More specifically, the study should seek to answer the following questions (but not limited to only these);

• What are the concentrations of heavy metals (Pb, Hg, As, Cd, Fe, Mn, Cu and Zn) in the bloodstream of inhabitants of selected communities exposed to galamsey polluted rivers?
• What are the average heart rates, blood pressures, renal functioning and mutations in study participants?
  • Are they hypertensive, have renal dysfunction or have genetic mutation/epigenetic modifications?
    • Are there sex differences in the disorders mentioned above?
• What extent (time) and concentration of heavy metals are required to trigger the pathogenesis of hypertension and renal damage in experimental animal models (preferably Sprague-Dawley [SD] rats)?
• What haemodynamic, immunological and molecular responses are elicited in Sprague-Dawley (SD) rats subjected to varying concentrations of heavy metals from galamsey water?
  • Are there sex differences in these responses?
• What are the exact hypertensive and renal pathologies heavy metals trigger in SD rats?
  • Are these pathologies similar to what is observed in humans?
• Could the ingestion of concentrations of heavy metals polluted water by pregnant SD rats affect foetal development and induce malformations/disorders?
  • Does it influence the occurrence of birth defects and epigenetic modifications?

Onsite studies: This part can include the following:

• Study site identification: Researchers must identify communities within districts most affected by galamsey. These communities should be near galamsey-polluted waterbodies where inhabitants depend on the water for domestic and/or commercial purposes. However, we propose the communities in the Prestea Huni Valley District of Ghana as preliminary study sites. This is because communities in this district meet the aforementioned criteria for the proposed study. Additionally, previous studies have been conducted in the district to assess heavy metal concentrations in polluted rivers and blood metals in individuals exposed to galamsey-polluted waterbodies (Armah et al. 2012, Obiri et al. 2016). To add to this, researchers must identify and select a comparative control district with communities whose inhabitants are served by a river or waterbody not polluted by galamsey activities. We suggest that, in the selection of a control community, researchers must consider similarities in demographics, environmental parameters and history and prevalence of hypertension and renal diseases.

• Sample size: Based on the reported 13.3% CKD prevalence in Ghana (Adjei et al. 2018), we employed Cochran's formula to propose a minimum of 177 participants per study site (whether target or control). This would ensure that the study has enough power to detect the true effect of residents' exposure to heavy metals on the development of kidney diseases and hypertension.

• Ethical Clearance: As per the declaration of the Helsinki Protocols, researchers are required to seek ethical approval for studies involving human participants (Kapp 2006). For the proposed study, ethical approval could be sought from the following authorities: the Ghana Health Service Ethics Review Board (preferably), the Institutional Review Board of the Council for Scientific and Industrial Research (IRB-CSIR) Ghana and/or the Research Ethics Committee of the University of Ghana.

• Recruitment of participants: This should be a two-stage recruitment process. Firstly, the study objectives should be explained to prospective participants both verbally and with documented handouts. If an individual agrees to participate in the study, they should be given a consent form to sign and be subjected to a questionnaire to determine their medical history, demographics, lifestyle and habits that may expose them to galamsey-polluted rivers within the community. Only participants with a history of contact with galamsey-polluted water should be recruited for the study.

• Data collection: Using the ambulatory blood pressure monitor, 24-hour blood pressure, heart rate and pulse should be measured in all participants.

• Sample collection:

  • Urine samples should be collected from all participants for proteinuria and urine albumin-creatinine ratio (uACR) tests.

  • Intravenous blood samples should be collected from all study participants for the following tests:

    • Using biochemistry analysers to measure serum creatinine for glomerular filtration rates.

    • Blood cell counts and serum immunoglobulin (Ig) M level.

    • Determination of blood metal concentrations using irradiation methods described by Armah et al. (2012). The confirmatory test can be carried out using mass spectrometry.

    • Immunoassays in ELISA should be employed in measuring TNF-α, a biomarker and mediator of hypertension (Ramseyer and Garvin 2013).

    • Molecular screening for polymorphisms and impairments in DSB DNA repair outcomes using methods described by Roy-Engel and colleagues (Morales et al. 2016). Additionally, research may investigate the presence of epigenetic modifications.

Experimental Studies: This is the second part of the proposed study that should investigate the pathophysiology of hypertension, renal damage and inherited renal defects due to galamsey-related heavy metal pollution in animal models. The Sprague-Dawley (SD) rat models should be the preferred choice for laboratory animals due to their predictable traits and their ability to develop similar physiological events that occur in human diseases (Lerman et al. 2005).

This part shall include:

• Ethical Clearance: The SD rats used in the study should be cared for and kept in humane conditions; standard operating procedures for appropriate care and use of laboratory animals could be obtained from the University of Ghana Institutional Animal Care and Use Committee (UG-IACUC). Ethical clearance could be sought from either the UG-IACUC or the IRB-CSIR.

• Husbandry and grouping of SD rats: A representative number of SD rats (per the discretion of researchers) should be used for this study. The SD rats (8–12 weeks old) could be obtained from colonies maintained at the Animal Experimentation Facility of the Noguchi Memorial Institute of Medical Research, University of Ghana. They should be housed and cared for in specialised breeding and experimentation rooms. All SD rats in the study should be housed in a temperature-controlled environment (20–23°C), maintained on standard rat chow and provided with clean water ad libitum.

• Data Collection (Vitals: BP, heart rate, temperature, blood flow and pulse): After one (1) week of provision of clean water ad libitum, SD rats should be divided into two (2) experimental groups for data collection:

  • BP Radiotelemetry group (BPRG): Rats in this group should be anaesthetised and surgically instrumented with BP radiotelemetry catheters as described by Polichnowski and colleagues (Polichnowski et al. 2020, Potter et al. 2021). After surgery, SD rats should be allowed to heal for 7 days (1-week post-surgery), then baseline vitals should be recorded continuously, 24 hours per day (10 seconds every 10 minutes at 500 Hz) for 3 consecutive days, during which rats should be administered purified water ad libitum. SD rats should then be divided into separate sub-experimental groups (later described) for vitals recording up to 14 weeks post-surgery. We recommend radiotelemetry as the primary BP measurement technique as several researchers have confirmed its efficiency (Harrison et al. 2024).

  • Tail Cuff Group (TCG): We propose non-invasive tail cuff BP measurement techniques to answer the specific research question "Could the ingestion of concentrations of heavy metals polluted water by pregnant SD rats affect foetal development and induce malformations/disorders?". Unlike the BPRG, the SD rats in TCG will be anatomically and physiologically fit for breeding and subsequent studies. Additionally, the TCG SD rats, free of extensive surgical instrumentation, will be ideal for longitudinal BP measurements throughout the entire study (baseline--heavy metal treatment--pregnancy--litter--post-weaning). In addition, another physiological activity that researchers may consider investigating is the levels of infiltration of immune cells into the kidneys of TCG rats vs. BPRG, since Harrison et al. (2024) stated that extensive surgical instrumentation may affect measures of inflammation. For the TCG, SD rats should first be trained for 2 to 3 days. Afterwards, rats should be restrained; and an inflatable cuff and a pulse sensor should then be fitted to the tail for BP measurements. As recommended by Harrison et al. (2024), multiple daily BP measurements should be recorded for 2 to 3 days weekly to improve the accuracy of the Tail cuff method. Baseline vitals should be recorded in week 1, during which rats will be administered purified water ad libitum. SD rats could be divided into separate sub-experimental groups (later described) for vitals recording up to 14 weeks post-baseline tail cuff measurements.

• Sample collection (blood, urine, organs; kidney, liver and spleen): SD rats in the sample collection groups (BPRG and TCG) can be categorised into three (3) sub-experimental groups each. Therefore, two (2) data collection groups should further be divided into six (6) sample collection groups. It should be ensured that groupings are random and all groups have significant gender representation. The sample collection groups could be as follows;

  • Galamsey heavy metals polluted (GHMP) water group: After one (1) week post-surgery or post-baseline tail cuff measurements, SD rats in this group should be fed with galamsey-related heavy metals polluted water samples collected from sites of human studies. Before this, collected water samples may be assessed for heavy metal concentrations using standard methods to examine wastewater as outlined by the American Water Works Association.

  • Laboratory-prepared heavy metals polluted (LPHMP) water group: After one (1) week post-surgery or post-baseline tail cuff measurements, SD rats in this group should be fed laboratory-prepared water induced with varying heavy metal concentrations. We propose this experimental group for the following reasons: i. to determine the concentrations of heavy metals required to trigger hypertension and renal damage in SD rats (since there are some difference in SD rat physiology as compared to humans). Separate groups of SD rats could be subjected to lower and/or higher concentrations of heavy metals as compared to the concentrations measured in GHMP water. Therefore, a positive control for the experimental study could be established from the LPHMP water group; ii. make up for physicochemical changes that may occur from the transportation and storage of galamsey heavy metals polluted water for the entirety of experimental studies; iii. additionally, the LPHMP water group will allow researchers to investigate the effects of singular or combinations of different heavy metals in triggering hypertension and renal damage (we believe this should be done, based on the discretion of the researchers).

  • Control group; After one (1) week post-surgery or post-baseline tail cuff measurements, SD rats in this group should be continuously fed with purified water throughout the study.

Table 1 shows all six proposed data and sampling groupings.

The following samples should be collected and their corresponding tests should be conducted:

• Urine samples should be collected each week post-surgery and post-baseline tail cuff measurements till week 14. At each time point, rats should be placed in metabolic cages and 24-hour urine samples should be collected for the assessment of proteinuria and uACR. Protein concentrations in the urine samples could also be determined using the Bradford method.
• Blood samples should be collected each week post-surgery and post-baseline tail cuff measurements till week 14. The tail vein sampling and the cardiac puncture (for rats to be euthanised) procedures as described by Parasuraman and colleagues could be used in collecting blood from various SD rat groups (Parasuraman et al. 2010). SD rat blood samples should be subjected to the aforementioned tests conducted for human blood samples.
• Organ excision: At each weekly post-surgery and post-baseline tail cuff measurements, relatively equal percentages of SD rats in each sampling group should be euthanised and subjected to nephrectomy (frequency of nephrectomy could be at the reasonable discretion of researchers). Excised kidneys should then be weighed, appropriately stained and treated for histological, immunohistochemical (flow cytometric analysis) and microscopic assessments to identify sclerosis, fibrosis, vascular injuries, cell death (types; necrosis or apoptosis) and infiltrating immune cells (Shimada et al. 2020, Abdelbary et al. 2022). A portion of the excised kidneys could be homogenised and subjected to the aforementioned molecular assessments in humans. Emphasis should be placed on determining gene expression mutations, single nucleotide polymorphisms, biochemical pathways, telomere lengths, alteration in DSB repair pathways and epigenetic modifications.

• Investigating the effects of ingestion of heavy metals polluted water on foetal development and progeny malformations (birth defects and epigenetic modifications): After 14 weeks of experimentation, one male and one female SD rat in the tail-cuff group should be selected from each sampling group (GHMP, LPHMP and Control) for breeding and subsequent studies. Rats may then be placed in separate breeding cages based on their groups and allowed to mate to produce litters. Based on the initial sampling group, water should be provided to both parent rats and litter ad libitum. By analysing the BP recordings from pregnant SD rats, researchers could explore investigating the incidence of pre-eclampsia in the study group as studies by Wang et al. (2020) have associated exposures to chromium, mercury and arsenic to increased pre-eclampsia prevalence. After the litters are weaned, parent rats could be euthanised and subjected to the earlier described tests. Litters should be weighed before weaning. Weaned rats should be maintained in separate cages, based on gender and parent sampling group. All litters should be thoroughly observed for anatomical malformations and birth defects. At 5 weeks post-weaning, vitals should be measured using the tail-cuff method (however, researchers may employ telemetry in a select group of rats for efficiency in BP measurement). Urine and blood samples (tail venipuncture) should be collected at 5-, 6-, 7- and 8 weeks post-weaning for tests previously mentioned. At eight (8) weeks post-weaning, all surviving SD rats should be euthanised, nephrectomised and subjected to anatomical, histological, immunohistochemical (flow cytometric analysis) and microscopic assessments. Molecular analysis should be carried out to investigate alterations in DSB repair pathways. Furthermore, we propose that researchers investigate DNA methylation and alteration of DNA and chromatin structure, as Bitto et al. (2015) indicate that heavy metals may trigger epigenetic modifications in foetal development and early childhood, posing significant health risks.