Therapy with 2'-O-Me Phosphorothioate Antisense Oligonucleotides Causes Reversible Proteinuria by Inhibiting Renal Protein Reabsorption

Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2'O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage

Therapy with 2'-O-Me Phosphorothioate Antisense Oligonucleotides Causes Reversible Proteinuria by Inhibiting Renal Protein Reabsorption

Antisense oligonucleotide therapy has been reported to be associated with renal injury. Here, the mechanism of reversible proteinuria was investigated by combining clinical, pre-clinical, and in vitro data. Urine samples were obtained from Duchenne muscular dystrophy (DMD) patients treated with drisapersen, a modified 2'O-methyl phosphorothioate antisense oligonucleotide (6 mg/kg). Urine and kidney tissue samples were collected from cynomolgus monkeys (Macaca fascicularis) dosed with drisapersen (39 weeks). Cell viability and protein uptake were evaluated in vitro using human conditionally immortalized proximal tubule epithelial cells (ciPTECs). Oligonucleotide treatment in DMD patients was associated with an increase in urinary alpha-1-microglobulin (A1M), which returned to baseline following treatment interruptions. In monkeys, increased urinary A1M correlated with dose-dependent accumulation of oligonucleotide in kidney tissue without evidence of tubular damage. Furthermore, oligonucleotides accumulated in the lysosomes of ciPTECs and reduced the absorption of A1M, albumin, and receptor-associated protein, but did not affect cell viability when incubated for up to 7 days. In conclusion, phosphorothioate oligonucleotides appear to directly compete for receptor-mediated endocytosis in proximal tubules. We postulate that oligonucleotide-induced low molecular weight proteinuria in patients is therefore a transient functional change and not indicative of tubular damage

pNS sera did not induce proteinuria in Balb/c^Thy-1.1 males.

Balb/cThy-1.1 males were injected intravenously, at 5 weeks ± 3 days old, with 2 mg serum protein from patients with recurrent FSGS, FSGS in the native kidneys, non-recurrent FSGS, or healthy controls. Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. No significant differences (p<0.05) compared to sera from healthy donors.</p

Different doses healthy plasma were injected in the Thy-1.1 mice with different backgrounds to determine maximum injection dose.

(A) Balb/cThy-1.1 males, (B) Balb/cThy-1.1 females, (C) C57BL/6Thy-1.1 males, (D) C57BL/6Thy-1.1 females were injected intravenously with various doses healthy plasma protein at 5 weeks ± 3 days old. (E) 129X1/SvThy-1.1 males, (F) 129X1/SvThy-1.1 females, (G) 129S2/SvPasThy-1.1 males, and (H) 129S2/SvPasThy-1.1 females received PBS vehicle (veh) injection only. Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. One-way ANOVA: * p≤0.05, ** p≤0.01, *** p≤0.001 versus vehicle.</p

Clinical characteristics of patients.

Clinical characteristics of patients.</p

Development of spontaneous proteinuria in Thy-1.1 mice with different backgrounds.

Development of spontaneous proteinuria in Thy-1.1 mice with different backgrounds.</p

<i>In vitro</i> and <i>in vivo</i> responses to simultaneously collected serum, plasma, and PP effluent.

(A) Cellular granularity of cultured human podocytes (hPod) exposed to simultaneously collected serum, plasma, and PP effluent, as measured by flow cytometry, is represented by the median side scatter relative to healthy controls. Relative granularity of two replicate experiments and their mean are presented. ** pB) Albuminuria of Balb/cThy-1.1 males injected intravenously, at 5 weeks ± 3 days old, with 2 mg protein from serum, plasma, or PP effluent from three FSGS patients during active disease (rec7, rec8, nat3) and serum and plasma from one FSGS patient not treated with PP, during active disease and remission (nat4). Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. Data of healthy donor sera derived from Fig 3 and pooled healthy plasma derived from Fig 1 are shown for comparison with patient samples. Although none of the patient sera induced proteinuria, the ages of the patients in this study do not match the ages of the healthy donors, which may limit interpretation of these data.</p

FSGS plasma induced proteinuria mostly in Balb/c^Thy-1.1 males.

FSGS plasma induced proteinuria mostly in Balb/cThy-1.1 males.</p

Development of spontaneous proteinuria of Thy-1.1 transgenic mouse strains.

Urine was collected weekly and urinary albumin was analyzed of (A) Balb/cThy-1.1 males, (B) Balb/cThy-1.1 females, (C) C57BL/6Thy-1.1 males, (D) C57BL/6Thy-1.1 females, (E), 129X1/SvThy-1.1 males, (F) 129X1/SvThy-1.1 females, (G) 129S2/SvPasThy-1.1 males, (H) 129S2/SvPasThy-1.1 females, (I) FVB/NThy-1.1 males, and (J) FVB/NThy-1.1 females. (DOCX)</p

Proteinuria of Balb/c^Thy-1.1 and C57BL/6^Thy-1.1 mice induced by FSGS plasmas.

(A) Balb/cThy-1.1 males, (B) Balb/cThy-1.1 females, (C) C57BL/6Thy-1.1 males, and (D) C57BL/6Thy-1.1 females were injected intravenously, at 5 weeks ± 3 days old, with 2 mg (Balb/cThy-1.1 males), or 12 mg (Balb/cThy-1.1 females, C57BL/6Thy-1.1 males and females) PP effluent protein from six patients with presumed CPF(s). Individual animal data and group means of urinary albumin/creatinine (alb/creat, mg/mg) ratio are presented. Data of healthy plasma injections from Fig 1 are shown for comparison with patient PP effluents. Mice were defined as responding to patient PP effluent if proteinuria exceeded the mean plus 3 times the standard deviation of proteinuria of healthy plasma-injected mice (indicated with dotted lines).</p