RU2453852C1 - Method for prediction of pesponse rate to immunosuppressive therapy of chronic glomerulonephritis with nephrotic syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: therapy is preceded by determining the urine nephrine content. If the values are less than 17 ng/ml, a response rate to immunosuppressive therapy is predicted.

EFFECT: use of the method allows predicting the response rate to immunosuppressive therapy in the patients with chronic glomerulonephritis combined with nephrotic syndrome.

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Description

The invention relates to medicine, in particular to therapy and nephrology.

In modern nephrology, clinical criteria (age, gender, presence of arterial hypertension, impaired renal function, duration of NS) are used to predict the course of chronic glomerulonephritis (CGN) with nephrotic syndrome (NS), the effectiveness of immunosuppressive therapy and the choice of tactics for managing a patient.

For example, Shiiki H. et al. Prognosis and risk factors for idiopathic membranous nephropathy with nephritic syndrome in Japan // Kidney Int. 2004; 65: 1400-1407 based on multivariate analysis, it was shown that the factors of poor prognosis in patients with CGN (membranous nephropathy) are male, over 60 years old, increased serum creatinine and tubulointerstitial changes in kidney biopsy.

However, in this case, clinical criteria are developed for only one variant of CGN in a specific population. To evaluate additional prognosis criteria - morphological (tubulointerstitial fibrosis, arteriolosclerosis, glomerulosclerosis in a kidney biopsy specimen, morphologically unfavorable forms of nephritis), an expensive invasive kidney biopsy procedure is necessary.

One of the biomarkers that reflect the degree of damage to podocytes (podocytic dysfunction) in patients with CGN is the level of urinary excretion of the structural protein of the slit diaphragm - nephrin (nephrinuria). To date, the study of nephrinuria (NU) was carried out mainly in the experiment. In the work of Nakatsue T., Koike H., Han G. D. et al. Nephrin and podocin dissociate at the onset of proteinuria in experimental membranous nephropathy. Kidney int. 2005; 67: 2239-2253 clarified the role of excretion of nephrine in the urine and reduction of its expression in the kidney tissue as an important reason underlying the development of proteinuria.

In humans, the method of assessing nephrinuria (Western blotting) was used by Patari A., Forsblom C., Havana M. et al. Nephrinuria in diabetic nephropathy of type 1 diabetes. Diabetes 2003; 52: 2969-2974. However, urinary excretion of nephrin was assessed qualitatively only for the purpose of early diagnosis of diabetic nephropathy in patients with diabetes mellitus; no precise values of nephrinuria were established to assess the prognosis.

The objective of the invention is a method for predicting the effectiveness of immunosuppressive therapy in patients with chronic glomerulonephritis with nephrotic syndrome.

The problem is solved by the method, which consists in the fact that in patients with chronic glomerulonephritis with nephrotic syndrome, the level of nephrin in the urine is determined before the start of therapy and when its value is less than 17 ng / ml, the effectiveness of immunosuppressive therapy is predicted.

In practice, the method is as follows:

1. 10 ml of morning urine is centrifuged to separate cells and sediment at 3000 vol. for 15 min, the supernatant is frozen at a temperature of -20 ° C.

2. Immediately before the test, urine samples are kept at room temperature until completely thawed.

3. Add 100 μl of a standard solution (biotin-conjugated polyclonal antibodies to nephrin) and a urine sample to each well of the kit, incubate for 2 hours at 37 ° C; after the liquid is removed without washing the wells.

4. Add 100 μl of reagent A (avidin conjugated with peroxidase), incubated for 1 hour at 37 ° C.

5. Wash 400 μl of the Wash solution 3 times, at the end remove the remaining solution with absorbent paper.

6. Add 100 μl of reagent B (TMB solution), incubate for 30 min at 37 ° C, and the solution turns blue.

7. Add 50 μl of a stop solution (sulfuric acid), incubate for 15-25 minutes (no more than 30 minutes) at a temperature of 37 ° C, when a stop reagent is added, the solution turns yellow.

8. The indicators are taken on a spectrophotometer with a wavelength of 450 nm.

9. The obtained spectrophotometer values are converted to ng / ml by constructing a standard calibration curve using the STATISTICA 6.0 program.

Nephrinuria indicators were obtained as a result of examination of 74 patients with various degrees of activity and progression of CGN (from 18 to 74 years), of which 38 patients with nephrotic syndrome (NS) were hospitalized in the clinic of nephrology, internal and occupational diseases of the First Moscow State Medical University them. I.M.Sechenova from 2007 to 2010.

A detailed analysis of urinary nephrin excretion was carried out in patients with NS - in the group with the clinically most pronounced damage to the glomerular filter. NU in this category of patients was characterized by high variability from 0.5 to 58 ng / ml. The timing of achieving remission of nephrotic syndrome in the appointment of immunosuppressive therapy was evaluated in 23 patients with initially different levels of nephrinuria. Having calculated the diagnostic sensitivity and specificity of the NU indicator for predicting the effectiveness of treatment in patients with NS, an ROC curve was constructed and a cut-off separation point of 17 ng / ml was determined. Groups of CGN patients with NU values above and below this level did not significantly differ in age, gender, severity of nephrotic syndrome, the presence of arterial hypertension and impaired renal function at the start of immunosuppressive therapy.

In 9 out of 11 (82%) patients with low (<17 ng / ml) urinary nephrin excretion, NS remission was achieved within six months of treatment (median was 4 months), and in 5 patients of this subgroup, NS was stopped quite quickly - within 4 months. At the same time, among 12 patients with high (> 17 ng / ml) NU indicators, only 2 to 6 months of treatment had a partial remission of NS; in 2 patients, NS managed to be stopped as a result of longer immunosuppressive therapy (within 10 and 27 months ) In 8 out of 12 (67%) patients with high NU, there was no response to adequate therapy with various immunosuppressive drugs (including the administration of ultra-high doses of prednisone and cyclophosphamide, treatment with cyclosporine and mycophenolate mofetil), which was carried out for a long time (median 27 months) from 9 months to 2.5 years (figure 1). Figure 1 shows two curves constructed by the Kaplan-Mayer method, reflecting the timing of achieving remission of the nephrotic syndrome in the appointment of immunosuppressive therapy. The abscissa axis shows the duration of immunosuppressive therapy in months, the ordinate axis represents the percentage (%) of patients with persisting NS in the group. The continuous curve reflects the timing of achieving NS remission (the effectiveness of immunosuppressive therapy) in patients with NU less than 17 ng / ml, and the dashed curve with NU more than 17 ng / ml. An empty marker (o) indicates the effectiveness of immunosuppressive therapy and the achievement of remission of the nephrotic syndrome, a cross (+) indicates a lack of response to ongoing immunosuppressive therapy and preservation of NS. The differences between the groups are statistically significant (Gehan test = -3.154, p = 0.002, logrank test = 2.97, p = 0.003).

Having calculated the odds ratios, it was shown that in the absence of the effect of immunosuppressive therapy after 6 months in patients with NU> 17 ng / ml, the chances of achieving remission of NS are further reduced by 7 times. On the contrary, in patients with NU <17ng / ml the probability of achieving remission of NS during the first 6 months of treatment is very high - 23 times higher than in patients with its high level (OS = 22.5, p <0.05).

Figure 2 shows the ROC curve, the ordinates show the sensitivity points of the method for determining nephrinuria to predict the effectiveness of immunosuppressive therapy, the ordinates show 1-specificity, the dotted line indicates the optimal sensitivity (80%) and specificity (85%) for the level of nephrinuria less than 17 ng / ml. The nature of the location of the ROC curve and the high area under the curve (AUC = 0.864) indicates that the level of NI is highly informative in predicting the response to treatment with immunosuppressive drugs.

The following examples illustrate the method of the invention.

Clinical example N1.

Patient R., 36 years old, was in the nephrology department of the clinic from 10/27/2009 to 02/17/2009 with a diagnosis of Chronic glomerulonephritis of the nephrotic type (morphologically - focal segmental glomerular sclerosis, biopsy from 11/18/2009) with intact function the kidneys.

Complaints upon admission: headaches, severe swelling of the legs, episodes of increased blood pressure to 150/100 mm Hg

From the anamnesis: from May 2009, noted an increase in blood pressure to 150/100 mm Hg, took antihypertensive drugs (ACE inhibitors), a few months later, swelling of the legs appeared (up to the upper third). Examined at the Russian Cardiology Center. Nephrotic syndrome was detected: SPU up to 3.92 g / day, total protein - 38.3 g / l, albumin - 15.8 g / l, kidney function was preserved (serum creatinine within normal limits - 95-88 μmol / l) . To conduct a kidney biopsy and resolve the issue of management tactics, the patient was referred to the clinic of nephrology, internal and occupational diseases of MMA named after I.M.Sechenova.

At admission: a state of moderate severity. The skin of normal color, clean. Peripheral lymph nodes are not enlarged. Severe swelling of the legs. In the lungs, vesicular breathing, no wheezing. NPV 16 min Heart sounds are muffled, the rhythm is correct. HELL 120/80 mm Hg, heart rate = Ps = 60 beats. in minutes The abdomen is soft, painless on palpation. S / m Pasternatsky negative on both sides. Dysuria, nocturia.

When examined in an. blood: hemoglobin 134-127 g / l, red blood cells - 4.4-4.0 × 10 ¹² / l, white blood cells - 4.2-4.48 × 10 ⁹ , ESR - 51-32 mm / h,

In an. urine: protein - 12.96-3.5 g / l, red blood cells - 0-3; 1-2 in s / sp., White blood cells - 1-3 in s / sp.

Daily proteinuria - 5.51-10

Biochem. an. blood: total protein - 4.3-5.1-5.2 g / dl, albumin - 2.3-2.4-2.6 g / dl, creatinine - 0.93-1.1 mg / dl, glucose - 4.2 mmol / l, uric acid - 312 μmol / l (149-416). Sodium - 141-123 meq / l, potassium 4.6-4.8 meq / l.

Reberg test: GFR - 108-78 ml / min, reabsorption - 99.0-98.0%

Electrophoresis of serum proteins: albumin: 46%, alpha-1 - 4.7%, alpha-2 - 21.6%, beta - 20.9%, gamma - 6.8%

Blood lipids: triglycerides - 3.23-1.74 mmol / L (0.57-2.28), total cholesterol - 14.55-7.87 (3.88-6.47) mmol / L, VLDL - 0.6-0.3 (0.114-0.342) mol / L.

Immunology: IgA - 275 mg / dl, IgM - 174 mg / dl, IgG - 620 mg / dl, RF - Neg., Complement - 31.2.

Coagulogram: APTT - 1.11 (0.75-1.25), PI - 110% (86-110), fibrinogen - 7.10 g / l (1.8-4.0).

ECG: EOS is located vertically. Sinus rhythm, correct. Moderately pronounced changes in the myocardium of the left ventricle.

Ultrasound of the kidneys: The kidneys are usually located, the contours are even, of normal size: the left is 102 × 57 mm, the thickness of the parenchyma is 18 mm, the right is 111 × 55 mm, the thickness of the parenchyma is 17 mm, the cortico-medullary differentiation is preserved. CLS is not expanded. Renal mobility during breathing is normal. With CDK, the blood flow is not changed, it can be traced to the peripheral departments. Kidney biopsy: Kidney biopsy is represented by a cortical layer (up to 14 glomeruli), 2 glomeruli are completely sclerotic. In 2 glomeruli, the areas of focal expansion and hyalinosis / sclerosis of the mesangium in the handle area, focal thickening of BMC, expansion and sclerosis of the mesangium, sclerosis of vascular loops, single synechia are determined. In the remaining glomeruli, minimal changes, plethora. Epithelium of convoluted tubules in a state of protein dystrophy and focal subatrophy, focal sclerosis and focal lymphohistiocytic infiltration of the stroma. Arteriolosclerosis. Amyloid not found. In the IG study, fixation of IgG, A, M, and C3 and fibrinogen on focal granular GBM was found. Conclusion: morphology fits into the picture of focal segmental glomerular hyalinosis.

A low level of nephrin in the urine was revealed - 14.4 ng / ml, which allows us to conclude that the prescribed immunosuppressive therapy will be effective.

Given the morphological variant of nephritis, cyclosporin therapy was prescribed 150 mg / day, metipred 24 mg per os. After 2 months of therapy, remission of the nephrotic syndrome was achieved - daily proteinuria was 1.1 g / day, total protein - 6.0 g / dl, albumin - 3.4 g / dl, kidney function was preserved (creatinine 1.1-1.06 mg / dl). Due to the development of infectious complications, cyclosporine was canceled, continued taking metipred at a dose of 12 mg / day. Nephrotic syndrome did not recur, proteinuria is maintained up to 1 g per day, kidney function is preserved.

Based on clinical features (long-term persistent nephrotic syndrome), an unfavorable morphological variant of CGN (FSH) with signs of emerging interstitial fibrosis, a progressive course and a high probability of no response to the therapy could be assumed in this patient. However, at the beginning of treatment, a positive effect was observed: proteinuria decreased, the level of total protein and serum albumin increased, and within 2 months, despite the need for a more rapid withdrawal of cyclosporine due to a recurrent infection, a disease remission was achieved. This was predicted by a low level of nephrinuria (<17 ng / ml), reflecting moderate podocytic dysfunction and the reversibility of violations of the filtration barrier with timely immunosuppression.

Clinical example N2.

Patient M., 23 years old, was in the nephrology department of the clinic from 09/26/2006 to 10/09/2006 with a diagnosis of Chronic glomerulonephritis of the nephrotic type (morphologically - mesangioproliferative variant, biopsy from 10/19/2006) with intact renal function.

From the anamnesis. Since January 2006, after an intestinal infection and taking immunomodulators, swelling of the legs appeared. During the examination, NS was detected (SPU was 2.6-3.5 g / day, total protein - 4.9-5.1 g / dl, albumin 2.8-3.1 g / dl, cholesterol - 319 mg / dl), creatinine 1.0-0.9 mg / dl, GFR - 66-104 ml / min. Therapy was carried out with ACE inhibitors, statins. She was hospitalized in a clinic for a kidney biopsy and development of patient management tactics.

Upon receipt, the condition is satisfactory. The skin is dry, acne on the face. Palpable submandibular l / y, painless, elastic. Slight swelling of the feet. In the lungs, vesicular breathing, no wheezing. NPV 16 min Heart sounds are clear, rhythmic. HELL 140/80 mm Hg, HCC = Ps = 70 beats. in minutes The abdomen is soft, painless on palpation. Pasternatsky’s syndrome is negative on both sides. Dysuria, nocturia.

When examined in an. blood: hemoglobin 134 g / l, red blood cells - 4.1.0 × 10 ¹² / l, white blood cells - 5.64 × 10 ⁹ , ESR - 35 mm / h,

In an. urine: Protein - 7.55 g / l, red blood cells - 3-6 in n / a., white blood cells - 3-5 in n / a.

Daily proteinuria - 3.9 g

Biochem. An. blood: total protein - 4.8 g / dl, albumin - 2.9 g / dl, creatinine - 0.9 mg / dl, glucose - 89 mg / dl, uric acid - 3.7 mg / dl. Sodium - 142 meq / l, potassium 4.2 meq / l.

Reberg's test: GFR - 81 ml / min, reabsorption - 99.5%. Electrophoresis of serum proteins: albumin: 50.9%, alpha-1 - 4.8%, alpha-2 - 16.2%, beta - 18.1%, gamma - 10%

Blood lipids: triglycerides - 142 mg / dl, total. Chs - 336 mg / dl, VLDL - 28.4 mg / dl.

Immunology: IgA - 500 mg / dl, IgM - 170 mg / dl, IgG - 84 mg / dl, RF - neg., Complement - 34.9, CRP - neg.

Ultrasound of the kidneys: The right kidney is lowered by 7 cm, dimensions: left 123 × 48 mm, parenchyma thickness - 16 mm, right - 115 × 50 mm, parenchyma thickness 17 mm, cortico-medullary differentiation is preserved. CLS is not expanded.

Kidney biopsy: A kidney biopsy is represented by a cortical layer (up to 8 glomeruli). In the glomeruli, focal proliferation of mesangiocytes, focal expansion of mesangium, focal thickening of BMC, sclerosis of individual vascular loops are noted. Epithelium of convoluted tubules in a state of protein dystrophy, focal sclerosis of the stroma. There are small lymphohistiocytic infiltrates. Amyloid not found. Conclusion: changes fit into the picture of mesangioproliferative GN.

A high level of nephrin in the urine was detected - 20.7 ng / ml, which allows us to predict the ineffectiveness of immunosuppressive therapy.

In November 2006, treatment of MP was started at a dose of 48 mg per day and by intravenous administration of ultra-high doses of MP and CFA. During the year, 9 combined “pulses” of MP (total dose of 7975 mg) and CFA (total dose of 4200 mg) were performed, however, NS (SPU - 4.5-6.96 g / day, total Protein - 4.9 g) remained / l (57-82), albumin - 27.2 (32-48) g / l, Xc - 359 mg / dl (150-250).

The patient had a moderate nephrotic syndrome with preserved renal function, mild arterial hypertension, which, in combination with a mild morphological variant of the disease, mesangioproliferative GN with a low degree of sclerosis in the tubulointerstitial structures of the kidney, revealed a favorable prognosis. However, despite the adequate dose and duration of immunosuppressive therapy, the expected effect was not obtained. A high risk of immunosuppressive therapy failure is indicated by a high level of nephrin in the urine detected in the patient, which reflects the severity of damage to the podocytes.

A method is proposed that allows to predict the effectiveness of immunosuppressive therapy in patients with CGN with nephrotic syndrome and is the starting point for choosing the treatment tactics for these patients.

Claims (1)

A method for predicting the effectiveness of immunosuppressive therapy of chronic glomerulonephritis with nephrotic syndrome, which consists in determining the level of nephrin in the urine before therapy, and when its value is less than 17 ng / ml, the effectiveness of immunosuppressive therapy is predicted.

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