WO2008129265A1

WO2008129265A1 - Diagnostics and therapeutics for diabetic nephropathy involving ccl18

Info

Publication number: WO2008129265A1
Application number: PCT/GB2008/001368
Authority: WO
Inventors: Frederick Wai Keung Tam
Original assignee: Imperial Innovations Limited
Priority date: 2007-04-21
Filing date: 2008-04-18
Publication date: 2008-10-30
Also published as: GB0707735D0

Abstract

A method for aiding in the assessment of diabetic nephropathy in a patient, the method comprising the step of determining the level of CCLl 8 protein and/or nucleic acid in a sample from the patient. The method may be for assessing the likely progression of diabetic nephropathy in the patient or for assessing the likely progression of response of the patient to treatment for diabetic nephropathy.

Description

DIAGNOSTICS AND THERAPEUTICS FOR DIABETIC NEPHROPATHY INVOLVING CCL18

The current invention relates to the diagnosis and treatment of diabetic nephropathy.

Background

Diabetes is the most common cause of end stage renal failure in the developed world, and inflammation and fibrosis are important mechanisms in the progression of this disorder.

There is a need to identify predictors of fibrosis in diabetic nephropathy and to understand how the disorder is able to progress despite the widespread use of antagonists of the renin/angiotensin system. Fibrosis of renal tissues is a critical event in the progression of renal failure from a variety of causes ranging from diabetic nephropathy, hypertensive nephropathy to immune mediatied nephritis. With deposition of extracellular matrix and loss of normal renal tissue, the renal structure is replaced by scar tissue. These processes result in irreversible loss of renal function. Severe renal fibrosis is frequently the final step of progression to end stage renal failure.

Pro-inflammatory and fibrotic cytokines monocyte chemoatractant protein- 1 (MCP-I) and connective tissue growth factor (CTGF) can be found in the urine of patients with diabetic nephropathy and may be markers for progression of diabetic nephropathy (Tarn et al. (2005) Renal Association abstract: RA5255).

CCLl 8 is synthesized by monocytes/macrophages and dendritic cells, is known to stimulate collagen production in pulmonary fibroblasts and is reported to cause fibrosis independent of TGF-β (Luzina et al. (2006) J. Cell Physiol 206: 221- 228). The potential role of CCLl 8 in the progression of renal diseases has not previously been investigated. In diabetic nephropathy, an increased number of macrophages are found (ref: Nguyen D et al Nephrology 11:226-231, 2006), but the number were far less than in glomerulonephritis. As CCLl 8 is produced by macrophages in fibrotic lung diseases, it will be logical to predict that CCLl 8 will have a more important role in the pathogenesis of glomerulonephritis than that of diabetic nephropathy (ref. Ferrario et al Kidney International 28:513-9, 1985). To our surprise, we found that urinary CCLl 8 correlated with the severity of diabetic nephropathy, but not with non-diabetic renal disease in our research project. A role for CCLl 8 in diabetic nephropathy was unexpected.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Description of the invention

We have identified a link between CCLl 8 levels in diabetic patients and diabetic nephropathy. This unexpected correlation of CCLl 8 and diabetic nephropathy has not previously been reported. Measurement of CCLl 8 in biological fluids provides quantitative data on disease activity, and provides biomarkers to monitor patients' response to treatment. This also provides a biomarker to assess the effects of new treatments for diabetic nephropathy. Furthermore, treatments that suppress production of CCLl 8, or its downstream effects, may be useful in prevention or treatment of diabetic nephropathy.

A first aspect of the invention provides a method for aiding in the assessment of diabetic nephropathy in a patient, the method comprising the step of determining the level of CCLl 8 protein and/or nucleic acid in a sample from the patient.

CCLl 8 is described in, for example, Schutyser et al. (2005) J. Leukoc. Biol. 78:14-26. It is preferred that the sample is a urine sample but the sample may also be, for example, a blood serum sample or a blood plasma sample. In the blood, there are peripheral blood monocytes, so measurement of mRNA for CCLl 8 may provide indications of synthesis of CCLl 8. Alternatively, detection of CCLl 8 protein by immunohistochemistry and mRNA by in situ hybridisation and RT-PCR from renal biopsied material may also be useful. Furthermore, studying of DNA polymorphism (of the coding DNA sequence or promoter region) may provide additional information about individual patients who are genetically prone to produce too much or too little CCLl 8. Using urine samples may be more convenient and may also be particularly informative, as the urine may accurately reflect renal conditions.

The method may be used for assessing the likely progression of diabetic nephropathy in the patient. The method may also be useful for aiding in the diagnosis of diabetic nephropathy in a patient. The method may also or alternatively be useful for aiding in the assessment of the likelihood or likely severity or likely progression of diabetic nephropathy in a patient.

The method may be useful for assessing and/or predicting the development of fibrosis in the patient. CCLl 8 may also be used as a surrogate marker for the development of fibrosis in diabetic nephropathy.

The screening of diabetic patients for changes in CCLl 8 protein and/or mRNA levels may be useful for diagnosing those patients that may have or may develop diabetic nephropathy.

By "fibrosis" is meant the damage to organs or tissues caused by the thickening and scarring of connective tissue. Production of collagen and extracellular matrix are the key components of the fibrous tissue. By "diabetic nephropathy" is meant the complex and often progressive injury of the kidney due to the direct effect of diabetes mellitus. The typical features of diabetic nephropathy include deposition of extracellular matrix, resulting in thickening of glomerular basement membrane, glomerulosclerosis and tubulo- intersitial fibrosis. The natural history of diabetic nephropathy is variable and a significant proportion of the diabetic patients progressed to renal failure.

The response of the patient to treatment for diabetic nephropathy may be assessed using the method of the current invention. Thus, the method may be useful in predicting future response of the patient to treatment for diabetic nephropathy.

The method of the current invention may also be used for assessing the likely progression of response of the patient to treatment for diabetic nephropathy. It may also be useful in prognosis or aiding prognosis. Measurement of CCLl 8 is considered to be most useful in assessing the macroalbuminuric subgroup of diabetic nephropathy patients.

The method of the current invention may comprise the steps of (i) obtaining a sample containing nucleic acid and/or protein from the patient; and (ii) determining whether the sample contains a level of CCLl 8 nucleic acid or protein associated with the development, progression or regression (after appropriate treatment) of diabetic nephropathy.

It will be appreciated that determining whether the sample contains a level of CCLl 8 nucleic acid or protein associated with diabetic nephropathy may in itself be diagnostic (or prognostic) of diabetic nephropathy or it may be used by the clinician as an aid in reaching a diagnosis or prognosis.

Thus, measurement of CCLl 8 levels may be performed or considered alongside other measurements or factors, for example, determining the level of albumin and/or creatinine and/or assessing the estimated glomerular filtration rate (eGFR) and/or HbAIc, in the sample from the patient and/or measuring the patient's blood pressure. Simultaneous measurement of other cytokines and growth factors is considered to be helpful. For example, urinary connective tissue growth factor/creatinine ratio and monocyte chemoattractant protein- 1 /creatinine ratio are prognostic for progression of early and late stages of diabetic nephropathy respectively.

It will be appreciated that determining whether the sample contains a level of CCLl 8 nucleic acid or protein associated with diabetic nephropathy may in itself be diagnostic (or prognostic) of diabetic nephropathy or it may be used by the clinician as an aid in reaching a diagnosis or prognosis. The clinician may wish to take in to account these or other factors, such as presence of reduced perfusion of the kidneys due to renovascular diseases, including renal artery stenosis, as well as consider the level of CCLl 8, before making a diagnosis. Measurement of CCLl 8 levels may provide more detailed information on the severity of individual disease mechanisms.

It will be appreciated that determination of the level of CCLl 8 in the sample will be useful to the clinician in determining how to manage diabetic nephropathy in the patient. For example, since our research has indicated that elevated levels of CCLl 8 are associated with diabetic nephropathy, the clinician may use the information concerning the levels of CCLl 8 to facilitate decision making regarding treatment of the patient. Because CCLl 8 is known to cause fibrosis directly, monitoring of CCLl 8 will provide direct information of ongoing renal fibrosis in the patient. Based on the CCLl 8 measurement, the use of inhibitors of renin angiotensin system and any new anti-fibrotic medication may be added or optimised.

The level of CCL 18 which is indicative of diabetic nephropathy may be defined as the increased level present in samples from diabetic patients with diabetic nephropathy as shown by macroalbuminuria (spot urinary albumin/creatinine ratio more than 25 mg/mmol or timed urine collection albuminuria of more than 300 mg per day) or the presence of deposition of extracellular matrix, resulting in thickening of glomerular basement membrane, glomerulosclerosis and tubulo- intersitial fibrosis. The level of said CCLl 8 protein may be, for example, at least 2 standard deviation higher in a sample from a patient with diabetic nephropathy than the control healthy volunteers.. The level of mRNA encoding CCLl 8 may be, for example, at least 2 standard deviation higher in a sample from a patient with diabetic nephropathy.

The level of CCLl 8 in a sample from the patient may be determined using any suitable protein detection or quantitation method, for example using methods employing antibodies specific for CCLl 8. Thus, immunoassay techniques, preferably quantitative techniques, may be used, for example an antibody array or captured ELISA technique, for example as described in the Examples. Preferred embodiments relating to methods for detecting CCLl 8 protein include enzyme linked immunosorbent assays (ELISA), radioimmunoassay (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al in US Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by reference. Other techniques include: Beads-based immunoassay using Luminex type machine; Antibody arrays (including membrane based, or glass based); Proteomic analysis (mass spectromotery, antibody coated biochips using SELDI-TOF technique).

It will be appreciated that other antibody-like molecules may be used in the method of the invention including, for example, antibody fragments or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs.

Bioassays may alternatively be used for measuring CCLl 8 activity. For example, CCLl 8 stimulates fibroblasts to produce collagen in vitro. See, for example, Luzina LG. et al. PKCalpha mediates CCL18-stimulated collagen production in pulmonary fibroblast. Am J Respir Cell MoI Biol 35:298-305, 2006. In one preferred embodiment of the invention it is determined whether the level of CCLl 8 nucleic acid, in particular mRNA, is a level associated with diabetic nephropathy. Preferably, .the sample contains nucleic acid, such as mRNA, and the level of CCL 18 is measured by contacting said nucleic acid with a nucleic acid which hybridises selectively to CCL 18 nucleic acid.

By "selectively hybridising" is meant that the nucleic acid has sufficient nucleotide sequence similarity with the said human nucleic acid that it can hybridise under moderately or highly stringent conditions. As is well known in the art, the stringency of nucleic acid hybridisation depends on factors such as length of nucleic acid over which hybridisation occurs, degree of identity of the hybridizing sequences and on factors such as temperature, ionic strength and GC or AT content of the sequence. Thus, any nucleic acid that is capable of selectively hybridising as said is useful in the practice of the invention.

Nucleic acids which can selectively hybridise to the said human nucleic acid include nucleic acids which have > 95 % sequence identity, preferably those with > 98 %, more preferably those with > 99 % sequence identity, over at least a portion of the nucleic acid with the said human nucleic acid. As is well known, human genes usually contain introns such that, for example, a mRNA or cDNA derived from a gene would not match perfectly along its entire length with the said human genomic DNA but would nevertheless be a nucleic acid capable of selectively hybridising to the said human DNA. Thus, the invention specifically includes nucleic acids which selectively hybridise to CCLl 8 mRNA or cDNA but may not hybridise to a CCLl 8 gene. For example, nucleic acids which span the intron-exon boundaries of the CCLl 8 gene may not be able to selectively hybridise to the CCLl 8 mRNA or cDNA.

Typical moderately or highly stringent hybridisation conditions which lead to selective hybridisation are known in the art, for example those described in Molecular Cloning, a laboratoiy manual, 2nd edition, Sambrook et al (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, incorporated herein by reference.

An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe nucleic acid is 500 bases or base pairs is:

6 x SSC (saline Na⁺ citrate) 0.5% Na⁺ dodecyl sulphate (SDS) 100 μg/ml denatured, fragmented salmon sperm DNA

The hybridisation is performed at 68 °C. The nylon membrane, with the nucleic acid immobilised, may be washed at 68 °C in 1 x SSC or, for high stringency, 0.1 x SSC.

20 x SSC may be prepared in the following way. Dissolve 175.3 g of NaCl and 88.2 g OfNa⁺ citrate in 800 ml of H₂O. Adjust the pH to 7.0 with a few drops of a 10 N solution of NaOH. Adjust the volume to 1 litre with H₂O. Dispense into aliquots. Sterilise by autoclaving.

An example of a typical hybridisation solution when a nucleic acid is immobilised on a nylon membrane and the probe is an oligonucleotide of between 15 and 50 bases is:

3.0 M trimethylammonium chloride (TMACl) 0.01 M Na⁺ phosphate (pH 6.8)

1 mm EDTA (pH 7.6)

0.5 % SDS

100 μg/ml denatured, fragmented salmon sperm DNA

0.1 % nonfat dried milk

The optimal temperature for hybridisation is usually chosen to be 5 ⁰C below the

T] for the given chain length. T₁ is the irreversible melting temperature of the hybrid formed between the probe and its target sequence. Jacobs et al (1988) Nucl. Acids Res. 16: 4637 discusses the determination of TjS. The recommended hybridisation temperature for 17-mers in 3 M TMACl is 48-50 °C; for 19-mers, it is 55-57 °C; and for 20-mers, it is 58-66 °C.

By "nucleic acid which selectively hybridises" is also included nucleic acids which will amplify DNA from the said CCLl 8 mRNA by any of the well known amplification systems such as those described in more detail below, in particular the polymerase chain reaction (PCR). Suitable conditions for PCR amplification include amplification in a suitable 1 x amplification buffer:

10 x amplification buffer is 500 mM KCl; 100 mM Tris.Cl (pH 8.3 at room temperature); 15 mM MgCl₂; 0.1 % gelatin.

A suitable denaturing agent or procedure (such as heating to 95 °C) is used in order to separate the strands of double-stranded DNA.

Suitably, the annealing part of the amplification is between 37 ⁰C and 60 °C, preferably 50 °C.

Although the nucleic acid which is useful in the methods of the invention may be

RNA or DNA, DNA is preferred, for example if assessing the patient for CCLl 8 polymorphisms. If assessing expression levels then mRNA may be preferred.

Although the nucleic acid that is useful in the methods of the invention may be double-stranded or single-stranded, single-stranded nucleic acid is preferred under some circumstances such as in nucleic acid amplification reactions.

The nucleic acid that is useful in the methods of the invention may be any suitable size. However, for certain diagnostic, probing or amplifying purposes, it is preferred if the nucleic acid has fewer than 10 000, more preferably fewer than 1000, more preferably still from 10 to 100, and in further preference from 15 to 30 base pairs (if the nucleic acid is double-stranded) or bases (if the nucleic acid is single stranded). As is described more fully below, single-stranded DNA primers, suitable for use in a polymerase chain reaction, are particularly preferred.

The nucleic acid for use in the methods of the invention is a nucleic acid capable of hybridising to the CCL 18 mRNA. Fragments of the CCLl 8 gene and cDNAs derivable from the mRNA encoded by the CCLl 8 gene are also preferred nucleic acids for use in the methods of the invention.

It is particularly preferred if the nucleic acid for use in the methods of the invention is an oligonucleotide primer which can be used to amplify a portion of the CCLl 8 nucleic acid, particularly CCLl 8 mRNA.

A further aspect of the invention provides a method for assessing a diabetic nephropathy treatment regime, the method comprising the step of determining the level of CCLl 8 protein or nucleic acid in a sample from patients receiving the treatment regime. The sample type is typically of the type discussed hereinbefore in relation to the first aspect of the invention, for example, a urine sample from the patient. The method may, for example, be used to provide information on the likelihood of the development of fibrosis in the patient. Thus, levels of CCL 18 may be used as surrogate markers in clinical trials of proposed treatments for diabetic nephropathy. Measurement of CCLl 8 may provide the overall assessment of how various factors affect the treatment of and progression of diabetic nephropathy.

A further aspect of the invention provides a method for identifying a compound useful in diabetic nephropathy, for example in treating or preventing diabetic nephropathy, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, CCLl 8 in body tissue or cells (for example leucocytes, dendritic cells or fibroblasts) from healthy volunteers or a patient with diabetes mellitus and b) selecting a compound which is capable of suppressing production of, or activity of, CCL 18 in body tissue or a cell sample from health volunteers or a patient with diabetes mellitus. Both healthy subjects and diabetic patients may be able to produce CCLl 8 in response to pathological stimuli. Other organ tissues may be more accessible for testing than the kidney. Alternatively, cells, such as circulating leucocytes, fibroblast from the skin may be used.

The method may comprise the step of determining whether a test compound is capable of suppressing production of, or activity of, CCL 18 in a sample, for example a urine sample from a patient, as discussed hereinbefore.

A further aspect of the invention provides an antagonist of CCL 18 protein and/or nucleic acid for the treatment of diabetic nephropathy.

The antagonist of CCLl 8 protein may comprise, for example, an antibody directed towards CCLl 8 protein. The antibody directed towards CCLl 8 protein may be a monoclonal antibody but may also be a polyclonal serum extracted from an appropriately immunised animal (such as a mouse, a rabbit, a goat or a horse). Alternatively, individual populations of antibodies directed to, for example, one epitope or a family of epitopes may be purified from said serum and used in this aspect of the invention. The antagonist may also comprise fragments of antibody or derivatives which retain their antigen-binding sites, synthetic antibody-like molecules such as single-chain Fv fragments (ScFv) and domain antibodies (dAbs), and other molecules with antibody-like antigen binding motifs. The antagonist may be a peptide inhibitor, such as mutated sequence of CCLl 8 or low molecular weight inhibitor of CCLl 8 receptor and its downstream pathway.

The antagonist of CCL 18 nucleic acid may comprise, for example, a short interfering RNA molecule directed towards CCLl 8 mRNA. Such molecules will hybridise selectively to CCLl 8 mRNA in vivo and lead to destruction of said mRNA and a reduction in the expression of CCL 18 protein.

A further aspect of the invention provides the use of an antagonist of CCLl 8 protein and/or nucleic acid in the manufacture of a medicament for the treatment of diabetic nephropathy. The antagonist may comprise any one of the entities described in the preceding aspect of the invention or any other appropriate compound.

A yet further aspect of the invention provides a method of treating a patient with diabetic nephropathy, the method comprising the step of administering to the patient an effective amount of an antagonist of CCLl 8. By antagonist of CCLl 8 is meant a compound which suppresses production of, or activity of CCLl 8, for example as determined by a method according to the aspect of the invention described hereinbefore for the provision of a method for identifying a compound useful in diabetic nephropathy.

The ability of CCL 18 to induce collagen synthesis depends on activation of protein kinase C alpha, ERK2, phosphorylation of transcription factor SpI and basal expression of Smad3 in lung fibroblast in cell culture study. See: Luzina et al. (2006) Am J. Resp. Cell MoI Biology 35: 298-305; and Luzina et al. (2006) J. Cell Physiol. 206: 221-228. Therefore, it is likely that inhibition of these pathways may inhibit production of CCLl 8 in diabetic nephropathy patients.

In all aspects of the invention the patient is typically a human. CCL 18 has not been identified in rodents yet, so an animal model may not be suitable. An in vitro model may be most appropriate. Examples are:

(1) Primary culture of renal cells/tissue from a patient with diabetic nephropathy;

(2) Human renal cell lines; (3) Primary culture human cells from uninvolved part of the kidney removed from patients with renal cancer, or donor renal material subsequently not suitable for transplantation;

(3) macrophage cell line; or

(4) peripheral blood leucocytes, fibroblast cell line, dendritic cells.

The invention will now be described in more detail by reference to the following, non-limiting, Figures and Examples. Any documents referred to herein are hereby incorporated by reference.

Figure Legends

Figure 1. Urinary CCL18 in diabetic nephropathy. Elevated amount of urinary CCLl 8 levels (expressed as CCL18/creatinine ratio) was detected in diabetic patients with macroalbuminuria (more severe stage of diabetic nephropathy) (p=0.004), but not in diabetic patients with normoalbuminuria or microalbuminuria.

Figure 2. Urinary CCL18 in non-diabetic renal diseases. A small amount of urinary CCL 18 levels (expressed as CCL18/creatinine ratio) was detected in some patients with non-diabetic renal diseases. Most of these were within normal range. There were no significant differences in urinary CCL18/creatinine between non- diabetic renal diseases with different degree of albuminuria. In our study of normal volunteers, urinary CCL18/creatinine ratio range from undetectable to 4.05 ng/mmol.

Figure 3. Relationship between urinary CCL18 and albuminuria in diabetic patients. There was a significant correlation between urinary CCL18/creatinine ratio and albumin/creatinine ratio in diabetic patients (Spearman correlation R=0.4, pO.0001)

Figure 4. Relationship between urinary CCL18 and albuminuria in non- diabetic renal diseases. There was no significant correlation between urinary , CCL18/creatinine ratio and albumin/creatinine ratio in diabetic patients.

Figure 5. Subgroup analysis of diabetic macroalbuminuric patients: relationship between CCL18 and urinary albumin/creatinine ratio. Data are presented as scattered plot with median values. Figure 6. Subgroup analysis of diabetic macroalbuminuric patients: relationship between CCL18 and renal function. Data are presented as scattered plot with median values.

Example 1: Novel cytokine present in urine of diabetic patients may be involved in the regulation of inflammation and fibrosis in diabetic nephropathy.

Methods

In this study, 100 patients with diabetes were examined. Among these patients, 29 had no renal disease, 27 had microalbuminuria (early stage of diabetic nephropathy) and 44 had macroalbuminuria (late stage of diabetic nephropathy). As controls 54 patients with renal diseases but not diabetes, who were attending the Hammersmith Hospital diabetic/renal outpatient clinics, were also examined. The concentrations of CCLl 8 from spot urine samples were measured by ELISA. The results were expressed as urinary CCL18/creatinine ratio. Demographic and clinical parameters were recorded for all patients. The presence of systemic and urinary tract infection was assessed by measurement of C-reactive protein and microscopy and bacterial culture of urine and these samples were excluded from the study.

Processing and storage of urine samples.

Spot urine samples were collected from patients in the clinic. Sediments were removed by centrifugation. The samples were aliqoted and stored at -80C until testing by specific ELISA.

Sandwiched ELISA for CCL18

Matched antibody pairs specific for human CCLl 8 and recombinant human CCLl 8 were purchase from R & D Systems, UK. The ELISA was set up and optimized following the general guidelines by the manufacturer. The sensitivity of the ELISA was 7.8 pg/ml. Results

Urinary CCL18 in different stages of diabetic nephropathy and non-diabetic renal diseases Urinary CCL18/creatinine ratio was significantly elevated in diabetic macroalbuminuric patients (p=0.004, Mann Whitey-U test) in comparison to both normoalbuminuric patients and microalbuminuric patients (Figure 1). In healthy volunteers, urinary CCL18/creatinine ratio is less than 5 ng/mmol.

Among the patients with other renal diseases not due to the diabetes, we found that there was no significant differences in urinary CCL18/creatinine ratio between three subgroups of patients (Figure 2).

This is an unexpected finding. Renal fibrosis is a common pathological process in a wide range of renal diseases. Furthermore, macrophage is known to be an important source of CCLl 8. In many types of glomerulonephritis, there are increased number of renal macrophages, in much larger number than diabetic nephropathy. These data suggested that CCLl 8 is a selective pro-fibrotic mediator in diabetic nephropathy.

Relationship between urinary CCL18 and albuminuria

The relationship between urinary CCLl 8 and the severity of albuminuria was further examined by non-parametric analysis.

Within the diabetic group of patients, urinary CCLl 8 was strongly correlated with albumin/creatinine ratio, see Figure 3 (Spearmen correlation, r=0.40 p<0.0001) and also had negative correlation with eGFR (Spearmen correlation, r=-0.36 p=0.0003) (Figure 3), but this was not the case in the patients with other renal diseases (Figure 4). These results showed that increased urinary CCLl 8 in diabetic nephropathy is not due to general effect of proteinuria. In fact, the clear differences between the urinary CCLl 8 results in diabetic nephropathy in contrast to other causes of renal diseases showed that CCLl 8 is a specific pathological mediator in diabetic nephropathy.

Relationship between CCL 18 and glomerular filtration rate (GFR)

Table 1. Non-parametric analysis of the relationship between CCLl 8 and eGFR using Spearman correlation test.

Estimated glomerular filtration rate (eGFR) was calculated using MDRD formulae. The correlation between eGFR and urinary CCL18/creatinine ration was assessed by non-parametric correlation. In patients with diabetic nephropathy, urinary CCLl 8 correlated with eGFR (Spearsman's correlation R= -0.35, p=0.0003) (Table 1). Therefore, diabetic patients with higher urinary CCL18/creatinine ratio had worse eGFR. In contrast, patients with non-diabetic renal diseases did not have significant correlation between urinary CCL18/creatinine ratio and eGFR (Table 1). These results further supported that CCLl 8 is relevant to the severity of diabetic nephropathy.

Relationship between CCL18 and other risk factor for diabetic nephropathy

Poor diabetic control and uncontrolled hypertension are known factors for progression of diabetic nephropathy. There were not any correlation between FIbAIc or blood pressure with urinary CCL18/creatinine ratio. Therefore, measurement of urinary CCL18/creatinine ratio provide extra information of ongoing renal damage which beyond traditional measurement of blood pressure and diabetic control.

Example 2: Subgroup analysis: CCL18 in the diabetic macro albuminuric subgroup.

Among diabetic patients with macroalbuminuria, there was a wide range of urinary CCL18/creatinine ratio, ranging from undetectable to very high level. Therefore, subgroup analysis were carried in patients with severe diabetic nephropathy (diabetic macroalbuminuric patients).

Urinary CCL18 and severity of albuminuria In this subgroup analysis, diabetic macroalbuminuric patients with high urinary CCL18/creatinine ratio has a significantly higher albumin/creatinine ratio than diabetic macroalbuminuric patients with undetectable urinary CCLl 8 (p=0.001) (Figure 5). This finding supported the initial conclusion that urinary CCLl 8 associated with more severe diabetic nephropathy.

Furthermore, detailed inspection of individual patients in this subgroup analysis, as shown in the scattered plot (Figure 5), urinary CCL 18 provided extra information about ongoing fibrotic process. Patients with similar urinary albumin/creatinine ratio may have either undetectable urinary CCLl 8 or high CCLl 8. Therefore, direct measurement of urinary CCLl 8 provides extra clinical information beyond current standard medical test of diabetic nephropathy.

In this analysis we compared diabetic macroalbuminuric patient with undetectable urinary CCLl 8 with diabetic macroalbuminuric patients with high urinary CCLl 8/creatinine ratio (>5 ng/mmol).

Patient with high urinary CCLl 8/creatinine ratio had significantly higher albumin/creatinine ratio than the patients with undetectable urinary CCLl 8 (Figure 5). Further inspection of individual patients showed that measurement of urinary CCLl 8/creatinine in individual patient provided additional information beyond standard measurement of urinary albumn/creatinine ratio. For example, some individual patients with low urinary albumin/creatinine ratio has high urinary CCLl 8 (Figure 5). Urinary CCL18 and impaired renal function

The patients with high urinary CCL18/creatinine ratio had lower GFR than those with undetectable urinary CCLl 8. This finding concern that CCLl 8 is associated with impaired renal function in patients with diabetic nephropathy. Furthermore, detailed inspection of individual patients in this subgroup analysis, as shown in the scattered plot (Figure 6), urinary CCLl 8 provided extra information about ongoing flbrotic process. Patients with similar GFR may have either undetectable urinary CCLl 8 or high CCLl 8 (Figure 6). Therefore, direct measurement of urinary CCLl 8 provides extra clinical information beyond current standard medical test of diabetic nephropathy.

Patient with high urinary CCL18/creatinine ratio had significantly lower eGFR and the patients with undetectable urinary CCLl 8. Further inspection of individual patients showed that measurement of urinary CCL18/creatinine in individual patient provided additional information beyond standard measurement of renal functions, such as GFR or plasma/serum creatinine concentration. For example, some individual patients has high urinary CCLl 8 although their eGFR at baseline overlaps with the patients with low urinary CCLl 8 (Figure 6). By measuring the urinary CCL18/creatinine ratio, the risk of further renal injury due to ongoing fibrosis may be assessed.

Applications of urinary CCL 18

The main principle is to intervene on a fibrotic mechanism in diabetic nephropathy before irreversible renal fibrosis happens.

Clinical management of diabetic patient

In additional to current clinical targets in blood pressure, diabetic control and urinary albumin/creatinine ratio, CCLl 8 level in the urine, blood or renal biopsies are assessed. In patients with high CCL 18 level, the dose of rennin/angiotensin antagonist or other antifibrotic therapy will be increased, even if other clinical parameters are within target. In this way, the patients at risk of progressive renal fibrosis will receive more vigorous therapy. Clinical trials of new therapy

Current clinical trials often focus on the severity of established injury as an assessment end point. This has the disadvantage of demanding long follow up period because the new therapy can be assessed. Furthermore, any failure of the new experimental therapy will take longer time to be identified, which is a potential ethical disadvantage for the patients. Monitoring of CCLl 8 during the clinical trial will provide a mechanistic disease marker for early assessment of the efficacy of the therapy. This is a particular advantage when the optimal.dose needs to be titrated to achieve the clinical benefit. When peripheral leucocytes, blood or urine are examined, this also has the additional advantage of a non-invasive disease marker, which is a safe option for serial monitoring.

Screening of potential drugs in vitro Cell lines or primary culture of cells (such as macrophages, dendritic cells, renal cells and fibroblast) may be used to identify drugs/chemicals which can inhibit the synthesis of CCLl 8, or the downstream effect of CCLl 8.

Claims

1. A method for aiding in the assessment of diabetic nephropathy in a patient, the method comprising the step of determining the level of CCLl 8 protein and/or nucleic acid in a sample from the patient.

2. The method of claim 1 wherein the sample is a urine sample.

3. The method of claim 1 or 2 wherein the method is for assessing the likely progression of diabetic nephropathy in the patient.

4. The method of claim 3 wherein the method is for assessing and/or predicting the development of fibrosis.

5. The method of claim 1 or 2 wherein the method is for diagnosing diabetic nephropathy in the patient.

6. The method of claim 1 or 2 wherein the method is for assessing the response of the patient to treatment.

7. The method of claim 6 wherein the method is for assessing the likely progression of response of the patient to treatment.

8. The method of any one of the previous claims further comprising the step of determining the level of albumin and/or creatinine and/or assessing the eGFR, in the sample from the patient.

9. The method of any one of the preceding claims wherein the patient has diabetic macro albuminuria.

10. A method for assessing a diabetic nephropathy treatment regime, the method comprising the step of determining the level of CCLl 8 protein or nucleic acid in a sample from patients receiving the treatment regime.

11. A method for identifying a compound useful in diabetic nephropathy, for example in treating or preventing diabetic nephropathy, the method comprising the steps of a) determining whether a test compound is capable of suppressing production of, or activity of, CCL 18 in renal tissue or a sample from a patient with diabetic nephropathy and b) selecting a compound which is capable of suppressing production of, or activity of, CCLl 8 in body tissue or cells (for example leucocytes, dendritic cells or fibroblasts) from healthy volunteers or a patient with diabetes mellitus; and b) selecting a compound which is capable of suppressing production of, or activity of, CCLl 8 in body tissue or a cell sample from healthy volunteers or a patient with diabetes mellitus.

12. An antagonist of CCLl 8 protein and/or nucleic acid for the treatment of diabetic nephropathy.

13. The antagonist of claim 12 wherein the antagonist is an antibody directed towards CCL 18 protein.

14. The antagonist of claim 13 wherein the antibody is a monoclonal antibody directed towards CCLl 8 protein.

15. The antagonist of claim 12 wherein the antagonist is a short interfering RNA molecule directed towards CCL 18 mRNA.

16. The use of an antagonist of CCLl 8 protein and/or nucleic acid in the manufacture of a medicament for the treatment of diabetic nephropathy.

17. A method of treating a patient with diabetic nephropathy, the method comprising the step of administering to the patient an effective amount of an antagonist of CCLl 8.