kidney functionand diseases

Kidney Function

and diseasesBy

Dr. Moustafa RizkProf. of Clinical Pathology

Faculty of Medicine, University of Alexandria.

Organ function course 16/10/20110/16/2010

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Objectives: Describe the macroscopic and microscopic anatomy of the

renal system.

Define the following terms Nephron –Glomerulus …….

List and describe the functions of the renal system

Understand the concepts og glomerular filtration rate and clearance

Unerstand the renal handling of electrolytes and water

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Objectives:

Describe the physiologic role of the kidney.

State the laboratory tests used to assess glomerular function.

Discuss the concept of renal clearance and how it is measured.

State the laboratory tests used to assess tubular function.

Record laboratory biochemical findings in some renal disorders (chronic renal disease-end stage renal disease-acute renal failure-acute nephritic syndrome-nephrotic syndrome-pyelonephritis-urinary tract obstruction

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Kidney Function and diseases

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Functional unit of the kidney is the

NEPHRON

1.2 x 106 nephra per kidney.

Each nephron about 50 mm long.

Combined lengths about 145 km (85 miles)

Renal corpuscle.

Proximal convoluted tubule (PCT)

Loop of Henle

Distal convoluted tubule (DCT).

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Glomerular Filtration Rate (GFR)

Kidneys receive 25% of the cardiac output

GFR = the amount of filtrate that forms in both

kidneys every minute.

In adults GFR is about :

105 ml.min-1 in females

125 ml.mi-1 in males

Daily volume of glomerular filtrate

150 L in females

180 L in males

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Tubular Reabsorption: One of the

important nephron functions99% of glomerular filtrate reabsorbed.

1% leaves body: 1-2 L per day.

Cuboidal epithelial cells with microvilli.

Osmosis, diffusion, active transport.

Materials reabsorbed include:

Water

Glucose

Amino acids, urea.

Na+, K+, Ca2+, Cl-, HCO3-

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Reabsorption of Selected Substances

Conserve valuable nutrients, e.g.

Amino acids

Glucose

Abnormally, glucose, amino acids, blood, ketones,

leukocytes, kidney stones, etc. might be found in

urine.

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Fluid Compartments

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Regulates blood volume and blood

pressureAdjust the amount of water lost or retained

Energy dependent NA+ extrusion

Antidiuretic hormone (ADH, or vasopressin)

Blood volume related to blood pressure

Regulation of blood pressure and blood flow

Renin-angiotensin-aldosterone pathway

Flow-rate through kidney can be adjusted.

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Renal Functions : Acid-Base balance

Stabilizes blood pH (in company

with buffer systems and

regulation of [CO2]

Regulates loss of H+ ions

(acid)

Regulates loss of HCO-3 ions

(base)

Normal plasma pH

7.35-7.45

Acidosis

pH < 7.35

Alkalosis

pH > 7.45

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For the bicarbonate buffering system, the Henderson-Hasselbalch equation becomes

pH= 6.1+ log[HCO3-]/ [H2CO3]

And at normal physiologic conditions,pH= 6.1+ log ([20]/[1])= 6.1+1.3= 7.4

HCO3-= 24mEq/L (22-28)

H2CO3= Pco2 x 0.03

Pco2 = 38-42 mmHg.


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Renal System

Reabsorption of HCO3

- in the proximal tubule


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Renal System

Mechanism of formation of

titratable acids in the distal nephron


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Renal System

Mechanism of formation of NH4+ in

the distal nephron segments(intercalated cell)

Glutamine

(PT)


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Renal System

Regulation of hydrogen secretion: There are a large number of factors that affect H+

secretion. The most important are:

1. Filtered HCO3-

2. Arterial Pco2:CO2+H2OH++ HCO3-

3. Aldosterone : next figure

4. PCT Na+ reabsorption

5. Plasma H+ concentration


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Renal System

Mechanism of Aldosterone

modulation of H+

secretion in renal tubule cell

Aldosterone

Aldosterone


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Interpreting Results of Blood Gases

pH 7.4= Absolute normal pH 7.35-7.45=Normal range

pH7.35= Acidemia

pH7.45= Alkalemia

Pco2 40mmHg=Absolute normal

Pco235-45mmHg= Normal range

Pco2 35mmHg=Resp. alkalosis

Pco2 45mmHg= Resp. acidosis

HCO3 24 mEql/L =Absolute normal

HCO322-28 mEql/L =Normal range

HCO322mEql/L =Metab. Acidosis

HCO328mEql/L =Metab. Alkalosis

Base Excess: reflects the pH buffers in blood. A positive BE indicates alkalemia, while a negative BE indicates acidemia. BE correlates directly with HCO3 . Normally BE = 02 mEql/L.


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Regulates plasma concentrations of electrolytes (e.g. Na+, K+, Cl-, and other ions)

Water retained or lost in response to plasma

osmolality (normal plasma osmolality 290 mOsm/kg

H2O).

ADH mechanism

Controls loss in urine

Contributes to plasma [Ca2+] regulator by vitamin D

(calcitriol) regulation

Renal activation of calcitriol increases Ca2+

uptake from gut.10/12/2017 5:11 AM 33


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Vit D

25-HCC (Liver)

Ca/PTH

1,25-DHCC 24,25-DHCC

(Kidney) (Kidney)


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Juxtaglomerular Apparatus

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Rennin-Angiotensin-aldosterone

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Overall function of

urine production is to

maintain homeostasis

by regulating the

volume and

composition of blood.

Re-absorption of selected

substances

Acid-base balance

Electrolyte balance

Red blood cell

formation

Regulate activated

vitamin D production

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Laboratory tests aiding in the evaluation

of kidney functions

1. General characteristics of urine.

2. Tests measuring glomerular filtration rate

Clearance tests (urea,inulin,creatinin)

Non protein nitrogenous compounds

3. Tests measuring tubular function

Urinary acidification test

Sodium and potassium excretion tests

Specific gravity of the urine

Osmolality of urine and serum

Concentration and dilution tests10/12/2017 5:11 AM 40

•Volume: (N: 700 – 2500 ml / day)

•Specific gravity: (N: 1.015 – 1.025)

•Osmolality: The number of dissolved solutes

/ kg body H2O.

(N: 500 – 800 mosmol /

kg body H2O)

•Protein.

•Microscopic examination: (cells and casts)

A- Complete Urine Examination:


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Specific Gravity:

SG of early morning and 24-hours urine: 1.015 –

1.025.

Randomly measured urinary SG: 1.002 –

1.060.

*SG can be measured by:

Urinometer.

Refractometer.

Urine reagent strips.

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• Urinometer:

It is a specially calibrated hydrometer.

The lower the concentration of solutes, the

further the urinometer will sink in the

urine.

It is calibrated at 15°C.

Every 3°C more or less than 15°C, add or

subtract 0.001 degree SG.

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Refractometer:

It measures the refractive index.

The measurement is based on the number of dissolved particles in the urine. The higher the concentration of particles, the greater the increase in refractive index.

In addition, some instruments can measure serum total proteins.

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Casts:They are moulds of renal tubular endothelial

lining:

Hyaline casts.

Blood and leucocyte casts.

Epithelial casts.

Granular casts.

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Casts are common in CRF.

In terminal stages of RF, casts are often veryscanty as most of the renal tissue has beendestroyed and fixed SG of the urine at 1.010produces rapid breakdown of any cells by anosmotic effect.

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S. Creatinine

S. urea

B-Glomerular function tests

GFR

glomerular permeability

(selectivity index)

Direct Indirect


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Test to estimate Glomerular Filtration Rate

(GFR)

1. GFR at which ultrafiltrate of plasma is formed;

is a “true” physiological estimate of renal

function. It assumes no secretion from blood

into tubule and no reabsorption once it is in

tubule.

2. Inulin: is an inert carbohydrate that is not

metabolized, secreted or reabsorbed; thus

ideal agent for GFR determination; thus is

used mainly for research since not practical. 10/12/2017 5:11 AM 56

Glucose UreaCreatinine

InulinPAHA

A B C D

Different ways by which renal tubules can handle

substances freely filtered at glomeruli.


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Creatinine Clearance (90-140 ml/min/1.73 m2)

1. Creatinine is primarily excreted via glomerualr

filtration with about 10-15% eliminated by

active tubular secretion.

2. CrCl is estimate of GFR

3. Clinical uses for obtaining CrCl :Assessing kidney function in patients with acute

or chronic renal failure

Monitoring patients on nephrotoxic drugs.

Determining dosage adjustments for renally

eliminated drugs.

I

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Definition of Clearance ( C ):

( ml / min)

It is the hypothetical volume of

plasma, that is completely cleared

from a certain substance per unit

time (minute).


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Calculating Creatinine Clearance

Direct measurement:

CrCl = (UV) (Ucr) / (SCr) (1440) x 1.73 m2 /

BSA

- UV = 24 hour urine volume in ml.

- Ucr = urinary creatinine conc. in mg/dl.

- SCr = serum creatinine in mg/dl at midpoint

of urine collection.

- 1440 is number of minutes per day

- CrCl is in ml/min. 10/12/2017 5:11 AM 60

Criteria for an ideal GFR marker

Produced inside the body i.e. Endogenous.

Constant production rate .

Elimination only via glomerular filtration with no

reabsorption or secretion.

No laboratory interference for its measurment.


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1- Inulin clearance:

- Reference clearance method.

- Not suitable for routine investigation.

- Exogenous material I.V. infused in such a

way to maintain plasma level steady during

the period of the test.

(N: 125 ml / min).

GFR can be measured by:


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2- Creatinine clearance:

- It is widely used in routine work.

- An endogenous substance.

- Its value correlates fairly closely with inulin

clearance.

- Plasma creatinine is used as a day to day

indication of changes in GFR. It is superior to

creatinine clearance ( error in all analysis based

upon timed collection of urine).

♂105 ± 20 ml/min

♀ 95 ± 20 ml/min

GFR can be measured by:

N:


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3- Urea clearance:

Not used nowadays.

It is 70% of GFR because part of urea in the

glomerular filtrate diffuses back into the

tubular cells and the amount reabsorbed

varies inversely with urine flow rate.


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Clearance ( C ) =

U X V

P

1.73

AX = 75 ml/min

Clearance ( C ) =

U X√ V

PX = 54 ml/min

1.73

A

Maximum urea clearance:

If the volume of urine / min = > 2 ml/min

Standard urea clearance:

If the volume of urine / min = < 2 ml/min


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• Para-amino hippuric acid ( PAHA ) is a

substance that can be filtered at the glomeruli and

excreted by the tubules.

• Such substance when infused at a low plasma

concentration, its clearance is very high.

• It measures the effective renal blood flow

(RBF). (N: 650 ml/min).

4- PAHA clearance:


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5-Cystatin C ( New marker for GFR)

Specific and sensitive parameter for glomerular filtration rate (GFR).

Independent of muscle mass, age and sex.

Only eliminated via filtration.

Not influenced by acute phase reaction.

Independent from urine collection, only one serum sample.

quick, simple and reliable.


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Assessment of Glomerular permeability

A. Selective proteinuria: the glomerular membrane has still the ability to prevent filtration of proteins of large MW and allow the filtration of small MW proteins ( Albuminuria).

B. Non selective proteinuria: as the damage proceeds ; proteins of larger molecular sizes pass through the membrane.

Selectivity is measured by comparing the clearance of 2 proteins of different MW; albumin or transferrin versus larger IgG.

Selectivity ratio = Clearance of IgG

Clearance of albumin

Lower ratio <0.16 = more selective proteinuria.


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Creatinine (0.7-1.5 mg/dl)

Is normal metabolic product of creatine

and phosphocreatine which are

constituents of skeletal muscle.

The daily production of creatinine is

determined by person’s muscle mass;

in normal patients, the rate of creatinine

production equal its excretion.

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Cause of true changes in S. creatinine

Unlike BUN, SCr is not influenced by changes in

renal blood flow or diet

A rise in SCr almost always indicates worsening

renal function (decreased GFR)

Since creatinine is byproduct of muscle

metabolism, severely decreased muscle mass

(cachexia) may decrease SCr

Vigorous exercise may temporarily increase SCr

by 0.5 mg/dly10/12/2017 5:11 AM 70


Cause of false serum creatinine

Depends on the test that lab uses.

If using Jaffe assay, larger amounts of non

creatinine chromogens (uric acid, glucose,

acetone, acetoacetate, pyruvic acid, ascorbic

acid) can increase the results of the test.

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Age-adjusted Standards for CrCl

CrCl declines with age at any given SCr.

An elderly with a “normal” SCr does not have

normal CrCl.

Estimate age-adjusted normal values of CrCl :

Males : CrCl = 133 - (0.64 X age)

Females : takes 93% of this value

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Blood Urea Nitrogen (BUN) (8-20 mg/dl)

Is concentration of nitrogen within urea in

serum; produced in liver.

Serum concentration depends on urea

production (liver) and tubular reabsorption as

well as glomerular filtration;

Thus by itself is not a useful indicator of GFR.

Used with other lab data; can assess hydration

status, renal function, protein tolerance, &

catabolic process

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Elevated BUN

Pre-renal causes

1. Decreased renal perfusion

Dehydration

Shock

Diuretics

Blood loss

Severe CHF

Note : BUN follows

sodium & water, if

increased reabsorption

of sodium & water then

BUN reabsorption also

increases

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Elevated BUN

Pre-renal causes

2. Increased protein breakdown

GI bleeding

High protein diets

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Elevated BUN

Renal causes of elevated BUN :

• Acute renal failure : drugs such as :

aminoglycosides, amphotricin B, cisplatin.

• Chronic renal failure : diabetes,

pyelonephritis. Chronic analgesics abuse

Post-renal causes of elevated BUN :

• Obstruction of ureter, bladder or urethra.

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• GF passes through tubules;

where:

1. Reabsorption of water and solute.

2. Excretion of certain substances.

3. Exchange of ions across cell wall.

• As a result, urine is excreted carrying waste

products and maintaining body homeostasis.

• The urine finally excreted has an entirely

different composition from GF.

C- Tubular Function Tests:Kidney Function and diseases

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UrineGF

1.0 ml/min120 ml/minVolume

800 mosmol/kg300 mosmol/kgOsmolality

6.87.4pH

AbsentPresentProtein

--- g/dl--- mg/dlUrea

AbsentPresentAmino acids

AbsentPresentGlucose


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Tests to assess tubular function

1. Urine concentration test (14-16 h water restrict)

• N. SG > 1.025 & > 800 mOsmol/kg

• Decreased RF < 1.020 & 400-600

mOsmol

• Severe Renal impairment , SG

approaches 1.010 &< 400 mOsmol

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2. If failure to concentrate urine to 800 mOSmol/kg

We do DDAVD test IM. Injection

(1-deamino 8-D-Arginint Vasopressin test)

• To distinguish causes of polyuria (inability

to concentrate urine)

DDAVP Fluid depr

Central Response No response

Psychogenic Response Response

Renal No response No response10/12/2017 5:11 AM 80

3-Urinary acidification test:

[H+] of urine is normally > [H+] of blood & of GF.In order to achieve this degree of acidification, the Kidney:

- Reabsorbs [HCO3-].

- Excretes [H+] partly as free [H+] and partly as NH4

Salt or in combination with anions principally inorg Ph.


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Urine acidification test

• 0.1 g/kg body weight of ammonium

chloride (NH4Cl) given by mouth.

• Urine collected/h. for 8 hours – falls below

(pH<5.3) in at least one specimen.Blood

specimens are collected befor the test

and 2 hours after ammonium chloride

intake for measuring total CO2 to ensure

satisfactory acidosis.10/12/2017 5:11 AM 82

Sodium and Potassium excretion:

♦ Sodium excretion:- About 70% of Na in the GF is reabsorbed by the PCT

(active reabsorption).

Serum Na:(N:135-145 mmol/L)

Urinary Na:(N: 40-220 mmol/d)

♦ Potassium excretion:- About 90% of K in GF is normally reabsorbed in the

PCT while the DCT secretes K.

Serum K: (N: 3.5-5.5 mmol/L)

Urinary K: (N: 25-125 mmol/d)


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4. Na excreation

• Giving diet containing 20 mmol Na+/day.

• Normally urinary sodium falls within a

week to the amount present in the diet.

• In diseased kidney, when dietary Na+

Na+ and water retained, by contrast

when dietary Na+ Na+ depletion

GFR aggravate the condition.

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Laboratory findings in tubular dysfunction

Plasma: Metabolic acidosis (¯ pH & ¯ HCO3-).

¯ K+

¯ Phosphorus

¯ Uric acid

Osmolality

- Normal urea and creatinine.

Urinevolume

- osmolality

sodium content

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Renal diseases commonly present with

proteinuria.

Since glomeruli filter 5 - 7 g of protein / 24 h

and only < 150 mg is excreted in urine / 24 h,

therefore, tubular reabsorption must be very

efficient.

Proteinuria


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♦ Mechanisms of Proteinuria:

1- Overflow.

2- Glomerular:

due to glomerular permeability,

e.g. albumin.

3- Tubular:

due to tubular reabsorption,

e.g. β2-microglobulin.

Proteinuria


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1. Orthostatic: (proteinuria after standing or walking).

2. Transient: (during pyrexia, CHF or intense exercise).

3. Systemic diseases: e.g. DM, SLE, MM….etc.

4. Renal disease: acute & chronic GN and NS.

Causes of proteinuria:

- Mild 1.0 g /d

- Moderate 3.0 g /d

- Severe 3 g /d

Grades of proteinuria:


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♦ Selective and non-selective proteinuria:

The concept of selectivity derives from the

fact that glomerular permeability to a

plasma protein depends largely on its MW;

small molecules being cleared more rapidly than

large molecules.

Selective: High MW proteins ( IgG & α2-MG ) tend to

be retained.

Non-selective: Both high, moderate (albumin) & low

( transferrin) MW proteins are excreted.

Proteinuria


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.

Glomerular filtration Tubular reabsor.

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DESCRIBE THE TYPES OF PROTEINS FOUND IN URINE.

Urine contains very little protein, up to 150 mg in a 24 hour period. Over the course of 24 hours, urine will contain from 1.0 to 15.0 mg of protein/dL. Proteins that are found in the glomerular filtrate have a molecular weight of 90,000 or less. Larger proteins are non-filterable. One estimate states that a given urine specimen will contain about 34% albumin and 66% globulins. Other resources state that albumin is a true small molecular weight protein and will be the dominate protein in urine. The urinary tract produces three proteins of interest: (1) Tamm-Horsfall protein, (2) urokinase (a fibrinolyticenzyme), and (3) secretory IgA (immunoglobulin of the renal tubular epithelial cells). Other proteins reported are from the prostate, seminal vesicles, and vagina.

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DISCUSS THE CLINICAL SIGNIFICANCE OF PROTEIN FOUND IN URINE.

Protein testing of urine is important because it tends to be denotative of renal disease. The fact that a test is positive does not mean the patient has a renal disorder but that additional testing is necessary. A positive protein test of a random urine specimen is more significant than a first morning specimen.The major reasons for pathologic proteinuria are: (1) damage to the glomerular membranes, (2) tubular disorders characterized by altered tubular reabsorptionmechanisms, and (3) increases in the serum levels of low-molecular weight proteins, which gives rise to several types of proteinuria: overflow, renal, glomerular, tubular, post-renal, and orthostatic. The amount of protein loss that can occur in the urine varies from 0.15 grams to 20.0 grams per day. In most cases of proteinuria, protein loss does not exceed 4.0 grams per day.

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DSCUSS ORTHOSTATIC PROTEINURIA

Also known as functional or postural proteinuria, it is a non-pathogenic condition associated with the upright position and disappearing when the horizontal or supine position is assumed. The daily protein loss usually does not exceed 1.0 gm, but has been reported at 1.5 gm. This is a disorder of young adults. To diagnose this disorder, collect a urine specimen immediately after rising. This will be negative for protein. Collect a second specimen 3 or 4 hours after rising. This will be positive. Patients should be monitored every six months and re-evaluated. This may be due to blood pressure phenomenon in the renal vein when in the upright position.

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DISCUSS OVERFLOW PROTEINURIA

Overflow proteinuria (also called "pre-renal" or "overload" proteinuria) is the consequence of increased amounts of low-molecular weight plasma proteins passing through the glomerular membranes into the urine. This phenomenon can be the result of a number of conditions: (1) hemolytic transfusion reaction episode, (2) muscle trauma that causes myoglobin to also appear in the urine, and (3) acute-phase reactant proteins due to surgery, myocardial infarctions, or bacterial septicemia. Note: Acute-phase reactant proteins are: hemoglobin, C-reactive protein, α1-antitrypsin, fibrinogen, and haptoglobin. These are normally occurring proteins. Other proteins are abnormal, low-molecular weight proteins from light-chain diseases (example: multiple myeloma).

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DISCUSS GLOMERULAR PROTEINURIA.

This is the most common and serious of the proteinuria's. Most of the protein found in this disorder is albumin and usually referred to as "albuminuria". There is an increase in glomerular permeability due to injurious effects upon the glomerular capillaries. Causes for such injuries includes (1) immune complexes resultant of multi-systemic diseases as "systemic lupus erythematosus" (2) primary glomerulardisease, as "minimal change disease" or "focal glomerulosclerosis"; (3) infectious diseases, as "hepatitis", malaria, or bacterial endocarditis"; (4) drug injury, as seen with penicillin, lithium, or chloramphenicol; (5) pre-eclampsia; and (6) transplant rejection. Glomerularproteinuria can be progressive and if progressive, the amount of protein loss increases, with losses up to 4.0 gm/day possible. If the loss of protein (albumin) >2.0 gm/day, then the patient may experience edema. This disorder can progress and develop into nephrotic syndrome or if the glomeruli are destroyed, then renal failure and proteinuria ceases.

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If the loss of protein is <1.0 gm/day and is due to a disturbance in the glomerular apparatus, with no evidence of renal disease, then this condition is designated as "functional proteinuria". Also called "benign proteinuria", this condition is thought to be caused by blood flow changes in the glomerulus or slight changes in permeability. This type of protein loss is seen in pyrexia, exposure to cold, heavy physical activity or exercise, congestive heart failure, emotional stress, hypertension, and atherosclerosis. This type of proteinuria usually resolves itself over time with care and treatment.

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EXPLAIN WHAT IS MEANT BY SELECTIVE PROTEINURIA

Selective proteinuria is associated with glomerular proteinuria. If the disease is "non-progressive" and the size of protein molecules being lost are correlated to the size and number of lesions in the glomerulus, then glomerularproteinuria is designated as "selective proteinuria". If severe proteinuria is present, with all kinds of sizes of protein molecules, then the proteinuria is designated as "non-selective".

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DISCUSS TUBULAR PROTEINURIA.

Results when the normal tubular protein reabsorptive functions are impaired. Small molecular weight proteins will appear in the urine. Examples of the more common small proteins that are lost are:

(1) β2-microglobulin [MW = 11,600], (2) lysozyme [MW = 14,500], (3) α2-microglobulin [MW = 27,000, (4) α1-acid glycoprotein [MW = 40,000], (5) retinol binding protein [21,000].

The protein loss per day is < 2.5 grams. Common causes of tubular proteinuria include:

(1) lupus erythematosus, (2) galactosemia, (3) heavy metal poisoning (mercury, cadmium, or lead), (4) antibiotics (penicillin, sulfonamides, or cephalosporins), (5) muscle trauma, (6) transfusion reaction, (7) renal tuberculosis.

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This type of proteinuria can be diagnosed by identifying the presence of lysozyme or β2-microglobulin. If this proteinuria is designated as the acute type, it is usually reversible with treatment. Acute tubular proteinuria may be seen in acute pancreatitis or burns. There is a chronic tubular proteinuria. Its prognosis is more serious and may not clear up with treatment. This form of proteinuria is seen in Fanconi'ssyndrome, chronic pyelonephritis, or sarcoidosis.

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DISCUSS POST-RENAL PROTEINURIA

A disorder that is associated with inflammation in any part of the urinary tract other than the kidney. This can result for tissue injury due infections, trauma, or tumors that allow proteins to "leak" into the urinary tract. A diagnostic feature of this proteinuria is the presence of "pus" cells and/or malignant cells in the urine specimen. Red blood cells are not a reliable indicator of this type of proteinuria.

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DISCUSS THE IMPORTANCE OF TESTING FOR MICROALBUMINURIA.

The use of the term "microalbuminuria" implies a proteinuriathat is not detected with the usual screening tests used in the urinalysis lab. A patient is designated as having "microalbuminuria" when tests can detect 30 to 300 mg of albumin over a 24 hour period in at least two of three specimens over a six month period. The value of detecting this disorder is its correlation to diabetes mellitus. If renal complications are "silent" and "insidious", being brought on by the presence of glucose in the urine, the detection of microalbuminuria provides the physician a clue to implement corrective treatment and better stabilize the diabetic patient. Failure to intervene will often lead to diabetic neuropathy. .

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BRIEFLY DISCUSS TESTING METHODS FOR MICROALBUMINURIA.

Sensitive testing methods include enzyme immunoassay, radioimmunoassay, and fluorescent technique. There are two commercial sensitive screening methods available.a. A tablet method (available from Ames) that can detect 4.0 to 8.0 mg albumin per dL. Urine and water are placed on top of the tablet and

observed for the appearance of a blue-green color. A color chart is provided for semi-quantification.b. An immunochemical strip reagent test (available from Boehringer-

Mannhein) can detect 1.0 to 2.0 mg albumin per dL.

10/12/2017 5:11 AM 102

DESCRIBE BENCE-JONES PROTEINS AND EXPLAIN WHY IT APPEARS IN THE URINE.

Bence-Jones protein is a low-molecular weight (MW is < 44,000), immunoglobulin para-protein (either a kappa or lambda monoclonal light-chain type) that is abnormally produced in patients with multiple myeloma, primary amyloidosis, lymphoreticular neoplasms, or macroglobulinemia disorder. This protein is readily filtered from the glomerular capillaries and possesses unique solubility properties

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DESCRIBE AND/OR PERFORM THE CLASSICAL HEAT SCREENING TEST FOR BENCE-JONES PROTEINS

Begin with clear urine (centrifuging or filtering if necessary) and transfer 5 to 10 mLs to a large test tube. Heat in a water bath to a temperature up to 60 oC and observe for turbidity. If Bence-Jones (BJ) proteins are present, flocculation will occur between 40 oC and 60 oC. Continue heating, bringing the bath to boiling temperature. The flocculated BJ protein will disappear and the urine will be clear. (NOTE: If flocculation is still present, there may be other interfering proteins and the sample should be filtered hot.) Allow the hot, but clear urine specimen to cool. Observe the behavior as the temperature cools to 60 oC. BJ proteins will re-flocculate between 40 oC and 60 oC. Electrophoresis is the best testing method for detecting BJ proteins.

10/12/2017 5:11 AM 104

DISCUSS THE REAGENT STRIP TEST REACTION FOR PROTEIN

The reagent strip test employs a indicator dye (tetrabromphenol blue or 3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrabromosulfonphthalein) in an acidic buffer to maintain a constant pH of approximately 3.0. Either dye at this pH value is yellow in color. Albumin is the principle protein measured by the strip, the globulins and non-albumin proteins have little or no influence. The strip will not detect BJ proteins. Albumin is a hydrogen ion acceptor and will remove hydrogen ions from the indicator dye causing it to change from yellow to blue-green. The intensity of the color change is proportional to the amount of protein in the urine specimen. This testing procedure is sensitive enough to detect 5 - 10 mg protein per dL. This testing principle is referred to as "the protein error of indicator". This means that at a certain pH, one color appears with protein present, but a different color if protein is absent. Test readings are reported out as: negative, trace (< 30 mg/dL), 1+ (30 mg/dL), 2+ (100 mg/dL), 3+ (300 mg/dL), and 4+ (2000 mg/dL).

10/12/2017 5:11 AM 105

LIST CAUSES FOR FALSE-POSITIVE AND FALSE-NEGATIVE RESULTS WITH PROTEIN REAGENT STRIP TESTING.

False-positives: (1) an elevated pH (≥9.0) will override the buffer system... urine specimen should be re-adjusted to 7.0 and retested, (2) quaternary ammonium compounds, (3) detergents, and (4) over wetting of the pad (which leaches out the buffer salts).False-negatives: (1) elevated specific gravity due to increases "salts" may override the buffer system and cause a lowering of the reading, (2) globulins and non-albumin proteins present and albumin is absent, (3) polyuria [dilutes out the protein is non-detectable levels],

10/12/2017 5:11 AM 106

DISCUSS HOW MYOGLOBIN APPEARS IN URINE AND ITS SIGNIFICANCE

Myoglobin is a small hemoprotein (MW = 17,000) that is rapidly cleared from blood and excreted in the urine. It is the result of muscle tissue trauma due to surgery, injury crushes, toxic action of ethanol or drug addiction, muscle tissue, electric shock, muscle disease, snake venoms, and idiopathic paroxysmal myoglobinuria. Myoglobin is readily reabsorbed by the proximal tubular cells.

Myoglobin has the potential to damage the kidneys. If urine is +ve for myoglobin pathological condition to be clarified.

10/12/2017 5:11 AM 107

DESCRIBE HOW MYOGLOBIN CAN BE IDENTIFIED AND CONFIRMED

A simple screening technique requires that 5.0 mLs of centrifuged urine (neutral pH) be placed in a test tube and add 2.8 grams of ammonium sulfate, then mix well. Allow the specimen to sit for five minutes. Filter and test the filtrate with a chemical test for blood. If the test is positive, then myoglobin is presumed to be present. The principle of this test lies in the fact that hemoglobin is precipitated out in an 80% ammonium sulfate solution. The best methods for identifying myoglobin is protein electrophoresis. Other reliable test procedures are radioimmunoassay, absorption spectrophotometry, and immunodiffusion.

10/12/2017 5:11 AM 108

WHEN GIVEN DATA, DIFFERENTIATE BETWEEN ERYTHROCYTES, HEMOGLOBIN, AND MYOGLOBIN IN URINE.

The following table of information will illustrate the parameters to differentiate the differences.

Descriptor Hemoglobin Myoglobin RBC'sReagent strip test positive positive positiveAppearance of urine clear/red clear smokeyAppearance of plasma pink/red normal normalRBC's in urine sediment 0 to few 0 to ew presentSerum creatine kinase (CK) elevated (*) elevated (#) normalSerum haptoglobin decreased normal normalSerum lactate dehydrogenase (LD) elevated elevated normalSerum LD-1 and LD-2 elevated normal normalSerum LD-4 and LD-5 normal elevated normal

(*) will be ≤ 10 times the upper reference limit.(#) will be ≥ 40 times the upper reference limit.Upper reference limit for serum CK in men = 130 IU/L and for women = 115

IU/L. Upper reference limit for serum LD for adults range from 40 to 90 IU/L

10/12/2017 5:11 AM 109

EXPLAIN THE IMPORTANCE OF TESTING FOR NITRITES

IN URINE

Nitrites are not normally detected in the urine of healthy individuals. This test provides a quick and convenient method of screening for urinary tract infections (UTI). One value that is inherent in this test is that a UTI may be asymptomatic or the patient may be complaining of vague symptoms that would not alert an physician to order a urine culture and sensitivity test. A word of caution.... this test depends upon the infecting bacteria to be nitrate reducers (have the enzyme "nitrate reductase) and not all bacteria that can cause a UTI have the nitrate reducing enzyme .

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Testing for nitrites can be a means for assessing the effectiveness of antibiotic therapy; the presence of a UTI (cystitis, urethritis, ureteritis, pyelonephritis, or glomerulonephritis); monitoring patients at high risk for a UTI; and screening urine specimens for UTI.

10/12/2017 5:11 AM 110

EXPLAIN THE CHEMICAL BASIS OF THE STRIP TEST FOR

NITRITES. This test is a modified Griess reaction (1879) and depends upon the presence of

nitrites in the urine. The test pad contains an aromatic amine (p-arsanilic acid or sulfanilamide) and buffers to maintain an acidic environment for the reaction to occur. Nitrite and the aromatic amine will chemically interact (called "diazotization") to form an intermediate, a "diazonium salt". The diazonium salt would then react with another chemical in the pad, a chromogen [3-hydroxy-1,2,3,4-tetrahydorbenz-(h)-quinolin] and the end product would a pink "azodye" molecule. The pink color (without regard to intensity) is interpreted as positive. See the reaction sequence in the following illustration.

Aromatic Amine–NH2 + Nitrite (NO2) ----> Aromatic Amine + +N=N +chromogen ------> Aromatic Amine–N=N - chromogen (a pink Azo-dye)

This test pad is "standardized" to be insensitive to the presence of < 100,000 microorganism/mL in order to eliminate false positive test results. If this test is positive, then there are > 100,000 microorganisms/mL. Any shade of pink represents a clinically significant number of microorganisms. This test will not work if there are no dietary nitrates in the diet of the patient. Fresh urine specimens (in particular a first morning specimen) should always be used when testing for nitrites.

10/12/2017 5:11 AM 111

LIST A MINIMUM OF EIGHT THINGS THAT CAN CAUSE A FALSE-POSITIVE OR FALSE- NEGATIVE NITRITE TEST.

False-positive: [1] presence of colored medications (pyridium), [2] contaminating bacteria in a stool that has stood too long or improper collection, [3] storage of reagent strip test in an open container.False-negative: [1] lack of dietary nitrate, [2] antibiotic therapy, [3] ascorbic acid at concentrations of 25 mg/dL or greater, [4] high urinary urobilinogen, [5] a urine pH < 6.0, [6] a urine specimen that did not stay in the bladder a sufficient amount of time, or [7] or bacteria that do not reduce nitrate.

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Biochemical Findings in Some


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Acute Renal Failure

• Prerenal conditions (shock, hemorrhage, heart

failure).

• Renal (acute tubular necrosis, glomerulonephritis).

• Post-renal (bladder obstruction).

60%of cases occur during or immediately

after surgery.

10%associated with obstetric problems.

30%medical conditions

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Early stage findings

• Scanty urine <50 ml/day.

• Usually bloody with specific gravity.

• Urine sodium >50 mmol/L.

• BUN by > 50 mg/dl/day.

• Creatinine, uric acid

• Hypocalcemia.

• Metabolic acidosis.

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Second week findings

• Urine becomes clear after several days with a

small daily increase in volume.

• Daily volume of 400 ml indicates onset of

recovery.

• BUN continues to rise after onset of diuresis.

• Metabolic acidosis

• Serum K (tissue injury – acidosis – failure of

excretion).

• Na is ¯. 10/12/2017 5:11 AM 116


Diuretic Stage

• Urine sodium is 50-70 mmol/L.

• Large urinary potassium excretion may

casue decreased serum potassium.

• Serum Na and Cl may due to dehydration

resulting from large diuresis if

replacement of water is inadequate.

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Chronic Renal Failure (CRF)

• It is the end result of progressive and

gradual destruction of the nephrons

e.g. chronic GN, chronic

pyelonephritis, chronic obstructie

uropathy.

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Biochemical features of renal failure

• Increased serum urea.

• Increased serum creatinine.

• Capacity of the kidney to concentrate urine is impaired.

• Capacity of the kidney to dilute urine remains

unaffected.

• Serum sodium tends to be low especially if Na+ intake

is diminished but in severe case ® sodium retention

occurs.

• Serum potassium: raised level is uncommon expect

when large load of K is given. 10/12/2017 5:11 AM 119



• Metabolic acidosis.

• Decreased serum Ca due to:

Decreased serum albumin.

Improper activation of Vit. D.

• Increased serum phosphorus.

We can susbect that patient with CRF can maintain Na+, K+

and water balance normal if that dietary load remains

close to normal daily requirement.

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Nephrotic Syndrome (NS)

Diagnostic criteria:Proteinuria ( > 3 g / d ).

Hypoalbuminemia ( < 3 g/dl).

Edema.

Etiology:I- Primary NS:

- Minimal change lesion.

- Membranous nephropathy.

- Proliferative GN.

II- Secondary NS:Secondary to systemic disease e.g. SLE, DM

and amyloidosis.


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Nephrotic Syndrome (NS)

Biochemical findings:

Serum:- Hyperlipidemia: CHOL & TG.

- Albumin.

- α2- macroglobulin & β- globulins.

- N. Urea & creatinine at presentation.

Urine:- Proteinuria ( > 3 g/d ).

- Mild MP hematuria.

N.B: Patients with NS who have selective proteinurina respond to

steroid therapy more than those with non-selective proteinuria.


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Conclusions

• The urinary system is more than a waste

disposal system.

• It regulates plasma volume and composition

from minute to minute.

• This in turn results in constant composition for

all the other fluid compartments.

• The three fundamental mechanisms of kidney

function are filtration, secretion and

reabsorption. 10/12/2017 5:11 AM 123

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kidney functionand diseases

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