN (SLE,PAN)
• Bacterial – Bacterial pyelonephritis
• Broad-Progressive renal failure with compensatory hypertrophy of nephrons
• Pseudocasts are composed of clumped urates, leucocytes and bacteria 

Crystals

• Triple phosphate [coffin lid shaped]and calcium oxalate crystals [envelope shaped] may be found even in normal urine and are not significant per se
• Other crystals identifiable in abnormal urine are of cystine, urate, sulphonamides, etc.

2. CONCENTRATION & DILUTION TESTS

• Aim-To evaluate functional capacity of renal tubules
• Ability of nephron to do so –dependent upon Functional activity of tubular cells in renal medulla & Presence of ADH
• Failure to achieve adequate urinery concentration due to Defects in renal medulla [NDI] Lack of ADH [CDI ]
• Traditionally concentration is determined by Specific gravity of urine gives rough estimate of osmolarity

WATER DEPRIVATION OR CONCENTRATION TEST

• To diagnose tubular disease in early stage
• Artificial fluid deprivation for > 14 hrs
• No food or water after 6 p.m on the night preceding the test.
• Discard any urine voided during the night & the first specimen- voided at 7.00 a.m
• A second specimen – at 8 a.m   & tested
• If the nephron is normal ,water is selectively reabsorbed & excretion of urine of high solute concentration [SG-1.025 or more] with an osmolality exceeds 850 mOsm/kg
• If tubular cells are non functional solute concentration remains constant regardless of stress of water deprivation
• The test should not be performed on a dehydrated patient

VASOPRESSIN TEST

• Depends only on renal tubular function
• At 8 pm-five units of vasopressin tannate is injected subcutaneously
• All urine samples are collected separately until 9 a.m. the next morning
• Satisfactory concentration is shown by at least one sample having a specific gravity above 1.020, or an osmolality above 800 m osm/kg
• This test will often detect impaired function when creatinine clearance is normal, as in hypertension or potassium deficiency

DILUTION TEST/WATER EXCESS TESTS

• After an overnight fast the patient  empties his bladder completely and is given 1000 ml of water to drink
• Urine specimens are collected for the next 4 hours, the patient emptying bladder completely on each occasion
• Unless there is renal functional impairment, the patient will excrete at least 700 ml of urine in the 4 hours, and at least one specimen will have a specific gravity less than 1.004.
• Kidneys which are severely damaged cannot excrete a urine of lower specific gravity than 1.010 or a volume above 400 ml in this time. There is a delayed diuresis
• Abnormal results are also found if there is delayed water absorption or adrenal cortical hypofunction
• If renal tubules are diseased the concentration of solutes in the urine remain constant irrespective of excess water intake
• The test should not be done if there is oedema or renal failure; water intoxication may result

DYE EXCRETION TESTS

• Using Phenolsulphonphthalein (phenol red) or Indigo-carmine
• Its excretion essentially tests for renal plasma flow and is therefore impaired early in conditions such as heart failure 

[3]   BLOOD CHEMISTRY
Impairment of renal function leads to elevation of end products of protein metabolism thus increased accumulation of urea, BUN, & creatinine in blood & azotemia results 

Urea

• End-product of protein metabolism chiefly excreted through the kidney
• It is filtered by the glomeruli and variably reabsorbed in the tubules
• The normal plasma concentration is 20-40 mg/dl
• Blood urea concentration is about 14% less than plasma concentration.
• Blood urea does not rise until a reduction of 50-60% of GFR
• The real urea concentration is BUN x 2.14
• Raised -High-protein diet , Hypercatabolic states,  Surgery, Infection ,Trauma , Steroid therapy , Tetracyclines , Hypotension, dehydration
• Low – Low-protein diet , Old age (reduced catabolism] 

BUN  [blood urea nitrogen]

• Normal BUN range is 8-25 mg/dL
• It is not possible to detect renal damage by a raised BUN until renal function has fallen by about 50 percent as measured by the creatinine clearance test
• Estimation is most useful for the assessment of the severity and progress of renal failure in Acute tubular necrosis, Acute glomerulonephritis, Chronic renal disease, Post-renal obstruction
• Decreased BUN -in Low protein diet,Liver damage,Dialysis

Creatinine

• The breakdown product of creatine phosphate released from skeletal muscle at a steady rate. It is filtered by the glomerulus.
• It is generally a more sensitive and specific test for renal function than the BUN.
• Normal range is 0.6-1.3mg/dL
• High levels of creatinine associated with high levels of beta 2 microblobulin in the serum as well as urine
• Increased-Impaired renal function,Very high protein diet, Anabolic steroid users, Vary large muscle mass: body builders, giants, acromegaly ,Rhabdomyolysis/crush injury.Athletes taking oral creatine drugs

Uric Acid

• Metabolite of purine metabolism
• Filtered by the glomeruli and both reabsorbed and secreted by the renal tubules.
• Normal value-2.4-7.0 mg/dl
• Increased -Renal failure,Gout,Liver and sweetbread gourmets,Lead poisoning,Thiazide diuretics
• High dose aspirin, Burns, Crush injuries, Severe hemolytic anemia, Myeloproliferative disorders
• Plasma cell myeloma, Tumor lysis: post chemotherapy

BUN/creatinine ratio

• Of > 15 is abnormal and indicates pre or post renal azotaemia
• It is also elevated in all conditions associated with urea overproduction.
• A low ratio is found in pregnancy, overhydration, severe liver disease, and malnutrition.
• The ratio is normal in renal azotaemia

[4] RENAL CLEARANCE TESTS
To assess GFR & renal blood flow

Renal clearance of a substance is the volume of plasma that is completely cleared of the substance by the kidneys per unit time

Clearance principle
If the plasma passing through the kidneys contains 1 milligram of a substance in each milliliter and if 1 milligram of this substance is also excreted into the urine each minute, then 1 ml/min of the plasma is “cleared” of the substance.

Thus, clearance refers to the volume of plasma that would be necessary to supply the amount of substance excreted in the urine per unit time.

Stated mathematically  ,        C_s × P_s = U_s × V

Re arranged as      C_s  =  U_s × V

                                              P_s

• C_s = clearance rate of a substance   ‘s’
• P_s = plasma concentration of the substance
• U_s = urine concentration of that substance
• V    = urine flow rate [volume in ml/min]

Thus, renal clearance of a substance is calculated from the urinary excretion rate (Us × V) of   that substance divided by its plasma concentration

Estimation of GFR
If a substance is freely filtered and is not reabsorbed or secreted by the renal tubules, then the rate at which that substance is excreted in the urine (Us ×V) is equal to the filtration rate of the substance by the kidneys (GFR  × Ps)

• Thus, GFR × Ps  = Us × V
• The GFR, therefore, can be calculated as the clearance of the substance as follows:
• GFR  = Us × V /Ps  = Cs     ; Thus here equal to clearance of that substance
• Normal GFR -120 + 25ml/min/1.73m² 

a. Inulin or mannitol clearrance test

Innulin

• Ideal substance
• Polysaccharide molecule
• Molecular weight of about 5200
• Not produced in the body, is found in the roots of certain plants
• IIts filtered from the glomerulus  & is excreted unchanged in urine
• IV infusion of inulin/mannitol is given to maintain constant plasma concentration
• Timed urine samples are collected

Creatinine clearance test
Normally released in to plasma by muscle metabolism and is cleared from the body fluids almost entirely by glomerular filtration.

Because measurement of creatinine clearance does not require intravenous infusion into the patient, this method is much more widely used than inulin clearance for estimating GFR clinically

Creatinine clearance is not a perfect marker of GFR because a small amount of it is secreted by the tubules so that the amount of creatinine excreted slightly exceeds the amount filtered

Easy method & routinely employed method of GFR estimation

Method
A careful and accurate 24 hour collection of urine is made at some time during the day (but not within 1-3 hours after a large meal)

A blood sample is taken for plasma creatinine analysis

Creatinine clearance =   U_cr  × volume [ml]      x 1.73/A

                                          P_cr × time [min]

• U_cr = Urine creatinine concentration
• P_cr = Plasma creatinine concentration
• V = Urine flow in ml/min
• A = Body surface area in m2 and
• 1.73 is the standard body surface area

Body surface area can be measured with the help of height and weight charts or with the help of following formula.       log A = 0.425 log Wt + 0.725 log Ht – 2.144

According to Cock Croft and Gaut formula – When Errors in 24 hours urine collection, creatininine clearance is calculated from plasma creatininine concentration which incorporates age, sex, and weight to estimate C_cr from plasma creatinine levels without any urinary measurements

Creatinine clearance =    140 – age (years) X wt (kg)    )          x  72  for men

                                            Serum creatinine (mg/dl)

For women, the estimated GFR is multiplied by 0.85 because muscle mass is less.

• This formula overestimates GFR in patients who are obese or edematous, and is most accurate when normalized for body surface area of 1.73 m².
• Normally creatininie clearance is 90-130 ml/min in an adult of normal size
• Approximately 100 mL/min/1.73 m² in healthy young women and 120 mL/min/1.73 m² in healthy young men.
• The C_cr declines by an average of 0.8 mL/min/yr after age 40 years as part of the aging process, but 35% of subjects in one study had no decline in renal function over 10 years.
• Values below 90 ml/min are indicative of diminished glomerular filtration rate seen with renal insufficiency
• Increased values in pregnancy,exercise etc
• The test has particular value in the general assessment of renal function in cases when plasma analyses are invalid, such as after dialysis, or when the BUN has been lowered by a low protein diet
• Creatininie clearance & GFR  are inversely proportional to plasma creatinine concentration
• If GFR suddenly decreases by 50%, the kidneys will transiently filter and excrete only half as much creatinine, causing accumulation of creatinine in the body fluids and raising plasma concentration
• If GFR falls to one-fourth normal, plasma creatinine would increase to about 4 times normal, and  a decrease of GFR to one-eighth normal would raise plasma creatinine to 8 times normal 

Urea clearance test

• No need of IV infusion
• Less sensitivity test Cz plasma concentration of urea is affected by number of factors
• Like dietary protein,fluid intake, inflammation, trauma, surgery, corticosteroids
• Partly reabsorbed from tubules
• Other substances for  GFR estimation- radioactive iothalamate and  EDTA and DTPA (=both derivates of acetic acid)  & cystatin C

 Para amino hippuric acid [PAH] clearance tests

• To measure renal blood flow
• When IV infusion of PAH, both filtration at glomerulus & secretion by tubules
• If a substance is completely cleared from the plasma, the clearance rate of that substance is equal to the total renal plasma flow
• Because the GFR is only about 20 per cent of the total plasma flow, a substance that is completely cleared from the plasma must be excreted by tubular secretion as well as glomerular filtration
• The percentage of PAH removed from the blood is known as the extraction ratio of PAH and averages about 90 per cent in normal kidneys
• Therefore, the clearance of PAH can be used as an approximation of renal plasma flow
• In diseased kidneys, this extraction ratio may be reduced because of inability of damaged tubules to secrete PAH into the tubular fluid
• Clearance of PAH = 585 ml/min
• Total renal plasma flow = Clearance of PAH/Extraction ratio of PAH
• Thus Total renal plasma flow is 650 ml/min
• If the hematocrit is 0.45 and total renal plasma flow is 650 ml/min
• Renal blood flow is calculated,  RBF = 650/(1 – 0.45) = 1182 ml/min

Other measures-

Renal failure indices

• Two important diagnostic indices are the renal failure index and fractional excretion of filtered sodium.
• Renal failure index (RFI) =  Urine sodium (mEq/L)    x  Plasma creatinine (mg/dl]
•                                                         Urine creatinine (mg/dl)
• Fractional excretion of filtered sodium (FENa) calculated as = Urine sodium (mEq/L) x plasma creatinine (mg/dl) x 100
• Plasma sodium (mEq/L) x urine creatinine (mg/dl)
• In prerenal failure, RFI and FENa are < 1

To see the structural integrity of renal system
Plain X-ray KUB, IVP , Cystoscopy ,Excretion urography ,Ultrasonography,CT,MRI, Antegrade pyelography , Retrograde pyelography ,Micturating cystourethrography (MCU), Aortography or renal arteriography ,Renal scintigraphy – dynamic and static, Transcutaneous renal biopsy etc can be done.

Summary

• Examination of the urine is the most important initial test for suspected renal damage, particularly glomerular diseases
• Search must be made for protein, erythrocytes and casts.
• The urine concentration test (or vasopressin test) is sensitive. It is possibly the most useful single test for confirming the presence of renal tubular impairment
• The creatinine clearance is quantitative for glomerular impairment and needs rarely be done unless simpler tests are abnormal.
• The estimation of plasma urea or creatinine should be done as a guide to progress and prognosis if there is severe renal damage or obstruction