Erlotinib preserves renal function and prevents salt retention in doxorubicin treated nephrotic rats

Abstract

Nephrotic syndrome is associated with up-regulation of the heparin-binding epidermal growth factor (HB-EGF). Erlotinib blocks the activation of the epidermal growth factor receptor (EGFR) in response to HB-EGF. This study investigates the effect of Erlotinib on the progression of proteinuria, renal dysfunction, and salt retention in doxorubicin treated nephrotic rats. Male rats were divided into 3 pair-fed groups (n = 13/group) as follows: Control rats (Ctrl); rats receiving intravenous doxorubicin (Dox); and rats receiving intravenous doxorubicin followed by daily oral Erlotinib (Dox + Erl). Upon establishment of high grade proteinuria, urine sodium and creatinine clearance were measured. Kidney tissue was dissected and analyzed for γ-epithelial sodium channel (γENaC), sodium-potassium -chloride co-transporter 2 (NKCC2), sodium chloride co-transporter (NCC), aquaporin 2 (AQP2), and EGFR abundances using western blot. Creatinine clearance was preserved in the Dox + Erl rats as compared to the Dox group (in ml/min: Ctrl: 5.2±.5, Dox: 1.9±0.3, Dox + Erl: 3.6±0.5). Despite a minimal effect on the degree of proteinuria, Erlotinib prevented salt retention (Urinary Na in mEq/d: Ctrl: 2.2±0.2, Dox: 1.8±0.3, Dox + Erl: 2.2±0.2). The cleaved/uncleaved γENaC ratio was increased by 41±16% in the Dox group but unchanged in the Dox + Erl group when compared to Ctrl. The phosphorylated EGFR/total EGFR ratio was reduced by 74±7% in the Dox group and by 77±4% in the Dox + Erl group. In conclusion, Erlotinib preserved renal function and prevented salt retention in nephrotic rats. The observed effects do not appear to be mediated by direct blockade of EGFR.

Figure 1. Doxorubicin-induced increases in water intake and urine output are reversed by Erlotinib treatment.

Shown are the changes in weight (A), urine output (B), and water intake (C) plotted over time in control rats (solid line), doxorubicin (Dox) treated rats (dotted line), and doxorubicin + Erlotinib (Dox + Erl) treated rats (dashed line). Data are expressed as means ± SE (n = 13, * p<0.05 for Dox or Dox+Erl vs. control at same time point, and for Dox or Dox+Erl vs. Dox or Dox+Erl at time 0).

Figure 2. Proteinuria is apparent by 10 days after doxorubicin.

The lines show the progression of the proteinuria plotted over time in control rats (solid line), doxorubicin treated rats (dotted line), and doxorubicin + Erlotinib treated rats (dashed line). Data are expressed as means ± SE (n = 13, * p<0.05 for Dox or Dox+Erl vs. control at same time point).

Figure 3. Erlotinib restores urine sodium excretion to control levels but only partially corrects creatinine clearance.

Left: Bar graph showing the average urine sodium excretion evaluated at the end of the observation period in control rats (Ctrl; white bar), doxorubicin treated rats (Dox; solid black bar), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bar). Right: Bar graph showing the average creatinine clearance evaluated at the end of the observation period in control rats (Ctrl; white bar), doxorubicin treated rats (Dox; solid black bar), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bar). Bottom: Bar graph showing the average fractional excretion of water (FE Water) evaluated at the end of the observation period in control rats (Ctrl; white bar), doxorubicin treated rats (Dox; solid black bar), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bar). Data are expressed as means ± SE (n = 13, * p<0.05, ** p<0.001).

Figure 4. The doxorubicin-induced decrease in phosphorylated EGRF/EGRF ratio is not corrected by Erlotinib.

EGFR and phosphorylated EGFR protein abundances in rat cortex tissue of control rats (Ctrl; white bars), doxorubicin treated rats (Dox; solid black bars), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bars). A: Representative immunoblots showing abundances of EGFR and phosphorylated EGFR in rat cortex tissue lysates. An equal amount of total protein from a different rat tissue sample was loaded into each lane. B: Densitometric analysis of western blots from 3 cohorts of animals. Data were normalized for the average densitometry of untreated animals in each group. Right: Total EGFR densitometric analysis. Middle: Phosphorylated EGFR densitometric analysis. Left: The ratio of phosphorylated EGFR to total EGFR densitometries. Data are expressed as means ± SE (n = 11, * p<0.05).

Figure 5. Cleaved γENaC subunit protein abundance is increased by doxorubincin relative to control or Erlotinib-treated animals.

Shown is the γENaC subunit in rat cortex tissue of control rats (Ctrl; white bars), doxorubicin treated rats (Dox; solid black bars), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bars). A: Representative immunoblots showing abundance of γENaC in rat cortex tissue lysates. Each lane was loaded with an equal amount of total protein from a different rat. B: Densitometric analysis of western blots from 3 cohorts of animals. Data were normalized for the average densitometry of untreated animals in each group. Separate densitometric analysis was performed for the total, cleaved, and uncleaved γENaC. Left: Total ENaC densitometric analysis. Right: The ratio of cleaved to uncleaved γENaC densitometries. Data are expressed as means ± SE (n = 11, * p<0.05).

Figure 6. NCC and NKCC2 show different responses to doxorubicin and Erlotinib.

Shown are NCC and NKCC2 protein abundances in cortex and outer medullary tissue of control rats (Ctrl; white bars), doxorubicin treated rats (Dox; solid black bars), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bars). A: Representative immunoblot showing abundance of NCC in cortex tissue lysates and NKCC2 in outer medullary tissue lysates. An equal amount of total tissue lysate protein from a different rat was loaded into each lane. B: Densitometric analysis of western blots from 3 cohorts of animals. Data were normalized for the average densitometry of untreated animals in each group. Data are expressed as means ± SE (n = 11, * p<0.05).

Figure 7. Doxorubicin decreases AQP2 in inner medulla (IM) and Erlotinib restores levels in IM tip.

Shown: AQP2 protein abundance in IM tip and base tissue of control rats (Ctrl; white bars), doxorubicin treated rats (Dox; solid black bars), and doxorubicin + Erlotinib treated rats (Dox + Erl; patterned bars). A: Representative immunoblots showing abundance of AQP2 in IM tip and base tissue lysates. Each lane containes samples from a different rat. B: Densitometric analysis of western blots from 3 cohorts of animals. Data were normalized for the average densitometry of untreated animals in each group. Data are expressed as means ± SE (n = 11, * p<0.05).