Familial Renal Glucosuria (FRG) Cell Models for Research

Familial Renal Glucosuria (FRG) is a rare autosomal recessive disorder characterized by persistent glucosuria in the absence of hyperglycemia, with an estimated prevalence of 0.1-0.5% in the general population (WHO, 2023). The condition results from mutations in the SLC5A2 gene, encoding the sodium-glucose cotransporter 2 (SGLT2), which is primarily expressed in the proximal tubule of the kidney. FRG is generally benign, with most individuals asymptomatic, but it can lead to polyuria, dehydration, and electrolyte imbalances in severe cases. The clinical impact is limited, but the disorder serves as an important model for understanding renal glucose handling and the pharmacology of SGLT2 inhibitors, which are widely used in the treatment of type 2 diabetes and chronic kidney disease. Research on FRG has provided insights into the molecular mechanisms of glucose reabsorption and the potential off-target effects of SGLT2 inhibitors.

FRG is an ideal model for studying the physiology and pathophysiology of renal glucose transport. The disease is monogenic, with over 100 mutations identified in SLC5A2, providing a clear genotype-phenotype correlation. This simplicity allows for precise genetic manipulation in cell models to dissect the functional impact of specific mutations. Public datasets, such as the Exome Aggregation Consortium (ExAC) and gnomAD, provide allele frequencies for SLC5A2 variants, facilitating the selection of clinically relevant mutations for experimental studies. Open questions include the molecular basis of differential SGLT2 inhibitor affinity, the role of SGLT2 in non-renal tissues, and the long-term renal consequences of chronic glucosuria. Gene-edited cell models are essential for addressing these questions, as they enable the creation of isogenic lines with specific mutations to study their effects on SGLT2 function, trafficking, and inhibitor binding.

FRG is not a cancer, but it involves dysregulation of glucose transport pathways. The primary pathway affected is the sodium-glucose cotransport system in the proximal tubule. The key steps are:

1. Glucose filtration: Glucose is freely filtered at the glomerulus.

2. Reabsorption: SGLT2 in the early proximal tubule reabsorbs the majority of filtered glucose (about 90%) in a sodium-dependent manner.

3. Secondary transport: SGLT1 in the later proximal tubule reabsorbs the remaining glucose.

4. Mutations in SLC5A2: Loss-of-function mutations reduce or eliminate SGLT2 activity, leading to glucosuria.

Other pathways that may be indirectly affected include the renin-angiotensin-aldosterone system (RAAS) and the sympathetic nervous system, which are involved in renal hemodynamics and sodium balance.

Data from ClinVar and gnomAD indicate that most SLC5A2 mutations are rare, with a few recurring variants such as p.Arg77His and p.Thr157Met. These mutations impair SGLT2 trafficking, substrate binding, or catalytic activity.

Although FRG primarily affects glucose transport, the resulting glucosuria can influence systemic metabolic signaling. Key networks include:

• • Insulin signaling: Chronic glucosuria may lead to a mild energy deficit, affecting insulin sensitivity and secretion.
• • Renal sodium handling: Reduced SGLT2 activity increases distal sodium delivery, activating tubuloglomerular feedback and affecting blood pressure regulation.
• • Inflammatory pathways: Persistent glucosuria may induce mild renal inflammation, though this is not well-characterized.

In cell models, SGLT2 knockout or knockdown can alter cellular energy metabolism, AMPK activation, and autophagy. These pathways are relevant for understanding the broader effects of SGLT2 inhibition.

Organoids derived from human induced pluripotent stem cells (iPSCs) can recapitulate proximal tubule structure and function, providing a more physiologically relevant model for studying SGLT2 mutations. Organoids can be gene-edited to introduce specific SLC5A2 mutations and used for drug testing.

Animal models for FRG are limited because mice with Sglt2 knockout exhibit a milder phenotype, likely due to compensatory mechanisms. However, they are useful for studying the physiological consequences of SGLT2 loss.

• • Sglt2 knockout mice: These mice exhibit glucosuria and increased urine output, but are otherwise healthy. They are used to study the role of SGLT2 in glucose homeostasis and the effects of SGLT2 inhibitors.
• • Zucker diabetic fatty (ZDF) rats: These rats are used to study diabetes and SGLT2 inhibitor efficacy, but they do not have SLC5A2 mutations.
• • Induced models: Pharmacological inhibition of SGLT2 with inhibitors like phlorizin can mimic FRG in wild-type animals.

Patient-derived xenografts (PDX) are not applicable for FRG as it is not a cancer.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with specific SLC5A2 mutations, providing powerful tools for studying FRG. Examples include:

• • SLC5A2 knockout cell lines: Complete loss of SGLT2 function, useful for studying the effects of total loss of glucose transport.
• • Point-mutation knock-in lines: Introduction of clinically relevant mutations (e.g., p.Arg77His) to study their impact on protein expression, trafficking, and function.

These models are commercially available from various sources, ensuring sequence verification and quality control. They are essential for drug discovery, as they allow screening of SGLT2 inhibitors in a physiologically relevant context and for studying the molecular mechanisms of inhibitor binding and resistance.

Gene-edited cell lines are used to validate the function of SLC5A2 and its variants. For example:

• • Knockout lines: Used to confirm the role of SGLT2 in glucose uptake and to identify downstream signaling pathways.
• • Knock-in lines: Used to assess the pathogenicity of specific mutations by measuring glucose transport activity, protein localization, and sensitivity to inhibitors.

These models enable high-throughput functional assays, such as fluorescent glucose uptake assays, to characterize mutant SGLT2 proteins.

Isogenic cell lines with different SLC5A2 mutations are valuable for drug screening. They can be used to:

• • Evaluate SGLT2 inhibitor efficacy: Test the inhibitory effect of compounds on glucose uptake in wild-type vs. mutant cells.
• • Study resistance mechanisms: Identify mutations that confer resistance to SGLT2 inhibitors, which may inform personalized treatment strategies.
• • Assess off-target effects: Use knockout lines to confirm target specificity of inhibitors.

These applications are critical for the development of next-generation SGLT2 inhibitors with improved selectivity and fewer side effects.

CRISPR-based screens using gene-edited cells can identify synthetic lethal interactions and biomarkers. For example:

• • Synthetic lethality screens: In SLC5A2 knockout cells, screen for genes that become essential for survival, potentially revealing novel therapeutic targets.
• • Biomarker identification: Compare gene expression profiles between wild-type and mutant cells to identify biomarkers of SGLT2 dysfunction.

These approaches can lead to the discovery of new biomarkers for renal diseases and improve patient stratification.