Lecture 17: Nephrotoxicity

What are the Key Functions of the Kidney?

• It is an essential organ, playing a major roles in

  • Excretion of waste products and unwanted substances e.g.

    • Urea, creatinine, uric acid (products from nitrogen metabolism)

    • Xenobiotics and their metabolites

  • Regulation of extracellular fluid

    • Retention of vital substances (proteins, glucose) → may otherwise be lost in urine

    • Conservation of water, electrolytes, amino acids and other solutes

    • Maintenance of acid-base balance

  • Endocrine activity

    • e.g. renin, calcitriol (Vitamin D3), erythropoietin

• It is an important organ → damaged to it caused by disease or toxicity can have signifcant effects on the body

What is the Nephron?

• The main functional unit of the kidney (~1-2 million present)

  • Tubular in structure

• Carries out excretion and reabsorption

• Divided into different segments, which contribute to the formation of urine and the removal of unwanted waste products

• These segments include:

  • Glomerulus and Bowmans Capsule

  • Proximal Convoluted Tubules

  • Loop of Henle

  • Distal Convoluted Tubule

  • Collecting Duct

Describe the flow of filtrate through the nephron and the role of the glomerulus and Bowman’s capsule

• Filtrate enters Bowman’s capsule (located proximally), which surrounds the glomerulus (a capillary network), where it then directly enters the nephron

• Flow of filtrate: These solutes pass through

  • 1. Proximal convoluted tubule (PCT)

  • 2. Loop of Henle → Major role in water and NaCl reabsorption

  • 3. Distal convoluted tubule (DCT)

  • 4. Collecting duct → forms urine

• The Glomerulus and Bowman’s capsule are important in the filtration and collection of solutes from the blood

What is the role of the Proximal Convoluted Tubule (PCT)?

• Plays a major role in the nephron’s function

• It is surrounded by lots of capillaries

• The main site at which the kidney is damaged through toxic injury

• It is involved in:

  • Reabsorption of all nutrients and most ions that enter the lumen of the tubule through filtration

  • Tubular secretion: movement of waste products, xenobiotics and metabolites from blood into the lumen

  • Recapture of filtered proteins (that enter the glomerular filtrate and enter the lumen of tubules) via endocytosis

What is the Function of the Distal Convoluted Tubule?

• It is involved in:

  • Hormone-controlled reabsorption of Na+ and Ca2+

  • Secretion of K+

What is the Function of the Collecting Duct?

• It is the site of:

  • Water absorption

  • Urea recycling

  • Ion homeostasis

Why is the Kidney Particularly Susceptible to Toxic Injury?

• Most toxicants are blood-borne → High blood flow to kidney (=high delivery of toxicants)

  • Blood flow to the kidney~2000 L/day

  • 180-200 L filtered every day by the kidney

    • Glomerular Filtration Rate (GFR): (the rate at which glomeruli filter the fluid to the interior of the nephron) → ~125 mL/min

• Bioactivation of Toxicants → High expression of metabolising enzymes (e.g. CYP450)

  • Leads to bioactivation of toxicants and toxic metabolite formation locally, making the kidney vulnerable to toxicity

• Powerful concentrating mechanisms (reabsorption of undesired substances and secretions of unwanted materials, e.g. metabolites and xenobiotics)

  • ~99% of filtrate is reabsorbed → strong concentrating effect

  • Only 1–2 L of urine is produced per day

  • Extensive tubular secretion and reabsorption (of unwanted substances and xenobiotics) → repeated cellular exposure (to cells that form kidney tubules)

• High expression of multiple membrane transporter proteins

  • Transporters allow for toxicants to be taken into tubular cells (giving rise to kidney vulnerability)

  • Leads to locally high intracellular toxicant concentration → readily enters kidney cells and can accumulate = significant damage

  • W/o specific transporters, toxicants can’t enter and pose no damage

• Proximal convoluted tubule (PCT) is particularly susceptible → major site of reabsorption and tubular secretion + high levels of transporter expression

What is Acute Kidney Injury?

• A broad medical and consensus term for renal failure, characterised by a rapid loss of excretory function

  • Used to describe the renal injury caused by toxicants/ toxic insults

• It occurs within hours or days → potentially fatal

• Manifestations include:

  • decreased Glomerular Filtration Rate (GFR),

  • decreased urinary output,

  • increased Blood Urea Nitrogen (BUN),

  • increased serum creatinine,

  • altered ion concentrations in blood

• Potentially reversible but associated with long-term changes in kidney function

• Can lead to chronic kidney disease (CKD) → progressive and irreversible kideny damage and can result in death

What are the Manifestations of Kidney Injury and Toxicity?

• Glomerular Injury

• Acute Tubular Injury

• Acute Interstitial Nephritis

• Insolubility of Drugs in Urine

How Does Glomerular Injury Manifest in Acute Kidney Injury and Toxicity?

• Damage to the Glomerulus,

• e.g. gentamycin (causes proliferation and contraction of mesangial cells in the glomerulus, reducing glomerular filtration)

• Results in the entry of proteins into the lumen of the nephron

How Does Acute Tubular Injury Manifest in Acute Kidney Injury and Toxicity?

• Occurs particularly in the PCT

• Toxicant enters kidney epithelial cells (especially PCT) and causes damage

• e.g. cisplatin, cadmium

How Does Acute Interstitial Nephritis Manifest in Acute Kidney Injury and Toxicity?

• T-cell-mediated immune response

• Inflammation of the kidneys, including infiltration of kidney by immune cells

• e.g. immune checkpoint inhibitor drugs (new cancer drugs), beta lactam antibiotics (e.g. penicillin)

How Does Insolubility of Drugs in Urine Occur in Acute Kidney Injury and Toxicity?

• Mechanical problems associated with the insolubility of some drugs and xenobiotics in the injury

• May crystallise (‘crystal nephropathy’) or form (insoluble) non-crystalline aggregates.

• Can block tubules and/or cause physical tubular damage and inflammation

  • e.g. methotrexate (crystallises out urine),

  • e.g. vancomycin (interacts with the kidney protein uromodulin and forms non-crystalline aggregates (casts), which can block the tubules).

• This is often worse in people with:

  • pre-existing renal insufficiency;

  • urine pH

  • flow rate

    • … These can have significant effects.

Why is the Proximal Convoluted Tubule A Major Site of Damage Following Toxicant Injury?

• PCTs express a large variety of transporters

• Transport of materials occurs in both directions via:

  • tubular secretion of unwanted substances (including xenobiotics) from blood into the lumen of tubules;

  • recapture of filtered substances from the lumen of the tubules

• PCTs are highly polarised → protein expression on the apical membrane (facing the lumen) differs from protein transporter expression on the basolateral membrane (in contact with blood and tissue fluid)

  • Many different transporters, with differing expression

• Specific transporters for recapture of essential materials from the lumen

  • High expression of transporters for metal ions → important roles as trace elements in the body

  • Cells recapture filtered proteins and some xenobiotics from the lumen via endocytosis e.g. megalin

What Transporters Mediate Apical Transport?

• Extrusion into the lumen is largely mediated by:

  • Multidrug and toxin extrusion proteins (MATEs)

  • Multidrug resistance (proteins) (MDR1 (PgP; MRPs)

  • Organic cation/carnitine transporters (OCTNs)

  • Breast cancer resistance protein (BCRP) – an active transporter that removes substances from cells (protective role)

What Transporters Mediate Basolateral Transport?

• Uptake from the blood is largely mediated by:

  • Organic Cation Transporters (OCTs)

  • Organic Anion Transporters (OATs)

  • Organic Anion Transporting Polypeptides (OATPs)

How do aminoglycosides (e.g. neomycin) cause nephrotoxicity in proximal convoluted tubule (PCT) cells?

• Powerful antimicrobial antibiotic used for serious gram-negative infections → notoriously nephrotoxic

• Enters PCTs from the glomerular filtrate via megalin-mediated endocytosis

• ~5% of the administered dose may accumulate in PCT cells (concentration mechanisms)

• Enters and accumulates in lysosomal lumen via uptake mechanisms (endocytosis of megalin and neomycin)

• Binds lysosomal phospholipids, compromising membrane function, leading to lysosomal leakage

• Lysosomes contain powerful digestive enzymes, which can leak into the cytoplasm and cause cell injury damage

• Aminoglycosides can also enters cytoplasm, causing mitochondrial damage, ROS generation and apoptosis of PCT cells

What is Megalin?

• An Endocytosis 600kDa apical membrane protein

• It recaptures filtered proteins and vitamins present in the glomerular filtrate by endocytosis

• This mechanism is effective in causing the accumulation of neomycin

What is Cadmium?

• A non-essential metal (no physiological role) and toxicant

• It is found widely in the environment as:

  • Anti-corrosive agent

  • Component of some batteries

  • Contaminant in food and tobacco

How is Cadmium Handled By The Body?

• Cd²⁺ is taken up by the liver

• It induced metallothionein (MT) production → Forms a protective complex with Cd²⁺

• Cd²⁺-MT is released into the blood and filtered in the glomerulus

• Complexes are endocytosed into the PCT cells via megalin from the filtrate

• It is processed via lysosomal degradation, degrading megalin in PCT cells and releasing Cd²⁺

• Cd²⁺ can induce MT expression in the kidney (protective effect), but if levels exceed a threshold, free cadmium accumulates and impairsthe cells ability to produce MT

• Cd²⁺ also enters cells via Zn transporteres and divalent metal transporters

  • Entry through endocytosis is one of the main mechanisms by which Cd²⁺ causes harm to the kidney

How Does Cadmium Cause Nephrotoxicity?

• If free in the cytosol, Cd²⁺ induces ROS generation and oxidative stress

• Cd²⁺ experimental administration in mice causes GSH depletion, increased lipid peroxidation

  • Demonstrates a high level of ROS production or a failure of ROS detoxification

• Initial changes (manifestations) in PCT cell adhesion, altered signalling and autophagy, then apoptosis

How Does Cadmium Cause Oxidative Stress?

• Cd2+ causes an increase in the production of ROS and reduces the cell’s ability to protect itself

• It inhibits mitochondrial complexes I and III → main mechanism of ROS generation

• Increases NOX expression in kidney → increases superoxide production

• Displaces copper and iron ions from carrier proteins → promotes Fenton reaction (HO^• production)

• Directly binds to GSH and Cystine (its precursor), via SH groups, leading to GSH depletion and reducing cellular protection

• Forms complexes with Selenium in the body and is excreted via bile → selenium deficiency

• If GSH and Se are depleted, peroxiredoxin, glutathione peroxidase, glutaredoxin and thioredoxin are unable to function → loss of protective antioxidant defence functions

  • Only SOD and catalse are available for enzymatic ROS detoxification

• Loss of this protection is likely the main reason for oxidative stress following cadmium exposure

What is the Nephrotoxicity Assocaited with Cisplatin?

• A widely used anticancer drug (member of the platins family), killing tumours through platinum-DNA adduct formation

• Causes dose-limiting nephrotoxicity in 1 in 3 patients and contributes to 20% of hospitalisations for acute renal failure

• Limits the dosage given to patients (+reduces effectiveness) → oncologists are unable to administer as much to treat the tumour due to side effects

• Main site of damage: PCT

  • Cisplatin can’t cross the membrane alone → requires a transporter

• It is also neurotoxic, causing otoxicity and sensory neuropathies

• Effective cancer drug but limited by side effects

What evidence shows that OCT transporters mediate cisplatin toxicity?

• Model: HEK293 cells induced to express human OCT1–3 (Transfection)

• Cells compared to increasing concentrations of cisplatin

• Toxicity is measured by cytoplasmic enzyme Lactate dehydrogenase (LDH ) release (a marker of cell damage, when present in the extracellular fluid)

• With increasing concentrations of cisplatin:

  • Little effect (minimal damage) in Vector Control or OCT3 cells

  • OCT1 cells: increased amounts of cell damage following cisplatin exposure

• OCT2 cells: greatest cell damage following cisplatin exposure

• Demonstrates OCT1 and especially OCT2 transport cisplatin into cell interior, causing toxicity and damage

How do membrane transporters contribute to cisplatin nephrotoxicity?

• Necessary for uptake and extrusion in PCT cells:

• Cisplatin uptake is mainly mediated by OCT2 and also via copper transporter (Ctr1)

• In humans, efflux (removal) is mainly mediated by MATE1 (MATE2K may also contribute)

• Cisplatin is a poor substrate for efflux transporters → readily enters the cell but is not efficiently removed

• Leads to its accumulation in proximal convoluted tubule (PCT) cells to unsafe levels

• Results in cell damage and nephrotoxicity

• Transporters influence both:

  • Toxicity and harmful effects (kidney damage)

  • Tumour sensitivity and drug efficacy (drug must enter tumour cells)

How does the SLC22A2 (OCT2) polymorphism affect cisplatin nephrotoxicity?

• OCT2 (encoded by SLC22A2 gene) mediates cisplatin uptake into renal cells

• Polymorphism present in this gene across the human population, e.g. SNP: c.808G>T (rs316019)

  • Causes amino acid change: Ser270 (G>G)→ Ala270 (G>T)

  • SNP at position 808 means Guanine can be Thymine

• This reduces OCT2 transport activity, decreasing uptake of cisplatin (+ OCT substrates) into proximal tubule cells (shown in Human study in patients receiving cisplatin for cancer treatment)

  • Previous studies show individuals have reduced transport of various OCT2 substrates due to this polymorphism

• Clinical consequence: reduced intracellular accumulation of cisplatin

• Polymorphism is suggested to offer protection against nephrotoxicity

How is Serum Creatine Used To Assess Kidney Damage e.g. Following Cisplatin?

• Used as a biomarker of kidney function

• A poor biomarker, but it has traditionally been used to monitor kidney function

  • Baseline levels are low prior to drug administration

• Cisplatin evidence (OCT2 polymorphism):

  • Individual with GG variant (Ser270): Large increase in serum creatinine after first round of chemotherapy indicating more kidney damage

  • Individuals with GT variant (Ala270): Smaller increase in serum creatinine (less pronounced) → reduced nephrotoxicity

• Supports role and implicates OCT2 in cisplatin-induced kidney damage and nephrotoxicity

What Must Be Considered When Using OCT Knockout Models to Study Cisplatin-Induced Nephrotoxicity?

• Caution with KO models: species differences affect interpretation

• Human PCT transporters:

  • OCT2 = main uptake transporter in basolateral membrane

  • MATE1 + MATE2K = main efflux/extrusion transporters in apical membrane

• Rodent PCT transporters:

  • OCT1 + OCT2 are both expressed at high levels in basolateral membrane

  • OCT1 has a minor role in cisplatin transport

  • MATE1 = main efflux/extrusion transporter in apical membrane

• Need to knock out both OCT1 and OCT2 in the mouse due to their contribution to cisplatin transporters, to examine the OCT role in nephrotoxicity

  • Must use OCT1/2 double knockout in mice → both contribute to cisplatin uptake

• Double KO mice are viable → able to survive and develop

• Rodents are not directly comparable to humans

What evidence shows that OCT1/2 knockout protects against cisplatin nephrotoxicity?

• OCT1/2 double knockout mice compared to wild-type (WT)

• Histology:

  • WT → tubular dilation + necrosis (significant damage)

  • OCT1/2 KO → minimal damage + less nephrotoxicity than WT (morphologically normal → loss of transporter expression has minimal effect)

    • Cisplatin is unable to enter cells → transporter mediating entry into cells is absent

• Kidney function (Blood Urea Nitrate levels):

  • WT → Significant increase in BUN (consistent with kidney damage)

  • KO → no significant increase in BUN

• Supporting evidence:

  • Cimetidine (an OCT2 inhibitor; competitive inhibition) acts as a substrate for OCT2, protecting kidney cells from cisplatin toxicity in WT mice

• Inhibiton of OCT offers protection → implicates OCT2 as having a major role in mediating cisplatin nephrotoxicity

Why is Nephrotoxicity of Clinical Importance and Concern?

• Drug nephrotoxicity is implicated in ~25% of all AKI cases

• ~25% of serious adverse drug reactions affect the kidney

• ~19% of candidate drugs are withdrawn in phase 3 clinical trials due to nephrotoxicity, but only~2.8% of candidate drugs are rejected due to nephrotoxicity in pre-clinical trials

• Most drugs that show nephrotoxicity in clinical trials make it through pre-clinical testing

• This suggests that better tests are required to identify nephrotoxicity earlier

How is Nephrotoxicity Currently Assessed?

• Following in vitro testing using cultured cells, animal testing typically uses 1 rodent and non-rodent species across a 28-day exposure period, with effects monitored using biomarkers (BUN and serum creatinine) and kidney histopathology

What Difficulty is Observed When Detecting Renal Injury/Disease?

• Only detectable when damage is severe, and function is impaired → excretion is unaffected until kidney damage is advanced

• Difficulty assessing damage directly → typically requires a biopsy (invasive process)

• Use of biomarkers for kidney injury, but traditional biomarkers (e.g. BUN, serum creatinine) are unreliable

  • Test kidney function rather than directly assessing damage

  • Only increases after significant damage has occurred

  • Many other factors affect them, e.g. nutrition

• Structural damage precedes functional impairment

  • Significant loss of healthy renal tissue is required before changes in function are detectable

• Traditional biomarkers only measure functional disturbance, rather than early damage → only report damage at high levels (low sensitivity)

What Efforts Have Been Made to Improve Nephrotoxicity Assessments?

• Focus for the past 10 years on the development of novel biomarkers

• Allow for the detection of kidney damage early on, before major levels of damage have occurred

What are Key Features of A Good Biomarker?

• They are ideally

  • Organ specific

  • Proportional to damage

  • Detectable before appreciable damage occurs

  • Urinary (less invasive than blood)

What is a Novel Urinary Biomarker Currenly Investigated for Renal Injury and Damage?

• Kidney injury molecule-1 (KIM-1), currently used to assess nephrotoxicity in drug development programmes

• When compared to serum creatinine in mice exposed to aristolochic acid (a plant nephrotoxin):

  • Both function as biomarkers

  • Serum creatinine: levels increase on day 3 post aristolochic acid administration; measured from a blood sample

  • KIM-1 levels increase >1 day post AA administration; measured from urine sample (sensitive and rapidly detects damage)

• Qualified by regulatory agencies (FDA & EMEA)

• Initially approved in rats and now used together with 5 other urinary biomarkers and traditional serum biomarkers (BUN, creatinine) in Phase I clinical trials.

Give Examples of Urinary Biomarkers Used to Assess Renal Toxicity

• Clusterin.

  • Molecular chaperone and cytoprotective protein.

  • Upregulated following kidney damage and released by damaged cells (glomerulus, tubules).

• Cystatin C (also measured in serum):

  • endogenous cysteine protease inhibitor.

  • Marker of glomerular injury and/or impaired tubular reabsorption.

• KIM-1.

  • PCT transmembrane protein with anti-inflammatory and phagocytic physiological functions.

  • Upregulated and cleaved following kidney injury: presence in urine due to damage to PCT.

• NAG (N-acetyl-beta-D-glycosaminidase)

  • lysosomal enzyme, a marker of acute oxidative stress, damaging PCT.

• NGAL (neutrophil gelatinase-associated lipocalin; also measured in serum)

  • Cytoprotective and anti-inflammatory effects.

  • Expression increased following distal tubular damage.

• Osteopontin

  • a multifunctional protein, including the regulation of immune responses.

  • Upregulated following tubular damage.

• Not all fully characterised in humans (data originally from rats).

Why are In Vitro Approaches Limited in Studying Renal Toxicity?

• Difficulty using cultured cells in in vitro approaches

• Kidney cells are highly polarised, with the apical surface experiencing different conditions from the basolateral surface

  Apical surface (exposed to lumen):

  • Exposed to a constant flow of fluid, causing mechanical stress, affecting the cytoskeleton’s conformation and transporter localisation

• Basolateral surface (exposed to interstitial fluid only)

  • Not exposed to mechanical stress

• Hard to replicate these conditions in 2D cultures and cells lose polarity and transporter expression

  • Reduces physiological relevance for toxicity testing

What In Vitro Approaches are Used for Assessing Renal Toxicity?

• 2D cultures

• Transwell culture

• Organoids

• Microfluidic Devices (Microphysiological systems)

How are 2D Cultures Used for Assessing Renal Toxicity In Vitro?

• Use of:

  • Cell lines (e.g., HK-2)

  • Primary renal cells (don’t divide well)

  • Immortalised cells (generated using viruses)

• Easy to grow and handle, but lacks polarity and fluid flow

• Not representative of in vivo kidney physiology (limited relevance)

  • Limited predictive relevance for toxicity

• More complex models (e.g., organ-on-chip) are being developed to address these limitations and overcome this problem

How are Transwell Cultures Used for Assessing Renal Toxicity In Vitro?

• A well with an insert with a porous membrane is used → creates 2 distinct chambers in a dish

• Allows for two different fluids to be in contact with the apical and basolateral surfaces of cells

• Maintains polarity but no fluid flow

• Attempts to overcome issues observed with 2D culture

How are Organoids Used for Assessing Renal Toxicity In Vitro?

• 3D cultures, where cells will differentiate → develop characteristics of glomerulus and tubules.

• Lack of perfused vascularisation is a challenge (unable to mimic capillaries), but can be transplanted into mice (which develop capillaries to supply the organoid) or co-cultured with endothelial cells to allow for capillary development.

  • Major impacts for toxicity testing in the future

How are Microfluidic Devices (MPS) Used for Assessing Renal Toxicity In Vitro?

• Example: “Kidney on a chip” → cells grown on ECM-coated membrane with fluid shear stress present → mimics physiological situation

• Use 3D bioprinting to model kideny organisation, including cells from glomerulus, PCT, DCT and collecting duct

• Fluid flow can be applied using microfluidic devices

How are organoids and kidney-on-a-chip models created, and why use human cells?

• Made from human iPSCs → can differentiate into different kidney cell types.

• Use of human material avoids the selective toxicity seen in animal models.

• Challenge: high variability between cultures → standardisation needed before use in pharma

What methods improve organoid realism, and what are their limitations?

• Fluid flow via microfluidics enhances kidney-like characteristics, but only lasts 2–4 weeks.

  • Limitations for long-term testing protocols

• Co-culture with endothelial & immune cells mimics vascularisation & immune response → aims to be a more authentic model

• Some renal injury biomarkers (e.g., KIM-1) are used to detect toxic damage.

What is the potential of organoids and chips in medicine and drug development?

• Chips can model kidney diseases → potential for patient-specific toxicity testing of various different compounds for personalised medicine

• Shows promise and significant potential as New Approach Methodologies (NAMs) in drug development.

• But requires validation to ensure accurate prediction of human kidney toxicity and other factors associated with the human kidney before replacing animal tests (ensure sutability as alternative before widespread uptake)