Impaired renal transporter gene expression and uremic toxin excretion as aging hallmarks in cats with naturally occurring chronic kidney disease

Abstract

Aging leads to nephron senescence and chronic kidney disease (CKD). In cats, indoxyl sulfate (IxS) has been previously quantified and associated with CKD, and little is known about tubular transporters. Two cohorts of cats aged 6 to 21 years were enrolled. Cohort 1 included 41 colony cats with 28 control and 13 CKD cats. Cohort 2 had 30 privately-owned cats with 10 control and 20 CKD cats. In cohort 1, serum concentrations of IxS, trimethylamine N-oxide (TMAO), p-cresol sulfate (PCS), and phenyl sulfate were higher in CKD vs. control cats (all P<0.05). This observation was independently validated in cohort 2. Renal cortical and medullar tissues were collected from a third cohort of cats euthanized for humane reasons unrelated to the study. We provided the evidence that renal tubular transporter genes, OAT1, OAT4, OATP4C1, and ABCC2, but not OAT3, were expressed in the kidneys of cats, and their expressions were downregulated in CKD (all FDR<0.1). Cats and humans share 90.9%, 77.8%, and 82.5% identities in OAT1, OATP4C1, and ABCC2 proteins, respectively. In healthy cats, circulating TMAO and IxS are significantly correlated with age. Our study reveals impaired uremic toxin secretion and tubular transporter expression in cats with CKD.

Keywords: ABCC2; OAT1; OATP4C1; indoxyl sulfate; trimethylamine N-oxide.

Figure 1

Serum concentrations of uremic toxins. The concentrations of uremic toxins were quantified in cohort 1 (A–D) and cohort 2 (E–I). Asterisks (*) indicate statistical significance from (A–D) Mann-Whitney test and (E–M) Dunn’s multiple comparisons test: *P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. The box and bars represent mean and SEM. TMAO, trimethylamine N-oxide; IxS, indoxyl sulfate; PCS, p-cresol sulfate; PS, phenyl sulfate; IAA, indole-3-acetic acid; CKD2, CKD stage 2; CKD3, CKD stage 3.

Figure 2

Urine levels of uremic toxins in cohort 1. The levels of urinary uremic toxins were normalized by creatinine (A–E). The box and bars represent mean and SEM. TMAO, trimethylamine N-oxide; IxS, indoxyl sulfate; PCS, p-cresol sulfate; PS, phenyl sulfate; IAA, indole-3-acetic acid; Cr, creatinine; CKD2, CKD stage 2. Asterisks (*) denote P-values from the Mann-Whitney test, *P<0.05, **P<0.01.

Figure 3

Uremic toxins in healthy senior cats. Healthy cats in cohort 1 were divided into two groups: those less than 12 years of age (YNG), and those aged 12 years or older (OLD). (A–E) The box and bars indicate means and SEM. P-values from the Mann-Whitney test are presented as asterisks: *P < 0.05. (F, G) Spearman’s correlation analysis between cat’s age and log concentration of TMAO and IxS, respectively. P-value and correlation coefficient are indicated in the plot.

Figure 4

Organic anionic transporter RNA-seq gene expressions. Medullar (A–D) and cortical (E–H) tissue expressions of OAT4, OAT1, OAPT4C1, and ABCC2, respectively. Illustration of transporters in the proximal tubule cell (I). OAT1 and OATP4C1 are the uptake transporters on the basolateral surface, while ABCC2 is the efflux transporter localized at the apical surface. OAT4 is thought to play a role in both reabsorption and secretion from the apical side. All genes were differentially expressed between control and across CKD groups in both cortex and medullar (FDR<0.1 in all cases). Bars indicate means. Asterisks indicate Dunn’s multiple comparisons tests: *P<0.05, **P<0.01. OAT1/4, organic anion transporter family member 1/4; OATP4C1, organic anion transporting peptide family member 4C1; ABCC2, ATP-binding cassette subfamily C member 2. CKD1/2: CKD stages 1 and 2; CKD3/4: CKD stages 3 and 4; Amyloid: CKD by amyloidosis. Panel I created with BioRender.com.

Figure 5

Alignment on mammalian OAT1 protein sequences. Mouse, rat, dog, and cat OAT1 protein sequences are compared to their human orthologous sequence. Dogs and cats share 91.3% and 90.9% OAT1 protein sequence identities with humans, respectively. “Cov” indicates the percentage of amino acids covered in the alignment, and “pid” indicates protein sequence identity compared to humans, consensus denotes consensus out of 80% of the sequences from the group. Red asterisks indicate the five glycosylation sites on the first extracellular loop, which is delineated by the two upward red arrows in positions 39 and 125, respectively. Humans share the same hydrophilic aspartate (D) with cats and dogs (marked by the 2^nd red arrow), but not rodents. Dog and cat’s OAT1 proteins are computationally predicted.