What is a chronic kidney disease model? Explanation of types, creation methods, and evaluation indicators.

The number of patients with chronic kidney disease (CKD) is increasing both in Japan and overseas, and the development of new therapeutic agents is urgently needed. This article describes typical types of chronic kidney disease models, methods of creation, and evaluation indices.

Chronic kidney disease (CKD) model overview

Background of the demand for CKD models

CKD is a general term for diseases that cause chronic decline in kidney function. It is estimated that about one in eight adults in Japan has CKD*, and the increase in medical costs associated with the growing number of patients on dialysis has become a matter of social concern. In response, the Ministry of Health, Labour and Welfare (MHLW) is accelerating its efforts to prevent severe disease through study groups and other means.

Against this backdrop, there is a high need for the development of new drugs that inhibit or ameliorate the progression of CKD, and accurate prediction of the efficacy and safety of candidate compounds is required at the non-clinical trial stage. The use of appropriate animal models makes it possible to obtain highly accurate data that contributes to the decision to move on to clinical trials (clinical studies).

List of typical types of chronic kidney disease models

The main CKD models used in nonclinical studies can be broadly classified into "induced models" by drug administration, "physical defect models" by surgical techniques, and "immunological models" by administration of antibodies and other means.

Since each model has its own advantages and limitations, the choice must be made according to the objectives of the study and the evaluation items. Selecting the right model for the purpose and constructing an appropriate evaluation system will lead to the success of nonclinical studies.

Characteristics and Methods of Making Adenine-Induced Chronic Renal Failure Models

The adenine-induced chronic renal failure model is a pathological model in which renal failure is induced by oral administration of adenine, a type of purine. Adenine metabolized in the body becomes 2,8-dihydroxyadenine, which is deposited as crystals in the renal tubules. This crystal deposition causes tubular obstruction and fibrosis of the interstitium, resulting in chronic renal dysfunction.

Since it does not involve surgery, it is less invasive to the animals and suitable for large-scale testing. The fact that it can be produced in both mice and rats also makes it highly versatile.

Procedures and conditions for the creation of adenine-induced models

There are two main methods of administration: "mixed feeding" and "forced oral administration.

• Mixed feeding: Adenine is mixed with regular feed. Less stressful on animals and suitable for long-term rearing.
• Forced oral administration: administered directly into the stomach using a sonde. This has the advantage of allowing strict control of the amount ingested per individual.

Typically, mice (such as C57BL/6J) or rats around 8 weeks old are used, and dosing protocols are set up for several weeks depending on the purpose of the study.

Correlation between adenine levels and pathology in mice

The concentration of adenine in the adenine admixture has a significant impact on the progression of the disease. It is essential to correctly understand the differences among concentrations and to set the conditions appropriate for the purpose of the study.

The following are general concentration conditions for mice

0.1% concentration

0.1% adenine admixture does not cause significant body weight changes or significant increases in blood BUN (urea nitrogen) and Cre (creatinine). It is believed that the condition is not sufficiently pathological to be used as a renal failure model.

0.15% concentration

In the 0.15% adenine mixture, BUN and Cre increase stepwise with gradual weight loss. This is a suitable condition for evaluation of drug efficacy, since stable renal function decline can be achieved while avoiding rapid deterioration. Another advantage is that the timing of administration of the test substance and the evaluation schedule can be flexibly set.

0.2% concentration

Rapid elevations in BUN and Cre and significant weight loss occur early after initiation of treatment. Due to the rapid progression of the disease, care must be taken in setting the timing of drug efficacy evaluation; when using the 0.2% concentration, the recommended protocol is to switch to a normal diet after 4 weeks of mixed feeding, followed by a 2-week recovery period before administering the test substance.

In pharmacopharmacological studies, the 0.151 TP3T concentration, which is easy to detect drug effects under stable pathological conditions, has been reported in many cases. When constructing an evaluation system, it is important to select a concentration that is appropriate for the animal species and the purpose of the study.

Characteristics of chronic renal failure model with renal artery ligation and nephrectomy

renal artery branch ligation

In renal artery branch ligation, the central branch of the left renal artery is first ligated under isoflurane anesthesia. One week later, the right kidney is removed, and a portion of the tissue of one kidney is ischemic or infarcted to reduce the number of functioning nephrons and induce chronic renal failure. The animals used are reported to be rats (Lewis strain, 10 weeks old).

The study group usually consists of three groups: 1K group (ligation of left renal artery branch + right nephrectomy), 2K group (ligation only and preservation of right kidney), and Sham group (sham surgery). 1K group shows a significant increase in BUN, Cre, and CysC (cystatin C) at 1 week after right nephrectomy.

The advantage of this condition is that it remains stable for at least 4 weeks, is less likely to cause weight loss or abnormalities in the general condition, and is less severe. It is positioned as a highly useful model for nonclinical studies because it allows evaluation of the drug effects of the test substance under stable renal function decline.

5/6 Comparison with nephrectomy model

The 5/6 nephrectomy model involves surgical removal of a large portion of the kidney and can reproduce severe renal dysfunction. However, the pathology may be excessively intense and carries the risk of significant deterioration in the animal's general condition. Careful study design is required due to the possibility of increased mortality during the study period.

The renal artery branch ligation technique has the advantage that the pathology is not as intense as in the 5/6 nephrectomy model, and it is easier to ensure a stable evaluation period. It is important to select the appropriate surgical model according to the purpose of the study and the severity of the required pathology.

Characteristics of Nephritis Models by Immunological Mechanisms

Unlike surgical techniques or models using drugs such as adenine, models in which specific "antibodies" are administered are used to reproduce nephritis caused by immune abnormalities (such as glomerulonephritis). Two typical examples are as follows

Anti-GBM antibody nephritis model

In this model, antibodies are administered that directly attack the glomerular basement membrane (GBM), which acts as a filter to filter the blood. This model is often used to reproduce the severe, rapidly progressive glomerulonephritis with rapidly worsening symptoms and crescent formation.

Anti-Thy-1 antibody nephritis model

This model targets and attacks "mesangial cells" that support glomerular capillaries. The model can reproduce mesangial proliferative nephritis similar to IgA nephropathy in humans, and is characterized by the ability to evaluate the process of spontaneous recovery (reversible lesions) from a once deteriorated condition.

Thus, even within the same "chronic kidney disease" framework, the model to be selected depends on the desired mechanism of action (i.e., which site of immune abnormality to target).

Evaluation indexes and key points for conducting studies in chronic kidney disease models

In pharmacopharmacological studies of chronic kidney disease models, multiple renal parameters are combined for a comprehensive evaluation. The following parameters are commonly analyzed from multiple angles in the evaluation of drug efficacy

• Blood biochemical indices: BUN, Cre, CysC (Cystatin C)
• Urinalysis indices: urine protein, NAG, urine volume
• Histopathologic indices: thickening of glomerular basement membrane, tubular dilatation, fibrosis of the interstitium, crescent formation
• Other: kidney weight, special stains (HE, PAS, Masson trichrome stain, etc.)

The timing of the evaluation is set according to the study protocol. Generally, the test substance is administered during the period of pathological stability after model creation, and changes over time are tracked after administration. Setting the appropriate evaluation time point determines the accuracy of drug effect detection.

When requesting a contract research organization, it takes about 2 months from inquiry to plan formulation, contract, implementation, and final report as a standard. By organizing "what kind of drug mechanism of action is being targeted" in advance, it is possible to select an appropriate model and draw up a schedule.

Challenges and Measures to Improve Clinical Predictability in CKD Nonclinical Studies

Novel biomarkers for early detection and evaluation of drug efficacy

Conventional assessment of renal function in nonclinical studies has relied primarily on measurement of serum BUN (urea nitrogen) and creatinine, as well as histopathological evaluation. However, these indices are manifested at a stage when renal function has already declined significantly, making it difficult to capture minute changes in the early stages of disease onset and the early improvement effect of drugs.

In recent years, the use of novel biomarkers that acutely reflect earlier tubular damage and inflammation is becoming the standard. Specifically, the incorporation of measurements such as KIM-1 (Kidney Injury Molecule-1), NGAL, L-FABP, and cystatin C into the evaluation panel allows for highly sensitive detection of early renoprotective effects at the cellular level and, conversely, potential renal toxicity.

Selection of a "complication (multi-organ linkage) model" that reflects realistic clinical conditions.

In actual clinical practice, most patients with CKD do not have a single disease, but are complicated by metabolic diseases such as diabetes, hypertension, and dyslipidemia. In particular, Cardiorenal Syndrome, in which chronic renal failure and heart failure mutually worsen, is a critical issue that affects life prognosis.

Therefore, in addition to simple renal injury models produced physically or chemically, there is a strong need to utilize models that mimic the complex background of humans, such as the ZSF1 rat (obesity and type 2 diabetes mellitus combined CKD model) and the STZ (streptozotocin)-induced diabetic nephropathy model. Selecting the right pathological model for the target population of a new drug is an important factor that contributes to improving the success rate in clinical trials.

The Rise of "MPS (Molecular Pattern System) and Image Analysis" to Transcend the Barriers of Species Differences

Non-clinical studies using animal models are an essential process, but there are "species differences" between humans and experimental animals in terms of kidney anatomy, speed of progression to fibrosis, and immune response. To bridge this gap, there are an increasing number of cases where biomimetic systems (MPS) such as "kidney organoids" derived from human iPS cells and "kidney-on-a-chip" that reproduces the microenvironment of glomeruli and tubules on a microfluidic device are incorporated into in vitro evaluations. The number of cases of in vitro evaluation of kidney chip is increasing.

Furthermore, in in vivo evaluation, functional MRI (e.g., BOLD method) technology has been developed to noninvasively measure renal blood flow and fibrosis status over time without euthanizing the animals. In selecting a contract research organization (CRO), whether or not they employ such advanced technologies is also an important checkpoint for obtaining high-quality data.

Summary

There are several types of chronic kidney disease models: drug-induced models such as adenine-induced models, surgical models such as renal artery ligation, and immunological models such as anti-GBM antibodies. Since each of these models has different production methods and pathological characteristics, it is important to select the appropriate model according to the research objectives and evaluation items.

When considering non-clinical studies in the field of chronic kidney disease, please consult with a contract research organization regarding the selection of a model suitable for the purpose of the study and the design of the evaluation system. We can conduct your study efficiently by providing consistent support from model creation to evaluation and report writing.