Recent study suggests that running is good for the memory because scientist measured the presence of Cathepsin B.

Cathepsin B is produced in muscle tissue during metabolism. It is capable of crossing the blood-brain barrier and is associated with neurogenesis, specifically in the mouse dentate gyrus.

And more…

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.

Although cathepsins are regulated at the transcriptional level, the main step in regulating their activity occurs in the lysosomes where cathepsins are stored as inactive zymogens before being cleaved into the active protease12. Pro- and mature CtsD as well as mature CtsB were increased in UUO kidneys. In contrast mature CtsL decreased in diseased kidneys (Fig. 2D). To define the time course of CtsD upregulation, protein and mRNA levels were measured 5, 7 and 10 days after UUO induction. Both protein and gene expression were significantly upregulated by day 5 UUO with further increase by day 7. CtsD mRNA expression rose further by day 10 while protein expression plateaued after day 7 (Supplementary Fig. S1A,B). Therefore, during UUO there is a differential regulation of aspartyl and cysteine cathepsins with an increase of CtsD and B protein expression and processing into mature forms and a decrease in mature CtsL.

Pepstatin A reduces kidney fibrosis in two different models of chronic kidney disease

Active CtsD and B act as pro-fibrogenic enzymes in liver fibrosis6. To investigate whether the increase in active enzymes was playing a role in the development of kidney fibrosis, Pepstatin A (CtsD inhibitor) or Ca074-Me (CtsB inhibitor) were administered three times a week for 15 days following UUO. To be more representative of human disease, surgery was performed and renal injury was allowed to develop for 5 days before starting treatment, by which stage an increase in α-SMA and Col1A1 gene expression was already evident in untreated UUO mice (Supplementary Fig. S1C,D). Consistent with Fig. 2D, CtsD activity was significantly increased in injured kidneys and reduced back to control levels by Pepstatin A administration. Ca074-Me had no effect on CtsD activity assessed by fluorimetric activity in kidney lysates (Fig. 3A). The inhibitory effect of Ca074-Me on CtsB activity was demonstrated in vivo in 10 days UUO kidneys by IVIS analysis using a CtsB activable fluorescent probe (Fig. 3B). Morphometric analysis of Sirius Red, collagen III and IV cortical staining, showed an increase in fibrosis and thickening of the tubular basal membrane in injured kidneys (Fig. 3C–E). Ca074-Me administration had no effect over Sirius Red or collagen IV and only a moderate effect over collagen III. However, Pepstatin A treatment significantly reduced cortical accumulation of Sirius Red, collagen III and IV in fibrotic kidneys (Fig. 3C–E).


Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).


Catalysis is achieved by one of two mechanisms:

Aspartic, glutamic and metallo- proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it.

Serine, threonine and cysteine proteases use a nucleophilic residue in a (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme.

In this study we clearly demonstrate that inhibition of aspartyl cathepsin D leads to a reduction in interstitial fibrosis in two models of renal disease. The number of patients with CKD is increasing and some of these patients will progress onto end stage renal disease and face life-long dialysis or organ transplant. Treatment options for patients with progressive disease are limited, thus there is an urgent need to find new therapeutic targets that could lead to drug development. Interstitial fibrosis is almost invariably seen in patients with progressive CKD. Dysregulation of extracellular matrix (ECM) homeostasis leads to a gradual replacement of the healthy nephrons by electrondense fibrotic ECM. Proteases play a crucial role in regulating this process; however, our knowledge of proteases biology and function in CKD is still very limited.

Lysosomal proteases have been implicated in the pathogenesis of fibrotic disease in the liver, (CtsB and D)6,7 lung, (CtsK)8,9 and heart, (CtsL)10,11 but very little is known about their function in renal disease. The only reports relate to the role of cysteine but not aspartyl cathepsins (CtsD family group). There is decreased activity in the kidney of the cysteine cathepsins B, H and L accompanied by an increase in their urinary secretion in rat polycystic kidney disease22, puromycin induced nephrosis23 and rat and human diabetic nephropathy24,25. However, not all cysteine cathepsins decrease during proteinuric kidney disease and CtsL26 increases in proximal tubular cells and podocytes. Therefore, the role of cathepsins is currently far from understood, pointing towards a cell and disease specific function.

Screening of a panel of human renal biopsies show for the first time the expression of CtsD and B in distal and proximal tubular cells respectively in human renal disease (Fig. 1A,B). In agreement with Goto et al.’s27 observations in human normal kidney, CtsD is mainly expressed in distal convoluted tubules. Our results point towards an increase of CtsD expression in areas of tissue damage with no change in CtsB expression. The number of patients screened in our study was insufficient to draw statistical conclusions and further investigations will be needed in a bigger cohort to determine an association between level of CtsD expression and disease outcome.

To investigate the role of CtsD and B in CKD we used an aspartyl or cysteine protease inhibitor, Pepstatin A or Ca074-Me, in a murine CKD model, UUO. Pepstatin A but not Ca074-Me diminished collagen accumulation and thickening of the tubular basement membrane (Fig. 3C–E) with no effect on collagen transcription (Fig. 5A–C) or myofibroblast (Fig. 6A) numbers. Pepstatin A effects on fibrosis were reproduced in a second model of CKD, 35 minutes/28 days IRI (Figs 4B–D,5D,F and 6B). Our results suggest that Pepstatin A reduces fibrosis by increasing collagen I and III degradation (Fig. 6C,D).

Despite Pepstatin A being the best inhibitor against CtsD available and in contrast to Ca074-Me, which is a rather specific inhibitor against CtsB28, Pepstatin A can also affect other proteases of the same family, thus additional effects on other proteases cannot be excluded. Indeed, this may contribute to the outcome of our study, as redundancy and compensatory mechanisms12 are common problems when targeting only one member of a protease family. CtsD knock-out mice die approximately 26 days after birth due to neurological disorders29 replicating human deficiency30,31. In our models Pepstatin A did not completely block CtsD activity, achieving a reduction back to physiological levels (Figs 3A and 4A), avoiding possible undesirable secondary effects.

Cathepsins can indirectly modulate ECM turnover by affecting other proteases. We investigated the relationship between CtsD and UPA as an example of how CtsD can affect extracellular protease activity. Urokinase (UPA) was upregulated in fibrotic kidneys after the treatment with Pepstatin A (Fig. 7A–C). In vitro, extracellular UPA was increased in human tubular epithelial cells treated with CtsD but not B inhibitor and siRNA against CtsD (Fig. 8A–C).

UPA belongs to the urokinase plasminogen activator system, the role of which in CKD remains controversial17,32,33. UPA is anti-fibrotic in lung34 and liver35 whereas surprisingly no difference in the severity of UUO was observed in UPA knock-out mice36. UPA is secreted extracellularly and activated upon binding to its surface receptor, UPAR. Then activates other proteins, preferentially plasminogen into plasmin, which can directly degrade ECM proteins37,38,39 and also activates MMPs18,19, triggering further ECM degradation. Despite several reports in the literature of a direct link between UPA, plasmin and MMP activation, further work is required to demonstrate a link to CtsD.

PAI-1, UPA’s natural inhibitor, covalently binds to the UPA:UPAR complex inhibiting UPA’s enzymatic activity. The UPA:UPAR:PAI-1 complex is then rapidly internalized upon binding to LDL receptor-related protein-1 (LRP-1) through clathrin dependent endocytosis pathway into the lysosomes21. There UPA and PAI-1 are degraded and UPAR is recycled back to the cell surface. We confirmed localization of UPA in clathrin endosomal vesicles by co-localizing UPA with AP2-μ1 adaptor protein, which is an essential protein for the clathrin-coated pit formation, in hDTC (Fig. 8D). We also proved co-localization of CtsD and UPA within the lysosomes in fibrotic kidneys (Fig. 7D, Supplementary Fig. S2). Previous work by van Kasteren SI et al. support our hypothesis of Pepstatin A affecting endo/lysosomal recycling as they described EGFR clathrin dependent endo/lysosomal degradation being impaired by a cystatin-pepstatin inhibitor (CPI)40. In order to further confirm a link between lysosomal degradation and the increase in UPA, we used chloroquine (CQ) as endo/lysosome inhibitor. Both Pepstatin A and CQ had similar effects in vitro, increasing the active extracellular UPA (Fig. 8E). In addition, CQ administration in the UUO model mimicked the effect seen with Pepstatin A administration, reducing collagen accumulation and fibrosis (Fig. 8F).

In summary, here we report for the first time the distribution of CtsD and B in human renal disease and show the effect of their inhibition in two mouse models of renal fibrosis. We propose a novel mechanism by which CtsD inhibition by Pepstatin A leads to an increase in extracellular protease activity, in particular UPA, due to altered lysosomal recycling. This can trigger a proteolytic cascade activating first plasminogen into plasmin and culminating possibly with the regulation and activation of MMPs18,19. Both plasmin37,38,39 and MMPs are able to degrade ECM proteins causing a net reduction in fibrosis. This situation can be further sustained by a positive feedback loop, as plasmin is also able to activate UPA20. Our model does not exclude the regulation by CtsD of other proteases that might be recycled through the lysosomal pathway and further investigation will be needed to clarify this. This work opens new and exciting prospects for the treatment of CKD by targeting lysosomal proteases.


 Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease

Scientific Reports 6, Article number: 20101 (2016)


Chronic kidney disease, Renal fibrosis

Cathepsin B is produced in muscle tissue during metabolism. It is capable of crossing the blood-brain barrier and is associated with neurogenesis, specifically in the mouse dentate gyrus.

A wide array of diseases result in elevated levels of cathepsin B, which causes numerous pathological processes including cell death, inflammation, and production of toxic peptides. Focusing on neurological diseases, cathepsin B gene knockout studies in an epileptic rodent model have shown cathepsin B causes a significant amount of the apoptotic cell death that occurs as a result of inducing epilepsy.[6]

Cathepsin B inhibitor treatment of rats in which a seizure was induced resulted in improved neurological scores, learning ability and much reduced neuronal cell death and pro-apoptotic cell death peptides.[7] Similarly, cathepsin B gene knockout and cathepsin B inhibitor treatment studies in traumatic brain injury mouse models have shown cathepsin B to be key to causing the resulting neuromuscular dysfunction, memory loss, neuronal cell death and increased production of pro-necrotic and pro-apoptotic cell death peptides.

In ischemic non-human primate and rodent models, cathepsin B inhibitor treatment prevented a significant loss of brain neurons, especially in the hippocampus.

In a streptococcus pneumoniae meningitis rodent model, cathepsin B inhibitor treatment greatly improved the clinical course of the infection and reduced brain inflammation and inflammatory Interleukin-1beta (IL1-beta) and tumor necrosis factor-alpha (TNFalpha).[13]

In a transgenic Alzheimer’s disease (AD) animal model expressing human amyloid precursor protein (APP) containing the wild-type beta-secretase site sequence found in most AD patients or in guinea pigs, which are a natural model of human wild-type APP processing, genetically deleting the cathepsin B gene or chemically inhibiting cathepsin B brain activity resulted in a significant improvement in the memory deficits that develop in such mice and reduces levels of neurotoxic full-length Abeta(1-40/42) and the particularly pernicious pyroglutamate Abeta(3-40/42), which are thought to cause the disease.

In a non-transgenic senescence-accelerated mouse strain, which also has APP containing the wild-type beta-secretase site sequence, treatment with bilobalide, which is an extract of Ginko biloba leaves, also lowered brain Abeta by inhibiting cathepsin B.[21] Moreover, siRNA silencing or chemically inhibiting cathepsin B in primary rodent hippocampal cells or bovine chromaffin cells, which have human wild-type beta-secretase activity, reduces secretion of Abeta by the regulated secretory pathway.