RU2422826C1 - Method for prediction of clinical course of chronic renal insufficiency in congenital urinogenital malformations - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: ploidy of peripheral blood cell is evaluated by a flow cytometry method. If observing aneuploidy, development of an intermittent or terminal stage of CRI is predicted.

EFFECT: method allows higher accuracy and reliability of the prediction of disease severity.

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Description

The present invention relates to medicine, namely to immunology, urology, and can be used in the complex diagnosis of the course of chronic renal failure (CRF) in congenital malformations of the genitourinary system (URMS) in children.

In recent years, in the Republic of Bashkortostan there has been a tendency to increase the number of children born with malformations of the genitourinary system [Murzabaeva S.Sh. et al., 2005]. According to M.S. Ignatova, Yu.E. Veltishchev (1989), the frequency of anatomical anomalies of the kidneys with various nephropathies is 33.5%. An X-ray contrast study of children with nephropathy revealed anatomical anomalies of the urinary system in almost all of them. For the most part, these are organ stigmas of dysembryogenesis: horseshoe-shaped kidney, doubling of the kidneys, 1/3 had a combination of several developmental abnormalities [Trukhina ON, 1994].

According to WHO, the number of children born with malformations has no downward trend. In this regard, at the present stage of development of neonatology, questions related to the organization of medical care for congenital malformations remain relevant [Bochkov NP, Zhuchenko NA, 1996; Green D.R. et al., 1998].

One of the most common complications of AMPS is the development of chronic renal failure of varying severity. In these situations, a radical treatment is kidney transplantation, and timely bilateral nephrectomy is essential for its successful implementation. In this regard, it seems to be very relevant to study the processes occurring in immunocompetent cells, including the determination of the ploidy and kinetics of the cell cycle of peripheral blood cells with VLPS, complicated by chronic renal failure of varying severity as a marker of the severity of the destructive process.

There is a method for predicting the severity of chronic pyelonephritis, which consists in identifying the relationship between individual increases in the excreted portion of calcium associated with inflammation and a decrease under the influence of blood plasma of patients with latent and active chronic pyelonephritis Ca - ATPase activity of the microsomal fraction of the cortex of intact rat kidneys with further use revealed dependence for predicting the severity of latent pyelonephritis and damage to calcium reabsorption in the kidney ah patients in the phase of clinical remission. An increase in the excreted portion of calcium with a latent phase of inflammation of more than 0.365% indicates the severity and complexity of the course of the disease, and when predicting calcium reabsorption in the phase of clinical remission, a decrease in this indicator below 96.8% indicates tubular damage in patients with chronic pyelonephritis that is not associated with inflammation ( patent RU 2055363, 1996).

A known method for determining the effectiveness of the treatment of chronic renal failure, characterized in that globulins are precipitated from the blood serum with ammonium sulfate, a sufficient amount of the DSP-6 probe solution is introduced into the supernatant, the luminescence intensity is recorded at a wavelength of 540 nm, and if the luminescence intensity is reduced, the therapy is considered effective (patent RU 2070323, 1996).

There is a method for early prediction of the chronic course of the disease in children who have had acute pyelonephritis, including determining in urine of patients with acute pyelonephritis upon admission and after antibiotic therapy, persistence factors of uropathogenic enterobacteria, namely, the microbial adhesion index (IAM), anti-lysozyme activity (ALA) and antiin (AIA) and in the presence of strains of enterobacteria with high rates of persistence factors IAM more than 4, ALA more than 5 μg / ml and AIA more than 3 units. against the background of bacteriuria below 100 CFU / ml upon admission, a chronic course of pyelonephritis is predicted (patent RU 2194281, 2002).

A known method for diagnosing the severity of endogenous intoxication syndrome in patients with chronic renal failure, characterized in that in patients with chronic renal failure, low and medium molecular weight substances are determined in plasma, on and in red blood cells, in urine, the effective concentration of serum albumin, index plasma antiprotease activity, the number of birefringent crystals of citrate plasma with a proteolysis inhibitor and an orthophenanthroline test, followed by calculation of the value three integral indices characterizing mild, moderate and severe severity endogenous intoxication. According to which of the calculated integral indicators has the maximum value, determine the appropriate severity of endogenous intoxication syndrome (patent RU 2276366, 2006).

The prototype of the invention is a method for predicting the development of nephrogenic hypertension in children with chronic pyelonephritis, which consists in a comprehensive study of biomedical, clinical, laboratory and functional parameters (patent RU 2234090, 2004).

The task of the invention is to develop an effective and easy-to-use method for assessing the course of chronic renal failure in patients with chronic obstructive pulmonary artery disease and for timely bilateral nephrectomy.

The technical result when using the invention is predicting the course of chronic renal failure in congenital malformations of the genitourinary system in children.

The proposed method is as follows. Peripheral blood sampling is performed in an amount of 5 ml in the morning on an empty stomach. The ploidy of peripheral blood cells is determined by flow cytometry. If aneuploidy is detected, the development of an intermittent or terminal stage of chronic renal failure is predicted and bilateral nephrectomy is indicated.

Research method

To characterize the ploidy and proliferative activity of cells, we used a method based on DNA binding with a fluorescent probe-propidium iodide, which allows you to determine the ploidy and proportion of cells in different phases of the mitotic cycle: G0 / G1, S and G2 + M. Trypsin buffer 250 μl, trypsin inhibitor with RNAse 200 μl and a solution of propidium iodide 200 μl with intermediate stirring and 10-minute incubations are successively added to a test tube with 100 μl of blood, then the sample is filtered through nylon tissue with 50 μm cells. Samples within 3 hours after addition of PI [Vindelov L., 1989] (at least 20,000 cells) are analyzed on a FACSCalibur flow cytometer, equipped with a duplet discrimination module in the ModFit program.

The invention is illustrated by the following figures: figure 1-2 shows a DNA histogram of peripheral blood cells of patient L. according to example 1; figure 3-4 - DNA histogram of peripheral blood cells of patient K. according to example 2.

When analyzing the results of DNA cytometry in a group of children with congenital malformations in the compensated stage of chronic renal failure, the cells had a diploid set of chromosomes, aneuploidy of peripheral blood cells was detected in the terminal stage of chronic renal failure. The obtained results became the basis for the recommendation to include DNA cytometry of peripheral blood cells in immunological monitoring before kidney transplantation to exclude aneuploidy. After bilateral nephrectomy, in some cases, the transformation of the aneuploid type of histograms into diploid was observed. Perhaps one of the causes of the transient change in cell ploidy that we identified is the destruction of renal tissue. Our assumption is based on the fact that previous morphological studies at the Institute of Pediatrics and Pediatric Surgery revealed the presence of destructive-dystrophic disturbances in the renal interstitium in the terminal stage of chronic renal failure with congenital malformations of the genitourinary system [Kazanskaya IV, 1987].

Example 1. Patient L., 16 years old, was admitted to the transplantation department with complaints of fever up to 39 ° C, pain in the lumbar region, urinary incontinence, weakness. Diagnosis: Neuromuscular dysplasia of the urinary system. Bilateral non-reflective megaureter. Chronic pyelonephritis. Renal hypertension. CRF, terminal stage.

History of morbi: Sick from an early age. For the first time, changes in urine tests (leukocyturia) were detected at the age of 1 year. It was examined and treated at the place of residence with a diagnosis of Chronic pyelonephritis. He was sent to the urological department of the RCCH, where after an additional examination, a congenital malformation of the genitourinary system (bilateral non-reflux megaureter) was established. For a long time was observed and received treatment in the RCCH. Recently, there has been a progression of renal failure (an increase in creatinine above 400 μmol / L, urea to 20-25 mmol / L, a decrease in CF to 25-30 ml / min).

At the next admission to the RCCH, a comprehensive examination was performed: a general analysis of peripheral blood: Eryth. - 4.12 × 10 ¹² / l; ESR-51 mm / h; Hb - 113 g / l; Lake - 11.5 × 10 ⁹ / l; fell / cores. - four%; seg / cores. - 82%; lymph - 10%; mon - four%.

General analysis of urine: color - s / f; turbidity - cloudy; the reaction is acidic; specific weight - 1003; protein - 2.4 g / l; lake. - in large. count erythritis. - 4-6-6 p / sp.

Immunograms: IgA - 1.98 g / l; IgM - 0.87 g / l; IgG - 11.0 g / l; CEC-96 c.u .; CD3-65%; CD4 / CD8 - 1.58; CD19 - 9%; CD4 - 38%; CD8 - 24%; CD16 - 3%.

According to the results of the examination, the patient showed a pronounced inflammatory reaction: accelerated ESR, leukocytosis, neutrophilia, lymphopenia, high levels of CIC, the presence of protein and white blood cells in the urine. With DNA cytometry (Fig. 1), aneuploid chromosome content was detected in 10.59% of peripheral blood cells, diploid content in 89.41%. Conservative therapy was performed. The patient's condition has stabilized. After 3 months, the child was re-admitted due to increasing azotemia (increase in creatinine above 600 μmol / L, urea - 30 mmol / L, CF below 10 ml / min) for renal replacement therapy by dialysis.

Results of dynamic observation:

General analysis of peripheral blood: Eryth. - 4.09 × 10 ¹² / l; ESR - 12 mm / h; Hb - 146 g / l; Lake - 7.0 × 10 ⁹ / l; eos. - 3%; seg / cores. - 66%; lymph 24%; mon - 7%.

General analysis of urine: color - s / f; turbidity - transparent; the reaction is neutral; specific weight - 1001; protein - 1.33 g / l; lake. - 1-0-1-0 in s / z .; salts - unit. in s / sp.

Immunograms: Ig A - 1.51 g / l; Ig M - 2.38 g / l; Ig G - 9.4 g / l; CEC - 54 c.u .; CD3 - 65%; CD4 / CD8 - 1.58%; CD19 - 9%; CD4 - 38%; CD8 - 24%; CD 16 - 3%.

Hematological and immunological parameters generally corresponded to the age norm. A deterioration in the biochemical parameters of blood was noted. When re-examining the ploidy of peripheral blood cells revealed a similar, as in the first examination, the percentage of aneuploid cells - 11% (figure 2).

The patient was constantly undergoing hemodialysis sessions throughout the year. For health reasons, a phased nephrectomy was performed, followed by a cadaveric kidney transplant (allotransplantation). In the postoperative period, there was no vascularization of the kidney and two days later the patient died.

In our opinion, destructive-degenerative changes in the kidneys have occurred in the patient’s body for a long time, which affected the chromosome apparatus of peripheral blood cells and manifested as aneuploidy. A confirmation of this is the data of histological examination of biopsy samples: Severe fibrosis of the stroma of the cerebral layer, justmedullary, median, cortical zones of the cortex. Most glomeruli are hyalinized, sclerotic.

Example 2. Patient K., 13 years old, was admitted to the transplantation department with complaints of weakness, headaches, fatigue, and increased blood pressure.

Diagnosis: Hypoplasia of both kidneys. Chronic renal failure, terminal stage.

Anamnesis morbi: Sick from birth. Repeatedly received inpatient treatment in the urological department of the RCCH for a congenital malformation of MPS (hypoplasia of both kidneys).

In a laboratory study, the following data were obtained: General peripheral blood test: Eryth. - 4.7 × 10 ¹² / l; ESR - 25 mm / h; Hb - 107 g / l; Lake - 12.2 × 10 ⁹ / l; eos. - one%; pal. / cores. - fifteen%; seg / cores. - 61%; lymph - eighteen%; mon - 5%. General analysis of urine: color - brown; turbidity - cloudy; the reaction is acidic; specific weight - 1005; protein - 1.22 g / l; lake. - 2-4-2 in the field; erythritis. - 1-3-2 in s / sp.

Immunograms: Ig A - 1.82 g / l; Ig M - 1.03 g / l; Ig G - 10.8 g / l; CEC - 26 cu; CD3 - 38%; CD4 / CD8 - 1.10%; CD19 - 23%; CD4 - 22%; CD8 - 20%; CD16 - 5%; CD25 - 36%.

Biochemical analysis of blood: creatinine - 366 μmol / l; urea - 25.9 mmol / l; CF - 0.75 ml / min.

When analyzing the results of the examination, an inflammatory reaction is noted: leukocytosis with a shift in the leukoformula to the left, accelerated ESR, turbid urine, protein is present, pronounced azotemia in the blood chemistry. The performance of the cellular link of the immune system is reduced. DNA cytometry of peripheral blood cells revealed cells with aneuploid DNA content (34.56%) (figure 3).

Conducted conservative treatment, as well as a course of hemodialysis. After discharge, the patient was under the supervision of doctors from the hemodialysis department and received basic therapy on immunological monitoring. According to vital indications, bilateral nephrectomy was performed. Data from a histological examination of the biopsy: Diffusively expressed infiltration of the stroma of the cortical and brain layers. Nephrocytes are sharply flattened, hyaline cylinders in the cavities. In the cerebral layer, stromal fibrosis, transitional epithelium of the pelvis with frequent inter-epithelial lymphocytes. The capsule of the kidneys is sharply sclerotic, about 96% of the glomeruli in the cortical and juxtamedullary zones are hyalinized (sclerotic).

After bilateral nephrectomy, the child underwent a course of hemodialysis 2-3 times a week. We present the survey data between dialysis courses.

General analysis of peripheral blood: Eryth. - 4.7 × 10 ¹² / l; ESR - 10 mm / h; Hb - 110 g / l; Lake - 8.2 × 10 ⁹ / l; pal. / cores. - 3%; seg / cores. - 63%; lymph - 28%; mon - 6%.

Immunograms: Ig A - 0.68 g / l; Ig M - 2.15 g / l; Ig G - 11.8 g / l; CEC - 38 cu; CD3 - 73%; CD4 / CD8 - 1.29%; CD19 - 9%; CD4 - 45%; CD8 - 35%; CD 16 - 9%; HLA-DR - 11%.

As you can see, hematological parameters, as well as the parameters of the humoral and cellular parts of the immune system were within the age norm. According to DNA cytometry (figure 4), peripheral blood cells had a diploid set of chromosomes (100%).

The revealed aneuploidy, in our opinion, was a transient and material substrate for the development of aneuploidy, and there were destructive-dystrophic processes in the kidneys. The results of dynamic observation after nephrectomy allowed to make a conclusion about the stabilization of the patient's condition: hematological, biochemical parameters, DNA cytometry and immune status data improved.

Thus, in children with congenital malformations in the terminal stage of chronic renal failure, peripheral blood cell aneuploidy was detected against the background of destructive-dystrophic changes in renal tissue interstitium, and therefore may be one of the indications for timely bilateral nephrectomy.

Claims (1)

A method for diagnosing the stage of chronic renal failure in congenital malformations of the genitourinary system in children, characterized in that the ploidy of peripheral blood cells is examined by DNA cytometry and, if aneuploidy is detected, intermittent or terminal stages of chronic renal failure are considered and bilateral nephrectomy is indicated.

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