Medicine

Biochemistry of kidneys and urine, urine formation

Biochemistry of kidneys and urine, urine formation

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Kidney functions in organism

Kidney – the couple organ, which is responsible for excreting of final products of metabolism and for homeostasis. They regulate water and mineral metabolism, acid-base balance, excreting of nitrogenous slags, osmotic pressure. Also they regulate arterial pressure and erhythropoesis.

Nephron – is the structural and functional unit of kidney.

Urine – fluid with different organic and inorganic compounds, which must be excreted (excess of water, final products of nitrogen metabolism, xenobiotics, products of protein’s decay, hormones, vitamins and their derivates). Most of them present in urine in a bigger amount than in blood plasma. So, urine formation – is not passive process (filtration and diffusion only).

In basis of urine formation 3 processes lay: filtration, reabsorbtion and secretion.

Glomerulal filtration. Water and low weight molecules go to the urine with help of following powers: blood hydrostatic pressure in glomerulas (near 70 mm Hg), oncotic pressure of blood plasma proteins (near 30 mm Hg) and hydrostatic pressure of plasma ultrafiltrate in glomerulal capsule (near 20 mm Hg). In normal conditions, as You see, effective filtration pressure is about 20 mm Hg.

Hydrostatic pressure depends from correlation between opening of a. afference and a. efference.

Primary urine formed in result of filtration (about 200 L per day). Between all blood plasma substances only proteins don’t present in primary urine. Most of these substances are undergone to the following reabsorbtion. Only urea, uric acid, creatinin, and other final products of different metabolic pathways aren’t undergone to the reabsorbtion.

For evaluate of filtration used clearance (clearance for some substance – it is an amount of blood plasma in ml, which is cleaned from this substance after 1 minute passing through kidney).

Drugs, which stimulate blood circulation in kidney (theophyllin), also stimulate filtration. Inflammatory processes of renal tissue (nephritis) reduce filtration, and azotaemia occurred (accumulation of urea, uric acid, creatinin, and other metabolic final products).

Reabsorbtion. Lenght of renal tubules is about 100 km. So, all substances important for our organism are reabsorbed during passing these tubules. Epithelium of renal tubules reabsorb per day 179 L of water, 1 kg of NaCl, 500 g of NaHCO3, 250 g of glucose, 100 g of free amino acids.

All substances can be divided into 3 groups:

1. Actively reabsorbed substances.

2. Substances, which are reabsorbed in a little amount.

3. Non-reabsorbed substances.

To the first group belong Na+, Cl-, Mg2+, Ca2+, H2O, glucose and other monosaccharides, amino acids, inorganic phosphates, hydrocarbonates, low-weight proteins, etc.

Na+ reabsorbed by active transport to the epithelium cell, then – into the extracellular matrix. Cl- and HCO3- following Na+ according to the electroneutrality principle, water – according to the osmotic gradient. Substances go from extracellular matrix to the blood vessels. Mg2+ and Ca2+ are reabsorbed with help of special transport ATPases. Glucose and amino acids use the energy of Na+ gradient and special carriers. Proteins are reabsorbed by endocytosis.

Urea and uric acid are little reabsorbable substances.

Creatinin, mannitol, inulin and some other substances are non-reabsorbable.

Henle’s loop plays important role in the reabsobtion process. Its descendent and ascendent parts create anti-stream system, which has big capacity for urine concentration and dilution. Fluid passes from proximal part of renal tubule to the descendent part of Henle’s loop, where concentration of osmotic active substances higher than in kidney cortex. This concentration is due to activity of thick ascendent part of Henle’s loop, which is non-penetrated for water and which cells transport Na+ and Cl- into the interstitium. Wall of descendent part is penetrated for water and here water pass into the interstitium by osmotic gradient but osmotic active substances stay in the tubule. Ascendent part continue to reabsorb salt hypertonically, even in the absence of aldosteron, so that fluid entering the distal tubule still has a much lower osmolality than does interstitial fluid.

Some substances (K+, ammonia and other) are secreted into urine in the distal part of tubules. K+ is changed to Na+ by the activity of Na+-K+ATPase.

Peculiarities of biochemical processes in kidney.

Kidney have a very high level of metabolic processes. They use about 10 % of all O2, which used in organism. During 24 hours through kidney pass 700-900 L of blood. Carbohydrates are the main fuel for kidney. Glycolysis, ketolysis, aerobic oxidation and phosphorylation are very intensive in kidney. A lot of ATP formed in result.

Metabolism of proteins also presents in kidney in high level. Especially, glutamine deaminase is very active and a lot of free ammonia formed. In kidney take place the first reaction of creatin synthesis.

Kidney have plenty of different enzymes: LDG (1, 2, 3, 5), AsAT, AlAT. Specific for kidney is alanine amino peptidase, 3rd isoform.

Utilization of glucose in cortex and medulla is differs one from another. Dominative type of glycolysis in cortex is aerobic way and CO2 formed in result. In medulla dominative type is anaerobic and glucose converted to lactate.

Two sources contribute to the renal ammonia: blood ammonia (is about one-third of excreted ammonia), and ammonia formed in the kidney. The predominant source for ammonia production within the kidney is glutamine, the most abundant amino acid in plasma, but a small amount may originate from the metabolism of other amino acids such as asparagine, alanine, and histidine. Ammonia is secreted into the tubular lumen throughout the entire length of the nephron. Secretion occurs both during normal acid-base balance and in chronic acidosis. Metabolic acidosis is accompanied by an adaptive increase in renal ammonia production with a corresponding increase in urinary ammonium excretion.

Kidney cortex like liver appear to be unique in that it possess the enzymatic potential for both glucose synthesis from noncarbohydrate precursors (gluconeogenesis) and glucose degradation via the glycolytic pathway. Gluconeogenesis is important when the dietary supply of glucose does not satisfy the metabolic demands. Under these conditions, glucose is required by the central nervous system, the red blood cells, and, possibly, other tissues which cannot obtain all their energy requirements from fatty acids or ketone body oxidation. Also, gluconeogenesis may be important in the removal of excessive quantities of glucose precursors from the blood (lactate acid after severe exercise for example). Although the biosynthetic pathways are similar, there are several important differences in the factors, which regulate gluconeogenesis in the two organs. 1) The liver utilizes predominately pyruvate, lactate and alanine. The kidney cortex utilizes pyruvate, lactate, citrate, α-ketoglutarate, glycine and glutamine. 2) Hydrogen ion activity has little effect upon hepatic gluconeogenesis, but it has marked effects upon renal gluconeogenesis. Thus, when intracellular fluid pH is reduced (metabolic acidosis, respiratory acidosis or potassium depletion), the rates of gluconeogenesis in slices of renal cortex are markedly increased. The ability of the kidney to convert certain organic acids to glucose, a neutral substance, is an example of a nonexcretory mechanism in the kidney for pH regulation.

Role of kidney in regulation of blood pressure. Regulation of urine formation.

In response of blood pressure decreasing juxtaglomerular apparatus of kidney secrete renin. This hormone activates angiotensinogen, which transformed to angiotensin I in liver. In lung angiotensin I transformed to angiotensin II – strong pressure agent, main actions of which are vessels contraction and stimulating of aldosteron secretion by cortex of adrenal glands. Aldosteron increase Na-reabsorbtion and decrease quantity of urine.

Na-uretic hormone (produced in heart) decrease reabsorbtion of Na+, and quantity of urine increased.

Decreasing of blood pressure is due to activity of kinines (blood plasma polypeptides, which can widen vessels and increase permeability of capillar walls). Source of kinines is kininogen which undergone to activity of different proteolytic enzymes. In result formed kalidin and bradikinin – main substances in this system.

Role of kidney in acid-base balance regulation.

Kidney have some mechanisms for maintaining acid-base balance. Na+ reabsorbtion and H+ secretion play very important role.

1. Primary urine has a lot of Na2HPO4 (in dissociated form). When Na+ reabsorbed, H+ secreted into urine and NaH2PO4 formed.

2. Formation of hydrocarbonates. Inside renal cells carboanhydrase forms from CO2 and H2O H2CO3, which dissociated to H+ and HCO3-. H+ excreted from cell into urine (antiport with Na+) and leaded with urine. Na+ connect with HCO3-, NaHCO3 formed and go to the blood, thereupon acidity decreased.

3. Formation of free ammonia. NH3 used for formation of NH4+ (H+ ion associated), and different acid metabolites excreted as ammonia salts.

Role of kidney in water balance regulation.

Excessive entrance of water leads to dilution of extracellular fluid. Decreasing of osmolality inhibits secretion of antidiuretic hormone. Walls of collective tubules stay non-penetrated to water and dilutive urine formed.

If volume of blood circulation increases, circulation in kidney increases also and hyperosmotic medium of kidney medulla removed. Some substances in these conditions return into blood. So, excess of water carried with urine and a lot of soluble substances are reabsorbed into blood. After water loading stopped, hyperosmolality in kidney medulla returns for previous stage during some days.

Physical and chemical characteristics of urine.

Urine amount (diures) in healthy people is 1000-2000 ml per day. Daytime diures is in 3-4 times more than nighttime. Decreasing of urine amount called oliguria (due to fever, diarrhea, vomiting, acute nephritis, cardiac insufficiency). Sometimes anuria occurred (amount of urine per day less than 50 ml). The main causes of anuria are lead’s or arsenic’s intoxication, nephrolytiasis, strong stress. Increasing of urine amount called polyuria (due to diabetes mellitus, another types of diabetes, usage of diuretic drugs, some another diseases).

Normal colour of urine is yellow (like hay or amber), what is due to presence of urochrom (derivate of urobilin or urobilinogen), some another colour substances are uroerythrin (derivate of melanin), uroporphyrines, rybophlavine and etc. Colour depends from urine concentration. Colour of urine can be changed due to different diseases and usage of some drugs. For example, red or pink-red colour can be due to massive haemolysis (hematuria and hemoglobinuria occurred) or usage of amidopyrin, santonin. High concentration of urobilin and bilirubin gives to urine brown or red-brown colour. Green or blue colour can be due to decay of proteins in the intestine.

Urine is transparent. This characteristic depends from amount of different salts (oxalates, urates, phosphates), amount of present epithelium cells and leucocytes. For differentiation of turbidity origin the following simple test is used. Add in urine acid product and heat it. Disappearing of turbidity means that main cause of this state were urates and phosphates of calcium and magnesium. If turbidity stays after heating we must think about inflammatory process in urinary tracts (turbidity in this case is due to presence of leucocytes, epithelium cells and another substances).

Density of urine depends from concentration of soluble substances. Borders of variation are from 1002 to 1035 g/l. Near 60-65 g of hard substances are excreted with urine per day. Increasing of density is due to intensive excretion with urine a lot of organic and inorganic compounds. Patients with diabetes mellitus have polyuria with increased density of urine due to presence in urine a lot of solved substances like glucose, ketone bodies etc. Patients with diabetes insipidus have polyuria with decreased density of urine due to insufficiency of antidiuretic hormone and disorders of water reabsorption in renal tubules. In case of severe kidney insufficiency urine has density near 1010 g/L (like primary urine). This state is called isostenuria.

In normal conditions urine has acid or weak acid reaction (pH=5,3-6,8). This depends from presence of NaH2PO4 and KH2PO4. A lot of meat and proteins in diet gives to urine acid reaction, plants give alkaline reaction. Alkaline reaction can be due to cystitis, pyelitis, after vomiting. Expressed acid reaction can be due to diabetes mellitus, fevers and starvation.

Fresh urine has a specific smell, which is due to presence of flying acids. But a lot of microorganisms, which are present in urine, split urea and free ammonia formed.

Organic compounds of urine.

Proteins. Healthy people excrete 30 mg of proteins per day. As a rule these are low weight proteins.

Urea. This is main part of organic compounds in urine. Urea nitrogen is about 80-90 % of all urine nitrogen. 20-35 g of urea is excreted per day in normal conditions.

Uric acid. Approximately 0,6-1,0 g of uric acid is excreted per day in form of different salts (urates), mainly in form of sodium salt. Its amount depends from food.

Creatinin and creatin. Near 1-2 g of creatinin is excreted per day, what depended from weight of muscles. This is the constant for each person. Men excrete 18-32 mg of creatinin per 1 kg of body weight per day, women – 10-25 mg. Creatinin is non-reabsorbable substance, so this test used for evaluating of renal filtration.

Amino acids. Per day healthy person excretes 2-3 g of amino acids (free amino acids and different low weight molecule peptides). Also products of amino acids metabolism can be found in the urine.

Couple substances. Hypuric acid (benzoyl glycine) is excreted in amount 0,6-1,5 g per day. This index increases after eating a lot of berries and fruits, and in case of protein’s decay in the intestines.

Indican (potassium salt of indoxylsulfuric acid). Per day excretion of indican is about 10-25 g. Increasing of indican’s level in urine is due to intensification of decay proteins in the intestines and chronic diseases, which are accompanied by intensive decomposition of proteins (tuberculosis, for example).

Organic acids. Formic, acetic, butyric, β-oxybutyric, acetoacetic and some other organic acids are present in urine in a little amount.

Vitamins. Almost all vitamins can be excreted via kidney, especially, water-soluble. Approximately 20-30 mg of vit C, 0.1-0.3 mg of vit B1, 0.5-0.8 mg of vit B2 and some products of vitamin’s metabolism. These data can be used for evaluating of supplying our organism by vitamins.

Hormones. Hormones and their derivates are always present in urine. Their amount depends from functional state of endocrinal glands and liver. There is a very wide used test – determination of 17-ketosteroids in urine. For healthy man this index is 15-25 g per day.

Urobilin. Present in a little amount, gives to urine yellow colour.

Bilirubin. In normal conditions present in so little amount that cannot be found by routine methods of investigations.

Glucose. In normal conditions present in so little amount that cannot be found by routine methods of investigations.

Galactose. Present in the newborn’s urine, when digestion of milk or transformation of glalactose into glucose in the liver are violated.

Fructose. It is present in urine very seldom, after eating a lot of fruits, berries and honey. In all other cases it indicates about liver’s disorders, diabetes mellitus.

Pentoses. Pentoses are excreted after eating a lot of fruits, fruit juices, in case of diabetes mellitus and steroid diabetes, some intoxication.

Ketone bodies. In normal conditions urine contains 20-50 mg of ketone bodies and this amount cannot be found by routine methods of clinical investigations.

Porphyrines. Urine of healthy people contains a few I type porphyrines (up to 300 mkg per day).

Inorganic compounds of urine.

Urine of healthy people contains 15-25 g of inorganic compounds.

NaCl. Per day near 8-16 g of NaCl excreted with urine. This amount depends from amount of NaCl in food.

Potassium. Twenty-four hours urine contains 2-5 g of K, which depends of amount of plants in the food.

Different drugs can change excretion of Na and K. For example, salicylates and cortikosteroids keep Na and amplify excretion of K.

Calcium. Twenty-four hours urine contains 0.1-0.3 g, which depends from content of calcium in the blood.

Magnesium. Content of magnesium in urine is 0.03-0.18 g. So little amount of calcium and magnesium in urine can be explained by bad water solubility of their salts.

Iron. Amount of iron in urine is about 1 mg per day.

Phosphorus. In urine are present one-substituted phosphates of potassium and sodium. Their amount depends from blood pH. In case of acidosis two-substituted phosphates (Na2HPO4) react with H+ and one-substituted phosphates (NaH2PO4) formed. In case of alkalosis one-substituted phosphates react with bases and two-substituted phosphates formed. So, in both cases amount of phosphates in urine increases.

Sulfur. Amount of sulfur in twenty-four hours urine is 2-3 g per day (in form of SO42-).

Ammonia. Ammonia is excreted in ammonium sulfates and couple substances. Ammonium salts make up 3-6 % of all nitrogen in urine. Their amount depends from character of food and blood pH.

 

Pathological components of urine, which are occur due to different metabolic disorders in organism.

Glucosuria. Occurs in two cases – when level of glucose in blood more than kidney threshold (8-10 mmol/l), so called extrarenal glucosuria (diabetes mellitus), and when the kidney cannot reabsorb even normal quantity of glucose, so called renal glucosuria (kidney insufficiency).

Ketonuria. Due to some diseases and pathological conditions (diabetes mellitus, starvation, severe heart weakness, when amount of fat in the food more than amount of carbohydrates) level of ketone bodies increases in a big amount (up to 20-50 g per day). This is the index of deep metabolic disorders, especially in carbohydrates metabolism.

Bilirubinuria. It occurs in case of hepatic parenhimatous inflammatory processes or in case of obstruction of gall bladder ductus. Urine has a colour as dark bear. After some times it stays yellow-green (bilirubin oxidized to biliverdin).

Urobilinuria. Increasing of amount of urobilin is due to haemolytic or parenhimatous hepatitis, when decomposition of mesobilinogen in liver is depressed.

Creatinuria. Amount of creatin in urine increases due to different pathological processes in muscles like myopathy and myodistrophy, starvation, hypovitaminosis E, radiation sickness, hyperthyreosis. Also this is present in small children and in women after delivery.

Indicanuria. Increasing of indican’s level in urine is due to intensification of decay proteins in the intestines, weaken of intestine peristaltic (atony, constipation), and chronic diseases, which are accompanied by intensive decomposition of proteins (tuberculosis, for example).

Phenylketonuria. Innate deficiency of phenylalanine hydroxilase in liver makes transformation of phenylalalnine in tyrosine impossible. Amount of phenylalanine in our organism increases more than 10 times and this amino acid utilized by another pathway with phenylbutyric and phenylacetate formation. These substances cannot be utilized and heaped up in blood and tissue. Growth of brain is stopped. With urine a lot of phenylbutyric and phenylacetate excreted. Fresh urine with FeCl3 gives olive-green colour. This test is wide spread in maternity homes, because special diet can prevent problems with child health.

 

Causes of changes of normal components’ content in urine.

Urea. Decreasing of urea in urine is due to deficiency of protein in diet, disorders of liver functions (especially, liver cirrhosis, phosphorus intoxication), acidosis (ammonia used for neutralization of acids), inflammatory or destructive processes in kidney (nephritis, when urea is not excreted and uraemia appeared). Increasing of urea in urine is due to excess of proteins in diet, and different diseases, which are accompanied by intensive proteins’ decomposition (diabetes mellitus, malignant tumors, infectious diseases with fever).

Creatinin. Amount of creatinin in urine is decrease in case of disorders of glomerular filtration (amount in blood increases in the same time). Increasing of creatinin in urine is due to intensive muscle work, intensive proteins’ decomposition, excess of creatinin in diet (meat).

Amino acids. Increasing of amino acids’ level take place in case of intensive decomposition of tissues’ proteins (trauma, burns, radiation sickness etc.). Also it indicates about liver function’s disorders, especially about depressing of proteins and urea formation.

Uric acids. Decreasing of uric acid in urine is present, when diet has mainly carbohydrates without purines. Meat, caviar, gland tissues, where a lot of nucleoproteins are present, can be cause of increasing level of uric acid in urine. Leucoses, gout, burns, radiation sickness, usage of aspirin and corticosteroids also can be causes of hyperuricuria.

Enzymes. Different enzymes can be present in urine according to disorders of organs’ functions and these dates can be used for precise topic diagnostic.

Inorganic compounds. Concentration of inorganic compounds depends from their amount in diet (particularly for sodium, potassium), from character of diet (plants contains a lot of potassium, and level of this ion in urine increases when diet consists of plants mainly; meat and other proteins can be cause of increasing of ammonia salts in urine), from blood pH (in case of alkalosis or acidosis level of  phosphates in urine increases), from some diseases and physiological stages (during pregnancy and in case of parathyroid glands hypofunction level of calcium in urine decreases).

General nitrogen. General nitrogen – it’s a sum of all compounds which contains nitrogen. Near 80-90 % of general nitrogen belongs to urea. Another parts – uric acid, creatinin and amino acids. So, level of general nitrogen depends from level of these substances.

 

Indeces of renal functions disorders.

Proteinuria. Proteinuria can be kidney and extralidney origin. Kidney proteinuria is due to damages of nephrons, when blood plasma proteins can pass through glomerular membranes. In this case albumins and globulins are present in urine. Extra kidney proteinuria is due to damages of urinary tracts and prostate.

Hematuria. This pathological component is due to damages of kidney or urinary tracts. In most cases nephrolytiasis is accompanied by hematuria. Some times hematuria is indicator of traumatical kidney damage.

Glucosuria. In most cases glucosuria is a symptom of diabetes mellitus, when level of glucose in blood more than kidney threshold (8-10 mmol/L). But sometimes glucose can be present in urine even its level in blood is normal. This is so called “renal glucosuria” which is due to disorders of glucose reabsorbtion in tubules.

Pyuria. In normal urine leucocytes are present in a very little amount. Due to different inflammatory processes of urinary tracts, urine bladder, prostate, also due to nephrolytiasis amount of leucocytes in urine increases and this situation is called “pyuria”.

Creatin. Decreasing of creatin in urine is index of kidney insufficiency.

 

Methods of proteins, glucose, ketone bodies, blood and bile pigments determination in urine. Clinical significance.

Proteins can be detected in urine after denaturation (during heating, because proteins under high temperature denaturated and stay visible, and after interactions with mineral acids – sedimentation with sulfosalicylic or nitric acid). Quantitative determination of proteins in the urine can be perfomed by Roberts-Stolnikov method. The method is based on the known fact that in adding of urine to nitric acid the white ring is formed on the border of two solution. This ring is formed within 2-3 min if the protein contents in urine is 0,033 g/l. Using variety of dilutions find that one forming the white ring within 2-3 min.

Glucose can be detected qualitatively with help of some reductive reactions – Felling (the reaction is based on the reduction of Cu2+ to Cu+. In this reaction the glucose is oxidized. Cuprous oxide (Cu+) has red color) or  Tromer reactions, special indicator paper “Glucotest”, and quantitatively with help of Althauzen’s method (in boiling of mixture of urine containing glucose with alkaline the different tints of brown color (from yellow to dark-brown) are formed. Tint depends from the glucose concentration in urine).

Ketone bodies can be detected in urine qualitatively by Legal or Herhardt (in adding of FeCl3 solution to urine containing ketone bodies the sediment of phosphates is formed) tests and quantitatively in reaction with acetic acid, ammonia or sodium nitroprussicum (if urine contains acetone the violet ring is formed in layer of ammonia on the urine containing natrium nitroprussicum and concentrated acetic acid. The velocity of ring appearance depends on the concentration of acetone in urine. The appearance of ring between 3 and 4 min means that concentration of acetone is 0,0085 g/l).

Blood in the urine can be detected qualitatively by benzidin test (blood peroxidase oxidizes benzidin in the presence of H2O2. Oxidized benzidin has the dark blue colour).

Fushe (after sedimentation by barium salts bilirubin is oxidized by FeCl3, which is a part of Fushe reactive, and give blue-green or blue colour), Gmelin (biliverdin and bilirubin are easy oxidized substances, which formed after oxydation different coloured products – yellow, red, violet, blue and green), Rosin (bilirubin oxidized into biliverdin under influence of iodine and gives a green colour), Florance (urobilin with hydrochloric acid formed a red-coloured substance) and Rosenbah (modification of Gmelin test) tests are used for qualitative detection of bile pigments in urine.

Clinical significance of presence in urine different pathological components – see above.