Method for detecting disease duration in children with chronic obstructive pyelonephritis (variants)

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

 

The invention relates to medicine, namely to clinico-morphological and laboratory diagnostic methods, and can be used in clinical pediatric Nephrology for the selection of appropriate therapy.

In the last decade more emphasis on diseases of the urinary system in children. Unfortunately, often the diagnosis of such diseases is not carried out immediately. If the stage of acute pyelonephritis (especially obstructive) have striking clinical picture of fever, back pain, dysuria, symptoms of intoxication, chronic pyelonephritis (CP), including chronic obstructive pyelonephritis, without exacerbation occurs with a very low symptoms, and only purposeful questioning allows to identify episodes of unmotivated fever, chills, pain in the lumbar region, disorders of urination (nocturia), decreased performance, fatigue, not usually associated with a particular disease.

Often the only symptom of CP is isolated urinary syndrome (leukocyturia, bacteriuria, proteinuria is not more than 0.5 g/day or a combination of bladder syndrome anemia in the absence of renal failure and hypertension (LT Ablaeva, V. Kirillov / To the topical question of diagnosis and treatment of urinary tract infections in children / PED is the atrium, No. 1, 1999, is pp.93-97).

Usually for confirmation of the diagnosis requires historical data on repeated episodes of urinary tract infection (UTI) or transferred bacteriuria and pyuria with their quantitative assessment. Often such information is not available, causing difficulty in determining the severity and duration of disease.

It is known that the duration of illness health is directly related to nephrosclerosis changes in the kidneys and, knowing the duration of the disease, can present realistic picture of the disease. Despite the large number of studies on structural and functional changes of kidney in obstructive pyelonephritis (soludo B. I. renal Pathology: (Clinico-morphological study). - L.: Medicine, 1983. - 296 S.; Maksimova I.G. Features sclerotic process in kidneys with hydronephrosis and pyelonephritis in children // Urology and Nephrology. - 1984. No. 5. - P.18-21.; FR VI ABOUT gidropaticheskim the nephrosclerosis // Brachen. case. - 1976. No. 6. - P.66-68), data on the correlation between the duration of disease chronic obstructive pyelonephritis and the results of clinical-morphological and laboratory studies in the literature could not be found.

At the same time the problem of early diagnosis and prognosis of sclerosing renal tissue Ave is obstructive uropathy is quite relevant.

The most objective picture of the state of the kidneys today give instrumental methods of analysis, such as the method of neprezentare, which is the closest to the claimed, as it allows for the received image to objectively assess changes in renal tissue and to determine the degree of development of nephrosclerosis, which in turn allows to some extent to judge the duration of the disease (Papayan AV, Savenkova / Clinical Nephrology children: a Guide for physicians. - SPb.: Sothis, 1997, - 718 S.)

However, the method requires expensive equipment and drugs and not always applicable in practical medicine today.

Numerous studies in recent years have clearly shown that one of the most important biological mediators that are involved in many physiological and pathophysiological processes is nitric oxide (NO). However, the published material mainly cover the participation of NO in the nervous, cardiovascular and respiratory systems (Eliseev E.V. Nitroxidergic regulation of the lungs. - Vladivostok: DVGU, 2001, - 176; Matakin P.A., geltzer B. I. Clinical and experimental pathophysiology of the lungs. - M.: Nauka, 1998, - 336 S.; Anggard E.Nitric oxide: mediator, murderer, and medicine // Lancet. - 1994 (May). - Vol.343. No. 8907. - P.1199-206; D.S. Bredt, S.H. Snyder Isolation of nitric oxide synthase, a calmodulin-requiring enzyme // Proc. Nat.Sci. U.S. - 1990. - Vol.87. - P.682-685).

It is known that nitric oxide plays an important role in the regulation of functional items kidneys. Violation of NO synthesis in different parts of the kidney leads to significant changes, primarily water-salt metabolism and the level of systemic blood pressure. It is established that in vivo long-term inhibition of NO synthesis stimulates the development of nephrosclerosis and hypertension. (Radomski M.W., Palmer R., Moncada S. An L-arginine / nitric oxide pathway present in human platelets regulates aggregation // Proc. Natl. Acad. Sci. USA. - 1990. - Vol.87. - P.5193-5197). Morphologically kidney damage develops vessels, consisting of focal adhesion and infiltration of mononuclear cells in the intima and the average shell preglomerular arteries and arterioles. These changes are associated with necrosis of the middle layer of blood vessels and partial or complete thrombosis of the lumen. Detected and ishemizirovanne the glomeruli (Verhagen, A., Rabelink So, Braam Century A receptor blockade alleviates hypertension and renal lesions associated with chronic nitric oxide synthase inhibition // J. Am. Soc. Nephrol. - 1998 (May). - Vol.9. - N.5. - P.755-762).

It is also known that the state of NO-synthase (NOS) in various organs and tissues of animals and man can be judged by the content of NADPH - diaphorase, which is an enzyme whose function is changed synchronously and simultaneously with nitroxylenes (NOS) (Neuronal NADPH-diaphorase synthasizes a second messenger: yes or no / V.T. Hope, G.J. Michael, K.M. Knigge, S.R. Vincent // Neurosci. Abstr. - 1990. - Vol.15. - P.538) (Malyshev IU Introduction to bi the chemistry of nitric oxide: Role of nitric oxide in regulation of major body systems // Rogur. gastroenterology, Hepatology, Coloproctology. - 1997. No. 1. 49 - 55).

Thus, published data suggest that nitric oxide, synthesized by nitroxylenes (NOS), contributes to the progression of kidney disease, but in different ways. The study nitrosodipropylamine kidney function and the participation of the NO in the pathogenesis of nephrogenic hypertension on the background of obstructive pyelonephritis in children and has led to the emergence of this decision.

An object of the invention delivered to the definition of disease duration in children chronic obstructive pyelonephritis by exploring NO-producing renal function in different parts of the nephron, the endothelium and the smooth vascular myocytes, as well as the collective tubules of the brain substance of kidneys and determine the correlation between the activity of NOS in the kidney and clinical and laboratory parameters that will allow to extend the diagnostic capabilities in the examination of children.

The problem is solved by the proposed method for determining the duration of the disease in children chronic obstructive pyelonephritis implemented in two versions.

The method according to the first embodiment is carried out by evaluation of enzymatic activity of NADPH-diaphorase histochemical method in units of optical density in the epithelial cells of the proximal, distal the tubules (DK), in vascular glomeruli, as well as in the endothelium and smooth myocytes vessels of the kidney and its subsequent comparison with the norm, while when exceeding the standards equal to 143 TU in the epithelial cells of the proximal tubules and 88.3 TU in the epithelium of the distal tubules 10% and 29.8%, respectively, and/or exceeding the standards equal 103,1 TU in vascular endothelium by 13% and the activity of NADPH-diaphorase in smooth vascular myocytes to a value of 65 TU diagnose disease duration up to 3 years; the reduction in the activity of NADPH-diaphorase in the epithelial cells of proximal and distal tubules to 131 and 83 TU, respectively, and/or in the endothelium and smooth myocytes vessels up to 82 and 38 TU, respectively, diagnosed with a disease duration of up to 7-8 years; activity of NADPH-diaphorase in the epithelium of the tubules 80 TU, and vascular glomeruli 35 EOP, while the norm in the last 61 TU diagnose the disease duration is 10-12 years, while values below these values is a disease duration of 13-15 years.

The method according to the second variant is carried out by conducting clinical and laboratory research of a sick child, compare the obtained results with the average rate for the corresponding age of the child and determine the duration of chronic obstructive pyelonephritis based on a comprehensive consideration of all the parameters, given the data in table 1, where the minus sign refers to the decrease, and (+) plus or to its increase:

The technical result of the invention is achieved due to the fact that the authors first determined the value of the activity of NADPH-diaphorase, reflecting the catalytic activity of NO-synthase (NOS)in various departments of the kidneys healthy and sick children and realized correlation of the obtained values with disease duration of chronic obstructive pyelonephritis, and conducted a comprehensive assessment of the correlation between the activity of NADPH-diaphorase in different parts of the kidney and clinical and laboratory parameters of the patient and a healthy child.

Reliably established that at the initial stages of the disease (up to 3 years) amplification of nitrosoproline renal function in epithelial cells of the distal and proximal tubules, as well as in the endothelium and smooth myocytes of renal vessels, but reduced activity of this enzyme, and hence the synthesis of nitric oxide, in the epithelium of the collecting tubules of the brain substance of kidneys. With the further development of the disease NO-producing kidney function more and more oppressed, which leads to decreased activity of nitroxides (NOS) in all departments of the kidneys is below normal, the degree of reduction of this activity speaks to the duration of disease.

Things the ity of the proposed solution in the first embodiment is that a sick child take sections of renal tissue (?), determine the activity of NADPH-diaphorase in Units of optical density (TU) largest density of sediment deformazione, compare the values obtained with the norm and make a conclusion about the duration of the disease.

The essence of the proposed solution according to the second variant of the proposed method lies in the fact that conduct clinical examination of the child, compare the obtained results with the average rate for the corresponding age of the child and on the comprehensive assessment of the deviations of all of the indicators shown in table 1, determine the duration of disease.

The advantage of the proposed method in the first embodiment is in the early and reliable diagnosis of the disease, and hence the correct choice of subsequent treatment due to the peculiarities of the histochemical localization and activity of NADPH-diaphorase in different parts of the kidneys in norm and pathology.

To determine the histochemical localization and activity of NADPH-diaphorase used sections of renal tissue obtained immediately after nephrectomy in 16 children 3-15 years (9 boys and 7 girls) with congenital hydronephrosis IV-V century, complicated chronic obstructive pyelonephritis, and compared them to normal. The rate was determined by the value obtained is fired in the study of autopsy material of a healthy kidney from 8 children of the same age, killed in the accident (road trauma). Research conducted not later than 6-8 hours from the moment of biological death.

For the second variant of the method had correlation between the activity of NADPH and clinical and laboratory parameters using statistical analysis to calculate the correlation coefficient between the mean values of the activity of NADPH in the various structures of the kidneys and the average values of laboratory parameters of blood and urine (22 indicators)identified in the survey the same 16 patients with secondary chronic obstructive pyelonephritis, who underwent nephrectomy. On the basis of the conducted analysis revealed statistically significant clinical and laboratory parameters that have a significant relationship with the activity of NADPH in the various structures of the kidney.

For a quantitative assessment of NOS in the physiological norm and at a pathology of kidneys was studied by using complex methods: obamaholics, histochemical, spectrophotometric and statistical analysis of the experimental material.

The morphological structure of the kidneys in norm and at pathology was studied by staining sections of material with hematoxylin and eosin according to standard procedures.

Because at the moment NADPH-diaphorase considered as KOF is rment NO-synthase or its paired enzyme (Neuronal NADPH-diaphorase is a nitric oxide synthase / V.T.Hope, G.J.Michael, K.M.Knigge, S.R.Vincent // Proc. Nat. Acad. Sci. U.S. - 1991. - Vol.88. - P.2811-2814) localization of NO synthase (NOS) was determined by the activity of NADPH diaphorase using histochemical method by the standard technique of Hope and Vincent (Hope V.T., Vincent S.R. Histochemical characterization of neuronal NADPH-diaphorase // Histochem. Cytochem. - 1989. - Vol.37. - P.653-661 (1989), which is the education of deformazione in the presence of endogenous NADPH and salts of tetrazole. NADPH-diaphorase carries out proton transfer from NADPH to microcinema tetrazolium (PCT). Restored CNT becomes insoluble product - diformate. The density of the sediment is directly proportional to the molecular content of NOS and, consequently, allows to judge about the activity and localization of this enzyme. Areas of human organs size 1,0×0.5 cm were isolated using a blade dipped in chilled prepared in 0.1 M phosphate buffer (pH 7.4), 4% paraformaldehyde, which from all class Diagoras remains active only NADPH-diaphorase. The material was fixed for 2 hours at t=+4°C, day washed at the same temperature in 15% sucrose solution, 7-8 times changing the solution. From tissue samples, frozen in the cryostat, produced slice thickness of 10 μm, mounted on glass slides and placed in the incubation medium. The composition and the final concentration of the medium were as follows: 50 mm Tris-buffer (pH 8.0), 1 mm NADPH (Sigma), 0.5 mm of NITR the blue tetrazole (Sigma) and 0.2% Triton X-100 (“Serva”). Incubation was carried out for 60 minutes in a thermostat at 37°C. Then the sections were rinsed in distilled water, was obezvozhivani and concluded in balsam on a common histology method.

When conducting histochemical studies, special attention was paid to compliance with the standardization of the conditions of sampling, preparation and processing of material for study.

The above method has studied the localization and activity of NADPH-diaphorase in the renal vessels of Taurus, the epithelium of the proximal, distal tubules and collecting tubules, as well as the endothelium and smooth myocytes vnutripochechnykh vessels in both groups of children.

Visualization images of all specimens on the computer received a vision system mounted on microdensitometer “Vickers-M85”. Digital image processing was performed using Adobe Photoshop 5.0 and Microsoft Excel 98. Enzyme activity and optical density expressed in units of optical density (TU).

Found that children in the comparison group (norm) NADPH-diaphorase determined in all investigated structures of the kidney, in addition to the smooth vascular myocytes. The highest enzyme activity was registered in the epithelial cells of the proximal tubules (143,0±2,9 TU), lower enzyme activity detected in the distal tubules (88,3±3,8 TU), moderate activity of NADPH-d is Afanasy, reflecting the catalytic activity of NOS, registered in the collecting duct epithelial cells of the tubules of the brain substance (78,6±3,2 TU), and the low basal activity of the investigated enzyme is fixed in the structures of the renal cells (vascular glomerulus) - 60,9±3,6 TU.

In figure 1 a, b shows a picture of a healthy person's kidneys, NADPH-diaphorase in the epithelium of the tubules and the glomeruli of the nephron. “and” - HC. 100x; “b” - HC. 400x. Method Hope, Vincent.

The presence of NADPH-diaphorase installed in the endothelium of blood vessels of the cortical substance (103,1±3,2 TU). Here the enzyme activity was significantly higher than in other structures of the kidney (p<0,001), but did not reach the level of one in the proximal tubule.

To study nitrosodipropylamine renal function in children, patients with chronic obstructive pyelonephritis, they were divided into 4 groups depending on the duration of the pathological process: I - children aged under 3 years (n=3); II - children aged 7-8 years (n=5); III - children aged 10-12 years (n=5); IV-13-15 years (n=3), received the following dynamic activity of the investigated enzyme.

Table 2 shows the change in the activity of NADPH-diaphorase in healthy children and patients with chronic obstructive pyelonephritis during the duration of the disease up to 3 years and 7-8 years.

Table 2
The group of patientsIndicators of activity of NADPH-diaphorase (TU)
Proximal tubuleDistal tubuleCollective tubulesVascular glomerulus
Norma143,0±2,9to 88.3±3,878,6±3,260,9±3,6
Up to 3 years157,3±1,2114,6±2,167,8±2,055,3±1,9
7-8 yearsto 130.6±1,683,1±1,365,8±1,834,7±1,1

Found that when the duration of the disease up to 3 years the activity of NADPH-diaphorase in comparison with normal increases in epithelial cells of the proximal tubules by 10% and is 157,3±1,2 TU (p<0,001).

Figure 2 shows a snapshot of the kidney of a patient with pyelonephritis on the background of congenital hydronephrosis with disease duration of 3 years, which is visible to the increased activity of NADPH-diaphorase in the proximal tubules and reduced activity in vascular glomeruli. Method Hope, Vincent. HC. 200x.

The epithelial cells of the distal tubules also demonstrate increased activity of the enzyme, which exceeds the norm by 29.8% (p<0,001).

In quantifying the enzymatic activity of NADPH-diaphorase in the glomeruli and the epithelium of going to the nutrient tubes in children up to 3 years compared to the norm has registered its reduction to 55.3±1,9 TU (p> 0.05) and 67,8±2,0 TU (p<0,001) (13.7%), respectively.

Further analysis of the state of NO-producing renal function showed a reduction in the disease progression (table 2).

In the age group of 7-8 years, the activity of NADPH-diaphorase in the epithelial cells of the PC is reduced by 9.9% as compared to that in healthy children (p<0.001) and 23.4% compared with the first age group and is to 130.6±1,6 TU (p<0,001) (figure 3 and 4). The enzyme activity in the epithelium DK also has a tendency to decrease compared to the norm. While in DC the decline in enzymatic activity of NADPH-diaphorase more significantly compared with that in children with disease duration of 3 years - 47.2% (p<0,0001).

Figure 3 shows a snapshot of the kidney of a sick child with disease duration of 7 years. In a single stored channel - moderate activity of NADPH-diaphorase Method Hope, Vincent. HC. 200x.

Children in this age group registered and a significant reduction in NO-producing functions in the vascular endothelium of the glomerulus to 34.7±1,1 TU (p<0,0001), which is 1.8 times lower than normal.

Figure 4 - the kidney of the patient with disease duration of 8 years. On NADPH-diaphorase react the epithelial cells of the proximal tubules. The decreased activity of NADPH-diaphorase in the modified distal tubules and vascular glomeruli kidney cells (Method Hope, Vincent. HC. 400x).

Cells epithelia Inoi lining the collecting tubules have almost the same activity of the enzyme compared with the first age group (65,8±1,8 TU), but significantly lower than normal (p<0,001).

In group III patients, there is a further reduction in activity nitroxylenes in all investigated renal structures (table 3). It should be noted that at a certain stage of the disease, namely in 10 years (III-IV age group) reliably distinguished with light microscopy proximal from the distal tubules is not possible (Kurkin A. Ultrastructural changes in the kidney in children with hydronephrosis // Health of Tajikistan. - 1977. No. 1. - P.14-18), therefore, table 2 presents the total activity in the epithelium of the tubular device.

The activity of the enzyme in this renal structure (the epithelium of the tubules) is 80,1±1,9 TU, which is 44% lower as compared to that in the proximal tubules and 10% lower as compared to that in the distal tubules in normal (p<0,001).

Figure 5 - snapshot of the cortical substance of the kidney of a patient with a disease duration of 11 years. The decreased activity of NADPH-diaphorase in the tubules (Method Hope, Vincent, UV).

In pyelonephritis smooth the glomeruli of the kidneys of children with disease duration 10-12 years the activity of the enzyme is virtually no different from the one in the previous group of patients and is of 34.7±1,9 TU.

Table 3
Group Bo is lnyh Indicators of activity of NADPH-diaphorase (TU)
The epithelium of the tubulesCollective tubulesVascular glomerulus
10-12 years80,1±1,944,5±1,334,7±1,9
13-15 years59,4±0,944,1±1,218,4±1,2

The decreased activity of the enzyme in the epithelium of the collecting tubules reaches 44,5±1,3 TU, which is 1.8 times less than normal (p<0,0001).

In group IV patients with duration of disease 13-15 years nitrosodipropylamine feature of all investigated structures of the kidney was minimal and is in the epithelial cells of the tubules - 59,4±0,9 TU, which is 58.5 per cent lower than in normal proximal tubules and 32.7% lower than in epithelial cells of distal tubules in normal (p<0,001).

Figure 6 is a kidney patient with pyelonephritis with disease duration of 15 years. NADPH-diaphorase with reduced activity can be detected only in some dystrophic changes of convoluted tubules (Method Hope, Vincent, UWH).

Structural elements wrinkled kidney glomerulus demonstrate the low activity of the enzyme is 18.4±1,2 TU, which is 3.3 times less than normal (p<0,0001).

The activity of NADPH-diaphorase dystrophic changes in the epithelial cells of the collecting tubules brain prophetic the STV kidney practically does not differ from that in group III children and is 44.1±1,2 TU (p> 0,5), which, however, is 1.8 times lower than in normal (p<0,001).

Changes in the activity of NADPH-diaphorase in the renal vessels in different age groups of children also characterized by a certain dynamics (table 4).

Table 4
The group of patientsIndicators of activity of NADPH-diaphorase (TU)
The vascular endotheliumSmooth vascular myocytes
Norma103,05±1,400,0±0,0
Up to 3 years118,5±1,964,8±1,1
7-8 yearsto 81.9±3,1538,2±1,6
10-12 years62,6±2,638,9±1,3
13-15 years32,3±1,331,3±1,3

So, with disease duration of 3 years there was a significant increase in enzymatic activity in the endothelium and smooth myocytes vessels up to 118.5±1,9 TU and 64,8±1,1 TU (OK - 103,05±1,4 TU 0,0 and TU, respectively, p<0,001).

7 is a kidney patient with pyelonephritis with disease duration of 3 years. On NADPH-diaphorase react endothelium and smooth myocytes of the vessel. Method Hope, Vincent. UVx.

Further, the enzyme activity decreased significantly in the endothelium: in group I - 1.3 times, but in the third age group 1.6 times in comparison with the norm and makes 81.9±3,15 TU and 62.6±2,6 TU (p<0,001), respectively. To 15 years of disease occurs more than three-fold inhibition of enzymatic activity nitroxylenes - to 32.3±1,3 TU.

In smooth vascular myocytes after increasing the activity of NADPH-diaphorase to 3 years in the future by the age of 8 and 12 years is a reduction of 1.7 times (up to 38,2±1,6 TU and 38.9±1,3 TU, respectively) compared with the first age group (p<0,001).

In group IV of the children the activity of NADPH-diaphorase in smooth myocytes minimum and is 31.3±1,3 TU, which is 2.1 times lower than in age group I. Significant differences of the optical density of the enzyme compared with the values in the second and third age groups is not received.

Indicators of activity of NADPH-diaphorase in vascular myocytes significantly higher than normal (p<0,0001).

Thus, the conducted research activity of NADPH diaphorase in different parts of the kidneys healthy and sick children and the analysis of the results showed that in chronic obstructive pyelonephritis in children within three years, there is a compensatory, a statistically significant increase in the activity of NADPH - diaphorase in the proximal and distal tubules and blood vessels. However, in the epithelium of the collecting tubules of the brain substance of kidneys that are already in the early hundred is s disease undergo atrophy of the violation of the outflow of urine and chronic pyelonephritis, statistically significant inhibition of the enzymatic activity of NOS, and, consequently, the formation of nitric oxide. To 13 years from the onset of the disease occurs total depletion of NO-producing functions in all renal structures that is associated with sclerotic processes in the body and is clinically manifested by severe arterial hypertension.

After analyzing histochemical activity of NADPH-diaphorase (as a marker of NOS) and making a conclusion about the duration of the disease, can assess the condition of compensatory renal function, and thus to provide appropriate preventive treatment, which will continue to reduce or slow down the development of end-stage chronic renal failure (i.e. wrinkling of the kidneys) and arterial hypertension.

Because it is not always possible histochemical analysis of renal tissue, the authors first conducted a study of the relationship between the activity of NADPH-diaphorase in the kidneys and laboratory blood and urine of patients with chronic obstructive pyelonephritis based on the calculated correlation coefficients, which may be considered as criteria for the activity of this enzyme in the clinic (table 5), and proposed a second variant of the method of determining the duration of the disease in children chronic obstructive pyelonephritis-based laboratory is atornic blood counts, urine and pressure values.

For this biological material (blood, urine)taken from the sick children of the previous group, investigated according to standard techniques in compliance with the rules of the fence bacteriological, microscopic and biochemical analysis using qualitative and quantitative methods, and compared the results of the study with the average rate and the activity of NADPH in the different departments of the kidneys and the duration of the disease. As laboratory parameters were taken into account clinical and biochemical blood indices, indicators common urine analysis, urine analysis on daily salt samples Rehberg, blood pressure, etc. just analyzed 22 indicator (table 5). All studies were performed in the clinical diagnostic laboratory Coastal regional children's clinical hospital, Vladivostok.

Mathematical processing of data was performed on a personal computer IBM PC / AT using a statistical software package patch BIOSTAT. Determined the arithmetic mean (X), standard error of the arithmetic mean (x), criteria of significance of difference (t). Based on the confidence coefficient t, using the number of degrees of freedom or the number of observations, that the persons of student-Fisher defined the ratio of the probability of error (p). The difference between the arithmetic mean was considered significant at p<0,05. (Afife A., Eisen, S. Statistical analysis: an approach using computer - M.: Mir, 1982. - 488 C.). The probability of type I error (the refutation of the true hypothesis) is related to the level of α (Urbach VY Biometric methods (statistical processing of experimental data in biology, agriculture and medicine). - M.: Nauka, 1964, of 415 C.). Was selected α=0,05, most often used in medical and biological research. Thus, all the indicators are 95-percent level of significance (lie in this interval in 95 cases out of 100).

Analysis of data was performed in the laboratory of mathematical modeling of ecological systems, Institute of automation and control processes far Eastern branch of the Russian Academy of Sciences.

The results are shown in table 5, show that there is a significant (p<0,01) the direct correlation between the activity of NADPH-diaphorase in the tubular apparatus of the kidney and its activity in the glomeruli (r=+0,75), in collective tubules (r=+0,84), vascular endothelium (r=+0,92).

Correlation between the activity of NADPH-diaphorase in the kidney, clinical and laboratory parameters and disease duration were conducted taking into account the age of the child for such blood as hemoglobi is h, lymphocytes, neutrophils, and specific gravity of urine and blood pressure, and other indicators that do not have explicit dependence on age, namely leukocytes, eosinophils, urea, β-lipoproteins, the level of glomerular filtration, the level of tubular reabsorption, the daily excretion of oxalate, blood pressure (systolic, diastolic, and pulse), the norm adopted the average performance (Wagasky, Hteller, Namarata, REVTECH-Yampolskaya. Morphofunctional constants of the child's body. - M.: Medicine, 1997, - 297 S.).

Found that there is a strong inverse correlation between the activity of NOS in a tubular apparatus of the kidney and the level of hemoglobin (r=-0,82; p<0.01), and number of blood eosinophils (r=-0,8; p<0.01), and the daily excretion of oxalate (r=-0,88; p<0,01), and systolic blood pressure (r=-0,75; p<0.05)and diastolic blood pressure (r=-0,76; p<0.05) and pulse pressure (r=-0,71; p<0,05).

The activity of NADPH-diaphorase in vascular glomeruli also showed a correlation with some laboratory and functional performance in children, and it is statistically significant (p<0,01) strong direct correlation with enzyme activity in the collecting tubules (r=+0,86) and vascular endothelium (r=+0,93). Silicatization correlation was identified with the number of peripheral blood leukocytes (r=-0,84; p<0.01), and the number of eosinophils (r=-0,92; p<0.01), and the level of urea in blood serum (r=-0,84; p<0.01), and the daily excretion of oxalate (r=-0,63; p<0.05), and also the systolic, diastolic and pulse pressure (r=-0,82 - 0,9; p<0,01).

A positive correlation between the activity of NOS in the epithelium of the collecting tubules and the renal vascular endothelium (r=+0,86; p<0.01), and specific gravity of urine (r=+0,68; p<0,05). In addition, identified a strong inverse correlation between the activity of NADPH-diaphorase in the specified structure of the kidneys and some laboratory parameters of blood and urine, in particular: the number of peripheral blood leukocytes (r=-0,76; p<0.05)and the number of eosinophils (r=-0,94; p<0,01), proteinuria (r=-0,72; p<0,05), the daily excretion of oxalate (r=-0,78; p<0.05)and the levels of urea and (β-lipoproteins in the blood (r=-0,71 and -0,82, respectively; p<0.05), and also systolic and diastolic blood pressure (r=-0,69 and -0,71 respectively; p<0,05).

The enzymatic activity in vascular endothelium in children with chronic obstructive pyelonephritis was negative correlational due to the number of peripheral blood leukocytes (r=-0,76; p<0.05)and the number of eosinophils (r=-0,9; p<0.01), and the level of blood urea (r=a-0.7; p<0,05), the daily excretion of oxalate (r=-0,74; p<0.05) and the systolic, diastolic and pulse D. the effect of the surveyed children (r=-0,82; r=-0,87 and r=-0,86 respectively; p<0,01).

The activity of NADPH-diaphorase in smooth myocytes renal vascular demonstrates a direct correlation correlation with the number of lymphocytes and neutrophils in peripheral blood (r=+0,69 and r=+0,8 respectively; p<0.01), and the level of blood urea (r=+0,73; p<0.05), and also a negative correlation with specific gravity of urine (r=-0,8; p<0.01), and the level of glomerular filtration rate (r=a-0.7; p<0.05) and reabsorption (r=-0,8; p<0,01). The relationship of the enzymes with the blood pressure here has not been established.

Thus, as can be seen from the statistical analysis of experimental activity values NADPH and laboratory blood and urine tests, statistically significant, showing a correlation with the activity of NADPH-diaphorase, as a marker of NOS activity, are hemoglobin, leukocytes, eosinophils, urea, β-lipoproteins, lymphocytes, neutrophils, the level of glomerular filtration rate (KF), the level of reabsorption, beats. weight of urine; the daily excretion of oxalate, blood pressure (systolic, diastolic, and pulse). The results in absolute values are shown in table 6.

Table 6
Clinical and laboratory findingsDisease duration
&x0200A; 3 years7-8 years10-12 years13-16 years
Hemoglobin, g/lto 121.5±1,2103,0±3,4137,0±3,3142,0±2,4
Leukocytes, ×109/l4,6±0,28,2±1,18,7±1,37,7±1,2
Eosinophils, %2,5±1,85,0±2,37,0±3,46,7±3,4
Urea, mmol/l4,6±0,86,7±0,96,4±0,76,1±1,3
β-lipoproteins, ED39,0±3,131,0±3,462,5±4,340,7±2,6
Lymphocytes, %32,0±1,836,0±1,337,0±1,533,0±2,1
Neutrophils, %62,0±2,855,0±4,147,0±1,859,0±3,7
The level of glomerular filtration rate (KF), ml/minto 96.9±3,8of 101.4±3,981,2±4,278,5±5,3
The level of tubular reabsorption, %97,0±0,697,0±0,596,0±0,295±0,5
UD. the weight of urine1011±21007,5±3,11010,5±1,11012,5±2,6
Daily excretion of oxalate, mEq/day 42,6±5,324,2±8,653,7±5,176,9±2,9
HELL, mm Hg

- systolic.
115,0±4,8145,0±4,0130,0±10,2190,0±20,3
- diastolic.75,0±5,495,0±5,188,0±6,8130,0±10,6
- pulse40,0±5,150,0±4,642,0±8,580,0±15,5

Because there is no reason to assume the existence of differences in the course of the disease depending on the geographical location of a sick child, we found the patterns presented in table 1 in percentage deviation from the norm, where a minus sign (-) refers to the decrease, and plus (+) to its increase, the average performance with respect to the age of the child were adopted following values: up to 3 years: Hemoglobin, g/l - 129; Lymphocytes, % - 50,5; - 144; 36; 53; 1022; and to 13-16 years 146; 28; 60; 1024 respectively (Wagasky, Hteller, Namarata, REVTECH-Yampolskaya. Morphofunctional constants of the child's body. - M.: Medicine, 1997, - 297 S.).

Thus, after the appropriate clinical and laboratory tests of a child with a diagnosis of chronic obstructive pyelonephritis and analyzing the results obtained taking into account identified by the authors of the data presented in table 1, do you have the od on the duration of the disease, i.e. ultimately about the state compensatory renal function, and in accordance with this conduct appropriate prophylactic treatment.

Clinical example of the application of the proposed method.

Patient Alexey G., age 11 (1999), entered uronefrologicheskijj Department PCDMB complaining of headaches, pain in the lumbar region, dull, constant, worse after exercise, periodic temperature rise to 37.0-38,0°C, nausea.

A state of moderate severity due to moderate symptoms of intoxication, hypertensive syndrome, pain and urinary symptoms. The temperature is 37.2°C. pale Skin, marked periorbital shadows, pastos century. The soft tissue turgor saved. Musculoskeletal-joint system - no visible pathology. From respiratory - without features. The cardiac region is not visibly changed. The left border of relative cardiac dullness on the left srednechrochnoy line. Heart sounds loud, accent II tone of the aorta. HELL: systolic blood pressure of 150 mm Hg (+44% from the norm), diastolic - 108 mm Hg (+58%), pulse - 42 mm Hg (+24%). Heart rate is 84 beats per minute. Belly symmetric, palpation of soft, painless in all departments. The liver, the spleen is not enlarged. The symptom of “tapping” positive on both sides, more with the Rav. Even royalty enlarged right kidney, painless. In the clinical analysis of a blood: erythrocytes - 4,15·1012/l b - 101 g/l (-22%), platelet - 190 thousand, leukocyte - 11,0·109/l (+22%), neutrophils (p/I - 2%, with/I - 56% (+16%), lymphocytes - 34% (12.8 percent), eosinophils - 7% (+250%), monocytes - 3%, ESR - 23 mm/h In the biochemical blood test: whole protein - 71,6 g/l, albumin - of 45.7 g/l, urea of 8.2 mmol/l (+76%), creatinine is 98.8 mmol/l, glucose - 4.6 mmol/l, cholesterol - 4.1 mmol/l, LDL - 32 units (+40%), liver function tests are normal. Blood group: A(II), Rh (+). In the coagulation tests: APTT - 42 seconds TV - 15 sec, PETIT 98%, fibrinogen - 4,6 g/l, ethanol test is negative, thrombotest - 6 degrees. In urinalysis: pH neutral, beats. weight - 1010, protein - no, epithelial cells - 1-2 p/SP., the leukocyte - 2-3 p/SP., erythrocytes - 1-2 p/SP., oxalates - +1. In the analysis of urine on Nechiporenko: leukocytes: 1750 (C/I - 86%, l - 14%), erythrocytes - 2000 in 1 ml of the urine culture is negative. In the analysis of urine on a daily salts and metabolites polar lipids (+), peroxide - (-), the excretion of uric acid - 342 mg/day excretion of oxalate - 24 mg/day (-25%). Antiretaliation normal diuresis - 1010 ml, pH - neutral. Sample of zimnitsky tests: Daily diuresis - 720 ml (tank weight - 1002-1012), nocturnal diuresis - 150 ml (tank weight - 1004-1016). Sample Rehberg: MD=1,4 ml/min, KF=97,0 ml/min (-19%), Reabsorption - 98,0% (-1%). A feces on eggs of worms and Giardia cysts is not detected by the ENES. Scraping on enterobiasis (repeatedly) - negative.

Ultrasound of the abdomen: without pathology. Ultrasound of the kidneys: echoprint of hydronephrosis of the right kidney. Excretory urography: hydronephrosis IV century on the right with a sharp decline in kidney function, pyelectasis left.

The ECHO-KG: heart cavity is not expanded, the valves are not changed. ECG: sinus rhythm with HR=96-80 per minute, RR=0,72; electrical position - poluvirtualnyh. R - 0,08; PQ - 0,14; QRS - 0,06; QT - 0,40. Conclusion: violation of the processes of ventricular repolarization, local disturbance of intraventricular conduction. TG: IWT: sympathicotonia, BP is normal. The opinion of the optometrist: angiopathy retinas on hypertonic type. Markers of viral hepatitis: detected HBsAg. Serological blood tests for opportunistic infections - negative. Urodynamic study: normolipidemic hypotonicity the bladder.

On the basis of clinical and laboratory findings diagnosis: Chronic latent pyelonephritis on the background of hydronephrosis of the fourth degree to the right.

In February 1999, held nephrectomy of the right kidney. Histochemical method determined the enzymatic activity of NADPH-diaphorase in the epithelial cells of the proximal tubules - 131 (130, 8mm) TU; in epithelial cells of distal tubules - 83 (83,1) TU; in vascular endothelium - 82 (81,9) TU; in smooth vascular myocytes - 38 (37,7) TU.

From the anamnesis is zabolevaniya: for the first time, headaches, pain in the lumbar region, nausea appeared at the age of 10. The examination revealed changes in the urine in the form of small pyuria (8-10 in the field of view). Ultrasonography detected gidroenergetichesky transformation of the right kidney. Sent to the regional medical facility.

The child from fifth pregnancy going on in the background preeclampsia, acute respiratory infections in the first half. Childbirth fifth, urgent. He was born at term weighing - 4200 g, body length - 52 see Cried at once. Estimation on Apgar scale of 7 points. The neonatal period proceeded without features. Artificial feeding with 1.5 months. With 7 years is in the dispensary with a diagnosis of Bronchial asthma, lung during. Genealogical history aggravated by hypertension, chronic pyelonephritis, diseases of the gastrointestinal tract. Objective: the boy right physique. The Quetelet index-II is 15.6 (25 percentile).

Comparative analysis of collected history initial treatment of the child at the age of 10 and integrating data from clinical and laboratory examination allows us to conclude that, despite the first signs of pyelonephritis year ago, the results of histochemical determination of enzymatic activity of NADPH-diaphorase, clinical examination, blood pressure (systolic, diastolic is about, pulse), laboratory tests (hemoglobin, leukocyte count, eosinophils, lymphocytes, neutrophils, urea, β-lipoproteins, the level of glomerular filtration, tubular reabsorption, specific gravity of urine, the daily excretion of oxalates) indicate the duration of the disease 7-8 years.

-0,32348 0,290053034
Table 5
 tubulesthe glomerulisobirat. pipes.indotel. container.myocytes SOS.Limp.NVEr.Lake.
Norma115,6760,9206349278,55555556103,050    
3 years136,301587355,2698412767,84126984118,564,8 32121,54,14,615
7-8 years127,936507934,6507936565,8253968381,938,2a 38.51033,33258,2
10-12 years80,095238134,6825396844,5238095262,638,936,51374,18,7
13-16 years59,4126984118,3492063544,0476190532,331,3331424,15the 7.65
 
 tubulesthe glomerulisobirat. pipes.indotel. SOS is D. myocytes soy.Limp.NoEr.Lake.
tubules1        
the glomeruli0,751       
sobirat. Tr.0,8426028940,8562451371      
indotel. 0,9212751910,9337093280,855158156 1     
myocytes0,230564848-0,145736339-0,2809489850,1446077291    
Limp.0,2095139250,2342685050,4080187240,125421742-0,6911293341   
NV-0,82341524-0,294190768-0,598771989-0,54426864-0,20748191-0,3910646731  
Er.-0,2843708880,39387858 0,0843447490,09094321-0,422378992-0,1717132770,7419241 
Lake.-0,558004347-0,836404043-0,761178875-0,7617191990,111107510,1577637540,064184-0,565131
Neutral.0,111983255-0,315004787-0,209398732-0,0391699530,794011129-0,799841823-0,17349-0,3885-0,01812
Eosin.-0,790497503-0,920301792-0,94047109-0,9002173070,144371417-0.1217363040,392658-0,307140,92975
Protein -0,581686329-0,325180698-0,72005959-0,3711896180,456742973-0,762404130,7709710,4093960,139883
Creatine.0,155312324-0,491500222-0,117852378-0,2337428030,2366996710,274308924-0,67951-0,973840,632404
Urea-0,412064503-0,840620715-0,7082703-0,677911580,3016334180,04183142-0,10477-0,729390,965348
In-PL-0,520278325-0,457079938-0,818467706-0,4402338090,468306905-0,3368648840,474898-0,0216 0,602116
The cholesterol.-0,430871391-0,516619171-0,24581924-0,512054419-0,07209561-0,4687700550,2812850,054633-0,01326
Erythrocyte sedimentation rate0,11085802-0,512776977-0,269898789-0,2414979720,4056863090,192474656-0,60069-0,971510,732755
Specific0,0291846670,5818475980,533682430,279276851-0,8012651340,3573843460,2984590,783063-0,64359
Protein0,361174503-0,3177426290,0782507170,0012718830,46123593-0,123597372 -0,71595-0,858710,222646
Day. The oaks.-0,878353881-0,629741626-0,787817092-0,7446582790,068068663-0,6341287510,8748930,40910,26124
Much. the-0,215695799-0,367414937-0,384778733-0,2239957710,631574134-0,9548912890,2720680,012603-0,09312
Simnick.0,100475406-0,481891997-0,326430997-0,1532989970,826616065-0,586970727-0,34417-0,693640,311743
KF0,1578027180,3286301640,6185066620,18145316 -0,6920581680,275551355-0,070650,341005-0,57802
Rehabs.-0,0533181840,5166798950,3517990670,197868255-0,8025884980,6077177490,303450,669983-0,31573
systolic-0,745045759-0,877790506-0,69258012-0,8606682690,043418934-0,4432742650,429145-0,116050,476878
diastolic-0,75569648-0,89459946-0,72817136-0,8702784590,083183995-0,4636863920,438272-0,129290,50543
Pulse-0,7081128-0,823629233 -0,594042821-0,824735019-0,056562015-0,3861237880,400395-0,081360,398919
Continuation of table 5
Neutral.Eosin.ProteinCreatineUreaIn-PLThe cholesterol.Erythrocyte sedimentation rateShareProteinDay. the oaks.
           
62,52,57180,54,6393,9510,510110,1325 42,6
555631466,75313,95231007,50,2524,2
46,5772,4589,056,4562,53,4514,51010,5053,7
58,756,7571,7394,336,140,754,85511,51012,50,1276,9
 
Neutral.Eosin.ProteinCreatine.UreaIn-PLThe cholesterol.Erythrocyte sedimentation rate ShareProteinDay. The eye.
           
           
           
            
           
           
           
            
           
1          
0,0783971         
0,299910,4481041         
0,2906660,377678-0,4805596741       
0,2086670,8728060,0989165810,7656761251      
0,0551370,7204790,77531021-0,0500271730,5414950471     
0,5174110,1800630,1215861090,0574627420,04983742 -0,3027756021    
0,292280,494551-0,2810256230,954941020,8555557930,222544892-0,1236680631   
-0,64195-0,57479-0,242431073-0,691377492-0,801410852-0,536973350,006553095-0,8295445591  
0,692250,055932-0,3998421130,8344073570,451190229-0,2872683230,3400361920,735811602-0,650251 
0,2504580,5952350,803684209-0,34642773 0,1895762830,4904099380,594926953-0,294976274-0,07242-0,2897354581
0,8932720,155160,594956541-0,0756494220,0543457520,1658485770,67196998-0,064816989-0,403140,3436485560,615237158
0,9188210,3030890,1808475720,6036746880,5376292410,1975320870,3440749330,640922522-0,87490,8237446390,11577852
-0,20474-0,57444-0,558590343-0,212016802-0,602724745-0,9064051660,471038086-0,4900778480,750184-0,012177657-0,149722315
-0,92824-0,197853946-0,590483426-0,53772528-0,188348072-0,402437211-0,6180729750,858018-0,818412016-0,166128382
0,4501040,6663850,3507726940,2336150760,4970660130,166224880,8529376590,163256257-0,309530,2855960870,776640141
0,46280,6971280,3873352070,2391907640,526665470,2184247390,8281198160,184892387-0,349050,2822305980,78931309
0,4121440,5803280,2545402430,2164461210,4162100660,0334669850,9031530990,106923555-0,206480,734215865
Continuation of table 5
       
       
1011,7296,90,96551157540
578,72101,40,96551459550
553,4181,20,9781308842
1049,21,75107,45 0,96725017080
 
Mock-TASimnick.KFRehabs.systolicdiastolicpulse
       
       
       
        
       
       
       
       
       
        
       
       
       
       
       
        
       
       
       
       
1      
0,7001697551     
-0,125213889-0,416660531    
-0,729836965-0,9978483360,3859849041   
0,6105638130,4334721320,038183372-0,4882941911  
0,6213837610,455607683-0,017545876-0,5088733150,9984215651 
0,575069790,3721502280,176931851-0,430103073 0,9901000680,9806539141

1. How to determine the duration of the disease in children chronic obstructive pyelonephritis by conducting laboratory tests, characterized in that to determine the enzymatic activity nitroxylenes in the epithelial cells of the proximal, distal tubules, vascular glomeruli, as well as in the endothelium and smooth myocytes of the renal vessels and compare them with the norm, while the activity nitroxylenes calculate the activity of NADPH-diaphorase, a specific histochemical method, and when exceeding the standards equal to 143 TU in the epithelial cells of the proximal tubules and 88.3 TU in the epithelium of the distal tubules, 10% and 29.8 per cent respectively, and/or exceeding the standards equal 103,1 TU in vascular endothelium, 13% and the appearance of the activity of NADPH-diaphorase in smooth vascular myocytes to a value of 65 TU, diagnose disease duration up to 3 years; decreased activity of NADPH diaphorase in the epithelial cells of proximal and distal tubules to 83 131 and TU, respectively, and/or in the endothelium and smooth myocytes vessels up to 82 and 38 TU respectively diagnose the disease duration up to 7-8 years; activity of NADPH-diaphorase in the epithelium of the tubules 80 TU, and vascular glomeruli 35 TU the norm in the last 61 TU, diagnose liternaturnaya 10-12 years, and at values below these values is a disease duration of 13-15 years.

2. How to determine the duration of the disease in children chronic obstructive pyelonephritis by conducting laboratory tests, characterized in that the laboratory examination includes the determination of hemoglobin, leukocytes, eosinophils, urea, β-lipoproteins, lymphocytes, neutrophils, level of glomerular filtration rate (KF), the level of tubular reabsorption, beats. weight of urine; the daily excretion of oxalate, systolic, diastolic and pulse arterial pressure, calculating the deviation of the results obtained from the average values of indicators taking into account the child's age and the subsequent definition of disease duration on the basis of comprehensive consideration of all these characteristics, shown in the following table, in which (-refers to the decrease, and (+) - to its increase relative to the average values:

% of normal

Indicators
Disease duration
3 years7-8 years10-12 years13-16 years
Hemoglobin-(6,0±0,5)-(22±0,5)+(5±0,5)+(3±0,5)
Leukocytes-(23±1)+(23±1)+(39±1)+(23±1)
Eosinophils+(130±5)+(285±5)+(440±5)+(440±5)
Urea+(25±1)+(75±1)+(50±1)+(50±1)
β-lipoproteins+(78±4)+(43±2)+(183±4)+(83±2)
Lymphocytes-(35±1)-(14±1)+(4±1)+(21±1)
Neutrophils+(63±2)+(17±2)-(10±2)0
The level of glomerular filtration rate (KF),-(23±1)-(20±1)-(36±1)-(38±1)
The level of tubular reabsorption-(2.2±0,1)-(2,2±0,1)-(3,5±0,1)-(4,3±0,1)
UD. the weight of urine-(0,2±0,05)-(1,1±0,1)-(1,2±0,1)-(1,2±0,1)
Daily excretion of oxalate+(22±1)-(24±1)+(51±1)+(110±2)
Blood pressure-systolic+(15±1)+(44±1)+(32±1)+(96±1)
end-diastolic+(26±1)+(59±1) +(50±1)+(121±2)
-pulse0+(24±1)+(10±1)+(107±2)



 

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