New Insights in the Distribution Profile of TRPC3 and TRPC5 in the Mouse and Human Kidney

doi:10.21203/rs.3.rs-3342412/v1

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New Insights in the Distribution Profile of TRPC3 and TRPC5 in the Mouse and Human Kidney

https://doi.org/10.21203/rs.3.rs-3342412/v1

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Several reports previously investigated Transient Receptor Potential Canonical subfamily channels 3 and 5 (TRPC3/TRPC5) in the kidney. However, most of the conclusions are based on animal samples or cell cultures leaving the door open for human tissue investigations. Moreover, results often disagreed among investigators. Histological description is lacking since most of these studies focused on functional aspects. Nevertheless, the same reports highlighted the potential differing key-roles of TRPC3 or TRPC5 in various renal disorders. Hence, our interest to investigate the localization of TRPC3 and TRPC5 in human kidneys. For this purpose, both healthy mouse and human kidney samples that were originated from tumor nephrectomies have been prepared for immunohistochemical staining using knockout-tested antibodies. Blocking peptides confirmed antibody specificity. A normalized weighted diaminobenzidine (DAB) area score between 0–3 comparable to a pixelwise H-score was established and employed for semiquantitative analysis. Altogether, our results suggest that glomeruli only express little TRPC3 and TRPC5 compared to several segments of the tubular system. Cortical and medullary proximal tubules are strongly stained. Intermediate tubules, however, are only weakly stained. The distal tubule was studied in three different localizations and depending on species and primary antibody, the staining was marked although slightly varying throughout the different localizations. Finally, the collecting duct was independently of primary antibody more stained in human compared to mouse tissue. We provide evidence that TRPC3 and TRPC5 are differently expressed in various localizations in both mouse and human samples. Especially, the TRPC5 distribution profile, we present here is completely new to our knowledge and raises questions, for instance its physiological relevance in the tubular system. We less verify results of previous studies than propose until now undescribed localizations of TRPC3 and TRPC5 in the mouse but especially and of greater interest in the human kidney. We thereby not only support the translational concept of TRPC channels as key players in physiology and pathophysiology of the human kidney but also present new potential targets to functional analysis.

The Transient Receptor Potential (TRP) superfamily was first described in Drosophila melanogaster and is, based on protein homology, subdivided into several subfamilies – TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPA (ankyrin), TRPML (mucolipin) and TRPN (no mechanoreceptor potential C)^1–3. Depending on associated proteins, TRP channels are critical in a wide range of physiological processes and appear in many different cell types^3–5,. The activation mechanisms of TRP channels are diversified and include ligand binding, alterations in temperature, mechanical stress and more^1,3,5,6. The TRPC subfamily accounts seven members of nonselective Ca²⁺-permeable cation channels that can be, based on their ability to form diverse functional heteromers, clumped into two different groups – namely TRPC3/6/7 and TRPC1/4/5 – TRPC2 being encoded by a pseudogene in humans^7–9. The structure of TRPC channels is tetrameric. Each subunit is composed of six transmembrane segments (S1-S6). The Ca²⁺ permeable cation-pore arises between the two final transmembrane segments (S5-S6) of each monomer and its gating activity can be meticulously regulated^2,10. Due to such properties, TRPC channels are eligible to function as key molecular players in multiple mechanisms including redox-sensitive currents, receptor-operated currents, store-operated currents and more as suggested in non-human experiments^6,9,11–15. Diacylglycerol (DAG) is a physiological prominent activator of TRPC3 and TRPC6 but not of TRPC5⁷. In addition, TRPC3 features an uncommonly long third transmembrane segment, that has been suggested to mediate the channel’s mechanosensitivity⁶. In 2002, Riccio et al.¹⁶ analyzed TRPC messenger ribonucleic acid (mRNA) distribution in peripheral tissues and revealed little TRPC5 mRNA but no TRPC3 mRNA in the kidney. However, later literature features reports on TRPC channels, including TRPC3 and TRPC5 in the kidney^6,13,17. Unfortunately, most of these studies that we depict below failed to find a consensus on the distribution profiles of the aforementioned channels. Hence, our genuine interest to finally provide such a description not only apprehending rodents, as mostly performed until now, but also extending to human tissue.

The kidney is divided into a renal cortex, an inner and outer medulla that is further subdivided into an inner stripe and outer stripe¹⁸ (Fig. 1). Each nephron – the functional unit of the kidney – is composed of a glomerulus and the corresponding tubular system. The glomerulus is a fenestrated capillary network surrounded by intraglomerular mesangial cells and podocytes that are also referred to as the visceral layer of Bowman’s capsule. The parietal layer represents the peripheral border of Bowman’s space^6,19. The tubular system is grossly segmented in a proximal convoluted and straight tubule, in an intermediate tubule with a thin descending and ascending leg as well as in a distal straight and convoluted tubule¹⁸ (Fig. 1). However, proximal, and distal tubules will be distinguished here depending on parenchymal localization (cortical/inner or outer stripe) not on morphology (straight/convoluted). While glomeruli are clear to recognize, identification of renal tubules type can be challenging. Proximal tubules are relatively large and feature a nearly lumen filling cuboidal epithelium that is equipped with a prominent brush border²⁰. Intermediate tubules, that can be distinguished in both the cortex and medulla, are characterized by a squamous epithelium²⁰. In contrast to proximal tubules, distal tubules can be detected in the entire outer medulla. Their epithelium is cuboidal, middle-sized and lacks an apical brush border²⁰. Collecting ducts, that do not belong to the nephron, possess a large lumen that is lined with clear definable cuboidal cells including principal and intercalated cells²⁰. General renal physiology is detailed elsewhere, since not the focus of this article^21,22.

Interestingly, TRPC channels are critical in both glomerular and tubular physiology and pathophysiology as suggested in mostly non-human experiments^1,11,17. For instance, Staruschenko et al.⁹ recently summarized the involvement of different channels, including TRPC members, in glomerular function, and both TRPC3 and TRPC5 have been detected in podocytes⁹. In contrast to TRPC6, little is known about TRPC3 in podocytes⁶. It was observed, however, that TRPC3 was upregulated upon TRPC6 knockout in podocytes, suggesting functional redundancy due to similar properties^23,24. TRPC3 expression was also increased under pathophysiological conditions such as angiotensin II-mediated hypertension^6,25. The first reports of TRPC5 in podocytes were associated with the small GTPase Rac1 (Ras-related C3 botulinum toxin substrate 1) and proposed a contractility-decreasing function in podocytes^26,27. Supported by TRPC5 knockouts and pharmacological inhibition, it was suggested that excessive Rac1 signaling, as observed in rare forms of focal and segmental glomerulosclerosis (FSGS), could be involved in dysfunctional cytoskeletal remodeling with subsequent proteinuria^1,26. Pharmacological TRPC5-inhibitors such as GFB-887 or GFB-024 are currently investigated in clinical trials including disorders suggested to be concomitant with an upregulated TRPC5-Rac1 pathway such as FSGS and diabetic nephropathy²⁸. However, other studies didn’t support the hypothesized pathogenic role of TRPC5, illustrating the need for further investigations¹. For instance, Polat et al.²⁹ recently demonstrated that TRPC5 inhibition or expression stimulation did not improve Rac1-mediated proteinuria and questioned its role as therapeutic target in podocyte-associated conditions²⁹.

Switching the glomerular cell type – TRPC3 but not TRPC5 was detected in contractile mesangial cells⁹. Whereas TRPC1 and TRPC6 are better investigated in mesangial cells, it is suggested that TRPC3 functions as Ca²⁺-sensing receptor (CaSR)-triggered receptor-operated channel to promote human mesangial cell proliferation³⁰. Combined with its high expression profile in pre-glomerular resistance vessels, it is supposed that TRPC3 is involved in GFR- and tubuloglomerular feedback maintainance⁶.

Until now, most of the scientific attention was paid to the glomerular localization and function of TRPC channels. However, their relevance in the renal tubular system is increasingly investigated and we recently summarized the current understanding of TRPC3 and TRPC6¹¹. Staying with the former, TRPC3 is for instance suggested to be involved in luminal osmosensation and vasopressin-induced aquaporin-2 (AQP-2) translocation in the collecting duct^31–33. In the proximal tubule TRPC3 is believed to have a nephroprotective function with respect to the sequence of hypercalciuria, nephrocalcinosis, and potentially chronic kidney disease^34–36. A pathogenic role in autosomal dominant proteinuric kidney disease (ADPKD) upon TRPP1/2 loss-of-function mutation has also been suggested^11,37. In contrast to TRPC3 and TRPC6, reports on TRPC5 in the renal tubular system are either very rare or not existent. There is therefore neither morphological nor functional understanding of this channel in this context. As to TRPC3, however, several open issues remain, since many discrepancies in results of functional investigations exist and convincing microscopical description is still lacking in contrary to TRPC6³⁸.

TRPC channels belong the best studied channels in glomeruli⁹. After a long period of glomerular studies, TRPC6 turned out to be involved in tubular ischemia/reperfusion injuries¹¹ and in the oncogenesis and tumor progression of renal cell carcinoma^39,40. As mentioned above, TRPC3 is also increasingly investigated in tubular cells. Similar may apply to TRPC5 in the future. Due to this, and to the wide relevance of these channels in Ca²⁺ homeostasis and signaling, there is a need for systematic investigation in mouse and especially human tissue to support and shape the increasing understanding of the involvement of TRPC3 but also the potential function of TRPC5 in human kidney – or more specifically tubular – function and dysfunction.

Since mouse sections present entire kidneys, gross morphological description of the staining distribution pattern is accessible. The samples we obtained from nephrectomies, on the other hand, represent only a fraction of an entire kidney cross section, ultimately impeding gross analysis. Normalized weighted diaminobenzidine (DAB) area score will usually be resumed as coloration in the following.

Glomeruli are weakly stained throughout the sections of each group (mean values < 1). DAB staining is, however, significantly higher when investigating TRPC5 (mean value _human = 0.9603, SD _human = 0.2343; mean value _mice = 0.8921, SD _mice = 0.1512) compared to TRPC3 (mean value _human = 0.3981, SD _human = 0.1280; mean value _mice = 0.4859, SD _mice = 0.08397), independently of species (P-value = 0.0286, Mann-Whitney U; Figs. 2A and 3). In contrast to human cortical proximal tubules, murine can be divided into two classes from their DAB staining behavior. As to the TRPC3 investigation, most proximal tubules are weakly stained, whereas a minority features a strong DAB staining (Fig. 3E). The difference in the TRPC5 group is less accentuated (Fig. 3C). For instance, the proximal tubules with enhanced DAB staining differ depending on primary antibody manifesting in increased staining in the TRPC5 group (mean value _TRPC5 = 2.227, SD _TRPC5 = 0.1499; mean value _TRPC3 = 1.644, SD _TRPC3 = 0.2308; P-value = 0.0286, Mann-Whitney U) (Fig. 2B). Human kidneys, however, show a rather homogenic staining of the cortical proximal tubules, that is similar to murine analogous structures in the TRPC3 group (mean value _human =1.678, SD _human = 0.3630; p-value = 0.4857, Mann-Whitney U) but not in the TRPC5 group (mean value _human =1.779, SD _human = 0.1954; P-value = 0.0286, Mann-Whitney U) (Fig. 2B). Proximal tubules of the outer stripe of the mouse outer medulla (mean value _TRPC3 = 1.492, SD _TRPC3 = 0.2453; mean value _TRPC5 = 2.284, SD _TRPC5 = 0.2365) were similarly stained in comparison with the strongly stained cortical analogues in mice (P-value _TRPC3 = 0.3429, Mann-Whitney U; P-value _TRPC5 = 0.8857, Mann-Whitney U) (Fig. 4A and 5). Thus, staining remained significantly stronger in the TRPC5 mice group compared to the TRPC3 mice group (P-value = 0.0286, Mann-Whitney U). Human outer stripe proximal tubules (mean value _TRPC3 = 1.235, SD _TRPC3 = 0.2384; mean value _TRPC5 = 1.654, SD _TRPC5 = 0.1530) featured a seemingly stronger TRPC5 than TRPC3 staining (P-value = 0.0571, Mann-Whitney U). Interspecies comparison is significantly different when considering TRPC5 (P-value _TRPC5 = 0.0286, P-value _TRPC3 = 0.1143, Mann-Whitney U) (Fig. 4A). Intermediate tubules appear to be similarly stained in mice (mean value _TRPC3 = 1.028, SD _TRPC3 = 0.1081; mean value _TRPC5 = 1.144, SD _TRPC5 = 0.05138) as in human (mean value _TRPC3 = 0.8657, SD _TRPC3 = 0.1233; mean value _TRPC5 = 1.210, SD _TRPC5 = 0.1780) sections (p-value _TRPC3 = 0.1143, Mann-Whitney U; P-value _TRPC5 = 0.4857, Mann-Whitney U) (Fig. 4B and 6). Staining is significantly increased in the TRPC5 investigation group compared to the TRPC3 group in human but not in mice samples (P-value _human = 0.0286, Mann-Whitney U; P-value _mice = 0.2000, Mann-Whitney U). Downstream, no significant difference in distal straight tubule staining was observed in mice between the inner (mean value _TRPC3 = 1.357, SD _TRPC3 = 0.2616; mean value _TRPC5 = 1.468, SD _TRPC5 = 0.1829) (Fig. 4C) and outer stripe (mean value _TRPC3 = 1.200, SD _TRPC3 = 0.2966; mean value _TRPC5 = 1.302, SD _TRPC5 = 0.03167) (Fig. 4D) of the outer medulla (P-value _TRPC3 = 0.4857, Mann-Whitney U; P-value _TRPC5 = 0.3429, Mann-Whitney U). In humans, distal straight tubule coloration seemed to differ between the inner (mean value _TRPC3 = 1.197, SD _TRPC3 = 0.02964; mean value _TRPC5 = 1.563, SD _TRPC5 = 0.2433) (Fig. 4C) and outer stripe (mean value _TRPC3 = 0.9772, SD _TRPC3 = 0.08878; mean value _TRPC5 = 1.574, SD _TRPC5 = 0.2879) (Fig. 4D) of the outer medulla when considering TRPC3 but not TRPC5 (P-value _TRPC3 = 0.0571, Mann-Whitney U; P-value _TRPC5 > 0.9999, Mann-Whitney U). The interspecies comparison shows no significant differences neither in terms of inner stripe (P-value _TRPC3 = 0.4000, Mann-Whitney U; P-value _TRPC5 > 0.9999, Mann-Whitney U) or outer stripe distal tubule (P-value _TRPC3 = 0.3429, Mann-Whitney U; P-value _TRPC5 = 0.3429, Mann-Whitney U). As to the cortical distal tubule, interspecies comparison is significantly different in the TRPC5 investigation group with superior score in human sections (P-value = 0.0286, Mann-Whitney U). TRPC3 (mean value _human = 1.350, SD _human = 0.5028; mean value _mice = 1.374, SD _mice = 0.1973) and TRPC5 (mean value _human = 1.686, SD _human = 0.1654; mean value _mice = 1.475, SD _mice = 0.03818) associated DAB staining are similar in the respective species (P-value _human = 0.3429, Mann-Whitney U; P-value _mice = 0.3429, Mann-Whitney U) (Fig. 4E). The last structure of interest we investigated, constitutes the collecting duct (Fig. 6). Again, TRPC3 (mean value _human = 1.744, SD _human = 0.2936; mean value _mice = 1.208, SD _mice = 0.2241) and TRPC5 (mean value _human = 1.862, SD _human = 0.1501; mean value _mice = 1.264, SD _mice = 0.1150) associated DAB staining are similar in the respective species (P-value _human = 0.6857, Mann-Whitney U; P-value _mice > 0.9999, Mann-Whitney U). However, the interspecies comparison is significantly different when considering TRPC5 staining (P-value _TRPC3 = 0.0571, Mann-Whitney U; P-value _TRPC5 = 0.0286, Mann-Whitney U) (Fig. 4F). A minority of human collecting duct cells was characterized by perinuclear halo-like figures, suggesting a decreased and/or localized TRPC3/5 expression (Fig. 6C and E). Beside all tubular structures, TRPC5 stained samples (Fig. 6C) featured an increased DAB staining of the interstitial tissue compared to TRPC3 stained samples (Fig. 6E).

Finally, sample staining with peptide-incubated secondary antibody featured decreased staining levels supporting a high antibody specificity (Figs. 2A and B, 3B and 5B). For instance, human TRPC5 blocking peptide-incubated cortical proximal tubule samples (normalized weighted DAB area score = 0.09443877) showed less colorations than non-peptide incubated analogues (mean value _human = 1.779, SD _human = 0.1954) (Fig. 2B). Corresponding negative control showed almost no staining (normalized weighted DAB area score = 0.0534761) (Fig. 2B). Negative control staining is shown in Fig. 6B.

Several studies have addressed the relevance of either TRPC3 or TRPC5 in renal physiology and distinct conditions including renal fibrosis, nephrocalcinosis and proteinuric kidney diseases^1,17,35. For this purpose, multiple animal and cellular experiments have been established. However, scientists remarkably often disagree on the expression patterns of TRPC3 and TRPC5 – that was until now restricted to the glomerulus – in the kidney as depicted below. Additionally, these brief and discordant descriptions refer in most cases to rodent tissue. Descriptions in human tissue remain rare. This highlights the relevance of our study in the translational concept of TRPC channels in human renal health and disease.

Originally, we investigated kidney tissue samples gained from anatomical dissections of the Institute for Anatomy and Cell Biology, Saarland University. However, autolysis was advanced in most cases and would therefore potentially impair the standardized semiquantitative analysis we performed here. For this reason, we accessed healthy kidney samples gained from surgeries such as tumor nephrectomies. The rarity of such healthy samples needs to be foregrounded at this stage, ultimately restricting the possible group size of investigation – a limitation of our study. Even though our samples are extracted from healthy poles of tumor-affected organs, it could be possible that the tumor entity also altered the long-range peripherical protein expression. However, based on here not shown robust investigations in non-tumor-affected cadaver tissue, we have no indication to believe that the expression profile of the here investigated proteins was altered by the tumorous pole. Mice tissue – that we used for interspecies comparison – is distinctly more available. We, however, preferred to adapt their group size to the human sample group size. Of course, protein distribution profile can vary between rodents such as mice or rats. We only used male mice due to better availability, eventually reducing generalizability of our results. In terms of immunohistochemistry, we employed knockout-validated antibodies and accessed negative controls but also antibody specificity by using blocking-peptides against primary antibodies making results trustable. Still, semiquantified levels of TRPC3 and TRPC5 cannot be necessarily reasonably compared one to one, since two different primary antibodies were used, and antigen affinities can ultimately differ between each other for instance. For instance, TRPC3 blocking-peptide incubated staining was less weakened than blocking-peptide incubated TRPC5 staining. An explanation may light in different antibody affinities, but also in differing protein expression. The normalized weighted DAB area score reflects the staining and is correlated with antigen presence but cannot be set equal to it.

In contrast to other studies^17,27,41,42, Goel et al.⁴ did not detect TRPC5 in kidneys of Sprague-Dawley rats, which might be due to lower expression level compared to detection level as authors commented. For instance, Reiser et al.¹³ revealed TRPC3 and TRPC5 mRNAs in isolated rodent glomeruli. Goel et al.⁴ detected TRPC3 in glomeruli and in specific tubular epithelial cells⁴. Referring to glomeruli, our study localized TRPC3 and TRPC5 in human and rodent glomeruli but did not allow secure distinction between the different glomerular cell types. A quick overview of the corresponding literature investigating the roles of different TRPC channels in podocytes and mesangial cells is presented in the introduction.

TRPC5 was especially investigated in glomeruli¹ and is not or minimally discussed in the tubular system in previous literature to the authors knowledge⁴². In this perspective, we contribute a completely novel description of the renal distribution of TRPC5. We highlight its wide distribution in the tubular system, demanding for further investigations to shape an understanding of the channel’s function in these tubular localizations.

Goel et al.⁴ showed that TRPC3 colocalized with aquaporin-2 (AQP-2), a collecting duct and connecting tubule marker, but not with aquaporin-1 (AQP-1), a proximal tubule marker. Since TRPC3 did neither colocalize with the sodium/calcium exchanger 1 (NCX1), a distal convoluted tubule and connecting tubule marker in rats, nor with peanut lectin, an intercalated cell marker, the authors restricted TRPC3 expression to principal cells of the collecting duct⁴. In 2012, Letarvernier et al.⁴³ stained a control biopsy from one single human specimen and confirmed that TRPC3 can be found in the collecting duct as suggested by Goel et al.⁴ in rats. Our results support TRPC3 expression in the collecting duct of mice and human tissue. However, we have no evidence for TRPC3 – and eventually also TRPC5 – expression restriction to principal cells in mice, since all collecting duct cells appeared stained. Another picture emerged in human tissue, considering that certain cells appeared unstained. The corresponding nuclei were similarly sized as neighbor’s cells ones, speaking against shrinking artefacts. The ratio of unstained cells to stained cell fits with the ratio of intercalated cells to principal cells of approximatively 1:2 to 1:3⁴⁴. On the other hand, a few cell membranes appeared disrupted, and if they were, the cell was often unstained. Similar patterns can also incidentally be observed in negative controls suggesting a cell membrane disruption with cytoplasmic evacuation in the early history of the sample acquisition. It remains elusive, while this phenomenon, if not attributable to protein distribution, was mostly observed in collecting ducts. An explanation might lay in biophysical aspects of the combination of large lumen and high cuboidal cell format of collecting duct cells, that is less pronounced in murine tissue. Altogether, even though unclear, our findings do not tend to support the restriction of TRPC3 to principal cells in the human collecting duct and even less in the murine collecting duct. An interesting feature was to observe that while the semiquantified signal was in most localizations higher when using the anti-TRPC5 antibody instead of the anti-TRPC3 antibody, the human collecting duct samples had a stronger signal independently of the employed antibody than the murine equivalents. These observations ultimately suggest an increased expression of TRPC3 and TRPC5 in human collecting duct compared to mice. As of 2009, Woudenberg-Vrenken et al.⁴⁵ reviewed the role of TRP channels in kidney disease highlighting that TRPC3-expression is restricted to podocytes and to principal cells of the collecting duct. In contrary, Ibeh et al.³⁵ detected TRPC3 only in the proximal convoluted and straight tubule of the renal cortex of mice and in LCC-PK1 cell lines (Pig Kidney Epithelial cells). Our study confirms the localization of TRPC3 in rodent proximal tubules and allows translation of these results to human tissue. Interestingly, only few of our mouse cortical proximal tubules were strongly stained for TRPC3 and TRPC5. One explanation could be, since the outer stripe proximal tubules were mostly strongly stained, that the straight segment of the proximal tubule expresses superior levels of TRPC3 and 5 compared to the convoluted segment. However, there is no evidence for this proposition. By 2020, Khayyat et al.⁶ also noticed the here depicted disagreement on the TRPC3 expression in the nephron among different working groups, but did not contribute to the morphological understanding, rather focusing on functional aspects. Awuah Boadi et al.³⁴ reported TRPC3 localization in distal tubular cells little later as supported by our immunostaining. Even though, almost no reports exist on TRPC3 and even less on TRPC5 in the distal tubule or its different segments, we reported the distribution in the different three segments for the sake of completeness. The small differences observed among the categories mice/human and TRPC3/TRPC5 are not consistent throughout the three subsegments and the relevance of the investigation in the distal tubules lies therefore in the overall detection of the proteins in this segment. As mentioned above, especially this localization is poorly investigated with respect to TRPC3. In analogy to the function of TRPC3 in the proximal tubule¹¹ and to the function of TRPV5 in the distal tubule^11,46, regulation of luminal [Ca²⁺] could be suggested as a channel’s function in this localization^11,34,35.

Altogether, we provide evidence supporting previously suggested TRPC3 expression in different localizations in mice and demonstrate a grossly similar distribution in human tissue. TRPC5 is widely present kidneys of in mice and humans and is accentuated in different segments of the tubular system, whereas literature only or mostly reports TRPC5 in glomeruli.

Briefly, glomeruli only express little TRPC3 and TRPC5 compared to several segments of the tubular system. Cortical and medullary proximal tubules are strongly stained. Intermediate tubules, however, are only weakly stained. The distal tubule was studied in three different localizations and depending on species and primary antibody, the staining was marked but not the same throughout all localizations. Finally, the collecting duct was independently of primary antibody more stained in human compared to mouse tissue.

While our description of TRPC5 is completely new, some authors have already worked on the contribution of tubular TRPC3 to the development or the prevention of certain renal disorders often ending in CKD. Since there is no satisfyingly available therapy for CKD, it is mandatory to further advance our knowledge of such diseases to develop targeted therapies including TRPC channel inhibitors or modulators^28,47.

Samples

Healthy human kidney samples are originated from tumor nephrectomies of the Department of Urology and Pediatric Urology of the Saarland University Medical Center. All patients had given their informed consent and the Ethical Committee of the Saarland Medical Association (Ethikkommission bei der Ärztekammer des Saarlandes) also gave its approval (141/14). Previous investigations in body donors (not shown) had also been consented prior to death by the donors themselves and approved by the Ethical Committee of the Saarland Medical Association (163/20). All investigations based on human tissue were conducted in accordance with the Declaration of Helsinki. Samples from four different patients were analyzed. The mean age was 60 with a standard deviation of 10.4 years. 75% were male and 25% female. Mouse kidney samples were obtained from four 4–5 months old male animals. We have a breeding permit according to § 11 for all animals and an annual disclosure statement for euthanized animals for scientific purpose according to § 4 paragraph 3 of the German Animal Welfare Act. The C57BL/6J and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice used here had no phenotypic burden. The breeding and keeping of these animals is not considered as an animal experiment and doesn’t require an additional permit. Animals were sacrificed after isoflurane inhalation by cervical dislocation. Kidneys were fixed with 4% buffered formalin and embedded in paraffin. Finally, all methods were performed in accordance with the relevant guidelines and regulations.

Immunohistochemistry

Hematoxylin and eosin-stained sections were prepared using routine techniques⁴⁸. For immunohistochemical staining of TRPC3 and TRPC5 paraffin was removed, and antigen recovery was performed with citrate buffer (60 minutes, 95°C). Overnight and at room temperature, the primary antibody (polyclonal anti-TRPC3, lyophilized, ACC-016, 1:50, Alomone Labs, Jerusalem BioPark, Israel; polyclonal anti-TRPC5, lyophilized, ACC-020, 1:50, Alomone Labs, Jerusalem BioPark, Israel) was applied. Rabbit serum was used for the negative control. Specificity was confirmed using blocking peptides (TRPC3 Blocking Peptide, lyophilized powder, BLP-CC016, Alomone Labs, Jerusalem BioPark, Israel; TRPC5 Blocking Peptide, lyophilized powder, BLP-CC020, Alomone Labs, Jerusalem BioPark, Israel). A peroxidase labeled secondary antibody (HRP anti-rabbit goat, A10547; Invitrogen AG, Carlsbad, CA, USA) and diaminobenzidine tetrahydrochloride (DAB) (SK-4103 Vector Laboratories, Burlingame CA, USA) were added, followed by counterstaining with hematoxylin (C. Roth, Karlsruhe, Germany). According to the manufacturer’s (Alomone Labs, Jerusalem BioPark, Israel) information, the anti-TRPC3 and 5 antibodies were knockout-validated and designed to detect the corresponding channels in mouse, rat and human tissue.

Evaluation

We performed a semiquantitative analysis of the DAB staining using the image analysis software from Visiopharm (version 09.2021, Visiopharm, Denmark). Initially, slides were digitalized using the Nano Zoomer S210 (Hamamatsu, Japan). Five sections of each structure (glomerulus, cortical proximal tubule, outer stripe proximal tubule, intermediate tubule, inner stripe distal tubule, outer stripe distal tubule, cortical distal tubule, collecting duct) were manually annotated as Region of Interest (ROI). DAB positive areas within the ROI were detected by thresholding (Fig. 2C and D). The value of threshold (0-160) was separated in three parts. Areas having the darkest DAB stain (value between 0–55) were attributed to 3+, the middle values (between 56–110) to 2 + and the lightest DAB-stained areas (value between 111–160) to 1+. Weighted DAB positive areas per substructure and slide were calculated by multiplying the DAB positive areas by their respective attributed value (1, 2 or 3) and summarizing all values. This value was normalized to the measured tissue area (sum of hematoxylin and DAB positive area) resulting in a normalized weighted DAB area score between 0–3 comparable to a pixelwise H-score⁴⁹. Values that lay between 0 and 1 are considered as negative or weakly positive.

Statistics

Statistical analysis was performed using the GraphPad Prism Software Package (GraphPad, Version 9.5.1.). The non-parametric Mann-Whitney U test was employed to compare the ranks of two independent groups. P-values were two-sided and considered significant when < 0.05.

Data Availability: Data can be apprehended after reasonable requests from the corresponding author.

Acknowledgements: We thank Noran Abdel Wadood, Monika Hollenhorst, Anja Beckmann, and Carola Meier for material support as well as Jörg Pekarsky and Friedrich Paulsen for their contribution to the creation of Figure 1. The authors also wish to sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially improve patient care and increase mankind’s overall knowledge. Therefore, these donors and their families deserve our highest gratitude.

Author Contribution Statement: T.T. and C.N.E. conceived the study. C.M.D. performed investigations. C.M.D, D.S. and C.N.E. analyzed and interpreted the data. T.T., G.K. and K.J. provided resources. C.M.D., D.S. and C.N.E. performed imaging. C.M.D. and C.N.E. wrote the manuscript. All authors read and approved the manuscript.

Ethics Declaration: Informed consent was obtained from all involved patients or their legal guardians. The Ethics Committee of Saarland also gave its approval (141/14). All investigations based on human tissue were conducted in accordance with the Declaration of Helsinki. The study is also reported in accordance with ARRIVE guidelines. A breeding permit according to §11 for all animals and an annual disclosure statement for euthanized animals for scientific purpose according to §4 paragraph 3 of the German Animal Welfare Act was obtained. The C57BL/6J and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice used here had no phenotypic burden. The breeding and keeping of these animals is not considered as an animal experiment and doesn’t require an additional permit.

Funding Statement: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Additional Information: The authors declare no competing interests.

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Competing interests: The authors declare no competing interests.

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New Insights in the Distribution Profile of TRPC3 and TRPC5 in the Mouse and Human Kidney

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