Lysosomal protease cathepsin D; a new driver of apoptosis during acute kidney injury

Abstract

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.

Figure 1. CtsD inhibition improves kidney function in FA nephrotoxic induced AKI.

Western Blot of pro- and mature CtsD and GAPDH in kidney lysates. (A) Representative pictures and magnified area of CtsD cortical staining in control and 48 hours FA treated kidneys. Red stars * point CtsD expressing cells. (B) BUN (C), serum creatinine (D), urine KIM-1 ELISA (E) in control and 48 hours FA vehicle and Pepstatin A treated kidneys. Animals were treated with vehicle or Pepstatin A (20 mg/Kg) 45 minutes before and 24 hours post-FA. N = 8, repeated measures of t-test, *P ≤ 0.05 or **P ≤ 0.01.

Figure 2. CtsD inhibition reduces tubular cell injury and apoptosis in FA nephrotoxic induced AKI.

Representative PAS pictures, damaged cells pointed with a black star *. (A) Percentage of tubular cell injury in cortex (B) as assessed by tubular dilatation, epithelial flattening and loss of brush border in FA vehicle or Pepstatin A treated kidneys. Average number of NIMP⁺ cells per field in FA vehicle or Pepstatin A treated kidneys. (C) Cleaved caspase-3 and GAPDH WB (D) of control vehicle and FA vehicle or Pepstatin A treated kidneys. Percentage of cortical TUNEL positive cells versus total cells and representative TUNEL only or DAPI merged pictures (E) in FA vehicle or Pepstatin A treated kidneys. White arrows point to TUNEL⁺ cells. Animals were treated with vehicle or Pepstatin A (20 mg/Kg) 45 minutes before and 24 hours post-FA. N = 8, t-test, *P ≤ 0.05 or **P ≤ 0.01.

Figure 3. CtsD is increased in IRI induced AKI following a similar trend than pro-apoptotic markers.

Western Blot of cleaved caspase-3, PARP-1 and cleaved PARP-1, pro- and mature CtsD and GAPDH (A) during increasing ischemic times (25, 35 and 45 minutes) and 24 hours reperfusion in control and IRI kidneys lysates. CtsD representative pictures and magnified area of CMJ or cortex of control kidneys and 25 minutes ischemic 24 hours reperfused kidneys. Red stars * point CtsD expressing cells (B,C). N = 6, t-test, *P ≤ 0.05 or **P ≤ 0.01.

Figure 4. Cathepsin D inhibition reduces tubular cell injury in IRI induced AKI.

CtsD fluorometric activity in kidney lysates assessed by the cleavage of a specific fluorescently labelled substrate (A) in sham and IRI vehicle or Pepstatin A treated animals. Percentage of tubular cell injury as assessed by tubular dilatation and granular cast formation in CMJ (B) of IRI vehicle or Pepstatin A treated kidneys. Representative PAS pictures, damaged cells pointed with a * (C) of sham or IRI vehicle or Pepstatin A treated kidneys. Ischemia was performed for 25 minutes and kidneys were reperfused for 24 hours. Animals were treated with vehicle or Pepstatin A 10 mg/Kg 1 hour before surgery and 4 hours post-surgery. N = 7, 1 way ANOVA or t-test,*P ≤ 0.05 or **P ≤ 0.01.

Figure 5. Pepstatin A reduces apoptosis with no alteration on neutrophil infiltration in IRI induced AKI.

Average number of NIMP⁺ cells per field with representative pictures in the CMJ of IRI vehicle or Pepstatin A treated kidneys. (A) CXCL-1 and CXCL-2 mRNA expression from sham and IRI vehicle or Pepstatin A treated kidneys. (B,C) Cleaved caspase-3 and GAPDH WB (D) of sham, control or IRI vehicle or Pepstatin A treated kidneys. Percentage of CMJ TUNEL positive cells versus total cells and representative TUNEL only or DAPI merged pictures (E) in IRI vehicle or Pepstatin A treated kidneys. Ischemia was performed for 25 minutes and kidneys were reperfused for 24 hours. Animals were treated with vehicle or Pepstatin A 10 mg/Kg 1 hour before surgery and 4 hours post-surgery. N = 7, 1 way ANOVA or t-test,*P ≤ 0.05 or **P ≤ 0.01.

Figure 6. Pepstatin A reduces hypoxic induced apoptosis in tubular epithelial cells.

CtsD and GAPDH WB of HKC-8 cells and CtsD and β-actin WB of hDTC. (A) HIF-1α immunostaining in HKC-8 cells under normoxic (20% O₂/5% CO₂) or hypoxic (1% O₂/5% CO₂) conditions for 48 hours. (B) White arrows point to HIF-1α located within the nuclei. Percentage of metabolically active viable cells assessed by MTT assay in HKC-8 cells (C) or hDTC passage 2 (D) treated with vehicle or Pepstatin A under normoxic (20% O₂/5% CO₂) or hypoxic (1% O₂/5% CO₂) conditions for 48 hours. Cleaved caspase-3 and GAPDH WB in HKC-8 cells under hypoxic conditions for 48 hours treated with vehicle or Pepstatin A. (E) Cleaved caspase-3 and β-actin WB in HKC-8 cells under hypoxic conditions for 48 hours treated with scramble or siRNA against CtsD. (F) N = 3, 1 way ANOVA,*P ≤ 0.05 or **P ≤ 0.01.

Figure 7. Pepstatin A pre-treatment reduces interstitial fibrosis development from IRI induced AKI.

CtsD fluorometric activity in kidney lysates assessed by the cleavage of a specific fluorescently labelled substrate. (A) Cleaved caspase-3 and GAPDH WB (B) in sham and IRI vehicle or Pepstatin A treated animals. Morphometric analysis of SR⁺ area/field (C) or α-SMA⁺ area/field (D) of kidney cortex from sham and IRI vehicle or Pepstatin A treated kidneys. Col1A1 (E) and Col3A1 (F) mRNA expression from sham and IRI vehicle or Pepstatin A treated kidneys. Ischemia was performed for 35 minutes and kidneys were reperfused for 28 days. Animals were treated with vehicle or Pepstatin A 20 mg/Kg 1 hour before surgery and from day 2 post-surgery three times a week up to 28 days. N = 6, 1 way ANOVA or t-test,*P ≤ 0.05 or **P ≤ 0.01.

Figure 8. CtsD is highly expressed in human ATN transplant biopsies.

Representative pictures of CtsD staining in human normal kidney and ATN transplant kidney biopsies. Percentage of CtsD positive area/total area is expressed as average ± SD. Black arrows point at CtsD expressing tubular cells. (A) CtsD/TUNEL dual staining in ATN transplant human kidney biopsy. Yellow arrow points to non-apoptotic and red arrow to apoptotic tubular cells. (B) Detail of CtsD distribution in a non-apoptotic tubular epithelial cell (TUNEL⁻/CtsD⁺), white arrows point to vesicular distribution. (C) Detail of CtsD distribution in an apoptotic tubular epithelial cell (TUNEL⁺/CtsD⁺). (D) A minimum of 6 different normal human kidneys and 9 different transplant ATN kidney biopsies were stained. t-test,*P ≤ 0.05 or **P ≤ 0.01.