Cathepsin B increases ENaC activity leading to hypertension early in nephrotic syndrome

Abstract The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for genetic and sporadic forms of nephrotic syndrome (NS). Patients with NS often present symptoms of volume retention, such as oedema formation or hypertension. The primary dysregulation in sodium handling involves an inappropriate activation of the epithelial sodium channel, ENaC. Plasma proteases in a proteinuria‐dependent fashion have been made responsible; however, referring to the timeline of symptoms occurring and underlying mechanisms, contradictory results have been published. Characterizing the mouse model of podocyte inactivation of NPHS2 (Nphs2∆pod) with respect to volume handling and proteinuria revealed that sodium retention, hypertension and gross proteinuria appeared sequentially in a chronological order. Detailed analysis of Nphs2∆pod during early sodium retention, revealed increased expression of full‐length ENaC subunits and αENaC cleavage product with concomitant increase in ENaC activity as tested by amiloride application, and augmented collecting duct Na+/K+‐ATPase expression. Urinary proteolytic activity was increased and several proteases were identified by mass spectrometry including cathepsin B, which was found to process αENaC. Renal expression levels of precursor and active cathepsin B were increased and could be localized to glomeruli and intercalated cells. Inhibition of cathepsin B prevented hypertension. With the appearance of gross proteinuria, plasmin occurs in the urine and additional cleavage of γENaC is encountered. In conclusion, characterizing the volume handling of Nphs2∆pod revealed early sodium retention occurring independent to aberrantly filtered plasma proteases. As an underlying mechanism cathepsin B induced αENaC processing leading to augmented channel activity and hypertension was identified.


| INTRODUC TI ON
The NPHS2 gene, encoding the slit diaphragm protein podocin, accounts for 43% of familial and 10% of sporadic forms of nephrotic syndrome (NS). 1,2 Conditional inactivation of podocin in adult mice is a novel model system for NS resulting from focal segmental glomerulosclerosis (FSGS), 3 which recapitulates human disease formation.
In the NS, the underlying dysregulation in volume homeostasis was shown to be an intrarenal defect 4 located beyond the distal convolutions in the renal connecting tubule and collecting ducts.
Abnormal high activity of the epithelial sodium channel (ENaC) was proven to be the reason for the increased transepithelial sodium reabsorption. 5 ENaC plays a key role in regulating extracellular fluid homeostasis and blood pressure. Numerous studies of animal models with proteinuria and sodium retention demonstrated increased full-length subunit expression of ENaC and proteolytical processing of the ENaC subunits alpha and gamma. [6][7][8][9] In animal models with NS, the increased expression level of ENaC was demonstrated to be independent of its hormonal stimulation. Various attempts in blocking hormones known to activate ENaC did not abolish volume retention. 7,10 Augmented ENaC activity also results from proteolytic processing of the large extracellular domain of α-and γENaC. A dual cleavage event in either subunit releases small intrinsic inhibitory tracts transitioning channels to a more active state. 11 While furin, an endogenous protease, was shown to cleave αENaC twice, it cleaves the γENaC only once. Additional proteases, including extracellular proteases, are needed for the second incision in γENaC to release the inhibitory tract. Several proteases processing γENaC were identified 12,13 including plasmin in the development of NS. 14,15 Regarding the timeline of the appearance of sodium retention and proteinuria, contradictory results have been published. In the rat model of PAN-induced nephrosis, sodium retention was shown to start before or at the same time as the onset of proteinuria. 7,16 Consequently, the question arises whether glomerular plasmin leakage is the only mechanism for ENaC-induced sodium retention. Both, We used Nphs2 fl/fl (control) and Nphs2 fl/fl crossbred with inducible podocyte-specific Cre recombinase transgenic mice, termed Nphs2 ∆pod hereafter and found that sodium retention and hypertension established before the onset of an unselective gross proteinuria. Increased ENaC channel activity, proteolytic processing of αENaC together with the appearance of proteases in the urine were encountered. Among several lysosomal enzymes identified by proteomic analysis, only cathepsin B was able to cleave αENaC and augment channel activity. Inhibition of cathepsin B influenced the development of hypertension demonstrating its important role in this disease model.

| ME THODS
Detailed methods are presented in the supplement files.

| Presentation of data and statistical analysis
Quantitative data are presented as means ± SEM. Statistical comparisons were performed with the GraphPad Prism Software Package 6 (GraphPad Software Inc, La Jolla, CA, USA). For statistical comparison, the non-parametric Mann-Whitney U test, the parametric two-tailed Student's t test and, where appropriate one-way ANOVA followed by Tukey, Dunnett or Newman-Keuls post hoc test was employed. P values of less than 0.05 were considered statistically significant.

| Podocin loss leads to nephrotic syndrome
Inactivation of podocin resulted in transiently reduced urinary sodium/creatinine ratio, which was significantly reduced between day 4 to 7 after tamoxifen administration ( Figure 1A). Systolic and diastolic blood pressure began to rise significantly at day 10 and day 12, respectively, after tamoxifen administration ( Figure 1B) and urinary protein/creatinine ratio augmented significantly at day 12 after tamoxifen administration ( Figure 1C). Blood pressure and protein/ creatinine ratio continued to increase until the end of the experiment at 28 days. Oedema was regularly encountered in all Nphs2 ∆pod at 3 weeks of the experiment. These results show in detail renal volume handling and proteinuria during the development of NS in Nphs2 ∆pod and confirm previous published data. 3,18

| Analysis of renal function, morphology and expression of ENaC and its cleavage products in nephrotic syndrome during sodium retention
To analyse renal alterations in more detail, additional animal experiments at two time-points after tamoxifen administration were chosen: day 5, during decreased sodium/creatinine ratio and day 9, when blood pressure starts to rise. Successful podocin deletion upon tamoxifen treatment was verified and confirmed ( Figure 2A).
Renal function analysis of mice at day 5 demonstrated reduced 24 hours sodium excretion, urinary Na/K ratio and fractional sodium excretion in Nphs2 ∆pod compared to control (Table 1). Urinary albumin excretion started to rise but did not reach statistical significance. At day 9, albuminuria and proteinuria developed significantly F I G U R E 1 Course of urinary sodium/creatinine ratio, blood pressure and urinary protein/creatinine ratio from Con and Nphs2 ∆pod . A-C, Daily urinary sodium/creatinine ratio (A), blood pressure (B) and urinary protein/creatinine ratio (C) of control (Con) and Nphs2 ∆pod during 28 days. Results are arithmetic means ± SEM of n = 5-7 per group; *P < 0.05. tam indicates the days of tamoxifen injection to induce Nphs2 knockout in relation to its full-length subunit. Increased cleaved αENaC was observed in medulla at 5, 9 and 17 days ( Table 2). The augmented expression level of full-length α-and γENaC was not due to transcriptional alteration, as mRNA of α-and γENaC at 5 and 9 days did not change ( Figure S1). Because apical Na + entry is tightly coupled to the basolateral Na + extrusion, we determined the basolateral αsubunit Na + /K + -ATPase (αNKA) expression level by measuring fluorescence intensity levels of cortical collecting ducts identified by aquaporin-2 expression ( Figure 3C). Semi-quantified αNKA abundance from micrographs similar to ( Figure 3C) after correction for background signal, cell area, and normalization to control values revealed significantly increased αNKA fluorescence at 5 and 9 days in Nphs2 ∆pod compared to the controls. This demonstrates that augmented transepithelial sodium reabsorption may involve both apical ENaC and basolateral NKA.

| Assessment of Nphs2 ∆pod at day 17
Additionally, we performed an animal experiment using control and Nphs2 ∆pod which we have stopped at 17 days after tamoxifen administration for renal analysis at a later time-point when NS was fully developed. Plasma analysis of Nphs2 ∆pod compared to control revealed reduced albumin and protein concentrations and increased creatinine levels (Table S2). Twenty-four hours urine analysis of Nphs2 ∆pod compared to control revealed increased albumin and protein excretion, reduced creatinine clearance and increased blood pressure. Ascites was regularly encountered in Nphs2 ∆pod in com- TA B L E 1 Renal functional data of control and Nphs2 ∆pod during sodium retention at 5 and 9 days after podocin inactivation Results are arithmetic means ± SEM of n = 5-7 per group. *P < 0.05, **P < 0.01.
F I G U R E 3 Assessment of ENaC subunit and collecting duct Na + /K + -ATPase expression levels during sodium retention. Western blots of α-, β-and γENaC from membrane fractions of kidney cortex and medulla at 5 (A) and 9 days (B). Specific bands are marked by green brackets. Densitometric evaluations are presented in the respective graphs below. Ponceau red staining and β-actin served as loading control. Results are arithmetic means ± SEM of n = 5-7 per group; *P < 0.05, **P < 0.005. Immunohistochemical double labelling of Na + /K + -ATPase (red) and aquaporin-2 (green) for the identification of collecting ducts (C). Collecting ducts are marked by an asterisk. Significantly increased collecting duct Na + /K + -ATPase expression is encountered in Nphs2 ∆pod at 5 and 9 days compared to control (Con) as depicted in the graph aside.

| Effects of amiloride in the nephrotic syndrome development
To see and prove whether ENaC is activated and responsible for the reduced urinary sodium/creatinine ratio early in the NS development, an additional animal experiment was conducted with amiloride ( Figure 4). As shown before, compared to their controls Nphs2 ∆pod / vehicle demonstrated significantly reduced urinary sodium/creatinine ratio between day 4 and day 7. Administration of amiloride to control increased urinary sodium/creatinine ratio at baseline and strongly augmented in Nphs2 ∆pod /amiloride between day 5 and day 8, proving increased ENaC activity early in NS development.

| Urinary protease excretion
Because of the early αENaC processing already at 5 days after FSGS induction, we analysed the urine of Nphs2 ∆pod and their controls from day 2 until day 16. In controls, a zymogram-positive band was observed at above 260 kDa, which also occurred in the urine of Nphs2 ∆pod with a constant intensity over the time ( Figure 5A).
Surprisingly, already at day 2 proteolytic activity was observed in the urine of Nphs2 ∆pod . With time, intensity of zymogram-positive bands increased as well as the number of proteases running at different molecular weight. At 2 days, a band at 140-160 kDa was found, at day 6 an additional band at 100 kDa was observed, at day 8 a band at 70 kDa was observed, and at days 10 and 12 bands at 85 kDa and 60 kDa was found respectively. Additionally, a band at 25 kDa from day 2 to 6 was observed. Then, we aimed to identify the protease responsible for the proteolysis of ENaC. Therefore, the urine of Nphs2 ∆pod from day 2 until day 9 was collected, HPLC-puri-  Table 3. The identified proteases were typically present in multiple HPLC fractions; hence they are summarized as a joint list.

| ENaC subunit processing and activation by lysosomal enzymes
Based on our results, we were focusing on αENaC cleaving enzymes and identified cathepsin B (see Table 3), which was shown previously TA B L E 2 Ratio of cleaved α-or γENaC in relation to its fulllength subunit. Calculated ratio of the abundance of cleaved ENaC in relation to its full-length subunit of cortical and medullary membrane fractions at 5, 9 and 17 days. Increased ratio of cleaved αENaC is found in the medulla at all time-points analysed  to activate ENaC currents by proteolytical processing of αENaC. 19 We have been suggested that there maybe also other lysosomal enzymes, as found and highlighted in Table 3, may be able to cleave αENaC. Performing in vitro protease assays using αENaC-GST fusion proteins, we found cathepsin B cleaved αENaC at the known 'furin' cleavage site with a resulting product at 30 kDa ( Figure 5B).  Surprisingly, a strong increase in cathepsin B in intercalated cells was detected at 5 and at 9 days ( Figure 6D and 6). Additionally, we followed the hypothesis that increased proximal tubular albumin uptake may activate endocytosis and that lysosomal enzymes may appear in the urine upon lysosomal spill over. Therefore, we determined endocytosed albumin in proximal tubular profiles, identified by double labelling with megalin ( Figure S3A). At 5 days, no difference in albumin fluorescence was observed between Nphs2 ∆pod and control. At 9 days and more pronounced at 17 days, endocytosed albumin increased significantly. As sodium retention occurs between days 4-7 it seems unlikely, that increased urinary cathepsin B stems from the proximal tubular lysosome spill over.

| Increased cathepsin B expression level in
Additionally, we performed coomassie staining of SDS-PAGE and Western blots from daily spot urine loaded after creatinine adjustment ( Figure S3B). Albuminuria was starting at days 4-6 and unselective gross proteinuria on days 13-14. Western blots of cathepsin B showed increased active sc-cathepsin B expression early between day 4 and 7, whereas plasminogen and plasmin started to increase at days 13-14. We also tested whether albumin may activate cathepsin B expression in cortical collecting duct cells and incubated mpkCCD cl14 cells with albumin at concentrations ranging from 0 to 20 mg/mL for 6 hours and at 10 mg/mL for 0, 3, 6 and 24 hours; however, no significant changes were observed (data are not shown).

| Impacts of cathepsin B inhibitor CA-074Me in Nphs2 ∆pod
To analyse whether cathepsin B may impact the disease progression in Nphs2 ∆pod , an additional animal experiment was performed with control and Nphs2 ∆pod receiving either CA-074Me (a membrane-permeable cathepsin B inhibitor) or vehicle for 14 days starting on the day after the first tamoxifen injection. As expected at day 9, Nphs2 ∆pod receiving vehicle developed significantly higher blood pressure, which remained high until the end of the experiment TA B L E 3 Proteases identified from HPLC-purified urine of Nphs2 ∆pod from day 2 until day 9 after podocin inactivation. Protein name, gene name and protein ID of the identified proteases are listed. Lysosomal proteases are marked in bold and known γENaC cleaving proteases are marked in cursive at 14 days ( Figure S4). Blood pressure of Nphs2 ∆pod receiving CA-074Me, however, remained at control levels. analysis of ENaC expression pattern was surprising. Increased occurrence of cleaved αENaC fragments and abundance of fulllength ENaC subunits without changes in aldosterone and vasopressin levels were observed. An increased ENaC abundance was found previously in many other proteinuric animal models. [6][7][8][9] Augmented ENaC function, however, seems to be unrelated to hormonal stimulation, as various hormonal blockades did not change the clinical outcome. 7,10 Transcriptional regulation can also be excluded as ENaC mRNA expressions levels neither vary at 5 nor at 9 days, similarly as previously reported. 20 In Nphs2 ∆pod , the augmented ENaC expression levels and or αENaC cleavage at 5 and 9 days is of functional relevance because NKA expression levels from the cortical collecting duct are consistently increased compared to control and other nephron segments in the same section. Supporting evidence for early increased ENaC activity was gained from daily administration of amiloride to control and Nphs2 ∆pod . Highest plasma values for amiloride occur 3-4 hours after amiloride administration, the time-point where sodium was determined from spot urine. From our experiment, we observed that between day 5 and 9 before sodium/creatinine ratio increased in control/amiloride and augmented strongly in Nphs2 ∆pod /amiloride suggesting that using this application procedure sodium/ creatinine ratio mirrors ENaC channel activity. These results support the overfill hypothesis where sodium retention is related to an intrinsic renal defect in sodium handling. we did not observe γENaC processing which may be due to differences in techniques and models used. The very faint cleavage of γENaC by cathepsin D at 70 kDa and legumain at 72 kDa corresponds to the increase in relative currents in mpkCCD cl14 cells at 2 hours. A more detailed analysis for further exploration needs to be performed in future.

| D ISCUSS I ON
In Nphs2 ∆pod during the time of early sodium retention between day 5 and 9, overall glomerular morphology remains largely intact, the glomerular filter however is altered and shows albuminuria/ selective proteinuria. Therefore, plasma proteases as shown for plasminogen are still unable to pass and do not account for the sodium retention during this period assuming that urinary proteases identified might stem from the kidney itself. Lysosomal cathepsin B activation and proximal tubular spill over into the primary ultrafiltrate was postulated to account for increased urinary cathepsin B activity in proteinuric diseases. 25 However, analysis of endogenous expression of taken-up albumin and β2-microglobulin, as a low-molecular weight protein, did not change at five days and was slightly increased at 9 days (data not shown);indicating that it cannot account for increased cathepsin B activity at least at 5 days. In summary, using the Nphs2 ∆pod mice, we were able to identify the chronology of the development of hallmarks of the NS. Early after genetic Nphs2 deletion, sodium retention occurred, followed by hypertension and gross proteinuria. This early sodium retention is based on augmented ENaC activity through proteolytical processing of αENaC. Cathepsin B was identified as αENaC cleaving enzyme where its blockade prevented hypertension.

ACK N OWLED G EM ENTS
We would like to thank Patricia Matthey, Danièla Grand-Habegger and Inka Geurink for expert technical assistance. This study was sup-

CO N FLI C T O F I NTE R E S T
There is no conflict of interest.