ES2745352B2 - GDF15 in urine as a biomarker of chronic kidney disease - Google Patents

Description

GDF15 in urine as a biomarker of chronic kidney disease

Technical field

The present invention relates to a method and a kit for the diagnosis and/or prognosis of kidney damage that comprises analyzing a urine sample obtained from a patient and determining the urinary levels of growth and differentiation factor 15 (GDF15).

Background of the invention

Chronic kidney disease (CKD) or chronic kidney failure (CKD) is a progressive (for three months or more) and irreversible loss of kidney functions, the degree of which is determined by a glomerular filtration rate (GFR) <60 ml/min. /1.73 m2. As a result, the kidneys lose their ability to eliminate waste, concentrate urine, and preserve electrolytes in the blood.

Symptoms of impaired kidney function are nonspecific and may include a feeling of general malaise and a reduction in appetite.

CKD is often diagnosed as a result of screening in people who are known to be at risk for kidney problems, such as those with high blood pressure or diabetes and those with relatives with CKD. Chronic kidney failure can also be identified when it leads to one of its recognized complications, such as cardiovascular diseases, anemia or pericarditis.

The most common causes of CKD are diabetic nephropathy, high blood pressure, and glomerulonephritis. Together, they cause approximately 75% of all adult cases of CKD. In clinical practice, most nephropathies progress slowly toward permanent loss of renal function. Historically, kidney diseases have been classified according to the part of the kidney anatomy that is involved:

- Vascular, includes diseases of large blood vessels, such as bilateral renal artery stenosis, and diseases of small blood vessels, such as ischemic nephropathy, hemolytic uremic syndrome, and vasculitis.

- Glomerular, covering a diverse group subclassified into: Primary glomerular disease, such as focal and segmental glomerulosclerosis and IgA nephropathy.

- Secondary glomerular disease, such as diabetic nephropathy and lupus nephritis.

- Interstitial tubule, including polycystic kidney disease, chronic tubulointerstitial nephritis induced by drugs or toxins, and reflux nephropathy.

- Obstructive, for example, with bilateral kidney stones and prostate diseases.

CKD patients suffer from accelerated atherosclerosis and have a higher incidence of cardiovascular diseases, with a poorer prognosis. Initially they do not present specific symptoms and can only be detected as an increase in blood plasma creatinine. As kidney function declines:

- Blood pressure is increased due to fluid overload and the production of vasoactive hormones leading to hypertension and congestive heart failure. - Urea accumulates, leading to azotemia and ultimately uremia (symptoms range from lethargy to pericarditis to encephalopathy).

- Potassium accumulates in the blood (known as hyperkalemia), with symptoms ranging from discomfort to fatal cardiac arrhythmias.

- Erythropoietin synthesis is decreased (leading to anemia and causing fatigue)

- Fluid volume overload, symptoms range from mild edema to life-threatening acute lung edema.

- Hyperphosphatemia, due to phosphate retention that leads to hypocalcemia (also associated with active vitamin D3 deficiency) and secondary hyperparathyroidism, which leads to renal osteoporosis, osteitis fibrosa and vascular calcification.

- Metabolic acidosis, due to decreased bicarbonate generation by the kidney, leads to uncomfortable breathing and then worsening bone health.

In the initial stages of CKD, when clinical manifestations and laboratory results are minimal or non-existent, the diagnosis can be suggested by the association of non-specific manifestations by the patient, such as fatigue, anorexia, weight loss, itching, nausea , hypertension, polyuria, nocturia, hematuria or edema. Other symptoms are: oliguria, weakness, vomiting, insomnia, cramps, skin pallor, xerosis, proximal myopathy, dysmenorrhea and/or amenorrhea, testicular atrophy, impotence, cognitive or attention deficits, confusion, drowsiness, obtundation and coma. However, there is still a need to identify and validate new, more precise biomarkers of kidney damage, whose determination is also quick, simple and without the need to biopsy the patient and that allow determining the cause of kidney damage, more specifically chronic kidney damage. .

Furthermore, it is noted that during the last decade separate conceptual models have been developed for CKD and acute kidney injury (AKI; also called Acute Kidney Injury or in English, Acute Kidney Injury; AKI) that facilitate organized approaches to research and clinical trials. However, recent epidemiological and mechanistic studies indicate that the two syndromes are not different entities but are closely interconnected—CKD is a risk factor for AKI, and AKI is a risk factor for the development of CKD; In turn, both syndromes are risk factors for cardiovascular disease. Chawla LS, Eggers PW, Star RA, Kimmel PL. Acute kidney injury and chronic kidney disease as interconnected syndromes. N Engl J Med. 2014 Jul 3;371(l):58-66 Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International Supplements 2013;3: 1-150. In this sense, the present invention provides new biomarkers of kidney damage, both CKD and acute kidney failure, these biomarkers have proven to be useful for diagnosing and prognosticating both syndromes by virtue of the interconnection that both seem to have.

Brief description of the invention

Based on the experimental results provided throughout the description, the present invention deals with the following aspects presented in a summarized and general manner:

to. urinary levels of GDF15 are altered, specifically they are increased, in several models of kidney damage, both acute and chronic, and therefore constitute a biomarker of kidney damage;

b. urinary GDF15 levels contribute to differentiating the cause of kidney damage in diabetic patients, differentiating between diabetic patients with diabetic nephropathy and without diabetic nephropathy; and

c. Urinary GDF15 levels identify patients already diagnosed with CKD who have a very high risk of death, specifically 100% of patients with urinary GDF15 >13.8ng/mg died during a mean follow-up of 30 months.

Brief description of the figures

Figure 1. Correlations of GDF15 in urine with GFR and Albuminuria (coc MAU/Cr = microalbuminuria/creatinine ratio).

Figure 2. GDF15 in urine of diabetic patients with a native kidney, that is, a kidney that has not been transplanted, with and without diabetic nephropathy.

Figure 3. Patient evolution classification tree: dialysis (D), alive (V) or dead (M).

Figure 4. ROC curve of GDF15 in urine, mortality, dialysis and dialysis death.

Figure 5. Behavior of GDF15 in serum as a predictor of mortality.

Figure 6. ROC curve of GDF15 in urine as a biomarker of diabetic nephropathy in diabetic patients. (N=13)

Description of the invention

The authors of the present invention, in a transcriptomic study of a preclinical model of kidney damage, determined that the most overexpressed GDF family gene was growth and differentiation factor 15 (see GDF 15 in Table 1 shown below). Furthermore, the authors of the present invention confirmed that said increase in GDF15 expression occurred in several models of acute and chronic kidney damage, of various etiologies (Tables 2, 3 and 4).

Table 1. Renal mRNA expression of GDF family genes in experimental acute renal failure induced by photic acid, analyzed by transcriptomics, Arrays technique. Data expressed as mean ± standard error of the mean. The mRNA levels were analyzed 72 h after the induction of damage.

Table 2. Renal mRNA expression of CDF family genes in the proteinurla kidney injury model ( Induced by administration of BSA or bovine serum albumin). Analyzed by Transcriptomics, RNA-seq technique. The mRNA levels ^{were analyzed} 7 ^{days after the induction of damage.}

Table 3. Renal mRNA expression of GDF family genes in inflammation, analyzed by transcriptomics, RNA-seq technique. Data expressed as mean ± standard error of the mean. The mRNA levels were analyzed 7 days after the ^{induction of damage by injection of the inflammatory cytokine Tweak.}

Table 4 GDF15 mRNA expression ( UA) in the whole kidney of MRL/lpr mice 6 weeks ( control) vs M RL/lprl4 mice -18 weeks ( lupus nephritis). Data expressed as mean ± standard error of the mean. LN: lupus nephritis

*Semiquantitative scale from+ to 4+

Based on these results, it was established that GDF15 was a biomarker of kidney disease, increasing the expression of this marker in the kidney in various models of acute and chronic kidney damage. Furthermore, GDF15 was confirmed to be increased in urine from patients with acute kidney injury. This is confirmed as illustrated below:

GDF15 levels in urine of patients with acute kidney injury (AKI)

n= 20 AKI

n= 7 Controls

Furthermore, in order to confirm these results also in chronic kidney damage, a prospective study was carried out, in which the levels of GDF15 were analyzed by ELISA in urine samples from 73 patients and plasma from 82 patients, who underwent to kidney biopsy between September 2012 and March 2017, at the Fundación Jiménez Díaz University Hospital. All of them had a diagnosis of CKD based on glomerular filtration rate and albuminuria. These values were correlated with baseline clinical data and with patient follow-up at 29 ± 17 months. As can be seen in Table 5, urinary GDF15 levels in urine were significantly increased in patients with CKD, compared to healthy controls.

Table 5 GDF15 values in urine, patients and controls. Data expressed as mean ± standard deviation.

(Patients=73 Subjects with CKD who were going to have a kidney biopsy) (Controls= 7 Control subjects)

CKD is diagnosed based on levels of Glomerular Filtration (GFR) and Albuminuria (A), which in turn identifies people at high risk of premature death. GDF15 levels in urine are not They modify depending on the Glomerular Filtration (GFR) or Albuminuria (A) (tables 6 and 7). Figure 1 shows the correlation with these variables.

Table 6 GDF15 urine by GFR-dependent CKD category: G l, G2, G3 and G4-G5. Data expressed as mean ± standard deviation.

Where G l, G2, G3, G4-G5 are the different levels of CKD stratification based on GFR (glomerular filtration rate). The categories G l-a G5 come from an international consensus (Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International Supplements 2013;3: 1-150) and are defined as follows: G l (>90); G2 (60-89), G3a (45-59), G3b (30-44), G4 (15-29), G5 (<15). The numbers in parentheses refer to glomerular filtration rate (GFR) expressed in ml/min/1.73 m2. Additionally, we would like to point out that on page 52 of the reference Rev Nefrol Dial Trasμl. 2017; 37 (1): 48-61), the stratification of diabetic nephropathy based on GFR levels and/or albuminuria is described (page 52).

Table 7 GDF15 urine by CKD category dependent on Albuminuria: A l, A2 and A3. Data expressed as mean ± standard deviation.

Where A l, A2, A3 are the different levels of CKD stratification based on A (albuminuria). Categories A to A3 come from an international consensus (Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International Supplements 2013;3: 1-150) and are defined as follows: A l (<30); A2 (30-300), A3 (>300). The numbers in parentheses refer to the albuminuria: creatinuria (creatinine in urine) ratio expressed in mg/g. We note that the values of different analytes in urine are normalized by mg or g of creatinine in urine, since the urinary elimination of creatinine is constant in 24 hours, as exemplified by the units of albuminuria (mg/g of creatinuria), as indicated by the international guidelines (Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International Supplements 2013;3: 1-150). Throughout the text, we have The urinary elimination of GDF15 was normalized in the same way, expressing the values in ng of urinary GDF15 per mg of urinary creatinine (ng/mg). In this sense, it is not recommended to express proteins (albumin, GDF15) in urine per unit of volume (eg mg/ml), since the volume depends fundamentally on water intake and varies from minute to minute (Kidney Disease: Improving Global Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney International Supplements 2013;3: 1-150; Ortiz A, Sanchez-Niño MD, Sanz AB. The meaning of urinary creatinine concentration. Kidney Int. 2011 Apr;79(7):791).

As a result of the results of the prospective study, the following aspects were identified:

1. There is an association between urinary GDF15 levels and the diagnosis of diabetic nephropathy in patients with diabetes.

It was observed that diabetic nephropathy is the cause of CKD in which there are higher levels of GDF15 in urine compared to other causes (table 8).

Table 8. GDF1S in serum ng/ml and urine ng/mg according to diagnoses, organized in order of frequency of diagnosis. Data ^{expressed as mean ± standard deviation (n). Values obtained from one or two patients are indicated in parentheses. The} data obtained from a patient does not have a standard deviation because it is a single value.

Table 9. GDF15 in urine in diabetic patients with native (non-transplanted) kidneys with diabetic nephropathy versus diagnoses other than diabetic nephropathy.

Likewise, it was observed that patients with a diagnosis of diabetes and CKD (Chronic Kidney Disease) based on glomerular filtration rate and albuminuria, who were diagnosed with diabetic nephropathy in the biopsy compared to patients biopsied with a diagnosis of diabetes and CKD ( Chronic kidney disease) based on glomerular filtration rate and albuminuria without diabetic nephropathy in the biopsy, had significantly higher levels of GDF15 in urine (figure 2 and table 9), so urinary GDF15 values contribute to differentiating the cause of the damage kidney in diabetic patients with CKD (Chronic Kidney Disease) based on glomerular filtration and albuminuria.

2. There is an association between urinary GDF15 levels and mortality

Classification trees were made to assess whether GDF15 values in serum and urine could predict the outcome of patients with survival without dialysis, survival with dialysis or death. According to this classification, the only variable that predicted the evolution of the patients was the value of GDF15 in urine, which if it was greater than 19.9 ng/mg was associated with increased mortality: mortality 83% among patients with urinary GDF15 values > 20 ng/mg creatinine in urine (figure 3). Additionally, to assess the contribution of GDF15 on patient outcomes, logistic regression models were performed (Table 10).

Table 10. Logistic regression models to predict outcome: death, dialysis or alive, with the ^{GDF1S variables in serum and urine assessed separately.}

Units in which the variables were measured: GFF15 serum ng/ml, GDF15 urine ng/ml. Age (years). Sex (mas/fem), MAU/Cr (albuminuria:creatinuria) mg/gr, GFR ml/min/l,73m2.

In all models: multivariable, classification trees and logistic regression, a relationship between GDF15 in urine and mortality is seen. In relation to GDF15 in serum, we only saw a relationship with mortality in the logistic regression model; this could be due to the fact that in the latter, GDF15 in serum was not included in the independent variables at the same time as GDF15 in urine, as in classification tree models. This indicates that the relationship with mortality is stronger with urinary GDF15 levels. To demonstrate this, we performed two additional multivariable models in which we included as independent variables: GDF15 in serum, GDF15 in urine and age, which were the variables associated with mortality in table 10. These models show how GDF15 in serum is excluded when compared with GDF15 in urine as a predictor of mortality (see table 11).

Table 11. Logistic regression models to predict outcome: death, dialysis or alive, simultaneously assessing the ^{GDF1S variables in serum and urine.}

Units in which the variables were measured: GFF15 serum ng/ml, GDF15 urine ng/ml. Age (years).

Given this important relationship between GDF15 in urine and unwanted mortality events, ROC curves were made to graphically represent the sensitivity and specificity of GDF15 in urine to predict the final state of the patient: death, dialysis, or death on dialysis (Figure 4 ). The ROC curves obtained indicate that GDF15 has a good diagnostic quality index for predicting mortality, with an area under the curve (AUC) of 0.952 (95% CI 0.9-1; p <0.001). Youden's index was used to decide the cut-off value that maximizes the difference between sensitivity and (1 - specificity). Using this index, the cut-off point was determined: GDF15 in urine 13.83 ng/mg. Which indicates that the test is positive if the value is greater than 13.83 ng/mg with a sensitivity of 100% and specificity of 81%. The area under the curve values are lower to predict dialysis mortality: AUC 0.777 (95% CI 0.6-0.9; p=0.001) and were not significant to predict dialysis separately: AUC 0.541 (95% CI 0. 3 0.7; p=0.695).

Therefore, and based on the two statistical analyzes explained in this section, we can conclude that the higher GDF15 in urine, the higher mortality. Explain that the traditional way of identifying a cut-off point is the ROC curve that identifies the optimal point associated with greater sensitivity and specificity. He cut-off point of 13.8 ng/mg identified in the preceding paragraph, this is the ideal cut-off point to identify all high-risk patients. The cut-off point of 19.9 ng/mg mentioned in the opening paragraphs of this section only identifies about 80% of those patients who will die.

In any case and given these findings, we can affirm that urinary GDF15 levels identify patients already diagnosed with CKD who have a very high risk of death. Specifically, 100% of patients with urinary GDF15 >13.8ng/mg died during a mean follow-up of 30 months.

Therefore, and based on the experimental results provided so far, we can conclude that:

to. urinary GDF15 levels are increased in several models of acute and chronic kidney damage and, therefore, constitute a biomarker of kidney damage;

b. urinary GDF15 levels contribute to differentiating the cause of kidney damage in diabetic patients, differentiating between diabetic patients with diabetic nephropathy and without diabetic nephropathy; and

c. urinary GDF15 levels identify patients already diagnosed with CKD who have a very high risk of death, specifically 100% of patients with urinary GDF15 >13.8ng/mg died during a mean follow-up of 30 months.

On the other hand, the choice to determine urinary GDF15 levels instead of serum GDF15 levels is not causal, as shown in the examples. GDF15 is a small 25KDa protein that easily crosses the glomerular filtration barrier. However, under normal conditions, filtered GDF15 should be reabsorbed by proximal tubular cells, and it should not appear in urine. The existence of GDF15 in urine could be due to the following reasons:

• That there is an exaggerated systemic production of GDF15 that exceeds the reabsorption capacity of the tubular cells.

• That urinary GDF15 comes from an increase in synthesis at a local level, secondary to kidney damage, just as it increases the synthesis of other cytokines and mediators in the context of the inflammatory state of uremia.

Without prejudice to the above, determining urinary levels of GDF15 instead of serum levels of GDF15, provides (see example 1) notably higher values of sensitivity and specificity as biomarker predictor of 30-month mortality in patients suffering from CKD. Patients with CKD have a high risk of premature mortality. However, within CKD different patients may have a different risk of premature mortality, the inability to identify these very high risk patients has a negative impact on the selection of patients for clinical trials. In general, clinical trials in patients with CKD who have a primary end point of serious events or mortality have failed. In at least some of them, the reason for failure was poor enrichment in high-risk patients. The possibility that GDF15 levels in urine offer to identify patients with a very high risk of death can help design clinical trials focused on these patients that allow the identification of new treatments that reduce mortality. In this sense, we must emphasize that GDF15 in urine predicted high mortality in patients followed up in nephrology units, that is, in patients treated, according to the current state of the art. This implies that current treatments are unable to avoid the mortality associated with high levels of GDF15 in urine.

On the other hand, determining urinary levels of GDF15 instead of serum levels of GDF15 allows us to differentiate between diabetic patients who suffer from diabetic nephropathy from those who do not, when serum levels of GDF15 do not allow this differentiation (see example 2). Therefore, and in this sense, the clinical usefulness of determining GDF15 in urine, to support or rule out the existence of diabetic nephropathy, would be of great interest, because it would avoid performing a kidney tissue biopsy, a highly invasive procedure, in patients with high clinical suspicion of diabetic nephropathy and a high urinary GDF15. On the other hand, a high clinical suspicion of diabetic nephropathy and a low GDF15 in urine could suggest performing a kidney biopsy to rule out another cause of CKD that requires a different treatment than the general management of CKD, such as immunosuppressive treatment.

Thus, the present invention provides a non-invasive method for the diagnosis and/or prognosis of kidney damage, easily usable in daily clinical practice, through a simple, rapid and economical test that allows the serial determination of a high volume of samples. .

Thus, in a first aspect, the present invention refers to the use of at least the urinary levels or concentration of the GDF15 protein, optionally before, after or simultaneously with the determination of the glomerular filtration rate and/or albuminuria, preferably after the determination of glomerular filtration rate and/or albuminuria, as a diagnostic and/or prognostic marker of kidney damage. By kidney damage in the present invention we refer, without limitation, to any functional or structural abnormality of diverse etiology, whether due to a decrease in renal or intrarenal perfusion, due to toxic aggression or obstruction of the renal tubule, due to inflammation. tubulointerstitial and edema or by a reduction in the filtration capacity of the glomerulus. In a more preferred embodiment of the present invention, the kidney damage that can be diagnosed and/or predicted is acute or chronic kidney damage, more preferably the kidney damage that can be diagnosed and/or predicted is chronic kidney damage (CKD). .

By acute kidney injury in the present invention we refer to kidney damage whose functional or structural abnormality that produces it is present for less than three consecutive months. More than three consecutive months of said functional or structural abnormality would produce chronic kidney damage (Kidney Disease: ImprovingGlobal

Outcomes (KDIGO) CKD Work Group. KDIGO 2012 Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney Disease. Kidney InternationalSupplements 2013;3:1-150).

It is noted that, according to any of the aspects of the present invention, GDF15 levels can be measured or determined by themselves or can be normalized with respect to urinary creatinine levels.

In a preferred embodiment of the first aspect of the invention, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate. and/or albuminuria, preferably the urinary levels or concentration of GDF15 are determined subsequent to the determination of glomerular filtration rate and/or albuminuria, as a diagnostic marker of diabetic nephropathy. Preferably, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate and/or albuminuria, preferably the levels or urinary GDF15 concentrations are determined after determining glomerular filtration rate and/or albuminuria, as a differential diagnostic marker of the etiological cause of kidney damage in diabetic patients. More preferably, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate and/or albuminuria, preferably the Urinary levels or concentration of GDF15 are determined after the determination of glomerular filtration rate and/or albuminuria, as a differential diagnostic marker of the etiological cause of kidney damage in diabetic patients, differentiating between diabetic patients with diabetic nephropathy and without diabetic nephropathy, where said patients Diabetics preferably had been previously diagnosed with CKD based on the determination of glomerular filtration rate and/or albuminuria.

In another preferred embodiment of the first aspect of the invention, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate. and/or albuminuria, preferably the urinary levels or concentration of GDF15 are determined after the determination of the glomerular filtration rate and/or albuminuria, as a prognostic marker of mortality for those patients or patients diagnosed with kidney damage, whether this damage is acute or chronic. Preferably, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate and/or albuminuria, preferably the levels or urinary concentrations of GDF15 are determined after the determination of glomerular filtration rate and/or albuminuria, as a prognostic marker of mortality in those patients diagnosed with chronic kidney damage. More preferably, the present invention relates to the use of at least the urinary levels or concentration of GDF15, optionally said urinary levels or concentration of GDF15 are determined before, after or simultaneously with the determination of the glomerular filtration rate and/or albuminuria, preferably the Urinary levels or concentration of GDF15 are determined after the determination of glomerular filtration rate and/or albuminuria, as a prognostic marker of mortality in those patients diagnosed with chronic kidney damage; where mortality prognosis means the death of the patient in a period of less than 5 years, preferably less than 3 years, more preferably less than 30 months, from the determination of the increased concentration of GDF15 in the patient's urine. Preferably, said patients had previously been diagnosed with CKD based on the determination of glomerular filtration rate and/or albuminuria.

In a second aspect, the present invention refers to a method for obtaining data useful in the diagnosis and/or prognosis of kidney damage that comprises determining the urinary levels or concentration of GDF15 in an isolated urine sample. In a particular embodiment, said level or concentration is determined by fluorometric analysis or by any type of immunoassay (including ELISA) or by mass spectrometry. In a preferred embodiment of the second aspect of the invention, the method is for obtaining data useful in the diagnosis of diabetic nephropathy. Preferably, the method is for obtaining data useful in the differential diagnosis of the etiological cause of kidney damage in diabetic patients. More preferably, the This method is for obtaining useful data in the differential diagnosis of the etiological cause of kidney damage in diabetic patients, providing data that helps in the differentiation between diabetic patients who suffer from CKD, preferably said diagnosis of CKD is made based on glomerular filtration. /or albuminuria, with diabetic nephropathy and without diabetic nephropathy.

In another preferred embodiment of the second aspect of the invention, the method is for obtaining data useful in the prognosis of mortality of those patients diagnosed with kidney damage, whether this damage is acute or chronic. Preferably, the method is for obtaining data useful in the mortality prognosis of those patients diagnosed with chronic kidney damage. More preferably, the method is for obtaining useful data in the mortality prognosis of those patients diagnosed with chronic kidney damage, preferably said diagnosis of CKD is made based on glomerular filtration and/or albuminuria, where for mortality prognosis, means the death of the patient in a period of less than 5 years, preferably less than 3 years, more preferably less than 30 months, from the determination of the increased concentration of GDF15 in the patient's urine.

In a third aspect, the present invention relates to a method for the diagnosis and/or prognosis of kidney damage that comprises analyzing a urine sample obtained from a patient, determining at least the urinary levels or concentration of GDF15, and comparing said level. or concentration with a control value, where the alteration of said level or concentration, where preferably said alteration constitutes an increase with respect to the control value, is indicative of kidney damage. In a preferred embodiment of the third aspect of the invention, the method is for the diagnosis of diabetic nephropathy, where at least the urinary levels or concentration of GDF15 are determined, and said level or concentration is compared with a control value, where the alteration of Said level or concentration, where preferably said alteration constitutes an increase with respect to the control value, is indicative of diabetic nephropathy. Preferably, the method is for the differential diagnosis of diabetic nephropathy in a diabetic patient diagnosed with CKD, preferably said diagnosis of CKD is made based on glomerular filtration and/or albuminuria, where at least the urinary levels or concentration of GDF15 are determined. , and said level or concentration is compared with a control value, where the alteration of said concentration level, where preferably said alteration constitutes an increase with respect to the control value, is indicative that the etiological cause of kidney damage in said diabetic patients is diabetic nephropathy.

In another preferred embodiment of the third aspect of the invention, the method is for the prognosis of mortality of those patients diagnosed with kidney damage, whether this damage is acute or chronic, which It comprises analyzing an isolated urine sample obtained from the patient, determining at least the urinary levels or concentration of GDF15, and comparing said level or concentration with a control value, where the alteration of said level or concentration, where preferably said alteration constitutes an increase with respect to at the control value, it is indicative of a prognosis of death of the patient in a period of less than 5 years, preferably less than 3 years, more preferably less than 30 months, from the determination of the increased concentration of GDF15 in the patient's urine. Preferably, the method is for the mortality prognosis of those patients diagnosed with chronic kidney damage, where preferably said diagnosis of CKD is made based on glomerular filtration and/or albuminuria.

On the other hand, the characterization of urinary GDF15 serves not only to elucidate the diagnosis or prognosis of kidney disease, help identify the mechanism of kidney damage, and/or indicate the site and severity of damage, but should also help to monitor the response to treatment, to screen or select patients and to treat those patients in accordance with the selection or screening carried out.

Therefore, a fourth aspect of the invention relates to a method of monitoring the response to treatment of a patient diagnosed with kidney damage and who is currently subject to treatment, which comprises analyzing a urine sample obtained from the patient, determining at least the urinary levels or concentration of GDF15 in said sample, and comparing said level or concentration with the level or concentration of GDF15 in a urine sample obtained at a previous moment in time from the same patient, where the alteration of said level or concentration of form That said alteration constitutes a reduction with respect to the previous value is indicative of a favorable progression of the patient; and where the alteration of said level or concentration so that said alteration constitutes an increase with respect to the previous value, is indicative of an unfavorable progression of the patient. In a preferred embodiment of the fourth aspect of the invention, the patient has been diagnosed with CKD. In another preferred embodiment of the fourth aspect of the invention, the patient is a diabetic diagnosed with CKD and diabetic nephropathy.

In a fifth aspect, the invention relates to a method for screening diabetic patients who suffer from or have been diagnosed with CKD, preferably said diagnosis of CKD is made based on the GFR and/or A, which comprises analyzing a urine sample. obtained from the patient, determine at least the urinary levels or concentration of GDF15, and compare said level or concentration with a control value, where if there is an alteration of said level or concentration and said alteration constitutes an increase with respect to the control value, the patient is classified as a diabetic patient suffering from diabetic nephropathy; and where if there is no alteration of said level or concentration or if This alteration constitutes a reduction with respect to the control value, the patient is classified as a diabetic patient who suffers from CKD other than diabetic nephropathy. Patients with CKD other than diabetic nephropathy will usually undergo a biopsy to determine the cause of the CKD. Those patients who suffer from diabetic nephropathy will not undergo a biopsy once the cause of CKD has been determined.

In a preferred embodiment of the fifth aspect of the invention, the patient who suffers from diabetes, whether diabetes mellitus type I or II and is classified according to the fifth aspect of the invention as a patient suffering from diabetic nephropathy, is treated with at least one pharmaceutical composition selected from the following: statins, antiplatelet drugs, sodium glucose cotransporter type 2 (SGLT2) inhibitors such as empagliflozin or canagliflozin, or glucagon-like peptide-1 (GLP-1) analogues such as liraglutide . Preferably if the patient suffers from type II diabetes mellitus and is classified according to the fifth aspect of the invention as a patient suffering from diabetic nephropathy, he is treated with sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin or canagliflozin.

A sixth aspect of the invention refers to a method of screening patients with a high risk of death and who suffer from or have been diagnosed with kidney disease, preferably CKD and where preferably said diagnosis of CKD is made based on glomerular filtration and/or or albuminuria, which includes analyzing an isolated urine sample obtained from the patient, determining at least the urinary levels or concentration of GDF15, and comparing said level or concentration with a control value, where if there is an alteration of said level or concentration and said alteration constitutes an increase with respect to the control value, the patient is selected as a patient with a prognosis of death in a period of less than 5 years, preferably less than 3 years, more preferably less than 30 months, from the determination of the increased concentration. of GDF15 in the patient's urine. Preferably, the method is for the mortality prognosis of those patients diagnosed with chronic kidney damage, where preferably said diagnosis of CKD is made based on the GFR and/or the A. In a preferred embodiment of the sixth aspect of the invention, the patient, diabetic or not, who suffers from kidney disease, preferably CKD and where preferably said diagnosis of CKD is made based on the GFR and/or the A, and is classified according to the sixth aspect of the invention as a patient with a prognosis of death. In a period of less than 5 years, it is treated with at least one pharmaceutical composition selected from the following: statins, antiplatelet drugs, sodium-glucose cotransporter 2 (SGLT2) inhibitors such as empagliflozin or canagliflozin, or with peptide analogues. similar to glucagon-1 (GLP-1) such as liraglutide.

In a seventh aspect, the present invention refers to a kit or device, hereinafter kit or device of the invention, for the diagnosis and/or prognosis of kidney damage according to any of the methods described above, which comprises the necessary reagents to determine the level or concentration of GDF15 in a urine sample such as anti-GDF15 antibodies, or fragments thereof or anti-GDF15 aptamers or fluorochromes. In a preferred embodiment, the kit comprises the substrates necessary to quantitatively determine the concentration or level of GDF15 by a fluorometric method or by any type of immunoassay (such as an ELISA) using anti-GDF15 antibodies or fragments thereof or aptamers. anti-GDF15. In a fifth aspect, the present invention refers to the use of said kit for the diagnosis and prognosis of kidney damage, preferably to carry out any of the methods described in the second to sixth aspects of the present invention.

The following examples merely illustrate, but in no way limit the present invention.

Examples

Example 1. GDF15 ROC curves

The ROC curves shown in Figures 4 and 5 justify that GDF15 in urine is a significantly better predictive biomarker of mortality than in serum.

The data in Figure 4 illustrate the behavior of GDF15 in urine as a predictor of mortality:

AUC: 0.952 (p<0.001)

Youden index: 0.810

Cutoff point: 13.83 ng/mg

Sensitivity: 1

1-Specificity: 0.190

The data in Figure 5 illustrate the behavior of GDF15 in serum as a predictor of mortality: AUC: 0.896 (p<0.001)

Youden index: 0.701

Cutoff point: 2.98 ng/ml

Sensitivity: 0.889

I-Specificity: 0.188

Example 2. Comparison between determining urinary levels of GDF15 instead of serum levels of GDF15, for the diagnosis of diabetic nephropathy

Serum and urine GDF15 values were analyzed, observing that patients with higher GDF15 values corresponded to patients with a diagnosis of diabetic nephropathy, in blood they were 2 times higher and in urine 3 times higher in patients with diabetic nephropathy compared with other diagnoses.

However, checking whether or not a patient suffers from diabetic nephropathy is especially useful in diabetic patients, in order to differentiate the cause of chronic kidney disease. Therefore, urinary GDF15 levels were compared between diabetic patients with diabetic nephropathy and diabetic patients without diabetic nephropathy: diabetic patients with diabetic nephropathy had urinary GDF15 levels 3 times higher and no difference was seen with serum GDF15 ( see figure 2 ^).

The clinical usefulness of determining GDF15 in urine, to support or rule out the existence of diabetic nephropathy without performing a renal biopsy, could be of great interest, because it would avoid performing the biopsy, an invasive procedure, in patients with a high clinical suspicion of diabetic nephropathy and a high GDF15 in urine. On the other hand, a high clinical suspicion of diabetic nephropathy and a low GDF15 in urine could suggest performing a kidney biopsy to rule out another cause of CKD that requires a different treatment than the general management of CKD, such as immunosuppressive treatment.

Figure 6 illustrates the ROC curve of GDF15 in urine as a biomarker of diabetic nephropathy in diabetic patients: (N=13)

AUC: 0.900 (p=0.0i9)

Youden index: 0.750

Cutoff point: 10,486 ng/mg

Sensitivity: 1,000

I-Specificity: 0.250

Claims (1)

1. In vitro use of urinary levels or concentration of growth and differentiation factor 15 (GDF15), normalized with respect to urinary levels or concentration of creatinine, greater than 13.8 ng/mg as a predictive marker of mortality in diagnosed patients with chronic kidney disease (CKD).