# **Kidney Inflammation, Injury and Regeneration 2020**

Edited by

Patrick C. Baer, Benjamin Koch and Helmut Geiger

Printed Edition of the Special Issue Published in *International Journal of Molecular Sciences*

www.mdpi.com/journal/ijms

## **Kidney Inflammation, Injury and Regeneration 2020**

## **Kidney Inflammation, Injury and Regeneration 2020**

Editors

**Patrick C. Baer Benjamin Koch Helmut Geiger**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Patrick C. Baer Nephrology Goethe University Frankfurt Germany

Benjamin Koch Nephrology Goethe University Frankfurt Germany

Helmut Geiger Nephrology Goethe University Frankfurt Germany

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *International Journal of Molecular Sciences* (ISSN 1422-0067) (available at: www.mdpi.com/journal/ ijms/special issues/kindey ijms 2020).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-2370-5 (Hbk) ISBN 978-3-0365-2369-9 (PDF)**

© 2021 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications.

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 He completed his studies in Biochemistry at the Technical University of Darmstadt and received his doctorate at the Goethe University of Frankfurt/M. P.C.B. has published 101 research articles, including three book chapters and three patents. P.C.B. has been working with cell culture models of proximal and distal tubular epithelial cells of the kidney for 25 years. The research areas of P.C.B. also focus on the isolation, culture and differentiation of mesenchymal stroma/stem cells (MSCs) and the transplantation of MSCs or their derivatives to improve renal regeneration.

#### **Benjamin Koch**

Benjamin Koch earned an MD with Prof. W. Wels at the Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, Frankfurt/M., Germany. His thesis work established RNAseq in its early days and integration with ChIPseq in order to identify novel target genes of an important transcription factor in leukemia. He then performed clinical training at the Goethe University Hospital (Division of Nephrology, Prof. Helmut Geiger). In addition to his clinical work in nephrology, he coordinates research projects in the lab of Prof. Patrick C. Baer and is directly involved in researching (1) pathogen-induced acute and chronic kidney injury as well as (2) novel blood filtration devices in inflammation and sepsis. His research tools include RNAseq, proteomics, and translatomics based on general molecular biology.

#### **Helmut Geiger**

Helmut Geiger is Professor of Medicine and head of the department of nephrology at the University Hospital Frankfurt/M. He has worked as a physician since 1983 in Wurzburg, Budapest, ¨ Nuremberg-Erlangen and Frankfurt/M. During this time, he has written more than 250 scientific publications and has received funding from the DFG, BMBF, BMWi and several foundations. His main research interests are renal diseases, kidney transplantation and hypertension. H.G. has been awarded the Nils-Alwall Prize of the German Working Group for Clinical Nephrology and the Franz Gross Science Prize of the German High Pressure League.

### *Editorial* **Kidney Inflammation, Injury and Regeneration 2020**

**Patrick C. Baer \* , Benjamin Koch and Helmut Geiger**

Division of Nephrology, Department of Internal Medicine III, University Hospital, Goethe-University, 60596 Frankfurt, Germany<EMAIL_ADDRESS>(B.K<EMAIL_ADDRESS>(H.G.) **\*** Correspondence<EMAIL_ADDRESS>Tel.: +49-69-6301-5554; Fax: +49-69-6301-4749

The kidneys play a vital role in the basic physiological functions of the body. Kidney dysfunction impairs these physiological functions and can lead to a wide range of diseases. Damage to the kidney cells can be caused by a variety of ischemic, toxic or immunological complaints that lead to inflammation and cell death, which can lead to organ damage and, ultimately, complete failure. Although the mechanisms underlying acute kidney injury (AKI) and chronic kidney disease (CKD) are quite distinct, clinical evidence suggests that the two conditions are inextricably interconnected [1]. AKI and CKD, regardless of the underlying cause, have inflammation and activation of the immune system as the common underlying mechanisms. Inflammation, a process aimed, in principle, at detecting and fighting harmful pathogens, is, therefore, a major pathogenic mechanism for both AKI and CKD [1]. While the kidney has the remarkable ability to regenerate after an acute injury and can recover completely, depending on the type of kidney lesion, the options for clinical interventions are currently limited to fluid management and extracorporeal kidney support. However, persistent chronic inflammation can trigger renal fibrosis and chronic kidney disease. The investigation of the molecular mechanisms involved in each individual injury is currently insufficiently understood.

In this context, we started a forum for the publication of new results on kidney inflammation, injury and regeneration, as well as for reviewing and discussing existing studies from this interesting research area. In 2019, we initiated the first edition of the Special Edition "Kidney Inflammation, Injury and Regeneration" with 29 articles [2]. The focus of this first edition was more on a summary of current results (represented by 17 review articles), along with 12 original articles from the current research. In the second Special Edition, presented here, the focus is now more on the current research results mainly from in vivo studies. This issue is accompanied by five review articles summarizing the current results on various nephrological diseases or issues. In this current Special Edition, thirteen original research articles are presented: twelve in vivo studies in a murine or rat model and one in vitro study [3]. Seven studies show results from AKI models [4–10], five from fibrosis models (or CKD models) [9,11–14] and two from a transplant rejection model [4,15] (two studies used two different in vivo models).

Steines and coworkers demonstrated that intrarenal tertiary lymphoid organs are sites of humoral immune activation within allografts during chronic rejection and that anti-B-cell activating factor treatment can hinder the formation of tertiary lymphoid organs in allografts [15]. The authors hypothesized that inhibition of the local alloresponses in chronic rejection with an anti-B-cell activating factor antibody represents a potential benefit to kidney transplant patients. Others evaluated the effects of a novel human fusion recombinant protein in two representative kidney inflammatory models: a renal ischemiareperfusion model (an AKI model) and an allogeneic kidney transplant model [4]. This study shows that targeting with that novel protein offers a good microenvironment profile to protect the ischemic process in the kidney and to prevent kidney rejection.

Another study using a renal ischemia–reperfusion (IR) model showed that preconditioning with cilastatin, a specific inhibitor of renal dehydrodipeptidase-1, attenuates renal IR injury via activation of the main hypoxia factor. The authors confirmed this effect

**Citation:** Baer, P.C.; Koch, B.; Geiger, H. Kidney Inflammation, Injury and Regeneration 2020. *Int. J. Mol. Sci.* **2021**, *22*, 5589. https://doi.org/ 10.3390/ijms22115589

Received: 29 April 2021 Accepted: 24 May 2021 Published: 25 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

through in vitro studies with immortal tubular epithelial cells [10]. Others used an IR model to show that the NOX1-selective inhibition attenuates kidney IR injuries via the downregulation of oxidative stress-mediated kinase signaling [8] or to investigate the autophagy dynamics during an IR injury, a potential treatment strategy due to removing damaged cells, macromolecules and organelles [6]. The effect of growth differentiation factor 15 (GDF15) was investigated in a murine model of anti-glomerular basement membrane glomerulonephritis [7]. The study showed that GDF15 is required for the regulation of T-cell chemotactic chemokines in the kidneys and demonstrated the protective effects of GDF15. The study revealed a novel mechanism limiting the migration of lymphocytes to the site of inflammation during glomerulonephritis [7]. The findings of Neži´c and coworkers investigated the molecular mechanism involved in the reno-protective effects of simvastatin in an endotoxin-induced AKI model [5]. The study indicated that simvastatin, a well-known lipid-lowering medication, has cytoprotective effects on induced tubular apoptosis, mediated by the upregulation of cell survival molecules and inhibition of the mitochondrial proteins. Therefore, the authors hypothesized that simvastatin has significant cell-protective effects in septic AKI [5].

Leong and coworkers showed that cyclophilin A, a damage-associated molecular pattern, promoted inflammation and acute kidney injury in a renal IR model but did not contribute to inflammation or interstitial fibrosis in a model of progressive kidney fibrosis (unilateral ureteric obstruction (UUO)) [9]. Other studies showed the effects of different proteins/peptides against UUO-induced renal injury, inflammation and fibrosis. The 20-amino acid peptide ND-13 protects against UUO-induced damage and is, therefore, a potential new therapeutic approach to prevent renal diseases [14]. Furthermore, the effects of verteporfin on UUO-induced renal tubulointerstitial inflammation, fibrosis and transforming growth factor-β1 regulation were investigated. The study showed that verteporfin decreases the UUO-induced increase in tubular injury, inflammation and extracellular matrix deposition in mice [12]. Son and coworkers investigated the attenuating effects of dieckol on hypertensive nephropathy in spontaneously hypertensive rats and hypothesized that dieckol could be beneficial for decreasing hypertensive nephropathy by decreasing EMT and renal fibrosis [11].

Others investigated the role of xanthine oxidase (XO) in CKD progression associated with hypercholesterolemia [13]. The authors used a murine model of uninephrectomy to induce CKD, in addition to a high-cholesterol diet with a XO inhibitor, and, also, evaluated the results in an in vitro model using immortal tubular epithelial cells. The study clearly showed that XO inhibition exerts reno-protective effects and identifies XO as a novel therapeutic target for hypercholesterolemia-associated kidney injury [13]. Finally, one in vitro study using cisplatin-injured primary tubular epithelial cells focused on the decisive role of the Lipocalin-2 iron load for its pro-regenerative functions [3]. The study detected a positive correlation between the total iron amounts in tubular epithelial cells and cellular proliferation. In conclusion, it was hypothesized that macrophage-released Lipocalin-2-bound iron is provided to tubular epithelial cells during toxic cell damage, whereby the injury is limited and recovery is favored [3].

In addition, five interesting review articles were included in this Special Edition summarizing the current state of knowledge of the treatment of IgA nephropathy [16], the role of endocan in kidney diseases [17] and the influence of inflammation on anemia in CKD patients [18]. It also discusses the mechanism of kidney injury in preeclampsia and the susceptibility of podocytes [19]. Finally, the review summarizes the role of Tolllike receptors in the pathogenesis of glomerulopathy and their role as potential marker molecules for the development of renal diseases [20].

**Author Contributions:** Writing, review and editing, P.C.B. and review and editing, B.K. and H.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors received no funding for this Editorial.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Attenuating Effects of Dieckol on Hypertensive Nephropathy in Spontaneously Hypertensive Rats**

**Myeongjoo Son 1,2 , Seyeon Oh 2 , Junwon Choi 2 , Ji Tae Jang 3 , Kuk Hui Son 4,\* and Kyunghee Byun 1,2,\***

**Abstract:** Hypertension induces renal fibrosis or tubular interstitial fibrosis, which eventually results in end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is one of the underlying mechanisms of renal fibrosis. Though previous studies showed that Ecklonia cava extracts (ECE) and dieckol (DK) had inhibitory action on angiotensin (Ang) I-converting enzyme, which converts Ang I to Ang II. It is known that Ang II is involved in renal fibrosis; however, it was not evaluated whether ECE or DK attenuated hypertensive nephropathy by decreasing EMT. In this study, the effect of ECE and DK on decreasing Ang II and its down signal pathway of angiotensin type 1 receptor (AT1R)/*TGFβ*/*SMAD*, which is related with the EMT and restoring renal function in spontaneously hypertensive rats (SHRs), was investigated. Either ECE or DK significantly decreased the serum level of Ang II in the SHRs. Moreover, the renal expression of AT1R/*TGFβ*/*SMAD* was decreased by the administration of either ECE or DK. The mesenchymal cell markers in the kidney of SHRs was significantly decreased by ECE or DK. The fibrotic tissue of the kidney of SHRs was also significantly decreased by ECE or DK. The ratio of urine albumin/creatinine of SHRs was significantly decreased by ECE or DK. Overall, the results of this study indicate that ECE and DK decreased the serum levels of Ang II and expression of AT1R/*TGFβ*/*SMAD*, and then decreased the EMT and renal fibrosis in SHRs. Furthermore, the decrease in EMT and renal fibrosis could lead to the restoration of renal function. It seems that ECE or DK could be beneficial for decreasing hypertensive nephropathy by decreasing EMT and renal fibrosis.

**Keywords:** epithelial-to-mesenchymal transition; E. cava extracts; dieckol; spontaneously hypertensive rats; renal fibrosis; angiotensin II

#### **1. Introduction**

The kidney is a major organ that is affected by hypertensive target organ damage: chronic kidney disease commonly occurs in around 16% of hypertensive patients [1].

The pathological features of hypertensive nephropathy include inflammation, glomerular sclerosis, tubular atrophy, and interstitial fibrosis [2]. The fibrotic tissue replaces the normal functional kidney tissue, which leads to renal failure [3,4]. Thus, renal fibrosis or tubular interstitial fibrosis is the main pathological lesion of hypertensive nephropathy, which induces end-stage renal disease (ESRD) [5]. Renal fibrosis shows the following characteristics: increased massive extracellular matrix (ECM) production, increased recruitment of fibroblasts to tissue injury sites, and increased phenotype changes from fibroblasts to α-smooth muscle actin (α-SMA)-expressing myofibroblasts [6]. The increased α-SMAexpressing myofibroblasts lead to the unnecessarily excessive deposition of collagen and

**Citation:** Son, M.; Oh, S.; Choi, J.; Jang, J.T.; Son, K.H.; Byun, K. Attenuating Effects of Dieckol on Hypertensive Nephropathy in Spontaneously Hypertensive Rats. *Int. J. Mol. Sci.* **2021**, *22*, 4230. https://doi.org/10.3390/ ijms22084230

Academic Editors: Patrick C. Baer, Benjamin Koch and Helmut Geiger

Received: 1 April 2021 Accepted: 16 April 2021 Published: 19 April 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

enhance the dysregulation of matrix metalloproteinases, which destroys the basement membrane and further accelerates fibrosis [6].

Recently, many studies have shown that epithelial-to-mesenchymal transition (EMT) is one of the underlying mechanisms of renal fibrosis [7,8]. By undergoing EMT, the epithelial cell gradually loses epithelial markers, such as E-cadherin, which is the main element of cell-to-cell junctions, and gains markers of mesenchymal phenotype, such as α-SMA [9]. By losing the cell junctions, the cells that underwent the EMT process easily move toward the interstitial space [9]. Moreover, the epithelial cells that acquired the properties of myofibroblasts express α-SMA and synthesize ECM proteins, such as collagen, eventually leading to tubular interstitial fibrosis (TIF) [9].

Angiotensin II (Ang II) acts as the main player in hypertension-induced fibrosis. As the main activator of the renin–angiotensin–aldosterone system (RAAS), Ang II causes severe vascular, glomerular, and tubulointerstitial injuries, which are accompanied by the increase in transforming growth factor-beta (TGFβ) via the angiotensin type 1 receptor (AT1R) [10]. The levels of Ang II and its receptor in the kidneys of the spontaneously hypertensive rats (SHRs) were higher than those of Wistar Kyoto (WKY) rats [11]. It is known that Ang II induces EMT via TGFβ-dependent signaling pathways [12]. The Ang II treatment for NRK52E cells (normal rat tubular epithelial cell lines) induces the expression of the TGFβ1/SMAD signaling pathway, which eventually increases the proapoptotic and fibrotic proteins [13]. When Ang II binds with AT1R, the SMAD3 signal pathway is activated and induces EMT in NRK52E cell lines [12].

Polyphenols from marine algae have been reported to function as Ang I-converting enzyme (ACE) inhibitors [14–17]. ACE is involved in the conversion of Ang I to Ang II, and increases Ang II as a potent vasoconstrictor, leading to hypertension [18]. Several phlorotannins, such as phlorofucofuroeckol A, dieckol (DK), and eckol, which are present in extracts from Ecklonia cava or Ecklonia stolonifera, show ACE inhibiting activities; thus, these polyphenols were expected to decrease blood pressure (BP) [15,19]. Even though E. cava extracts (ECE) and DK show ACE inhibiting activities, no report has investigated whether ECE or dieckol is involved in the EMT of the kidney or renal fibrosis that is induced by hypertension. Thus, the effect of ECE and DK on decreasing Ang II and its down signal pathway of AT1R/TGFβ/SMAD2/3, which is related with the EMT of the kidney and restoring renal function in SHRs, was investigated in this study.

#### **2. Results**

#### *2.1. ECE and DK Reduced Systolic BP and Serum Level of Ang II in SHRs*

The systolic BP of the SHRs was significantly higher than that of the WKY rats and was significantly decreased by the administration of either ECE or DK. The decreasing effects of 50, 100, and 150 mg/kg/day of ECE and DK administration were not significantly different (Figure 1a). The diastolic and mean BPs of the SHRs were significantly higher than those of the WKY rats. The administration of either ECE or DK did not significantly decrease the diastolic and mean BP (Figure 1b,c).

The serum level of Ang II in the SHRs was significantly higher than that of the WKY rats and was significantly decreased by the administration of either ECE or DK. Moreover, the most prominent decreasing effect was observed in the group treated with 150 mg/kg/day of ECE (Figure 1d).

#### *2.2. ECE and DK Attenuated the Expression of AT1R in the Kidney of SHRs*

The expression of AT1R was evaluated through qRT-PCR and staining. The mRNA expression of AT1R in the medulla and cortex of the kidney was significantly higher in the SHRs than that of the WKY rats. Such expression was decreased by the administration of either ECE or DK (Figure 2a). In the medulla, the decreasing effect was most prominent in the group treated with DK. In the cortex, 150 mg/kg/day of ECE had the most prominent decreasing effect. The expression level of AT1R in the cortex and medulla of the SHRs, which was evaluated through staining, was significantly increased and was significantly

decreased by the administration of either ECE or DK. Moreover, the most prominent decreasing effect was observed in the group treated with DK (Figure 2b,c).

**Figure 1.** Comparative analysis of ECE and DK administration on the reduction of systolic blood pressure and serum angiotensin II level in SHRs. (**a**) Systolic blood pressure, (**b**) diastolic blood pressure, (**c**) mean artery blood pressure, and (**d**) serum angiotensin II level were measured prior to sacrifice. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) were oral administrated for 4 weeks and 2.5 mg/kg/day DK also oral administrated for 4 weeks. Means denoted by a different letter indicate significant differences among groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol.

To validate ECE and DK attenuation of AT1R expression in kidney tubules, a mouse proximal tubule cell line (TCMK-1) was activated by angiotensin II in an in vitro model [20]. The expression level of AT1R protein in angiotensin II-treated TCMK-1 cells with ECE (5, 25, 50 ug/mL), DK or inhibitor was decreased compared to only angiotensin II treatment (Figure 2d).

#### *2.3. ECE and DK Reduced the Expression of TGFβ, SMAD2/3, and Snail2 in the Kidney of SHRs*

The expressions of TGFβ in the medulla and cortex of the SHRs were significantly higher than those of the WKY rats, which were decreased by the administration of ECE or DK. In the medulla, the decreasing effect was most prominent in the groups treated with 150 mg/kg/day of ECE and DK. In the cortex, the decreasing effect was most prominent in the group treated with DK (Figure 3a). The expressions of SMAD2 in the medulla and cortex of the SHRs were higher than those of the WKY rats, which were significantly decreased by the administration of ECE or DK. The decreasing effect in the medulla was most prominent in the groups treated with 150 mg/kg/day of ECE or DK. In the cortex, the decreasing effects of 50, 100, and 150 mg/kg/day of ECE or DK were not significantly different (Figure 3b). The expression of SMAD3 in the medulla and cortex of the SHRs was significantly higher than that of the WKY rats, and was significantly decreased by the administration of ECE or DK. The most prominent decreasing effect was observed in the group treated with 150 mg/kg/day of ECE (Figure 3c). The expression of Snail2 in the medulla and cortex of the SHRs was significantly increased by the administration of ECE or DK. The most prominent decreasing effect was observed in the group treated with DK (Figure 3d). In the in vitro model, the expression level of TGFβ and pSMAD2/3 protein in angiotensin II-treated TCMK-1 cells with ECE (5, 25, 50 ug/mL), DK or inhibitor was decreased compared to the only angiotensin II treatment (Figure 3e,f).

**Figure 2.** Comparative analysis of ECE and DK administration on the reduction of expression of AT1R in the kidney of SHRs and in angiotensin II-treated TCMK-1 cells. (**a**) AT1R mRNA levels in cortex area, and in medulla of the kidney were measured by qRT-PCR analysis. (**b**) AT1R protein expression levels in cortex area, and in medulla of kidney were measured by immunohistochemistry and (**c**) the protein levels were quantified by Image J software. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) were orally administrated for 4 weeks and 2.5 mg/kg/day of DK were also orally administrated for 4 weeks. (**d**) AT1R protein expression levels in angiotensin II-treated TCMK-1 cells (renal epithelial tubular cells) with 5, 25, 50 ug/mL ECE, 2.5 ug/mL DK or inhibitor were measured by Western blotting. Scale bar = 25 µm. Means denoted by a different letter indicate significant differences among groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol; Inhibitor, 1 µmol/L telmisartan.

#### *2.4. ECE and DK Reduced the EMT in the Kidney of the SHRs*

The expression of the mesenchymal cell marker, such as vimentin and α-SMA, of the SHRs was significantly higher than that of the WKY rats and was significantly decreased by the administration of either ECE or DK (Figure 4). The decreasing effect on the expression of vimentin in the cortex of the SHRs was most prominent in the group treated with DK, whereas the decreasing effect on the expression of vimentin in the medullas of the SHRs was most prominent in the group treated with 150 mg/kg/day of ECE. Moreover, the decreasing effect on the expression of vimentin in the cortex of the SHRs was most prominent in the group treated with DK (Figure 4a,b). The expression of α-SMA in the cortex and medulla of the SHRs was most significantly decreased in the group treated with 150 mg/kg/day of ECE (Figure 4c,d). The expression of the epithelial cell marker, such as E-cadherin, in the cortex and medulla of the SHRs was significantly lower than that of the WKY rats (Figure S1) and was significantly increased by the administration of either ECE or DK.

**Figure 3.** Comparative analysis of ECE and DK administration on the reduction of expression of TGFβ, SMAD2/3, and Snail2 in the kidneys of SHRs. (**a**) TGFβ, (**b**) SMAD2, (**c**) SMAD3, (**d**) Snail2 mRNA levels in cortex area, and in medulla of kidney were measured by qRT-PCR analysis. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) were orally administrated for 4 weeks and 2.5 mg/kg/day DK were also orally administrated for 4 weeks. (**e**) TGFβ, SMAD2/3, pSMAD2/3 protein expression levels in angiotensin II-treated TCMK-1 cells (renal tubular epithelial cells) with 5, 25, 50 ug/mL ECE, 2.5 ug/mL DK or inhibitor were measured by Western blotting and (**f**) the protein levels were quantified by Image J software. Means denoted by a different letter indicate significant differences between groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol; Inhibitor, 1 µmol/L telmisartan.

**Figure 4.** Comparative analysis of ECE and DK administration on the reduction of EMT in the kidney of the SHRs. (**a**) Vimentin protein expression levels in cortex area, and in medulla of kidney were measured by immunohistochemistry and (**b**) the protein levels were quantified by Image J software. (**c**) α-SMA protein expression levels in cortex area, and in medulla of kidney were measured by immunohistochemistry and (**d**) the protein levels were quantified by Image J software. Scale bar = 25 µm. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) was oral administrated for 4 weeks and 2.5 mg/kg/day DK also oral administrated for 4 weeks. Means denoted by a different letter indicate significant differences between groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol.

#### *2.5. ECE and DK Reduced the Renal Fibrosis and Glomerular Sclerosis in the Kidney of SHRs*

The fibrosis area in the cortex and medulla of the kidney of the SHRs was significantly higher than that of the WKY rats (Figure 5a,b) and was significantly decreased by the administration of either ECE or DK. In the cortex, the most prominent decreasing effect was observed in the group treated with DK, whereas in the medulla, the most prominent decreasing effect was observed in the group treated with 150 mg/kg/day of ECE. The GSI of the SHRs was significantly higher than that of the WKY rats (Figure 5c,d) and was significantly decreased by the administration of ECE or DK. Moreover, the most prominent decreasing effect was observed in the group treated with DK.

**Figure 5.** Comparative analysis of ECE and DK administration on the reduction of renal fibrosis and glomerular sclerosis in the kidney of SHRs. (**a**) Masson's trichrome stained cortex area and medulla area of kidney showing fibrosis area (blue color) and (**b**) fibrosis area was quantified by Image J software. (**c**) PAS-stained cortex area and medulla area of kidney showing glomerular sclerosis and (**d**) the sclerosis were evaluated using a semi-quantitative scoring method (Grades 0–4). Scale bar = 25 µm. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) were orally administrated for 4 weeks and 2.5 mg/kg/day of DK was also orally administrated for 4 weeks. Means denoted by a different letter indicate significant differences between groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol; GSI; glomerulosclerotic index.

#### *2.6. ECE and DK Attenuated Renal Function Aggravation in SHRs*

The amount of water intake within 24 h was not significantly different among all groups (Figure 6a). The urine volume of the SHRs within 24 h was significantly lower than that of the WKY rats (Figure 6b). It was significantly increased by the administration of 100 and 150 mg/kg/day of ECE and DK. The urine sodium/potassium (Na/K) ratio was not significantly different among all groups (Figure 6c). The albumin level in the urine of the SHRs was significantly higher than that of the WKY rats and was significantly decreased by the administration of ECE or DK. The decreasing effects of 50, 100, and 150 mg/kg/day of ECE and DK were not significantly different (Figure 6d). The ratio of urine albumin/creatinine of the SHRs was significantly higher than that of the WKY rats and was decreased by the administration of ECE or DK. The decreasing effects of 50 and 100 mg/kg/day of ECE and DK were not significantly different (Figure 6e).

**Figure 6.** Comparative analysis of ECE and DK administration on the attenuation of renal function aggravation in SHRs. (**a**) Water consumption, (**b**) urine volume, (**c**) urine Na/K ratio, (**d**) urine microalbumin level, (**e**) urine albumin-to-creatinine ratio (UACR) were measured prior to sacrifice. Three doses of ECE (50 mg/kg/day, 100 mg/kg/day and 150 mg/kg/day) were orally administrated for 4 weeks and 2.5 mg/kg/day of DK was also orally administrated for 4 weeks. Means denoted by a different letter indicate significant differences between groups (*p* < 0.05). - means the same group. ECE, Ecklonia cava extract; DK, dieckol; NS, not significant.

#### **3. Discussion**

Hypertension is the second leading etiology of ESRD after diabetes [21]. It is hard to predict the severity of hypertensive renal fibrosis with BP, since renal fibrosis could severely progress even when the patient's BP is not extremely high [21]. Although antihypertensive treatments with ACE inhibitors, such as angiotensin receptor blockers, renin inhibitors, and aldosterone antagonists, could reduce the severity of hypertensive kidney disease, they are not enough to prevent the progression of hypertensive nephropathy [22]. Even though the recommended target BP is achieved at below 130/80 mmHg, the treatment strategy for decreasing BP cannot delay the progression of hypertensive nephropathy [23]. Although hypertensive nephropathy is typically described as nephroangiosclerosis and glomerular hyalinosis [24,25], recently, it was revealed that the interstitium of the kidney, which is involved in TIF, is related to disease progression, as well as the glomerular and vascular compartments [26,27]. Since TIF is a main pathophysiology of hypertensive nephropathy, it is essential to the development of new agents directed to modulate TIF to prevent the progression of hypertensive nephropathy. In recent years, EMT has been known as a crucial player in renal fibrosis [28,29].

Ang II induces vasoconstriction and consequently induces hypertension [30]. In hypertension, RAAS is activated systemically, and the upregulation of RAAS is accompanied in several organs, such as the kidney [31,32]. The hyperactivation of intrarenal RAAS is an important mechanism of hypertension and chronic kidney disease [31,32]. Both TGFβ1 and Ang II induce the activation of the intrarenal RAAS and enhance the expressions of angiotensinogen, renin, ACE, and AT1R, which induce EMT [33]. In our study, the systolic BP of the SHRs was significantly decreased by the administration of either ECE or DK. The serum level of Ang II of the SHRs was significantly decreased by the administration of either ECE or DK. The expression of AT1R in the cortex and medulla of the SHRs was significantly decreased by the administration of either ECE or DK.

The upregulation of RAAS, such as the increased expression of AT1R, activates the down signaling pathway of TGFβ1 [34]. Activated by RAAS, TGFβ1 induces EMT via the SMAD-dependent and SMAD-independent pathways [33]. As the SMAD-dependent signaling pathway, TGFβ1 signaling is transduced via TGFβRII and TGFβRI and leads to the

activation of SMAD2/3. [10]. SMAD4 binds to SMAD2/3 and promotes the translocation of the SMAD complex into the nucleus [10]. Translocated SMADs upregulate the expressions of EMT genes, such as Snail, Twist, and ZEB [34]. In our study, the expression of AT1R was significantly higher in the cortex and medulla of the SHRs than that of the WKY rats, and was decreased by the administration of either ECE or DK. The expression of TGFβ and SMAD2/3 in the cortex and medulla of the SHRs was decreased by the administration of either ECE or DK.

The EMT is characterized by the loss of an epithelial marker, such as E-cadherin, and the attainment of mesenchymal markers, such as α-SMA, vimentin, and fibronectin [35]. E-cadherin is the most expressed cadherin in the epithelial cells and is, thus, frequently used as an epithelial marker [36]. α-SMA is a phenotypic marker of myofibroblast cells, and its expression is a feature of the advanced stages of EMT [37]. α-SMA-expressing myofibroblasts, which undergo EMT, induce the synthesis of excessive ECM and abnormal ECM remodeling and renal fibrosis [37].

Hypertensive nephropathy induces glomerulosclerosis and interstitial fibrosis, which induces the decrease in renal function and significant proteinuria [6]. In our study, the expression of vimentin and α-SMA in the medulla and cortex of the SHRs was increased and was decreased by the administration of either ECE or DK. The amount of fibrosis area in the medulla and cortex of the SHRs was increased, and it was decreased by the administration of either ECE or DK. The index of glomerular sclerosis of the SHRs was increased, and it was decreased by the administration of either ECE or DK.

The urine volume of the SHRs progressively decreased as compared with that of the WKY rats of the same age [38]. It is known that the greater the excretion of urine sodium, the less the urinary potassium excretion. Moreover, in human studies, the urine Na/K ratio is associated with the increase in BP and the fast impairment of renal function [39]. In our study, the urine volume of the SHRs was lower than that of the WKY rats, even though the water intake amount was not different between the SHRs and WKY rats. The urine Na/K ratio of the SHRs was significantly different among the WKY rats, SHRs, and ECE- or DK-treated groups. The urine Na/K ratio was used as a marker of dietary sodium and potassium intake in human studies [39]. The BP increased with the increase in sodium intake. However, increased potassium intake decreased the BP by increasing the excretion of sodium into the urine [40]. Therefore, the urine Na/K ratio is associated with the BP in humans [40]. In our study, all animals were not fed with a high-sodium diet, which explains why the urine Na/K ratio among the groups did not show any significant difference.

Hypertension tends to increase proteinuria [37]. It is known that the urine albuminto-creatinine ratio is associated with the decrease in the glomerular filtration rate [41]. Previous studies have shown that the development of microalbuminuria in SHRs is caused by predominant tubular injury, which induce the urinary loss of low molecular weight proteins [42,43]. In our study, the urine albumin-to-creatinine ratio of the SHRs was higher than that of the WKY rats, and was decreased by the administration of either ECE or DK.

Previous studies showed that several polyphenols have an inhibiting effect on ACE activity [14–17]. However, these studies only reported ACE inhibitory activity as an IC50 value, which is the 50% inhibition concentration of a positive control, and they did not show an antihypertensive effect or protecting effect against hypertensive nephropathy in the animals. One study showed that ECE decreased BP in the 2-kindney 1-clip Goldblatt hypertensive rats [44]. Pyrogallol-phloroglucinol-6,6-bieckol decreased BP in the high fat diet-induced hypertension animal model by modulating vascular dysfunction [45]. However, these studies showed only decreasing BP and did not evaluate whether ECE showed any effect of attenuating hypertensive nephropathy. Our study showed that ECE and DK decreased the expression of AT1R/TGFβ/SMAD2/3 in the kidney and decreased the serum level of Ang II. Additionally, ECE and DK decreased EMT, renal fibrosis and glomerular sclerosis. It seems that ECE and DK might be helpful in attenuating hypertensive nephropathy.

#### **4. Materials and Methods**

#### *4.1. Preparation of ECE and Isolation of DK*

The ECE were obtained from the Aqua Green Technology Co., Ltd. (Jeju, Korea). The E. cava was thoroughly washed with pure water and air-dried at room temperature for 48 h. It was ground, and 50% ethanol was added, followed by heating at 85 ◦C for 12 h. The ECE were filtered and then, when concentrated, sterilized by heating at a high temperature for 40–60 min and then spray-dried. Dieckol, which is one of the representative phlorotannins of E. cava, was then isolated. Briefly, centrifugal partition chromatography was performed using a two-phase solvent system that mixed pure water, ethyl acetate, n-hexane, and a methanol mixture (ratio 7:7:2:3). The organic stationary phase was filled in the column and the mobile phase was filled in the column, following the descending order of flow rate (2 mL per minute) and was used for separation. We followed the methods of Dr. Son et al. (2019) [45].

#### *4.2. Hypertension Animal Model*

Male SHRs (8 weeks old) and male WKY rats (8 weeks old) were bought from the Orient Bio (Sungnam, Korea) and kept at a constant temperature of approximately 24 ◦C, relative humidity of 50–55%, and light/dark cycle of 12 h/12 h. The rat sublimation was conducted for 1 week. The rats were randomly divided into six groups (five rats per group):

(i) WKY group, in which the rats were orally administered with drinking water for 4 weeks;

(ii) SHR group, in which the rats were orally administered with drinking water for 4 weeks;

(iii–v) SHR group, in which the rats were orally administered with ECE of (iii) 50 mg/kg/day, (iv) 100 mg/kg/day, and (v) 150 mg/kg/day for 4 weeks;

(vi) SHR group, in which the rats were orally administered with 2.5 mg/kg/day DK for 4 weeks. After 4 weeks, water consumption, urine volume and urine chemical analysis were validated with metabolic cage for 24 h. After completing the metabolic analysis, all rats were sacrificed according to the ethical principles of the Institutional Animal Care and Use Committee of Gachon University (approval number: LCDI-2019-0121).

#### *4.3. Immunohistochemistry*

Kidney tissue paraffin blocks were sectioned at 8 µm, placed on gelatin-coating slides, and dried at 37 ◦C for 3 days. The kidney tissue slides were deparaffinized and then incubated with 0.3% hydrogen peroxide (Sigma-Aldrich, MO, USA) for 10 min. Afterward, the slides were rinsed three times with phosphate-buffered saline (PBS), and incubated with an animal serum to inhibit antibody nonspecific binding. Moreover, they were incubated with primary antibodies (anti-AT1R, anti-E-cadherin, anti-α-SMA, and antivimentin) and rinsed with PBS three times. The tissue slides were then incubated with the biotinylated secondary antibodies from the ABC kit (Vector Laboratories, Burlingame, CA, USA), incubated for 3 h with a blocking solution, and rinsed with PBS three times. The tissue slides were developed with 3,3-diaminobenzidine as a substrate for 3 min, mounted with a xylene-based DPX solution (Sigma-Aldrich), and visualized via light microscopy (Olympus Optical Co., Tokyo, Japan).

#### *4.4. RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)*

The RNA from the rat kidney tissues was isolated using the RNAiso Plus reagent (TAKARA, Japan) according to the manufacturer's instructions. The kidney tissues were first divided into two parts, namely, the cortex part and the medulla part. The kidney tissues were finely cut and crushed into powder, and the powders were resuspended with 1 mL of RNAiso Plus reagent, mixed with 0.1 mL of pure glade chloroform (Amresco, Solon, OH, USA), and then centrifuged at 13,000× *g* for 20 min at 4 ◦C. After centrifugation, the supernatant was mixed with 0.25 mL of pure glade isopropyl alcohol, and the extracted RNA pellets were washed with 70% alcohol and centrifuged at 7500× *g* for 5 min at 4 ◦C. The dried pellets were dissolved in 10–30 µl diethylpyrocarbonate-treated pure water, and RNA was quantified and checked using the NanoDrop 2000 (Thermo Fisher Scientific, MA, USA). Complementary DNA (cDNA) was prepared from RNA using a cDNA synthesis kit (PrimeScript™, TAKARA). A quantitative real-time polymerase chain reaction (qRT-PCR) determined the RNA levels from the kidney tissues. The forward and reverse primers were mixed with distilled water, and then 10 µl mixtures were placed in a 384-well qRT-PCR plate. The cDNA and SYBR green (TAKARA) were subsequently added and then validated using a qRT-PCR machine (Bio-Rad, Hercules, CA, USA). The genes of interest are listed in Supplementary Table S1. We followed the methods of Dr. Son et al. 2019 [45].

#### *4.5. Enzyme-Linked Immunosorbent Assay (ELISA)*

To confirm the serum Ang II level, an aliquot of the withdrawn blood (1.5 to 1.7 mL) was added in serum separator tubes (Becton Dickinson, Franklin Lakes, NJ, USA) and then centrifuged at 2000× *g* for 20 min at room temperature. Afterward, the separated serum was moved into a new tube and stored in a deep freezer. The Ang II ELISA kit (MyBioSource, San Diego, CA, USA) was used for analyzing the presence of serum according to the manufacturer's instructions. Ang II was measured within 1 week after blood collection from the animals.

#### *4.6. Histological Analysis*

#### 4.6.1. Masson's Trichrome (MT) Stain

Masson's trichrome (MT) stain validates the fibrosis of the kidney. Blocks of paraffinembedded kidney tissue were sectioned to a thickness of 8 µm, placed on a coating slide, and dried at 37 ◦C for 3 days. The tissue slides were deparaffinized with xylene and alcohol and were re-fixed with Bouin's solution for 24 h and rinsed with running tap water for 3 min. The slides were submerged with Weigert's iron hematoxylin solution for 10 min and Biebrich scarlet-acid fuchsin solution for 15 min and then differentiated with phosphomolybdic–phosphotungstic acid solution for 10 min. Finally, the slides were transferred to aniline blue solution for 3 min and then washed with water. The stained slides were mounted with xylene-based DPX solution (Sigma-Aldrich) and visualized via light microscopy (Olympus). The collagen fiber appears to be blue in color in the fibrosis area, whereas the renal epithelium appears to be red in color. MT-stained fibrosis areas were measured using the ImageJ software. Original images of the MT-stained kidney were converted into RGB images, and then these images were deconvolved using the ImageJ software using the color deconvolution plugin.

#### 4.6.2. Periodic Acid–Schiff (PAS) Stain

A periodic acid–Schiff (PAS) stain validated the glomerular damage of the kidney. Blocks of paraffin-embedded kidney tissue were sectioned to a thickness of 8 µm, placed on a coating slide, and dried at 37 ◦C for 3 days. The tissue slides were deparaffinized with xylene and alcohol, hydrated with water, oxidized with 0.5% periodic acid solution for 5 min, and washed with water. The slides were submerged with the Schiff reagent for 15 min and then washed with lukewarm tap water for 5 min. Finally, the slides were transferred to Mayer's hematoxylin solution for 1 min and then washed with water. The stained slides were mounted with xylene-based DPX solution (Sigma-Aldrich) and visualized via light microscopy (Olympus).

Glomeruli were randomly selected, and the glomerular damage was evaluated using a semi-quantitative scoring method (Grades 0–4) using PAS-stained kidney tissue slides.

The glomerulosclerotic index (GSI) was calculated using the following equation: (4 × n4) + (3 × n3) + (2 × n2) + (1 × n1) / n4 + n3 + n2 + n1 + n0, where n (x) is the number of renal glomerular sclerosis in each grade [46]. This analysis was carried out in the treatment groups by a masked observer.

#### *4.7. Systolic Blood Pressure, Diastolic Blood Pressure, and Mean Arterial Blood Pressure Measurements*

The systolic BP, diastolic BP, and mean arterial BP were measured using a noninvasive CODA tail-cuff system (Kent Scientific Corp., Torrington, CT, USA). The rat sublimation was conducted for 20 min for 5 days, and the BP was measured on the last day.

#### *4.8. In Vitro Modeling Using TCMK-1 Cells*

TCMK-1 cells, a mouse proximal tubule cell line, were purchased from American Type Culture Collection (Washington, DC, USA). High glucose Dulbecco's Modified Eagle's medium (Gibco; Grand island, NY, USA), 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin were used as culture mediums. To validate the inhibitory effects of ECE and DK in angiotensin II-treated TCMK-1 cells, we treated ECE (5, 25, 50 µg/mL) and DK (1.8 µg/mL) with 100 nmol/L angiotensin II (Sigma-Aldrich, St Louis, MO, USA) for 12 h. Telmisartan (1 µmol/L, Sigma-Aldrich) was used for AT1 receptor antagonist and pretreated before angiotensin II for 3 h [47].

#### *4.9. Preparation of Protein and Western Blotting*

To isolate protein from angiotensin II-treated TCMK-1 cells, the cells were scraped using the RIPA lysis buffer with phosphatase and proteinase inhibitor (EzRIPA; ATTO; Tokyo, Japan) and incubated on ice for 20 min. After centrifuging at 13,000× *g* for 20 min, at 4 ◦C, clean supernatants were moved to a new tube and the concentration of supernatants was analyzed with a bicinchoninic acid assay kit (BCA kit; Thermo Fisher Scientific, Inc.; Waltham, MA, USA). To validate protein expression from TCMK-1 cells, blotting was conducted. An equal amount of lysate proteins (30 µg/lane) was separated by 10% sodium dodecyl sulfate polyacrylamide gel using electrophoresis. Then, running proteins were transferred to polyvinylidene fluoride membranes, which were incubated with diluted primary antibodies (Anti-β-actin, AGTR1, TGF-β, SMAD2/3 and pSMAD2/3) at 4 ◦C. The incubated membranes with antibodies were thoroughly washed using Tris-buffered saline with 0.1% Tween 20 and incubated with secondary antibodies for 2 h at room temperature. All membranes were developed by enhanced chemiluminescence (LAS-4000s; GE Healthcare, Chicago, IL, USA). The antibodies information used in this study can be found in Table S2.

#### *4.10. Statistical Analysis*

The results are presented as mean ± SD, and *p*-values of <0.05 means it is statistically significant. The Kruskal–Wallis test was used to validate the differences among the groups, and the Mann–Whitney U test was used in the SPSS ver. 22 software (IBM Corporation; NY, Armonk, USA) completed post hoc comparisons. Means denoted by a different letter indicate significant differences between groups.

#### **5. Conclusions**

In conclusion, AT1R was upregulated in the SHRs, and the signal pathway increased TGFβ and SMAD2 or 3, which induced EMT. Either ECE or DK downregulated the signal pathway and decreased EMT. Therefore, ECE or DK decreased the mesenchymal cell markers such as the expression of vimentin and α-SMA, the fibrotic tissue of the kidney, and the ratio of urine albumin/creatinine and attenuated the EMT. Furthermore, renal fibrosis led to the restoration of renal function (Figure 7).

**Figure 7.** Schematic image for effects of ECE and DK administration in SHRs. ECE or DK downregulated AT1R/TGFβ/SMAD2 or three molecule expression and EMT marker expression in SHRs.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/ijms22084230/s1.

**Author Contributions:** Conceptualization, K.H.S. and K.B.; formal analysis, M.S. and S.O.; funding acquisition, K.B.; project administration, K.B.; resources, J.T.J.; validation, M.S., S.O. and J.C.; writing—original draft, M.S. and K.H.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was part of a project entitled "Development of functional food products with natural materials derived from marine resources" (no. 20170285), which was funded by the Ministry of Oceans and Fisheries, Republic of Korea.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Institutional Animal Care and Use Committee and approved by the Institutional Ethics Committee of Gachon University (LCDI-2019-0121).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data are contained within the article.

**Acknowledgments:** The authors are grateful to Aqua Green Technology Co., LTD (Jeju, South Korea) for assisting in the preparation of the E. cava extract and DK.

**Conflicts of Interest:** The authors declare no competing financial interests by the sponsoring/ commercial partners.

#### **References**

## *Article* **Dual and Opposite Costimulatory Targeting with a Novel Human Fusion Recombinant Protein Effectively Prevents Renal Warm Ischemia Reperfusion Injury and Allograft Rejection in Murine Models**

**Jordi Guiteras 1,2 , Laura De Ramon 1 , Elena Crespo 1 , Nuria Bolaños 1 , Silvia Barcelo-Batllori 3 , Laura Martinez-Valenzuela 1,4, Pere Fontova 1 , Marta Jarque 1 , Alba Torija 1 , Oriol Bestard 4,5, David Resina 6 , Josep M Grinyó 5,\* and Joan Torras 4,5,\***

**Abstract:** Many studies have shown both the CD28—D80/86 costimulatory pathway and the PD-1— PD-L1/L2 coinhibitory pathway to be important signals in modulating or decreasing the inflammatory profile in ischemia-reperfusion injury (IRI) or in a solid organ transplant setting. The importance of these two opposing pathways and their potential synergistic effect led our group to design a human fusion recombinant protein with CTLA4 and PD-L2 domains named HYBRI. The objective of our study was to determine the HYBRI binding to the postulated ligands of CTLA4 (CD80) and PD-L2 (PD-1) using the Surface Plasmon Resonance technique and to evaluate the in vivo HYBRI effects on two representative kidney inflammatory models—rat renal IRI and allogeneic kidney transplant. The Surface Plasmon Resonance assay demonstrated the avidity and binding of HYBRI to its targets. HYBRI treatment in the models exerted a high functional and morphological improvement. HYBRI produced a significant amelioration of renal function on day one and two after bilateral warm ischemia and on days seven and nine after transplant, clearly prolonging the animal survival in a life-sustaining renal allograft model. In both models, a significant reduction in histological damage and CD3 and CD68 infiltrating cells was observed. HYBRI decreased the circulating inflammatory cytokines and enriched the FoxP3 peripheral circulating, apart from reducing renal inflammation. In conclusion, the dual and opposite costimulatory targeting with that novel protein offers a good microenvironment profile to protect the ischemic process in the kidney and to prevent the kidney rejection, increasing the animal's chances of survival. HYBRI largely prevents the progression of inflammation in these rat models.

**Keywords:** costimulation; coinhibition; ischemia-reperfusion injury; kidney transplant; SPR; protein binding affinity; innate immunity; adaptive immunity; inflammation

#### **1. Introduction**

Delayed graft function (DGF) is the clinical manifestation of ischemia-reperfusion injury (IRI) in human renal transplantation. The clinical impact of DGF on transplant-

**Citation:** Guiteras, J.; De Ramon, L.; Crespo, E.; Bolaños, N.; Barcelo-Batllori, S.; Martinez-Valenzuela, L.; Fontova, P.; Jarque, M.; Torija, A.; Bestard, O.; et al. Dual and Opposite Costimulatory Targeting with a Novel Human Fusion Recombinant Protein Effectively Prevents Renal Warm Ischemia Reperfusion Injury and Allograft Rejection in Murine Models. *Int. J. Mol. Sci.* **2021**, *22*, 1216. https://doi.org/10.3390/ijms22031216

Academic Editor: Patrick C. Baer Received: 30 December 2020 Accepted: 20 January 2021 Published: 26 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

related outcomes is an increase in the risk of rejection, inferior allograft function, difficult management of immunosuppression in the early post-transplant phase, and the need for repeated renal biopsies with their related associated complications [1–3].

IRI causes a series of humoral and cellular responses, producing complex and diverse pathophysiological pathways of damage to the organ [4,5]. The occlusion of the arterial blood supply leads to tissue hypoxia and the imbalance of metabolic demand, thus activating cell death programs, endothelial dysfunction, transcriptional reprogramming, the expression of cytokines, leukocyte-endothelial adhesion, macrophage and lymphocyte activation, and the generation of small amounts of reactive oxygen species (ROS) [6–9]. Additionally, the restoration of blood flow and reoxygenation is frequently associated with an exacerbation of tissue injury and intense inflammatory response [10]. During reperfusion, the restoration of oxygen levels further damages cells exposed to previous ischemia, leading to the production of large amounts of ROS that contribute to cell membrane and DNA damage. Reperfusion damage results in an autoimmune response, which includes mostly natural antibody recognition and complement cascade activation [11]. This induces cell death and the activation of the innate and, afterwards, the adaptive immune system [6]. Regarding adaptive immunity, multiple roles of T cells have been described in studies of ischemia, showing evidences in both antigen-specific and antigen-independent mechanisms of activation [1,12,13].

Given the crucial role that costimulatory signals play in T cell-mediated immune responses, several costimulatory pathways have been targeted using monoclonal antibodies and fusion proteins to induce immunosuppression. Currently, it is well known that the so-called costimulatory pathways constitute a complex set of stimulatory and inhibitory signals which together fine-tune cell responses. To induce immunosuppression, several of these targets have been directed [14–17]. Some studies reported that CTLA4 blocks the costimulatory CD28 pathway by binding to the ligands CD80 and CD86. This blockade prevents the clonal expansion of effector T cells, leading them to an anergy or apoptotic state [18–20]. The costimulatory blocker Belatacept (a mutated version of CTLA4Ig) was approved for immunosuppression in renal transplantation in the early 2000s. The observed advantages of Belatacept over cyclosporine include better graft function, the preservation of renal structure, and improved cardiovascular risk profile [21]. Concerns associated with Belatacept are a higher frequency of cellular rejection episodes, but protocol biopsies at 1 year showed a lower incidence of chronic allograft nephropathy with Belatacept compared to cyclosporine [22]. According to Parsons et al., Belatacept can also reduce Human leukocyte antigen (HLA) class I antibodies in a significant proportion of highly sensitized recipients [23].

Recent studies have shown the immunosuppressive effects of exacerbating the coinhibitory pathway formed by PD-1 and the ligands PD-L1 and PD-L2 [24–28]. Early PD-1 expression has been demonstrated in renal allograft, which was considered essential to modulate T cell expansion and cytokine production. Blocking the PD-1/PD-L1 coinhibitory pathway during the first week after transplantation doubled the number of PD-1–expressing CD8 and CD4 cells infiltrating the graft, mainly in the interstitium [29]. Contrasting to PD-L1, PD-L2 is mainly expressed in the Antigen presenting cells (APC), does not link to CD80, and seems to be more significant in terms of protection against an ischemic or allogeneic insult [24,30–32]. Moreover, this pathway has been recently related to the regulatory T cell induction setting, a cellular subset implicated in the immune and inflammatory response regulation [33–35].

According to these T cell-modulating pathways, the concurrent blockade of the CD28— CD80/86 costimulatory pathway with CTLA4 and the stimulation of the PD-1 coinhibitory pathway with PD-L2 in the cell synapses could exert better immunosuppressive or antiinflammatory effects [24,36]. In this regard, our group designed a novel human fusion protein construct to target these dual and opposite costimulatory signals. The production of the molecule consists of gen synthesis, cloning, and transient transfection with a stable Chinese hamster ovary (CHO) mammalian cell line. This recombinant protein design

consists of an IgG1 FC linked to a CTLA4 molecule which is also bound to two PD-L2 molecules by polypeptide links (Figure 1).

**Figure 1.** HYBRI protein structure.

This dual targeting may promote T cell anergy and apoptosis, enhance T cell exhaustion and T-reg expansion, and downregulate the CD80/86 expression in antigen-presenting cells [37–39]. This might suggest that lower concentrations of CTLA4 would be needed to inhibit the CD28—CD80/86 costimulatory synapsis. This potential dual targeting may close a synergistic circle to exert potent anti-inflammatory and immunosuppressive effects. We studied two experimental settings, the inflammatory environment of renal warm IRI and the renal allogeneic transplantation with cold ischemia, to evaluate the effect of our HYBRI protein.

#### **2. Results**

#### *2.1. Analysis of HYBRI Binding Affinity to Murine CD80 and PD-1 Proteins Using the Surface Plasmon Resonance (SPR) Technique*

SPR analysis was used to characterize the affinity and kinetics between the HYBRI construct and PD-1 and CD80. HYBRI was covalently bound to the CM5 sensor surface and the analytes (CD80 and PD-1) were injected in solution over the surface at concentrations ranging from 15.6 to 2000 nM. Sensorgrams (Figure 2) show a dose-dependent direct interaction between the analytes and HYBRI. Affinity constants determined in the steady state reflected a high affinity for both CD80 and PD1 binding to HYBRI (Table 1). Kinetics analysis revealed rapid association (Ka) and dissociation (Kd) for both CD80 and PD1. From these kinetic constants, affinity constants could be calculated (KD) and were in good agreement with the affinity constants determined from the steady-state analysis. Experiments were also conducted to assess the effect of the presence of PD-1 on CD80 binding to HYBRI, and there was found to be a lack of competition between the two molecules for HYBRI [40].

**Table 1.** CD80 and PD1 bind to HYBRI. Affinity constants (KD) were calculated from SPR analysis in the steady state and from the kinetics analysis.

**Figure 2.** Representative sensorgrams of CD80 (**a**) and PD1 (**b**) binding to HYBRI. HYBRI was bound to CM5 chip and CD80 and PD1 serial dilutions (15.625, 31.25, 62.5, 125, 250, 500, 1000, and 2000 nM) represented in distinct colors were injected for 90 s. Experimental data are shown as color traces and black traces represent the fitted data according to the 1:1 Langmuir binding model.

#### *2.2. Bilateral Warm Ischemia Model in Native Rat Kidneys* 2.2.1. Effects of HYBRI on Renal Function

HYBRI therapy produced a significant improvement in kidney function the following three days after warm ischemia, with a reduction in creatinine at 24 h from 2.85 mg/dL to 1.5 mg/dL (*p* = 0.0002) and a reduction in urea from 192.8 mg/dL to 129.2 mg/dL for the non-treated group to the HYBRI group, respectively (*p* = 0.0335, Figure 3). Interestingly, the major difference between groups was observed on the second day after ischemia, when both parameters began to decrease in the HYBRI group but peaked in the non-treated group. At this point, the creatinine level of the non-treated group increased to 3.02 mg/dL compared to the reduction to 0.89 mg/dL (*p* = 0.001) for the group treated with the HYBRI protein. Furthermore, in the case of blood urea the slope on the second day continued increasing, with a significant difference from 281.3 mg/dL in the non-treated group to 76.4 mg/dL in the HYBRI group (*p* = 0.0022). There were no serum creatinine and urea level fluctuations in the sham group.

#### 2.2.2. Renal Histopathology and Immunohistochemistry

A significant reduction in histological damage was observed from a total inflammatory score of 6.75 in the non-treated group to 3.2 in the HYBRI group (*p* = 0.0021), while there was no inflammation in the sham group (Figure 4).

Despite no significant differences, the HYBRI group showed a reduction of almost half in CD3 infiltrating cells (from 13.2 in the non-treated group to 8 in the HYBRI group). An enrichment of FoxP3+ infiltrating cells (Tregs) in the kidney was observed (from 0.7 in the non-treated group to 1.3 in the HYBRI group, with a trend to significance (*p* value= 0.075)). Even more, creating a ratio between infiltrating FoxP3+ cells and infiltrating CD3+ cells, animals treated with HYBRI showed a percentage of 39.1% in contrast to 8.9% in nontreated animals (*p* value= 0.069). The treatment also revealed a significant reduction in CD68 infiltrating cells from 3.3 in the non-treated group to 1.8 in the HYBRI group (*p* < 0.0001).

#### 2.2.3. Peripheral Blood and Spleen Cell Populations

There were no significant differences between groups in a flow cytometry analysis in spleen cell subpopulations. Regarding the Peripheral blood mononuclear cell (PBMC) analysis, the only differences seen were in the percentage of FoxP3+ cells (Tregs) in the CD4+ cells. These cells were expanded from 5% to 13% the next day after ischemia in the HYBRI group. In contrast, in the non-treated group, the difference was lower, with an initial 4% to 9% twenty-four hours after ischemia. Similar trends were observed in the percentage of CD25+ cells in both the CD3+ and CD4+ cell subsets.

**Figure 3.** Functional renal parameters throughout the ischemia-reperfusion model for all treatments, including serum creatinine (**a**) and serum urea (**b**). Values are expressed in mg/dL. Data are expressed as mean.

#### 2.2.4. Circulating Inflammatory Cyto/Chemokines

The serum levels of MCP1 and RANTES (also known as CCL5) at the end of the study decreased significantly with the HYBRI treatment. In addition, concerning the IP-10, IL-12, and MIP-1α levels, treatment with HYBRI in ischemic kidneys significantly reduced those cytokines to values similar to those of sham rats. A partial but non-significant reduction in the serum IL-2 levels was also observed in the HYBRI group compared to the non-treated rats (Figure 5).

#### 2.2.5. Renal Gene Expression

After analyzing both the TaqMan low density array (TLDA) and Polymerase chain reaction (PCR) techniques with a CT method analysis, a few significant differences were observed comparing the HYBRI group with the non-treated group. In this regard, the CTLA4 (*p* = 0.0133) and FoxP3 (*p* = 0.0435) gene expression were significantly reduced in the non-treated group compared to the HYBRI group.

**Figure 4.** Renal histopathology parameters for ischemia-reperfusion injury in the three studied groups. Semi-quantitative inflammatory score values (**a**) for Hematoxylin-Eosin stain. Cells per hpf values for renal immunofluorescence CD3+ (**b**) and FoxP3+ (**c**) kidney infiltrate, and semiquantitative score for CD68+ surface intensity (**d**). Data are expressed as a mean ± SEM. \*, *p* < 0.05 vs. non-treated; \*\*, *p* < 0.01 vs. non-treated.

In HYBRI animals, there was a trend for lower expressions of Ccl7, Ccr5, Cxcl10, Fcnb, Ptprc, S100a6, Trl2, Tlr4, HGF, RORC, and VCAM genes compared to non-treated animals. Interestingly, the degree of reduction in the expression of those genes in the group treated with HYBRI led to values closer to those of the sham group, and, contrarily to non-treated rats, they were not statistically different, thus bringing the treated kidneys closer to a healthier genotype.

The Pathwax database results for all these genes showed that the HYBRI treatment affected nine molecular pathways related to cellular processes, such as apoptosis, necroptosis, and focal adhesion (see Supplementary Material). It also affected twelve environmental information processing-related pathways, such as cytokine–cytokine receptor interaction; cell adhesion molecules; and NF-kappa B, MAPK, JAK-STAT, and TNF signaling pathways as the most relevant. Of note, HYBRI also affected thirty-three pathways related to human diseases, with the PD-1 expression pathway as the most significant. Twenty-one organismal systems-related pathways such as NK cell-mediated cytotoxicity; Th1, Th2, and Th17 cell differentiation; the chemokine signaling pathway; T and B-cell and toll-like receptor signaling pathways were also affected. The gene expression results showed that almost all the analyzed genes are regulated in the IRI setting, but just thirteen of them are significantly addressed by the HYBRI treatment.

#### *2.3. Rat Allogeneic Kidney Transplant Model*

#### 2.3.1. Effects of HYBRI on Renal Function and Survival

HYBRI treatment resulted in an improvement in renal function in the allogeneic transplant model. At the early ischemic peak 24 h after kidney transplantation, despite no significant differences HYBRI treatment reduced the serum creatinine from 1.4 to 1.1 mg/dL and the serum urea levels from 185.8 to 157.2 mg/dL. Furthermore, HYBRI treatment showed a significant reduction in creatinine levels on day 7 after kidney transplantation (*p* = 0.0171), when kidney function begins to decline due to allogeneic kidney rejection (Figure 6).

**Figure 5.** Circulating IL-2, IL-12, IP10, MCP1, MIP-1α, and RANTES in renal warm ischemia model measured by Luminex assay. Data are expressed as a mean ± SEM. \*, *p* < 0.05 vs. non-treated; \*\*, *p* < 0.01 vs. non-treated.

There were also significant mortality differences between the studied groups. The initial number of twenty-one non-treated animals was reduced to seven at the 21st day, representing a 33% survival rate (Figure 7). Among the surviving animals, only one lived to the end of the study at day 90 (4.8%). The mean survival of this group was 23 days. On the other hand, the administration of the HYBRI protein during the firsts 6 days of the study significantly improved the animal survival up to 57 days, more than double that in the group without treatment. This data was significant in the Log rank Mantel Cox test of the Kaplan Meier curve. On the 21st day of the study, a survival rate of 80% of the group was seen (*p* = 0.021), and this finally decreased to 30% at the end of the study at day 90, a higher percentage than the almost 5% corresponding to the untreated group (*p* = 0.005).

#### 2.3.2. Renal Histopathology and Immunohistochemistry

As seen in Figure 8, conventional kidney histology reflected a significant score reduction from 8.1 in the non-treated group to 3.5 in the HYBRI group (*p* < 0.001). Regarding kidney infiltrating cells, there were significant differences in CD3+, where the mean of 24.9 cells/hpf in the non-treated group was reduced to 10.3 cells/hpf in the HYBRI-treated group (*p* = 0.0237). Macrophage infiltration was also significantly reduced with HYBRI treatment, where the mean semi-quantitative score of 2.97 in the non-treated group was decreased to 1.94 with HYBRI treatment (*p* = 0.0015). Humoral effect was analyzed with

**Figure 6.** Functional renal parameters throughout the allogeneic transplant model for HYBRI treatment and non-treated groups. (**a**) Serum creatinine and (**b**) serum urea levels for the initial 21 days of the study. Values are expressed in mg/dL. Data are expressed as means.

#### 2.3.3. Circulating Inflammatory Cyto/Chemokines

A significant reduction of up to three-fold in IP-10, RANTES, and MCP1 was observed in rats treated with HYBRI compared with non-treated rats. Animals treated with HYBRI had reduced MIP1α values, but they were not statistically different because there was a huge dispersal in non-treated rats. Finally, non-significant reduction in IFNγ and IL-12 was seen (Figure 9).

**Figure 8.** Renal histopathology parameters for allogeneic transplant model in the two studied groups. Semi-quantitative inflammatory score values (**a**) for Hematoxylin-Eosin stain. Cells per hpf values for renal immunofluorescence CD3+ (**b**) and semi-quantitative score for CD68+ surface intensity (**c**) and C4d kidney deposition (**d**). Data are expressed as a mean ± SEM. \*, *p* < 0.05 vs. Non-treated; \*\*, *p* < 0.01 vs. Non-treated.

**Figure 9.** Circulating IFNγ, IL-12, IP10, MCP1, MIP-1α, and RANTES in allogeneic transplant model measured by Luminex assay. Data are expressed as a mean ± SEM. \*\*, *p* < 0.01 vs. non-treated.

#### **3. Discussion**

Ischemia reperfusion damage is a deleterious response in acutely injured kidneys in several clinical situations. In this setting, costimulatory lymphocyte signals have been involved or modulated successfully [41–45]. We here show a positive modulation of both warm renal ischemia and allograft response by means of a dual and opposite costimulatory targeting, combining two coinhibitory receptors, CTLA4 and PDL2.

After the theoretical design of the construct and the genetic transient insertion in the CHO mammalian cell line, HYBRI was synthesized and its in vitro activity was assessed in the mixed lymphocyte reaction [46] with successful inhibition of T cell response. As a first validation, the well-known SPR technique showed the great affinity of our newly designed HYBRI protein with the CD80 and PD-1 targets, thus confirming that the binding sites of the proteins that make up the structure do not condition their binding avidity. In this regard, HYBRI protein presents its effective binding to both CD80 and PD-1 simultaneously, which indicates that HYBRI can block the CD28-CD80 costimulatory pathway while stimulating the PD-1-PD-L2 coinhibitory pathway. These bindings are effective despite the approximately 70% protein homology shared between species, given that HYBRI is a fully human protein [47,48]. Consequently, these results support the protein affinity to its designed targets, appearing as an interesting option in the inflammatory animal models addressed in the present study. Despite the reported similar KD values of the CD80 and

HYBRI CTLA4 interaction, the KD values of the PD-1 and HYBRI's PD-L2 interaction are higher than the values found in the literature [31,49–52]. Similarly to the increased affinity of the modified PD-L2 described by Philips et al. [51], PD-L2 molecules in the HYBRI construct may have suffered conformational changes, which might account for its increased binding to PD-1. Nevertheless, in both rat models HYBRI shows therapeutic efficacy, which suggests that the binding is high enough to exert biological effects.

The dual and opposite costimulatory targeting conferred by HYBRI have induced a protective effect against the kidney warm ischemia model, as the renal function and structure after the insult is greatly improved in the treated rats. This effect may be based on CD80 costimulatory blocking, which reduces the macrophage and T cell migration and, also, may promote T cell apoptosis [19,53–56]. The PD-1/PD-L2 pathway may have also produced Treg clonal expansion and diminished the T effector cell expression [24,34,57]. These pathways appear to be well addressed by the protein, although the expressions of these molecules and their ligands are not exclusive to the APC and T cells and, in some cases, can be bidirectional [34]. For example, renal tubular epithelial cells (RTECs), podocytes, and other renal cells also express CD80 and PD-L2 on their surface [58–60]. This may suggest that the desired homeostatic effect is not specific to APCs but can also be exerted on other semi-professional cells in antigen presentation. As ischemia is present in both groups, it can be seen that the expansion of Tregs promoted by the administration of HYBRI plays an essential role in homeostasis. Thus, the observed reduction in macrophages and T cells in the kidney together with the increase in regulatory T cells indicates the cellular modulation by the HYBRI treatment of post ischemic renal inflammatory damage. In addition, the significant decline in circulating inflammatory-related mediators reflects the efficacy of HYBRI in modulating the inflammatory response at the systemic level [61–64].

A daily peripheral cytometric study showed only changes between study groups for Tregs expansion on the first day after ischemia. The results suggest that the CD25+ population experienced clonal expansion early after ischemia injury, also including the FoxP3+ subset, but that the Tregs subset is additionally expanded with the HYBRI treatment. At the end of the study, peripheral and spleen population assessment did not reveal significant cell differences between the groups despite a significant reduction in renal inflammatory infiltrate being found with HYBRI treatment. Thus, peripheral blood or spleen compartments may not reflect organ parenchymal changes where there is a real inflammatory environment with myriads of attractive and activation signals. The HYBRI treatment modulated the kidney inflammatory status towards a more attenuated inflammatory response and the enrichment of the Tregs population. In the allogeneic setting with a stronger immune response, the effect of HYBRI treatment may have offered two benefits. On the one hand, the reduction in the inflammation derived from cold ischemia reperfusion damage, but also an intense modulation of the adaptive response of T and possibly B cells given the reduction in C4d deposition.

A gene array analysis showed differences between the sham group and ischemiareperfusion groups, but few significant differences between the non-treated and HYBRI groups. Thus, the regulation of some of the studied genes can be observed. These genes are related to both the inflammation and expression of cell surface proteins such as CD80 and CD86. In this regard, the HYBRI treatment may produce a reduction in CD80 and CD86 cell surface expression, an important point in this immunomodulation strategy [19,53,54]. It is also remarkable that the protein HYBRI modifies only some of the pathways related to IRI, despite showing a large and significant therapeutic effect in vivo.

Regarding early cold ischemia injury in the allogeneic transplant model, a slight amelioration in renal functional parameters is observed in the group treated with HYBRI. In this complex surgical model, it is hard to discern ischemic damage among all surgical attritions, contrarily to the easier native warm ischemia. In this second proposed model, however, the effect of the HYBRI protein plays an essential role in kidney rejection, where the treated group improved the renal function on days 7 and 9 after transplantation and prolonged the survival of animals to more than double that of non-treated rats. Recently, it

has been shown that there is early PD-1 expression in renal allograft dynamics. Blocking this PD-1/PD-L1 negative costimulatory pathway during the first week doubled the number of PD-1-expressing CD8 and CD4 cells, causing terminal acute rejection [29]. These findings with an approach completely opposite to our PD-1, which activates this negative pathway, suggest the essential role of this pathway in modulating T cell expansion and cytokine production in the allograft setting. Similar studies with PD-L1 and PD-L2 blocking antibodies in a model of IRI also aggravated the renal damage, again suggesting the protecting role of this coinhibitory pathway [24].

To summarize, the SPR study has shown the exquisite binding affinity of HYBRI to both targets and the advantage of binding simultaneously in vitro. Regarding in vivo studies, the HYBRI protein may effectively mitigate damage from warm ischemia reperfusion. In the context of the kidney transplant model, the HYBRI protein also prevents kidney rejection even by treating animals only during the first week. Therefore, this dual and opposite protein effect on costimulatory pathways can promote several immunomodulatory mechanisms, leading to protecting the kidney from multiple immunoinflammatory states.

#### **4. Materials and Methods**

#### *4.1. HYBRI Characterization and Binding Affinity Assessment with Surface Plasmon Resonance (SPR) Technique*

Surface plasmon resonance (SPR) analyses were performed at 25 ◦C on a Biacore T200 (GE Healthcare BioSciences AB, Uppsala, Sweden). The HYBRI construct was diluted in acetate buffer pH 5 (50 µg/mL and immobilized on a CM5 chip (GE) by amine coupling in the active flow channels (Fc) at two different ligand densities. Briefly, the surface was activated with a mixture of 1:1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.1 M EDC (3-(N,N-dimethylamino) propyl-N-ethylcarbodiimide) at a flow rate of 5 µL/min, followed by the injection of ligand or buffer for the active or reference channel, respectively. Ethanolamine solution was injected in both channels in order to block the remaining reactive groups of the surface. Analytes (recombinant murine PD-1 and CD80, R&D systems) were prepared in PBS-P in serial dilutions (0, 15.6, 62.5, 125, 250, 500, 1000, and 2000 nM) and were injected in parallel in two active and reference channels at 30 µL/min flow for 90 s and a dissociation time of 240 s. Blanks were included for double referencing. Experiments were also conducted to assess the effect of PD-1 presence on the CD80 binding to Hybri. For this, concentrations series of CD80 (0–2000 nM) in the presence of PD-1 (250 nM) were included. Kinetic and affinity constants were calculated using the Biacore T200 evaluation software 2.0 after reference and blank subtraction, and sensorgrams were fitted according to the 1:1 Langmuir model.

#### *4.2. Animals and Surgical Procedures*

All the procedures were performed following the Guidelines of the European Committee on Care and Use of Laboratory Animals and Good Laboratory Practice. Rats from Charles River, Spain, were housed with 12 h dark/light cycles and at a constant temperature. The animals were fed *ad libitum* with standard diet and water. For both ischemia and transplant surgical models, a combined anesthesia based on ketamine (75 mg/kg), atropine (0.05 mg/kg), and valium (5 mg/kg) was used. A single intramuscular injection of ciprofloxacin (5 mg) was administered after the surgery.

#### *4.3. Renal Warm Ischemia Model*

In this model, Lewis rats received intraperitoneally one single dose of 20 mg/kg of HYBRI administered 24 h before bilateral renal ischemia of 40 min (*n* = 10, Hybri). Nontreated animals were used as control group (*n* = 10, Vehicle), and they received one single intraperitoneal administration of 300 µL of PBS 24 h before the bilateral renal ischemia of 40 min. The sham group received neither treatment nor ischemia and only laparotomy as a technique control group was performed (*n* = 3, Sham).

Blood samples were obtained from the tail vein at time zero and on daily basis until sacrifice on day 7 after ischemia. Animals were euthanized under anesthesia on the seventh day, blood was obtained by aortic puncture, and the spleen and kidneys were processed.

#### *4.4. Allogeneic Transplant Model (Cold Ischemia)*

In a model of renal allotransplantation, binephrectomized Lewis rats received a single kidney transplant from a Wistar donor rats. HYBRI group (*n* = 10) animals were treated with administration of 500 µg of HYBRI intraperitoneally 2 h before transplantation and the six following days. Non-treated (*n* = 21) animals were used as a control group with the administration of 200 µL of PBS with the same schedule.

Blood samples of both groups were obtained from the tail vein at time zero and on days 1, 3, 5, 7, 9, 11, and 14 and weekly after the transplant. Surviving animals at day 90 were euthanized under anesthesia, blood was obtained by aortic puncture, and the spleen and kidneys were processed.

#### *4.5. Renal Function*

Renal function was analyzed daily on warm ischemia model and on days 0, 1, 3, 5, 7, 9, 11, and 14 and weekly on the allogeneic transplant model. Serum creatinine and urea measurements were performed following Jaffe's and GLDH reactions (Olympus Autoanalyzer AU400, Hamburg, Germany) in the Veterinary Clinical Biochemistry Laboratory of Universitat Autonoma de Barcelona.

#### *4.6. Histological and Immunohistochemistry Studies*

Coronal kidney slices 1–2 mm thick were fixed in buffered 4% of formalin, paraffined, and stained with H&E. Kidney sections from the ischemia-reperfusion study were evaluated by a blinded pathologist, examining the tubular necrosis, dilation, interstitial edema, and cellular infiltrate. Abnormalities were scored on semiquantitative scale from 0 to 4 as follows: 0, no abnormalities; 1, changes <25%; 2, changes 25–50%; 3, changes 50–75%; and 4, changes >75%. A mean group score was attained from all the individual parameters.

For the allogeneic study, sections were analyzed by a blinded pathologist for tubulitis, interstitial infiltration, vasculitis, glomerulitis, tubular necrosis and glomerular necrosis following the Banff criteria for acute/active lesions scoring, held at the Ninth Banff Conference on Allograft Pathology in La-Coruna, Spain, on 26 June 2007.

For immunohistochemistry techniques, paraffin tissue sections were stained for CD3 (Abcam, Cambridge, UK), C4d (Hycult Biotech, Uden, Netherlands), CD68 (AbD Serotec, Raleigh, NC, USA), and FoxP3 (Novus Biologicals, CO, USA). Sections were immune peroxidase labeled and revealed by diaminobenzidine (Sigma, Madrid, Spain). For the CD3, CD68, and FoxP3 measurement, at least 10 hpf were taken to analyze and count. For the C4d measurement, a semiquantitative scale from 0 to 3 was employed. Negative controls from immunostained-matched sections without primary antibodies were used.

#### *4.7. Peripheral Blood and Spleen Cell Subsets Characterization by Flow Cytometry*

Daily peripheral blood from warm ischemia model and blood before transplant and on days 7 and 21 for the transplant model was collected in heparin tubes and separated in cytometry tubes to add the antibodies and lysis buffer needed to continue with the cytometry protocol.

Splenocytes were preserved at −80 ◦C after its isolation using the Ficoll (GE Healthcare) density gradient. Standard methods were used to thaw, wash, and recover the cells. An incubation of 25 min in the dark at room temperature with different monoclonal antibodies was performed for the cytometry technique using FACS Canto II Cytometer and the subsequent analysis using FACS DIVA software (BD Biosciences, San Jose, CA, USA). The antibodies were titrated, mixed, and formulated for optimal staining performance.

Cocktail T/B/NK (BD 558495) containing anti-CD3 APC, anti-CD45RA FITC, and anti-CD161 PE for T, B, and NK cell detection; anti-CD43 PE (Biolegend 202812), anti-

CD161 AF647 (AbD Serotec MCA1427A647), and anti-ED9 FITC (AbD Serotec MCA620F) for monocytes detection; and anti-CD3 PerCP efluor (eBioscience 46-0030-80), anti-CD4 FITC (eBioscience 11-0040-85), anti-CD25 APC (eBioscience 17-0390-82), and anti-Fox P3 PE (eBioscience 12-4774-42) for Tregs detection. Cell membrane and nucleus permeabilization was needed for Tregs cytometric detection.

#### *4.8. Measurement of Serum Levels of Inflammatory Factors by Luminex Fluorescent Assay*

The determination of IFNγ, IL-2, IL-12/IL-23p40, MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10), and MCP-1 (CCL2) concentrations in serum was conducted using Luminex ProcartaPlex™ Multiplex Immunoassay (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. Results were calculated from the calibration curves and expressed in pg/mL.

#### *4.9. Quantification of Gene Expression in Kidneys from Warm Ischemia Model*

Snap-frozen rat kidneys from the ischemia-reperfusion study were stored at—80 ◦C. RNA samples with an A260/280 ratio of ≥1.8 were extracted using the PureLinkTM RNA MiniKit (Invitrogen, Madrid, Spain) following the manufacturer's instructions. For the reverse transcription, High-Capacity cDNA reverse Transcription Kit (Applied Biosystems, Madrid, Spain) was used following the manufacturer's instructions.

The tissue expression of immune-inflammatory mediators was quantified by Taqman Low Density Array microfluidic cards (ABI-PrismH-7700, Applied Biosystems, Madrid, Spain) using the comparative CT method: Emp1/Emp3/Lgals1/Lgals3/Reln/S100a6/ S100a8/S100a9/Socs3/Socs5/Tnfrsf12a/Fcnb/CD4/CD40/CD80/CD86/Tnf/Nfkb1/Ctla4/ Tlr2/Tlr4/Tlr6/Ccl2/Ccl3/Ccl5/Ccl7/Cxcr3/Cxcl10/Cxcl11/Ccr2/Ccr3/Ccr5/Ptprc/Junb/ Igsf6/Ido2/Il2/Il9/Il10/Il15/Il17a/Tfcp2/Mapk9/Spp1/Bcl6 and eukaryotic 18S as an endogenous reference.

Moreover, the expression of genes validated in recent studies from our group [1] were quantified by TaqMan real-time PCR (Applied Biosystems, Madrid, Spain) using the comparative CT method: C1qa/C1qc/C1r/C1s/CD19/CD276/CD44/Cxcl3/Cxcr4/Fas/FoxP3/ Hgf/Ifng/IL-10ra/IL-11/RORC/Socs1/Sox9/TLR8/TLR9/Tnfrsf1b/Vcam and GAPDH as endogenous reference. Controls, which were composed of distilled water, were negative for the target and reference genes.

A pathway analysis of single gene sets, it was set up using the online PathwaX.sbc.su.se web server, which applies the BinoX algorithm to KEGG pathways and FunCoup networks [65].

#### *4.10. Statistical Analysis*

For a statistical analysis, the Student's t-test compared two conditions, whereas the ANOVA was employed for the comparison of multiple conditions. Repeated measures ANOVAs were used to analyze differences in various parameters due to HYBRI treatment throughout the follow-up. Nonparametric analysis was used as needed. For the comparative CT method, the value shown is the one obtained by performing the Wilcoxon rank sum test when comparing the data of the study treatment with the data of the control treatment. The Kaplan-Meier method was performed and analyzed using the Mantel Cox test for a comparison of the survival distributions in the two groups. A value of *p* < 0.05 was considered significant. Data are given as a mean ± s.e.m.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1422-006 7/22/3/1216/s1, Figure S1: PathwAX gene array results; Table S1: PathwAX list of the pathways related to gene array.

**Author Contributions:** Conceptualization, O.B., J.M.G. and J.T.; HYBRI design, J.M.G.; HYBRI recombinant synthesis, D.R.; methodology, J.G., L.D.R., E.C., N.B., S.B.-B., L.M.-V., P.F. and M.J.; formal analysis, J.G., J.M.G. and J.T.; writing—original draft preparation, J.G., L.D.R., J.M.G. and J.T.; writing—review and editing, J.G., J.M.G. and J.T.; visualization, J.G., A.T., O.B., J.M.G. and J.T.; supervision, J.M.G. and J.T.; project administration, O.B., J.M.G. and O.B.; funding acquisition, J.G., O.B., J.M.G. and J.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from the Instituto de Salud Carlos III (Madrid, Spain) PI17/00277 and PI19/01710, co-funded by FEDER funds/European Regional Development Fund -a way to build Europe. The study was also supported by Fundación Renal Iñigo Alvarez de Toledo (20PSJ003).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by "CEEA: Animal Experimentation Ethic Committee", the Institutional Ethics UB Committee for Animal Research (201/18, April 9th, 2018).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We thank Serveis Científico-Tècnics (UB, Campus Bellvitge) for technical support, Cristian Tebé from Unitat de Bioestadística of IDIBELL for the gene analyzing help and Josep Gardenyes (IDIBELL Scientific-Technical Services) for technical support with SPR experiments. We thank CERCA Programme/Generalitat de Catalunya for institutional support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Review* **Emerging Modes of Treatment of IgA Nephropathy**

#### **Dita Maixnerova \* and Vladimir Tesar**

1st Faculty of Medicine, General University Hospital, Department of Nephrology, Charles University, 128 08 Prague, Czech Republic<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +42-02-2496-6790

Received: 20 October 2020; Accepted: 19 November 2020; Published: 28 November 2020 -

**Abstract:** IgA nephropathy is the most common primary glomerulonephritis with potentially serious outcome leading to end stage renal disease in 30 to 50% of patients within 20 to 30 years. Renal biopsy, which might be associated with risks of complications (bleeding and others), still remains the only reliable diagnostic tool for IgA nephropathy. Therefore, the search for non-invasive diagnostic and prognostic markers for detection of subclinical types of IgA nephropathy, evaluation of disease activity, and assessment of treatment effectiveness, is of utmost importance. In this review, we summarize treatment options for patients with IgA nephropathy including the drugs currently under evaluation in randomized control trials. An early initiation of immunosupressive regimens in patients with IgA nephropathy at risk of progression should result in the slowing down of the progression of renal function to end stage renal disease.

**Keywords:** IgAN; proteinuria; CKD; progression; ACEI; corticosteroids

#### **1. Introduction, Diagnosis, Pathogenesis**

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide with potentially serious renal outcome leading, in 30–50% of patients, to end stage renal disease (ESRD) within 20 to 30 years of follow-up [1,2].

Diagnosis of IgAN is currently based on evaluation of renal biopsy specimens with the demonstration of mesangial IgA1-dominant or co-dominant immunodeposits [3,4].

Clinical risk factors predicting poor renal outcome in patients with IgAN include time-averaged proteinuria, hypertension, decreased estimated glomerular filtration rate (eGFR) [2,5] at presentation or during follow up as well as histological findings evaluated using C-MEST classification [6,7].

IgAN is an autoimmune disease arising from a multi-hit pathophysiological process and is believed to be caused by the interaction of genetic and environmental contributing factors [2,8,9]. Genome-wide associated studies indicated a pathogenetic role of the intestinal immunity abnormalities in IgAN and confirmed a direct link of IgAN with the risk of inflammatory bowel disease or maintenance of the intestinal epithelial barrier [9].

A key role in the pathogenesis of IgAN is played by aberrantly glycosylated forms of IgA1 with galactose-deficient *O*-glycans (galactose-deficient IgA1; Gd-IgA1), which are recognized by antiglycan autoantibodies of IgG and/or IgA1 isotype, resulting in the formation of circulating immune complexes [10,11]. These complexes are deposited in the glomerular mesangium with subsequent mesangial-cell activation and local stimulation of the complement system, proliferation of mesangial cells, and production of extracellular matrix and cytokines, which could alter podocyte gene expression and glomerular permeability in clinical presentation of proteinuria and tubulointerstitial changes in IgAN [12–15]. If unabated, the damage progresses to glomerulosclerosis and interstitial fibrosis with impaired renal function with subsequent end-stage renal disease.

#### **2. Treatment**

Which goals do we have in treatment of patients with IgAN? We have to respect the possibility of induction of clinical remmission, reduction in proteinuria and hematuria, stabilization of renal parameters without further decline of GFR, a decrease in the rate of progression, prevention of the necessity of renal replacement therapy as well as the risk of adverse events in patients with corticosteroids and other immunosuppressive regimens. The balance of risks and benefits needs to be taken into account in all individual cases.

#### *2.1. Low Risk Patients with IgAN*

Low risk patients with minor urinary abnormalities (proteinuria < 0.5 g/d and/or isolated microhematuria), normal glomerular filtration rate (GFR), no hypertension and without histological activity are at low risk of progression, do not require treatment and should be checked annually for at least 10 years according to KDIGO guidelines (Table 1) [16]. Supportive management such as diet modification, weight optimisation and smoking cessation should be taken into consideration. The ideal treatment of the initial phase of IgAN is focused on the decrease in or inhibition of production of Gd-IgA1 by means of a low cost drug with minimal adverse effects. Intestinal-associated lymphoid tissue and mucosal immunity show attractive targets [17,18]. Intestinal microbiota and/or diet regimens including gluten-free diet were suggested in an experimental mice model and human pilot studies [17,19].

Low protein diets were reported to decrease renal function decline [20]. The restriction of sodium was associated with sodium sensitivity of blood pressure and correlated with renal ultrastructural damage [21]. The decrease in proteinuria was shown even in normotensive patients with IgAN due to low-sodium diets [21]. The damaging effects of heightened sodium sensitivity are mediated due to the renin-angiotensin system and it was confirmed that sodium restriction improves the antiproteinuric effects of RAS inhibition in patients with IgAN [22]. Increased morbidity and mortality in patients with chronic kidney disease were associated with extreme body mass index [23]. The relation between body mass index and the probability of end stage renal disease was shown in patients with IgAN in a Chinese study [23]. Increased body mass index associated with lower remission of proteinuria subsequent to treatment was confirmed in a Japanese study [24]. It was assumed that obesity increased proteinuria in connection with hypertension and other parts of metabolic syndrome. Moreover, the advantages of losing weight in overweight patients with IgAN through protein/sodium restriction, and by attaining maximal control of hypertension using treatment with inhibitors of the renin-angiotension system, were confirmed [25].

In addition, the benefits of quitting smoking on slowing down the progression of renal function decline in patients with IgAN were assessed [26].

#### *2.2. Intermediate Risk Patients with IgAN*

Intermediate risk IgAN patients with proteinuria > 0.5–1 g/d, and/or hypertension and a reduced GFR (without active histological findings in renal specimens) should obtain optimized supportive treatment with the inhibitors of the renin-angiotensin system (RAS) with up-titration of the drug depending on blood pressure to achieve proteinuria < 1 g/d, and should be thoroughly monitored [16]. The suggested therapeutic goals of blood pressure in patients with proteinuria<1 g/d are<130/80 mmHg, and <125/75 mmHg in patients with initial proteinuria >1 g/d (Table 1) [16]. RAS inhibition was demonstrated to reduce proteinuria and may possibly also reduce the progression of chronic kidney disease in patients with IgAN; at least part of this effect is probably mediated by improved control of blood pressure. High risk of mortality of patients with IgAN might be caused by chronic kidney disease [27–29].

It was suggested that patients with persistent proteinuria ≥ 1 g/d, despite 3–6 months of optimized supportive care (including RAS and blood pressure control), and GFR > 50 mL/min per 1.73 m2 , should receive a 6-month course of corticosteroid therapy (Table 1) [16]. The 10-year renal survival and median proteinuria were significantly better in patients who received a 6-month regimen of corticosteroids compared to patients with a symptomatic treatment (97% vs. 53%, *p* = 0.0003; median proteinuria 1.9 g/24 h at baseline, 1.1 g/24 h after six months and 0.6 g/24 h after a median of seven years) [30]. Other randomized and observational trials supported the potential effect of corticosteroids in IgAN [27,31]. The retrospective analysis of the VALIGA study showed, in patients treated with steroids and RAS blockers (RASB), significant reduction in proteinuria, renal function decline and increased renal survival, not only in patients with normal renal function but also in patients with eGFR < 50 mL/min per 1.73 m2 (reaching proteinuria <1 g/day in 74% patients with corticosteroids and RASB vs. 37% in patients with RASB; slope of eGFR −0.3 ± 6.2 mL/min/1.73 m2 /year in patients with corticosteroids and RASB vs. −4.8 ± 7.4 mL/min/1.73 m2 /year in patients with RASB; *p* = 0.001) [32]. Nevertheless, little information is available about the doses, the duration of steroid treatment and the adverse events caused by corticosteroids [27].

The TESTING study, a randomized study by Lv et al., included 262 patients with persistent proteinuria 1 g/day and estimated GFR 20–120 mL/min/1.73 m2 , who were randomly assigned to receive 0.6–0.8 mg/kg/day of oral methylprednisolone or matching placebo [33]. The temporary results demonstrated a reduction in time-averaged proteinuria and a decreased rate of progression of CKD in the steroid-treated arm (1.37 vs. 2.36 g/day (42% lower), *p* < 0.01; −1.7 vs. −6.8 mL/min/1.73 m2 /year, *p* = 0.031) [33]. However, after a median follow-up of 1.5 years, serious adverse events occurred in patients with corticosteroids vs. placebo groups (14.7% vs. 3.2%, HR 4.95 (95% CI 1.87–17.0), *p* = 0.03) [33]. The additional long-term follow-up might reveal the balance of risks and benefits of steroid treatment [33]. Multiethnic trials such as the ongoing TESTING Low Dose trial (NCT01560052), should evaluate this issue further [34].

The recent STOP-IgAN Clinical Trial showed that the addition of immunosuppressive therapy (corticosteroids and cyclophosphamide followed by azathioprine) to intensive supportive care (ACEI-inhibitors or ARB) in patients with high-risk IgA nephropathy induced full remission of proteinuria (OR 4.82 (95% CI 1.43–16.30), *p* = 0.01) but did not significantly improve renal function (OR O.89 (95% CI 0.44–1.81), *p* = 0.75) and more adverse effects were observed among the patients with immunosuppressive regimens with no change in the rate of decrease in the eGFR (total number of infectious events in steroid treatment arm 182 vs. 111 in supportive care) [35]. However, of 309 patients who completed the 6-month supportive care run-in phase, 106 responded to supportive care (proteinuria level, <0.75 g of urinary protein excretion per day after the end of the run-in phase) and were not eligible for randomization. It needs to be highlighted that one third of patients were no longer suitable for randomization at the end of the run-in phase and the importance of RAS blockade was emphasized. The kidney function loss in the control group was 4 times slower in the STOP-IgAN trial than in the TESTING trial, suggesting a lower-risk population and/or differences in supportive therapy. Thus, it was assumed that low risk population of patients with IgAN with excellent prognosis with supportive care was selected in the STOP-IgAN trial. Nonetheless, detailed assessment of included patients with specific evaluation of histological renal findings and prolonged follow-up would be required for the elucidation of the results [35]. Included patients with inactive form of the disease might predominate in case of absent histological evaluation of renal specimens in the trials and the treatment of patients with inactive forms is useless. Recently, after ten years of follow-up from this study, the significant number of patients in both arms (supportive care plus immunosuppression and supportive care alone) reached the end-point with no benefits seen from the immunosuppression arm [36].

The enteric budesonide was evaluated for the treatment of IgAN in a European multicenter RCT [37]. In the NEFIGAN trial, a novel targeted-release formulation of budesonide was evaluated, designed to deliver the drug to the distal ileum with suspected suppression of B cells and inhibition of

production of Gd-IgA1 moleculs transported to blood in patients with IgAN [37]. The results of the Nefigan study showed a significant reduction in proteinuria with full stabilization of eGFR without any serious side-effects [37]. A confirmatory phase 3 trial is currently underway (NCT 03643965). Other potential protease treatment with selective cleavage of IgA1 reverses mesangial deposits and hematuria in animal model and on human kidney biopsies [38,39].

Among patients with chronic kidney disease, regardless of the presence or absence of diabetes, the risk of a composite of a sustained decline in the estimated GFR of at least 50%, end-stage kidney disease, or death from renal or cardiovascular causes was significantly lower with dapagliflozin than with placebo. IgAN made up a significant proportion of the non-diabetic subgroup and the HR was an impressive 0.79 for IgAN. Unlike corticosteroid, the side effect profile is likely more favourable [40].

#### *2.3. High Risk Patients with IgAN*

Nevertheless, high risk patients with a rapid decrease in the GFR and crescentic glomerulonephritis should be treated in addition to supportive treatment with combined immunosuppression (corticosteroids and cyclophosphamide) in a regimen for induction treatment of ANCA vasculitides (Table 1) [16]. Extreme conditions with crescents involving >50% of glomeruli were considered by the KDIGO guidelines [16]. However, these cases are very rare. Just a few crescents are evaluated in majority of forms of crescentic IgAN, for which the need for aggressive treatment is doubtful. Moreover, a few glomeruli in biopsy specimens are sometimes detected with difficulties for calculation of a valid percentage of crescents. Furthermore, the percentage of glomeruli with crescents changes within a few days which makes comparison impossible among individual patients within cohort studies. Crescentic lesions were not found to have a prognostic value in the Oxford and VALIGA studies, but there was a bias in favour of using of corticosteroid/immunossupression treatment [4,41]. Another international study [7] demonstrated that C correlated with E1 and was associated with the use of CS/IS. The revised Oxford classification [42] suggested considering C1 (1–24% of glomeruli) and C2 (≥25% glomeruli with crescents). Crescents in 16% glomeruli increased the risk of renal function decline in untreated cases and crescents in 25% glomeruli predicted an unfavourable outcome independent of treatment.

Undoubtedly, crescents are a marker of histological activity but crescents can regress and do not require treatment in case of involvement of low percentages of glomeruli [43]. However in untreated patients, the negative effect of crescents on the renal function decline is well known [43]. Treatment with corticosteroids/immunossuppresion might to be initiated in case of involved crescents lesions >25% of the glomeruli (C2) and in patients with C1 involving >16% of glomeruli with other signs of disease activity such as endocapillary hypercellularity (E1 according to the Oxford classification) [43]. The importance of identifying patients at risk of progression was detailed mentioned and the task remains to avoid of exposure to immunosuppression regimen unnecessarily in patients with low risk of progression of renal function. The new international risk-prediction tool was recently developed in patients with IgAN [44,45].

Other immunosuppressive agents including calcineurin inhibitors, azathioprine, mycophenolate salts (MMF) or high-dose immunoglobulins failed to show an evident benefit or manifested with toxicity and therefore they were not recommended in clinical practice (Table 1) [46–49]. A Chinese trial randomized patients to 6 months of full dose steroids or lower dose steroid with MMF [50]. Complete proteinuria remission was similar between the two groups after one year but with fewer steroid-related adverse events in the arm with MMF [50]. Nevertheless, it was not a multiethnic study population, not all patients were treated by means of RAS-blockade and the time of follow-up was too short to evaluate the effect on renal parameters [50]. Although a possible positive effect of mycophenolate mofetil treatment in IgAN was noticed in a randomized controlled trial in China [51] with a significant reduction in the percentage of patients positive for histological change of E1 [50], the effect in Caucasian was not assessed [52] apart from the recent study on the effect of MMF therapy with a significant histological reduction in E1 score in repeated renal biopsies after 24 months (*p* < 0.0001) [53]. Further studies are needed for the assessment of treatment with mycophenolate

mofetil in patients with IgAN. On the other hand several authors suggested potential benefits of rapamycin in animal and cell models of IgAN [54].

Moreover, Hydroxychloroquine in addition to optimized RAAS inhibition significantly reduced proteinuria in patients with IgAN over 6 months without evidence of adverse events. Undoubtadly, these findings require confirmation in larger treatment trials [55].

The question of the efficacy of fish oil in IgAN is uncertain (Table 1) [16]. Many randomizedclinical trials testing the efficacy of fish oil in patients with IgAN provided contrasting results [16]. It was reported that daily treatment with fish oil for 2 years may reduce the progression of renal function with few side effects in patients with IgAN [56]. Fish oil was also recommended by KDIGO guidelines if it was tolerated [16,34].

The role of adrenocorticotropic hormone (ACTH) was considered in the treatment of resistant glomerular diseases [57]. The potential involved mechanisms imagine stimulation of endogenous steroid production, activation of melanocortin receptors on inflammatory cells and direct binding to melanocortin receptors on the podocyte [57]. Administration of a 6-month course of ACTH gel in patients with IgAN at high risk of progression (proteinuria > 1 g per 24 h despite documented ACEI/ARB therapy and adequate blood pressure control for >3 months, 24-h creatinine clearance >30 mL/min/1.73 m2 ) was prospectively investigated in an open-label pilot study (NCT 02282930). A significant decline in 24-h urinary protein (2.6 to 1.3 g; *p* = 0.007) with no significant changes in eGFR (65.5 to 61.1 mL/min, *p* = 0.1) was detected at 12-month follow-up in patients with IgAN treated with 6 months of ACTH [58].

A systematic review and meta-analysis of 14 studies indicated that tonsillectomy may induce clinical remission and decrease the rates of ESRD in IgAN patients [59]. Another multicenter controlled trial did not show a beneficial effect of tonsillectomy combined with steroid pulse therapy over steroid pulses alone to increase the incidence of clinical remission [60]. In the large VALIGA cohort (the European validation study of the Oxford classification of IgAN) of 1.147 European subjects with IgAN, no significant correlation was found between tonsillectomy and renal function decline [61].

A therapeutic option targeting B-cell pathway treatment against the production of Gd-IgA1 and its specific antibodies, such as rituximab, was involved [62,63]. A multicenter trial of 34 adult patients wih biopsy-proven IgA nephropathy and proteinuria > 1 g per day, maintained on ACEIs or ARBs with well-controlled blood pressure and eGFR < 90 mL/min/1.73 m2 , were randomized to receive supportive therapy either alone or with rituximab. Rituximab effectively depleted B cells (a monoclonal antiCD20 antibody) but neither serum levels of Gd-IgA1 nor its antibodies were reduced and the addition of rituximab failed to improve eGFR decline and proteinuria reduction [62]. CD20 positive B cells were targeted by rituximab but IgA positive plasma cells secreting antibodies were not affected therefore the treatment with rituximab was not efficient in patients with IgAN.

The activation of complement plays an important role in the pathogenesis of IgAN [64–70]. It was suggested that the process occurs both systemically on IgA-containing circulating immune complexes, and also locally in glomeruli, and is mediated through both the alternative and lectin pathways [13]. Pathway components were presented in the mesangial immunodeposits, including properdin and factor H (alternative pathway) and mannan-binding lectin, mannan-binding lectin-associated serine proteases 1 nad 2, and C4d (lectin pathway) [13]. Deletion of complement factor H-related genes 1 and 3 was identified as protective against the disease in GWAS [9,67].

C5a is a potent local inflammatory mediator and the presence of C5a in the kidney correlates with histological severity and proteinuria in IgAN [71]. Targeting C5a enables the suppression of local inflammation, contributing to progressive renal disease including the maintaining of the formation of C5b-9 (membrane attack complex), which plays a crucial role in the elimination of gram negative bacteria [71]. Avacopan (CCX 168), an inhibitor of the C5a receptor [72], was evaluated in an open-label Phase II trial in patients with IgAN. At the end of twelve weeks, proteinuria reduced in 6 of the 7 patients and a significant improvement of UPCR < 1 g/g was detected in 3 of the 7 patients. Undoubtedly, larger studies with longer follow up are needed in IgAN. Nevertheless, the efficacy of avacopan was

confirmed in patients with ANCA-associated vasculitis where avacopan allowed the replacement of high-dose corticosteroids with respect of adverse effects of hepatic dysfunction and an increased risk of infection in a small amount of patients [73].

The alternative pathway forms an essential amplification mechanism for the activation of the classical and lectin pathways, resulting in enormous opsonisation and generation of the terminal lytic pathway [74]. The two proteases of Factor D and Factor B are elementary for this amplification process [74]. Selective reversible inhibitors of Factors B and D were developed to efficiently block the activation of alternative pathways [74]. A phase II trial of LNP023, a first in the class of oral inhibitor of Factor B, was recently recruited in patients with IgAN (Table 2). The key component of the lectin pathway demonstrates Mannose-binding lectin associated serine protease 2 (MASP-2), which elevates the production of C3 convertase and leads to further inflammatory consequences. Targeting MASP-2 seems to be promising in terms of reducing the activation of the glomerular lectin pathway and not affecting the formation of C3 convertase through the classical and alternative pathways. Presently, the MASP-2 inhibitor OMS 721 is being assessed in Phase II and Phase III studies in IgAN (Table 2).

**Figure 1.** Therapeutic options related to the different targets. Target 1 **(Hit 1)**: B-lymphocyte activation results in the production of Gd-IgA1 (IgA1 poorly O-glycosylated at the hinge region). **Hit 2**: B-cell production of anti-Gd-IgA (IgG). Inhibitors of BAFF/APRIL:Atacicept, blisibimod; Spleen tyrosine kinase inhibitor: Fostamatinib;Gut mucosa modulation: budesonide; Proteosome inhibitor: bortezomib, microbiome modulation (may modulate B-lymphocyte activity with the reduction of Gd-IgA1). **Hit 3**: IgA1 specific protease. **Hit 4**:Spleen tyrosine kinase inhibitor:Fostamatinib; Corticosteroids: Acthar/Prednisolone, Complement mediation:Avacopan (C5a), OMS721 (Lectin pathway), LNP023 (Alternative pathway); IgA1 specific protease; Sparsentan; Dapagliflozin.

#### **Table 2.**Clinical trials in patients with IgA nephropathy—recruiting.

Abbreviation: wks—weeks, yrs—years, mth—months.

The clinical effect of atacicept was investigated for treating systemic autoimmune diseases [75]. Studies in patients with IgA nephropathy investigate the contribution of dysregulation of IgA1 secretion in the intestinal epithelium to see if intestinal immunity or mucosal immunity inhibitors of overexpression of APRIL and B cell-activating factor would have a clinical effect [17,76,77]. Additional potential immunosuppressive regimens (such as blisibimod(NCT02062684)—A selective antagonist of the B-cell activating factor, atacicept (NCT02808429)—An inhibitor of B-cell activating factor (BLyS) and a proliferation-inducing ligand (APRIL), bortezomib—A proteasome inhibitor, fostamatinib–an inhibitor of spleen tyrosine kinase, leflunomide—A pyrimidine synthesis inhibitor) are currently being tested in ongoing trials (Table 2) [78–83].

BAFF (B-cell activating factor) and April (a proliferation inducing ligand) are members of the tumour necrosis factor family which mediate B-cell function and survival [84]. BAFF and April levels are elevated in the serum of patients with IgAN and correlate with the disease activity [85]. BAFF and April are bound by TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor), which mediates their downflow effects through the NF-kB pathway. Blisibimod is a selective antagonist of BAFF and atacicept is a fusion protein containing the extracellular ligand binding domain of TACI and is able to block the downflow effects of BAFF and April.

The clinical effects of atacicept and blisibimod were investigated for treating systemic autoimmune diseases, including SLE and rheumatoid arthritis [75,86,87]. Studies in patients with IgAN investigate the contribution of dysregulation of IgA1 secretion in the intestinal epithelium to see if intestinal immunity or mucosal immunity inhibitors of overexpression of APRIL and B cell-activating factor would have a clinical effect [17,76,77].

Bortezomib, a plasma cell proteasome inhibitor, is applied in the treatment of multiple myeloma [88]. Proteasomes are indispensable intracellular protein complexes which destroy useless and impaired proteins by proteolysis [88]. Proteasomes are switched to immunoproteasomes but the dysregulation of the proteasome:immunoproteasome axis was confirmed in mononuclear cells in patients with IgAN with overexpression of the immunoproteasome, increased nuclear translocation of factors related to the NF-kB pathway, and more severe disease symptoms including higher proteinuria [89]. Nowadays, the treatment with bortezomib is more common in amyloidosis, lymphomas, tumours and antibody mediated allograft rejection [90]. A clinical trial to assess the safety and efficacy of bortezomib in patients with IgAN is currently underway (Table 2) but the use of bortezomib in mostly young asymptomatic patients with IgAN would probably be limited by the adverse effects of bortezomib (thrombocytopenia, rash, peripheral neuropathy, fatigue and anorexia) [90].

Tyrosine kinase pathways were asserted in the homeostasis of many diseases, and targeting of the tyrosine kinase signalling pathways was considered for treatment of immune-mediated glomerulonephritides [91]. Spleen tyrosine kinase is a non-receptor tyrosine kinase which might regulate the amount of key pathogenic pathways in IgAN [80]. Spleen tyrosine kinase is a signal transducer following B-cell receptor activation, arranging downflow signalling and promoting B-cell maturation and survival [80]. Spleen tyrosine kinase phosphorylation with the release of pro-inflammatory mediators was activated by stimulation of mesangial cells in vitro with IgA1 acquired from patients with IgAN [80].

Moreover, higher renal expression of spleen tyrosine kinase was confirmed in patients with endocapillary hypercellularity compared to patients without this sign in renal biopsy [92]. Fostamatinib, a selective inhibitor of spleen tyrosine kinase, was investigated in patients with rheumatoid artritis with a favourable effect on disease activity compared to placebo but with frequent adverse effects mostly involving diarrhoea and hypertension [93]. A Phase II trial of fostamatinib in patients with IgAN was recently completed.

Endothelin-1 (ET-1) is a growth factor for mesangial cells [94] and was shown to play a role in the progression of kidney disease in transgenic animals. In human subjects, urinary excretion of endothelin-1 correlates with the severity of kidney disease [95]. Expression of endothelin-1 and endothelin B receptor (mediating vasodilation and natriuresis), but not endothelin A receptor

(mediating vasoconstriction and cell proliferation) was demonstrated in patients with IgAN and high-grade proteinuria [96] suggests that activation of the endothelin system in renal tubular cells may partly be a response to protein overload. We demonstrated association of the progresson of IgAN with polymorphisms of the ET-1 gene [97]. Association of ET-1 expression with the progression of IgAN was also confirmed using molecular profiling [98]. A specific ET-receptor antagonist (FR 139317) was shown to suppress the development of histologic lesions and proteinuria in ddY mice with IgAN [99]. Sparsentan, a dual inhibitor of the angiotensin II type 1 (AT1) and endothelin type A (ET-A) receptors was recently shown to significantly decrease proteinuria compared to irbesartan in patients with focal segmental glomerulosclerosis [100] and is currently tested in patients with FSGS in a phase 3 trial [101]. Recently, the PROTECT Study (Phase 3 NCT 03762850) evaluating the long-term nephroprotective potential of sparsentan for the treatment of IgAN was initiated. Activation of Nrf2/Keap 1 by bardoxolone methyl results in the suppression of the main proinflammatory transcription factor NFkappaB and activation of some antioxidative pathways [102]. In patients with type 2 diabetes and CKD4 bardoxolone, methyl was shown to increase the glomerular filtration rate by almost 50% [103]. Recently released data (PHOENIX–NCT03366337) demonstrated a significant increase in eGFR of 8 mL/min/1.73 m2 in patients with IgAN treated with bardoxolone [104]. The increase in eGFR is, however, associated with a proportional increase in albuminuria with uncertain impact on the long-term outcome of the patients [105]. The updated KDIGO guideline will soon be released [34] and the current recommendation from KDIGO came from 2012 prior to STOP-IgAN, TESTING, Nefigan and DAPA-CKD. This is a rapidly evolving field and hence the importance of this review on currently recruiting trials was emphasized.

#### **3. Conclusions**

In conclusion, IgAN is a disease with variable clinical course. Validated urinary or serum biomarkers, able to provide information on the activity of the disease and the extent of fibrosis, are needed to stratify high risk patients with the necessity of use of immunossuppressive regimens with respect to possible adverse events. Nevertheless, IgAN patients with proteinuria < 1 g/d, and/or hypertension and a reduced GFR should obtain optimized supportive treatment with the inhibitors of the RAS. In IgAN patients with persistent proteinuria ≥ 1 g/d, despite 3–6 months of optimized supportive care (including RAS and blood pressure control), and GFR > 50 mL/min per 1.73 m2 , a 6-month course of corticosteroid treatment is indicated. Combined immunosuppression with corticosteroids and cyclophosphamide is reserved for high risk patients with a rapid decrease in the GFR and crescentic glomerulonephritis. Nowadays, many specific biological regimens in RCT are evaluated with expected common use in the future (Table 2). The search for an effective and well tolerated treatment of IgAN, directly targeting its pathogenetic mechanisms, continues (Figure 1).

**Funding:** The authors received funding from grants PROGRES Q25/LF1 and DRO VFN 64165 from the Ministry of Health of the Czech Republic.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the result.

#### **References**

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## *Article* **Inhibition of Yes-Associated Protein by Verteporfin Ameliorates Unilateral Ureteral Obstruction-Induced Renal Tubulointerstitial Inflammation and Fibrosis**

**Jixiu Jin 1 , Tian Wang 1 , Woong Park 1 , Wenjia Li 1 , Won Kim 1,2, Sung Kwang Park 1,2,\* and Kyung Pyo Kang 1,2,\***

Received: 15 October 2020; Accepted: 31 October 2020; Published: 31 October 2020

**Abstract:** Yes-associated protein (YAP) activation after acute ischemic kidney injury might be related to interstitial fibrosis and impaired renal tubular regeneration. Verteporfin (VP) is a photosensitizer used in photodynamic therapy to treat age-related macular degeneration. In cancer cells, VP inhibits TEA domain family member (TEAD)-YAP interactions without light stimulation. The protective role of VP in unilateral ureteral obstruction (UUO)-induced renal fibrosis and related mechanisms remains unclear. In this study, we investigate the protective effects of VP on UUO-induced renal tubulointerstitial inflammation and fibrosis and its regulation of the transforming growth factor-β1 (TGF-β1)/Smad signaling pathway. We find that VP decreased the UUO-induced increase in tubular injury, inflammation, and extracellular matrix deposition in mice. VP also decreased myofibroblast activation and proliferation in UUO kidneys and NRK-49F cells by modulating Smad2 and Smad3 phosphorylation. Therefore, YAP inhibition might have beneficial effects on UUO-induced tubulointerstitial inflammation and fibrosis by regulating the TGF-β1/Smad signaling pathway.

**Keywords:** kidney fibrosis; inflammation; myofibroblast activation; extracellular matrix; Hippo pathway; verteporfin

#### **1. Introduction**

Chronic kidney disease (CKD) is a heterogeneous condition characterized by reduced glomerular filtration rate, glomerular sclerosis, tubular atrophy, and interstitial fibrosis with inflammatory cell infiltration [1]. As human life expectancy has increased, CKD has become one of the most common non-communicable diseases [2,3]. End-stage renal disease that requires renal replacement therapy such as dialysis or kidney transplantation is associated with a decrease in residual life expectancy compared with healthy individuals [3]. Despite plenty of health resources in developed countries, CKD's global burden is steadily increasing, and CKD-related mortality is also increasing [4]. A nationwide cohort study in Korea found a higher mortality rate in patients with CKD than in healthy controls or even in patients with diabetes or hypertension but without CKD [5]. Therefore, strategies to enable the early recognition and prevention of CKD are needed to decrease the global health burden.

Organ fibrosis is characterized by a dynamic process of non-resolving inflammatory reactions [6] that cause the deterioration of organ function. After an ischemic or toxic injury to the kidneys causes damage to glomerular, vascular, and tubular cells, an inflammatory reaction can increase the deposition of the extracellular matrix (ECM) in the renal parenchyma [7]. Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine related to cell growth, differentiation, apoptosis, and wound repair [8]. Histologically, CKD is characterized by excessive ECM production from activated myofibroblasts, which causes renal tubulointerstitial fibrosis and organ dysfunction. TGF-β1 is a potent mediator in this fibrotic process [8,9]. Activated TGF-β1 binds to type I and type II cell surface receptors and phosphorylates the Smad2 and Smad3 proteins to activate the canonical (Smad-dependent) TGF-β signaling pathway [8,10]. TGF-β also activates other, non-canonical (Smad-independent) pathways, such as mitogen-activated protein kinase, phosphatidylinositol-3-kinase, and Rho-like GTPase [11]. Therefore, modulation of the TGF-β1 signaling pathway is an important therapeutic target for preventing progressive CKD.