Table 3.

Protein-bound substances increased in CKD and examined for causal effects on cardiovascular calcification or associated readouts.

Effect	Substance	Species	Tissue or Cell Type	Type of Study		Analysis of Calcification or Calcification-Associated Process * by	Reference	Plasma Level in CKD	Classified as Uremic Toxin **
				In vitro	Animal
INDUCTION	Indoxyl sulphate (IS)	Human	VSMC	V $		Calcium content Alizarin red staining Upregulation of CBFA1/RUNX2, ALP, BMP2 and OPN Downregulation of α-SMA and SM22-α	Bouabdallah [30]	↑ [§]	V
		Human	VSMC	V		Alizarin red staining Upregulation of CBFA1/RUNX2, ALP and OPN Downregulation of α-SMA and SMTN	Chen [51]
		Human	VSMC		V	Alizarin red staining Upregulation of CBFA1/RUNX2 and OPN	Zhang [52]
		Human	VSMC	V		Alizarin red staining Upregulation of CBFA1/RUNX2 and OPN	Chen [53]
		Human	VSMC	V		Alizarin red staining ALP activity Downregulation of α-SMA Upregulation of CBFA1/RUNX2	He [54]
		Human	VSMC	V		Upregulation of p53, p21, and prelamin A	Muteliefu [55]
		Human	HepG2	V		Downregulation of fetuin-A	Ochi [56]
		Rat	Aorta	V $		Calcium content Alizarin red staining	Bouabdallah [30]
		Rat	Aorta		V 1	Von Kossa staining Upregulation of OPN, CBFA1/RUNX2, ALP and osteocalcin	Adijiang [57]
		Rat	Aorta		V 2	Von Kossa staining	Adijiang [58]
		Rat	Aorta		V 3	Alizarin red staining Downregulation of α-SMA and SMTN Upregulation of CBFA1/RUNX2, ALP and OPN	Chen [51]
		Rat	Aorta		V 4	Calcium content Von Kossa staining Activation of inflammation and coagulation pathways	Opdebeeck [59]
		Rat	Aorta		V 2	Upregulation of 8-OHdG and MDA in the calcification area	Muteliefu [55]
	p-Cresyl sulphate	Rat	Aorta		V 4	Calcium content Von Kossa staining Activation of inflammation and coagulation pathways	Opdebeeck [59]	↑ [§]	V

ALP—alkaline phosphatase; BMP2—bone morphogenetic protein 2; CBFA1/RUNX2—core-binding factor subunit alpha-1/runt-related transcription factor 2; MDA—malondialdehyde; 8-OHdG—8-hydroxyl-2’-deoxyguanosine; OPN—osteopontin; α-SMA—alpha-smooth muscle actin; SM22-α—smooth muscle protein 22-alpha; SMTN—smoothelin; VSMC—vascular smooth muscle cells. * Readouts of calcification-associated processes, such as cell signaling and gene/protein expression, are shown in italic. [§] As described in comprehensive uremic toxins reviews [19,20]. ** As described in comprehensive uremic toxins reviews [19,20,21]. ^$ In pro-calcifying medium. ¹ In vivo hypertensive rat model: (1) Dahl salt-resistant normotensive rats; (2) Dahl salt-sensitive hypertensive rats; (3) Dahl salt-sensitive hypertensive IS-administered rats for 30 weeks. ² In vivo hypertensive rat model: (1) Dahl salt-resistant normotensive rats; (2) Dahl salt-resistant normotensive IS-administered rats; (3) Dahl salt-sensitive hypertensive rats; (4) Dahl salt-sensitive hypertensive IS-administered rats for 32 weeks. ³ In vivo uremic rat model: rats subjected to 5/6 nephrectomy were injected with IS at a dosage of 100 mg/kg/48 h for 24 weeks. Control rats with 5/6 nephrectomy received the same volume of phosphate-buffered saline injection every 48 h for 24 weeks. ⁴ In vivo uremic rat model: Male Wistar rats were exposed to a 10-day adenine sulphate treatment via daily oral gavage (600 mg/kg per day) to induce CKD. CKD rats fed a phosphate-enriched diet (1.2% Pi and 1.06% Ca) were randomly assigned to three treatment groups: (1) vehicle, (2) 150 mg/kg IS, or (3) 150 mg/kg p-cresyl sulphate; ↑ indicates increased plasma levels in CKD patients.