RU2452959C1 - Diagnostic technique for renal parenchymal injury in children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: urine is examined for quantitative measures of π-glutathione-S-transferase (π-GST), and if observing its value exceeding 0.86 mcg/l, the renal parenchymal injury is diagnosed.

EFFECT: technique enables early diagnosis of the renal parenchymal injury in the absence of injury signs according to a standard integrated examination, coupled with non-invasiveness, accuracy and simplicity.

2 ex

Description

The invention relates to medicine, namely to pediatrics, and can be used in urology and nephrology.

Despite the large number of studies on the pathogenesis of tubulointerstitial fibrosis, a number of unresolved issues remain, primarily regarding minimally invasive diagnostic methods for nephrosclerosis.

Regarding the diagnosis of sclerotic changes in the kidneys, the arsenal of methods is small. Moreover, each of the methods has its drawbacks, which must be taken into account, especially for patients of a younger age group.

A method of excretory urography is well known, based on an X-ray assessment of the state of a functioning parenchyma, however, the sensitivity of this method is very low in assessing the degree of nephrosclerosis. In addition, this method is invasive, and allergic and toxic reactions to the administration of iodine-containing drugs limit the use of this method [Roth K., Koo N., Spottswood S., Chan J. Obstructive uropathy: an important cause of chronic renal failure in children /TO. Roth, H. Koo, S. Spottswood, J. Chan // Clin. Pediatr. - 2002 .-- Vol.41 (5). - P.309-314]. In a study by Zakharkina E.B. it was shown that when using radiopaque agents of different osmolarity, there is a significant effect on the tubular epithelium, which is accompanied by an increase in the release of membrane-bound enzymes (N-acetyl-β-D-glucosaminidase) [Zakharkina, E.V. The effect of radiopaque drugs on kidney function in children with pyelonephritis. / E.V. Zakharkina // Pediatrics. - 2008. - T.87, No. 4. - S. 27-31].

Due to its relative safety, speed, cost-effectiveness, high accuracy, ultrasound (ultrasound) is widely used in children to identify the structure and size of the parenchyma and the collective kidney system [Becker A.M. Postnatal evaluation of infants with an abnormal antenatal renal sonogram / A.M. Becker // Current Opinion in Pediatrics. - 2009 .-- Vol.21. - R. 207-213]. However, ultrasound can detect sclerotic changes in the kidneys when they are sufficiently pronounced [Gallo F., Schenone M., Giberti C. Ureteropelvic Junction Obstruction: Which Is the Best Treatment Today? / F. Gallo, M. Schenone, C. Giberti // J. Laparoendoscopic advanced surgical techniques. - 2009 .-- Vol.19 (5). - R.657-662].

Ultrasound of the kidneys in the color Doppler mapping mode allows you to determine the resistance index (IR) on the interlobar arteries, thereby indicating sclerotic changes in the parenchyma [Vrublevsky SG Diagnosis and treatment of children with expansion of the pyelocaliceal system // Questions of Practical Pediatrics. - 2008. - T.3. - Number 3. - p. 35-39]. However, in recent years it has been shown that in patients with a severe degree of reflux nephropathy (PH), accompanied by a significant decrease in kidney size and the number of functioning parenchyma, there is a significant variety of resistive indicators of renal blood flow. The expected increase in IR in children with sclerotic changes in the renal parenchyma does not always occur. This phenomenon is associated with arteriovenous blood shunting as a universal pathophysiological mechanism, which is a means of pathological compensation of increased peripheral resistance, which determines the absence of a direct relationship between the degree of sclerosis of the renal parenchyma and the peripheral resistance of arterial renal blood flow [Olkhova EB Ultrasound assessment of chronic pyelonephritis in children with vesicoureteral reflux. / E.B. Olkhova, E.M. Krylova, I.I. Efremova // Nephrology and dialysis. - 2001. - No. 2. - S.299]. In addition, in patients with obstructive uropathy, the speed characteristics of renal blood flow change significantly and are interpreted incorrectly, which does not allow reliable diagnosis of scars. These methods are subjective, depend on the qualifications of the specialist performing them, and do not provide information on the functional state of the kidneys.

Radioisotope diagnostic methods make it possible to determine the degree and volume of a functioning parenchyma by the integral capture index of a radiopharmaceutical [Khvorostov I.N., Zorky S.N. Smirnov I.E. Tactics for the treatment of vesicoureteral reflux in children // Bulletin of the Volgograd Scientific Center RAMS 4/2009; Fomin D.K. Diagnosis of obstructive uropathy in children by nuclear medicine methods: Abstract. dis. Doct. honey. sciences. - M., 2008. - 44 p.]. The disadvantages of this method are invasiveness, a high degree of radiation damage to other organs, as well as the high cost of using isotope equipment. In addition, anesthetics are required for children of a younger age group. All this limits the use of this method for the diagnosis of cicatricial changes in the kidney.

The method of percutaneous puncture biopsy of the renal parenchyma, followed by histological examination of the biopsy, allows you to see the proliferation of collagen fibers in the interstitium. The negative point of this method is, first of all, invasiveness, complexity, high risk of trauma, complications, lack of an exact choice of tissue site. These shortcomings determine the limitations of this method in the diagnosis of sclerotic changes in the kidney.

Closest to the proposed method is a method for determining damage to the renal parenchyma by examining microalbuminuria. Microalbuminuria is the level of albumin in the urine of 30-300 mg / day, which is not determined using classical research methods. According to available literature data, information on determining the level of nephron damage by the presence of albumin in urine is ambiguous. A number of authors found that the albumin level in the range of 30-300 mg / day is an early and affordable marker of proximal tubule damage and concluded: the detection of microalbuminuria allows you to diagnose early stages of nephrosclerosis that are not determined by other available methods [Herget-Rosental S., Poppen D ., et al. Prognostic Value of tabular Proteinuria and Enzymuria in nNonoliguric Acute Tubular Necrosis. / S. Herget-Rosental, D. Poppen, et al. // Clin. Chem. - 2004. - Vol. - 50. - P.552-558]. It should be noted that albumin is a medium-molecular protein (molecular weight - 66 kDa). Its appearance in the urine indicates damage primarily to the glomeruli of the kidneys. Glomerular damage develops as a compensatory mechanism in response to the death of a significant number of nephrons. Hyperfiltration of the remaining glomeruli leads to the appearance of a diagnostically significant level of this protein in the urine. Thus, microalbuminuria indicates severe damage to the renal parenchyma and appears in the late stages of nephropathy. The data presented by other authors showed high sensitivity of the test for determining the level of microalbuminuria of 19.1 mg / l or more for the diagnosis of pH as the main marker of fibrosis [Makhachev BM, Korsunsky A.A et al. The clinical significance of the determination of proteins in urine for the early diagnosis of reflux nephropathy in children // Nephrology and Dialysis. - 2005. - T7, No. 1. - S.88-93]. It should be noted that with retrograde urine flow, the damage to the distal tubules with subsequent involvement of the glomerular apparatus is primary, which indicates the occurrence of microalbuminuria only with gross changes in the renal parenchyma. Also, it should be noted that microalbuminuria is possible in the absence of a pathological process in the kidneys. So, its appearance may be due to the consumption of a large amount of protein or physical activity. In another study, it was found that the level of microalbuminuria of 15.1 ± 0.23 mg / l / 24 hours is characteristic of patients with MTCT, but without nephrosclerosis, while in the presence of signs of pH this level was within 31.7 ± 0. 62 mg / l / 24 [Zorin I.V., Vyalkova A.A. Parameters of microalbuminuria in patients with MTCT and reflux nephropathy // Materials of the International School and Scientific and Practical Conference on Pediatric Nephrology “Actual Problems of Pediatric Nephrology.” - Orenburg. - 2010. - S.301-302]. The question remains how to evaluate the level of microalbuminuria, which is in the range from 14.87 mg / l / 24 to 32.32 mg / l / 24. After analyzing the work to determine the urinary excretion of albumin, the level of albumin remains unclear, starting with which we can talk about the signs of nephrosclerosis. In addition, a number of researchers have proved that the determination of microalbuminuria does not allow to distinguish it as an early marker of pH [Konda R., Sato N., Sakai K., et al. Urinary excretion of vascular endothelial growth factor is increased in children with reflux nephropathy. / R. Konda, H. Sato, K.Sakai, et al. // Nephron Clin Pract. - 2004 .-- Vol. 98 (3). - P.73-8].

Thus, the information regarding urinary excretion of albumin, which claims to be considered as an early marker of damage to the renal parenchyma, is ambiguous.

The objective of the invention is to develop a non-invasive, accurate, simple method for early diagnosis of renal parenchyma damage in children.

As a result of our study, for the first time, quantitative determination of π-glutathione-s-transferase (π-GST) in urine was performed for healthy children and children with obstructive uropathies. It was found that π-GST is directly associated with damage to the renal parenchyma in children.

π-GST is a multifunctional family of glutathione-8-transferase enzymes that plays a significant role in the catalysis of carcinogens, lipids, oxidative stress products, and genotoxic metabolites by conjugation and neutralization of their electrophilic sites by the glutathione SH group. As a result of the catalytic reaction, water-soluble components are formed with their subsequent elimination from the cell. Such detoxification plays an important role in ensuring the resistance of cells to lipid peroxidation and protein alkylation. π-GST is an indicator of damage to the distal tubules in response to various factors. This factor is produced in response to minimal hypoxia, which may result from various pathological conditions [McMahon, Blaithin A. Urinary glutathione S-transferases in the pathogenesis and diagnostic evaluation of acute kidney injury following cardiac surgery: a critical review. / McMahon, A.Blaithin // Current Opinion in Critical Care. - 2010 .-- Vol.l6 (6). - P.550-555].

Our study is based on the analysis of the level of urinary excretion of π-GST in 20 practically healthy children without pathology from the urinary system, as well as in 63 children with obstructive uropathies. The average age of all children was 2.6 ± 2.5 years.

All patients underwent a π-GST study using enzyme-linked immunosorbent assay using the ARGUTUS MEDICAL test systems (Ireland, Dublin) using an enzyme-linked immunosorbent analyzer Stat Fax 2010 (Stat Fax USA). Urine sampling was performed prior to treatment. Urine samples were centrifuged at 5000 rpm for 15 minutes, and the supernatant was frozen and stored at -18 degrees Celsius until analysis. Similarly, freezing of urine samples from the control group was carried out.

Statistical analysis of the results was carried out using the Statistica 8.0 for Windows application software package from StatSoft-Russia Corporation (2009).

After analyzing the results of the study, we first determined the quantitative indicators of π-GST in healthy children, which were as follows: M = 0.48 μg / L, SD = 0.25. Values ranged from 0.01 to 0.86 μg / L.

We examined 63 children with obstructive uropathies. All patients underwent a comprehensive clinical laboratory and X-ray examination.

An ultrasound of the kidneys and bladder was considered the screening method for diagnosing the pathology of the urinary system. For detection of reflux, cystourethrography was used. To assess the morphofunctional state of the kidneys, excretory urography was used. From four years of age, the method of dynamic nephroscintigraphy with the determination of glomerular filtration rate was used to assess kidney function. All patients underwent renal blood flow assessment using ultrasound of the kidneys in color Doppler mapping. It should be noted that in a third of patients with obstructive uropathies, signs of damage to the renal parenchyma according to the standard examination complex were not detected. As a rule, these children were examined in connection with a history of pyelonephritis attacks. However, in all patients with obstructive uropathy, including patients with no data for damage to the renal parenchyma according to the standard examination, π-GST values were significantly higher than 0.86 μg / L.

The data obtained can be included in the algorithm for examining patients with obstructive uropathy for an objective assessment of damage to the renal parenchyma using a non-invasive, accurate, simple and early method, which is extremely important for pediatric practice.

Clinical example:

1. Feklistov Dmitry, 5 years old, was admitted to the children's planned surgical department with a diagnosis of Inguinal hernia on the left. Initial clinical and laboratory data: OAK: erythrocytes - 4.79 × 10 ¹² / l, hemoglobin - 114 g / l, hematocrit - 32.1, leukocytes 7.1 × 10 ⁹ / l, stab - 4%, segmented - 57 %, eosinophils - 1%, lymphocytes - 30%, monocytes - 7%, platelets - 287 × 10 ⁹ / L, LII = 0.05, INN = 0.09, LI = 0.48, IIR = 4.4; biochemical analysis of blood: total protein - 82 g / l, albumin-globulin coefficient - 0.97; OAM: reaction - acidic, density - 1012, red blood cells 0 in the field of view, white blood cells 0-1 in the field of view, the protein is negative. The level of urinary excretion of markers of damage to the renal parenchyma: π-GST - 0.84 μg / L. According to ultrasound of the kidneys and bladder, the size of the kidneys and abdominal systems is within the age norm.

From the above example, it is clear that the child has no pathology from the urinary system. This is confirmed by the absence of changes in the values of l-GST in the urine.

2. Korotenko Ekaterina, 7 months old, was admitted to the pediatric urology department with a diagnosis of Congenital malformation of the urinary system. Bilateral active and passive vesicoureteral reflux of the III degree. Chronic obstructive pyelonephritis, inactive phase. Background pathology: lymphatic-hypoplastic diathesis. Rickets II, subacute course. Concomitant pathology: mild polydeficiency anemia. Initial clinical and laboratory data: OAK: erythrocytes - 3.9 × 10 ¹² / l, hemoglobin - 95 g / l, hematocrit - 38.9, leukocytes 7.3 × 10 ⁹ / l, stab - 1%, segmented - 27 %, eosinophils - 0%, lymphocytes - 67%, monocytes - 2%, platelets - 254 × 10 ⁹ / L, LII = 0.2, INS = 0.04, LI = 3.3, IIR = 35; biochemical analysis of blood: total protein - 62.2 g / l, albumin-globulin coefficient - 0.97; OAM: the reaction is weakly acidic, density = 1015, red blood cells 0 in the field of view, white blood cells 1-2 in the field of view, the protein is negative. The level of urinary excretion of markers of damage to the renal parenchyma: π-GST - 12.1 μg / L. When performing mixed cystourethrography, bilateral passive and active reflux was detected on both sides of the III degree with an expansion of the distal ureter on the right to 0.8 mm, on the left to 0.5 mm. According to the ultrasound of the kidneys and bladder, the size of the kidney on the left is 55.9 mm × 26.0 mm, the parenchyma is 9.6 mm, the dimensions of the pelvis are 2.3 mm × 2.4 mm; on the right, the size of the kidney is 54.4 mm × 25.2 mm, the parenchyma is 9.3 mm, the dimensions of the pelvis are 5.7 mm × 6.2 mm. According to the Doppler study of the vessels of the kidneys, there are no hemodynamically significant disorders. According to excretory urography, the elimination of contrast is slowed down on both sides.

The above example shows that despite a significant degree of reflux on two sides, signs of damage to the renal parenchyma were not detected by ultrasound of the kidneys with dopplerometry. Only according to excretory urography, a slowdown in evacuation function is noted. The level of π-GST was 12.1 μg / L, which is significantly higher than the upper limit of this factor in practically healthy children, which indicates damage to the renal parenchyma.

Thus, as can be seen from the description of the method and the above examples, the proposed method is non-invasive, simple, accurate and early in the diagnosis of damage to the renal parenchyma. For the first time, π-GST values were given in practically healthy children, ranging from 0.01 to 0.86 μg / L and it was proved that values in the π-GST urine above 0.86 μg / L indicate damage to the renal parenchyma. Our data allow us to identify changes in the renal parenchyma in children with obstructive uropathy in the early stages, objectively evaluate the treatment, improve the treatment of children with uropathy and prevent the formation of renal failure in early childhood, and clarification of the mechanisms of initiation of nephrosclerosis will open up new directions and prospects in the treatment of patients with congenital anatomical and functional disorders of the urinary tract.

Claims (1)

A method for diagnosing damage to the renal parenchyma in children, including the determination of protein fractions in the urine, characterized in that π-glutathione-s-transferase (π-GST) is determined in the urine and, when its value is above 0.86 μg / l, damage to the renal parenchyma is diagnosed.