Current Concepts on the Reno-Protective Effects of Phosphodiesterase 5 Inhibitors in Acute Kidney Injury: Systematic Search and Review

Acute kidney injury (AKI) is associated with increased morbidity, prolonged hospitalization, and mortality, especially in high risk patients. Phosphodiesterase 5 inhibitors (PDE5Is), currently available as first-line therapy of erectile dysfunction in humans, have shown a beneficial potential of reno-protection through various reno-protective mechanisms. The aim of this work is to provide a comprehensive overview of the available literature on the reno-protective properties of PDE5Is in the various forms of AKI. Medline was systematically searched from 1946 to November 2019 to detect all relevant animal and human studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement. In total, 83 studies were included for qualitative synthesis. Sildenafil is the most widely investigated compound (42 studies), followed by tadalafil (20 studies), icariin (10 studies), vardenafil (7 studies), zaprinast (4 studies), and udenafil (2 studies). Even though data are limited, especially in humans with inconclusive or negative results of only two clinically relevant studies available at present, the results of animal studies are promising. The reno-protective action of PDE5Is was evident in the vast majority of studies, independently of the AKI type and the agent applied. PDE5Is appear to improve the renal functional/histopathological alternations of AKI through various mechanisms, mainly by affecting regional hemodynamics, cell expression, and mitochondrial response to oxidative stress and inflammation.


Introduction
AKI is considered a complex disorder with increased morbidity, prolonged hospitalization and mortality especially in high risk patients that may be attributed to various causes (pre-renal; renal, i.e., intrinsic to the renal parenchyma; and post-renal), including the use of nephrotoxic medications such as contrast media (CM), dehydration, sepsis, renal surgery, renal ischemia, ischemia-reperfusion (IR) renal injury, and urinary tract obstruction [1]. Criteria used for the diagnosis of AKI vary widely among studies in humans [2], including percent change in the baseline serum creatinine (sCr) levels (e.g., an increase of variously 25-50%) and absolute elevation from baseline sCr level (e.g., an increase of variously 0.5-2.0 mg/dL) [3]. These variable definitions have been addressed by two consensus groups, namely the Acute Dialysis Quality Initiative (ADQI) proposing the RIFLE (Risk, Injury, Failure, Loss and End-stage kidney disease) system [4] and more recently the Acute Kidney Injury Network (AKIN), which have attempted to standardize the diagnosis of AKI irrespective of etiology. According to the AKIN diagnostic criteria [5], AKI is an abrupt (within 48 h) reduction in human kidney function defined as occurrence of any of the following after a reno-toxic event: (a) absolute increase in sCr ≥ 0.3 mg/dL (≥ 26.4 µmol/L) or a percentage increase in sCr ≥ 50% (1.5-fold from baseline), which is known or presumed to have occurred within the prior seven days [6]; or (b) a reduction in urine output (documented oliguria of < 0.5 mL/kg/h for more than 6 h). This definition is in accordance with the current Clinical Practice Guideline for AKI by "Kidney Disease: Improving Global Outcomes" (KDIGO) [6]. Nevertheless, a recent systematic review evaluating the methods used to investigate AKI biomarkers showed that results are difficult to interpret, not comparable, and not consistently reproducible due to the impact of the variable AKI definitions still used to determine the outcome of interest in human studies (38.0% of the studies used the AKIN; 21.4% used the RIFLE; 20.3% used the KDIGO; and 20.3% used another definition) [2]. Similarly, variable definitions of AKI have been used in animal studies, a fact that has been recognized as an important limitation in translating preclinical findings in clinical studies [7,8] among others [9]. Several reviews of available animal models, including their advantages and disadvantages, have been discussed [10]; however, the types of models are often incomplete and many details, such as model techniques and modeling time, are not mentioned. Currently proposed AKI models include, among others: IR renal injury, including shock wave lithotripsy (SWL); injection of drugs, toxins, or endogenous toxins; ureteral obstruction, contrast-induced nephropathy (CIN); trauma such as burn; etc. [10][11][12][13][14][15][16].
Depending on the insult type, there are various mechanisms leading to renal damage such as renal vasoconstriction [17], vascular endothelial damage, cytokine expression [18], increase of IL-18, mediating acute tubular necrosis, caspase activity stimulation, p53 up-regulation [19], accumulation of toxic metabolites [20], mast cells/neutrophils activation, reactive oxygen species (ROS) generation causing lipid peroxidation that leads to cellular membrane destruction, excessive intracellular DNA breakdown, energy depletion, intracellular Ca2+ elevation, higher inducible nitric oxide (NO) synthase (iNOS) expression, NO deficiency, intra-parenchymal hemorrhage [21], fibrosis, direct cellular toxicity, tubular obstruction, vascular congestion, activation of angiotensin II axis [22], mitochondrial dysfunction [23], cell cycle arrest in G2 phase, ATPase activity inhibition, and cellular transport modification. ROS activate pro-apoptotic proteins eventually promoting Bax translocation (regulated by PI3K/Akt pathway) to the outer mitochondrial membrane, causing the release of cytochrome c in the cytosol [24]. Bax is also responsible for caspase 9 activation that activates caspase 3, triggering apoptosis. The tubular component of AKI consists of injured, necrotic/apoptotic cells falling into the lumen that cause obstruction/back leak of the filtrate to the interstitial space, inducing inflammation.
CIN is a real, albeit rare, entity in current clinical medical practice that represents a serious iatrogenic AKI form, occurring 24-72 h after administration of iodinated contrast media (CM) during angiographic or other procedures, such as urography [3,25]. The exact pathophysiology of CIN is not fully elucidated but oxidative stress is considered a major mechanism in CIN [26], and the identification of novel biomarkers that may more accurately detect renal function changes, reflect kidney damage, assist monitoring, and elucidate pathophysiology have attracted considerable scientific attention nowadays [27]. CM passing through the kidney results in an intense tubular transport that increases the activity in the thick ascending limb of Henle's loop. This process increases oxygen consumption/metabolic activity of outer renal medulla, exacerbating the marginal hypoxic conditions. Prostanoids and NO are mainly responsible for the medullary vasodilatory response [28]. Therefore, any NO deficit may contribute to an additional hypoxic renal insult. CIN and IR renal injury share common pathways regarding the vasodilatory potential of NO. IR renal injury is a common complication during renal transplantation/artery angioplasty, partial nephrectomy, cardiopulmonary/aortic bypass surgery, and others [29]. In the IR renal injury setting, however, there are conflicting results reported, with some studies suggesting that NO induces cytotoxicity, and others showing that increased NOS activity is linked to increased renal blood flow in the ischemic region [30]. NOSs are a family of enzymes catalyzing the production of NO from L-arginine. There are three isoforms: the endothelial NOS (eNOS), the neuronal NOS (nNOS), and the iNOS involved in immune response. In the IR renal injury, endogenous NO is synthesized by eNOS and iNOS [31], while it is found that eNOS-mediated NO production plays a pivotal protective role in IR-induced AKI [1]. IR renal injury is also closely linked to ROS generation/apoptosis. Prevention and/or management of the various AKI forms, such as CIN, is mainly supportive at present, consisting of intravenous hydration [32]. Even though the potential beneficial effects of many agents with antioxidant properties have been tested, the value of such substances other than sodium bicarbonate remains controversial [32,33]. Phosphodiesterase 5 (PDE5) inhibitors (PDE5Is) are currently recommended as first-line therapy of erectile dysfunction (ED) by enhancing the vasodilatory effects of NO [34]. Acting via the selective inhibition of cyclic guanosine monophosphate (cGMP)-specific PDE5 that metabolizes cGMP, the principal mediator of NO-induced smooth muscle relaxation, PDE5Is cause vasodilatation in the corpora cavernosa promoting erection ( Figure 1). This class of drugs has shown beneficial potential through various mechanisms in some CIN animal models [33]. The aim of this paper is to provide a comprehensive overview of the available literature on the potential reno-protective properties of PDE5Is in the various forms of AKI.
J. Clin. Med. 2020, 9, x FOR PEER REVIEW 3 of 45 angiographic or other procedures, such as urography [3,25]. The exact pathophysiology of CIN is not fully elucidated but oxidative stress is considered a major mechanism in CIN [26], and the identification of novel biomarkers that may more accurately detect renal function changes, reflect kidney damage, assist monitoring, and elucidate pathophysiology have attracted considerable scientific attention nowadays [27]. CM passing through the kidney results in an intense tubular transport that increases the activity in the thick ascending limb of Henle's loop. This process increases oxygen consumption/metabolic activity of outer renal medulla, exacerbating the marginal hypoxic conditions. Prostanoids and NO are mainly responsible for the medullary vasodilatory response [28]. Therefore, any NO deficit may contribute to an additional hypoxic renal insult. CIN and IR renal injury share common pathways regarding the vasodilatory potential of NO. IR renal injury is a common complication during renal transplantation/artery angioplasty, partial nephrectomy, cardiopulmonary/aortic bypass surgery, and others [29]. In the IR renal injury setting, however, there are conflicting results reported, with some studies suggesting that NO induces cytotoxicity, and others showing that increased NOS activity is linked to increased renal blood flow in the ischemic region [30]. NOSs are a family of enzymes catalyzing the production of NO from L-arginine. There are three isoforms: the endothelial NOS (eNOS), the neuronal NOS (nNOS), and the iNOS involved in immune response. In the IR renal injury, endogenous NO is synthesized by eNOS and iNOS [31], while it is found that eNOS-mediated NO production plays a pivotal protective role in IR-induced AKI [1]. IR renal injury is also closely linked to ROS generation/apoptosis. Prevention and/or management of the various AKI forms, such as CIN, is mainly supportive at present, consisting of intravenous hydration [32]. Even though the potential beneficial effects of many agents with antioxidant properties have been tested, the value of such substances other than sodium bicarbonate remains controversial [32,33]. Phosphodiesterase 5 (PDE5) inhibitors (PDE5Is) are currently recommended as first-line therapy of erectile dysfunction (ED) by enhancing the vasodilatory effects of NO [34]. Acting via the selective inhibition of cyclic guanosine monophosphate (cGMP)-specific PDE5 that metabolizes cGMP, the principal mediator of NO-induced smooth muscle relaxation, PDE5Is cause vasodilatation in the corpora cavernosa promoting erection ( Figure 1). This class of drugs has shown beneficial potential through various mechanisms in some CIN animal models [33]. The aim of this paper is to provide a comprehensive overview of the available literature on the potential reno-protective properties of PDE5Is in the various forms of AKI. Figure 1. PDE5I-induced smooth muscle relaxation in the corpora cavernosa. cGMP is the principal mediator of NO-induced smooth muscle relaxation/vasodilation [35]. cGMP propels a series of intracellular changes including inhibition of Ca 2+ entry into the cell, Ca 2+ shift into the endoplasmic Figure 1. PDE5I-induced smooth muscle relaxation in the corpora cavernosa. cGMP is the principal mediator of NO-induced smooth muscle relaxation/vasodilation [35]. cGMP propels a series of intracellular changes including inhibition of Ca 2+ entry into the cell, Ca 2+ shift into the endoplasmic reticulum, activation of K + channels leading to membrane hyperpolarization, and stimulation of a cGMP-dependent protein kinase that activates a myosin light chain phosphatase. All these actions promote smooth muscle relaxation. NO penetrates the cytoplasm of smooth muscle cells binding to guanylyl cyclase (sGC), which catalyzes the enzymatic conversion of GTP to cGMP. Elevation of cGMP stimulates cGMP-dependent protein kinase G leading to PDE5 phosphorylation/activation. PDE5 hydrolyzes cGMP in the cavernosal tissue. Inhibition of PDE5 results in smooth muscle relaxation with increased arterial blood flow, leading to compression of the sub-tunical venous plexus followed by penile erection [36].

Experimental Section
Medline (Ovid Medline Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Ovid MEDLINE(R) Daily, and Ovid MEDLINE(R) 1946 to November 2019) was systematically searched to detect all relevant animal and human studies in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement [37], using the following keyword combinations (Medical Subject Headings; MeSH): PDE5i or avanafil or benzamidenafil or dasantafil or icariin or lodenafil or mirodenafil or sildenafil or tadalafil or udenafil or vardenafil or zaprinast combined with renal or kidney or nephrotoxicity or contrast or CIN or AKI or nephrotoxic or cisplatin or aminoglycoside or trauma or acute kidney injury or NSAIDS or non-steroidal or shock or sepsis or hypoperfusion or hypovolaemia or hypovolemia or renal artery stenosis or obstruction or acute tubular necrosis or glomerulonephritis or nephritis or renal failure or adenine or cyclosporine. The specific literature search strategy used is available in Appendix A. The reference lists of selected studies were screened for other potentially eligible studies. After excluding duplicates, citations in abstract form, and non-English citations, the titles/abstracts of full papers were screened for relevance, defined as original research focusing on the topic "nephropathy AND effects of phosphodiesterase 5 inhibitors". Studies focusing on alterations of renal function and/or structure for >3 months (conventionally considered as following the KDIGO definition of chronic kidney disease (CKD) were excluded [6]). Two review authors (G.G. and IE.Z.) independently scanned the title and the abstract content, or both, of every record retrieved to determine which studies should be assessed further evaluated and extracted all data. Disagreements were resolved through consensus or by consultation with a third author (C.M.). A final draft of the manuscript was prepared after several revisions and approved by all authors.
The reno-protective action of PDE5Is was evident in the vast majority of studies (n = 81), independently of the AKI type and the agent applied. Only one human study on sildenafil [39] and one animal study on zaprinast [40] failed to reveal any reno-protective action of PDE5Is, showing a neutral effect. PDE5Is appeared to be beneficial in AKI of various etiologies by improving renal functional/histopathological alternations through various mechanisms, such as affecting regional hemodynamics, cell expression, and mitochondrial response to oxidative stress and inflammation. The reno-protective action of PDE5Is was evident in the vast majority of studies (n = 81), independently of the AKI type and the agent applied. Only one human study on sildenafil [39] and one animal study on zaprinast [40] failed to reveal any reno-protective action of PDE5Is, showing a neutral effect. PDE5Is appeared to be beneficial in AKI of various etiologies by improving renal functional/histopathological alternations through various mechanisms, such as affecting regional hemodynamics, cell expression, and mitochondrial response to oxidative stress and inflammation.

Discussion
PDE5Is have received a lot of attention since the first drugs were launched in the market. Four potent selective agents (avanafil, sildenafil, tadalafil, and vardenafil) have been approved by the European Medicines Agency (EMA) and the Food and Drug Administration (FDA) for the treatment of ED [111,112]. ED can be managed successfully with currently available treatment options, but it cannot be cured and most patients will be treated without cause-specific options, such as the use of PDE5Is [34]. Exceptions are psychogenic, post-traumatic arteriogenic in young patients, and hormonal causes (e.g., hypogonadism) of ED, which are potentially curable with specific treatments that might be employed first, when such causes are detected [34]. Consequently, treatment strategy of ED should be tailored depending on invasiveness, efficacy, safety, cost, and patient preference of the currently available options; in the context of this strategy, PDE5Is are currently recommended strongly as first-line treatment option given that lifestyle changes are initiated/risk factors are modified prior to or at the same time as initiating ED treatment [34].
Other EMA/FDA approved indications of PDE5Is include pulmonary arterial hypertension (PAH) (sildenafil and tadalafil) and management of men with moderate to severe LUTS secondary to benign prostatic obstruction with or without ED (tadalafil) [34,[113][114][115]. Besides the aforementioned agents, there are other non-EMA/FDA approved PDE5Is including benzamidenafil, dasantafil, lodenafil, mirodenafil, and udenafil, some of which are commercially available in a few countries (lodenafil in Brazil; mirodenafil in South Korea; and udenafil in South Korea, Russia, and Philippines) [113]. Other agents with weak PDE5I properties include icariin and zaprinast [116]. Icariin, a prenylated flavonol glycoside extracted from plants of the Epimedium genus, has demonstrated PDE5I activity in vitro, enhancement of NO, and antioxidant activity [116]. It has been widely used in Chinese traditional medicine. It shows peak concentration levels at 1 h and should be avoided in patients with bleeding disorders, hypotension, arrhythmias, and hormone-sensitive cancers (breast, ovarian, or prostate). Zaprinast is an inhibitor of PDE5, PDE6, PDE9, and PDE11. In the past, it has been used for the treatment of PAH and inhibition of malaria parasites. Zaprinast activates the G-protein coupled receptor, GPR35, that plays a crucial role in cardiovascular disease, pain, regulation of inflammation, hypertension, diabetes, and irritable bowel disease [117,118]. The main characteristics of PDE5Is are summarized in Table 7 -ID  ID  ID  ID  ID  ID   Icariin  No  -ID  ID  ID  ID  ID  ID   Zaprinast  No  -ID  ID  ID  ID  ID  ID Abbreviations: AKI, acute kidney injury; BP, blood pressure; Cmax, serum maximum concentration; CHF, chronic heart failure; CKD, chronic kidney disease; CVD, cardiovascular disease; ED, erectile dysfunction; ID, insufficient data; NO, nitric oxide; NYHA, New York Heart Association; OD, once daily; PAH, pulmonary arterial hypertension; PDE5I, phosphodiesterase 5 inhibitor; Tmax, transport maximum.
PDE5Is interfere selectively with cGMP hydrolysis by PDE5, increasing intracellular cGMP, which results in smooth muscle relaxation/raised arterial blood flow improving penile erection. PDE5 belongs to a superfamily of enzymes that convert intracellular cAMP/cGMP into the consonant nucleotides. It is a cytosolic protein with three isoforms expressed in various organs apart from the penis (corpora cavernosa), including kidney (vessels, glomeruli, inner medullary collecting ducts, and cortical tubules) that specifically degrades cGMP [66]. In particular, PDE5A1 and PDE5A2 are widely expressed in tubular epithelial cells of the renal proximal tubule and medullary collecting duct, as well as in vascular smooth muscle cells, platelets, brain, and lung, while PDE5A3 is only expressed in vascular smooth muscle cells [126].
Cyclic nucleotide signal transduction pathways represent an emerging research field in kidney disease, with selective PDE5 inhibition attracting ongoing interest nowadays [127]. Current evidence supports the notion that regulation of the cGMP -dependent protein kinase 1-PDE signaling pathway may be reno-protective and that its regulation might provide novel, therapeutic strategies for chronic kidney disease with selective PDE5Is having shown potential in treating kidney fibrosis, while possessing antithrombotic and anticancer activity [128]. In this respect, PDE5Is represent a potential but still understudied/controversial option against various forms of AKI such as CIN [28], given that NO/cGMP are crucial mediators in renal vasculature and NO is an essential endogenous vasodilator for medullary oxygenation [33].
The mechanism of action of PDE5Is in the prevention and management of AKI is still not fully elucidated. Multiple mechanisms have been proposed to play a role in counteracting the cascade of changes caused by the renal injury. Stimulation of NO production through NOS, medullary blood flow improvement, protection against vascular endothelial damage, Bcl2/Bax ratio reversal, ERK phosphorylation, mitochondrial biogenesis activation, renal progenitor cell upregulation, and the regulation of multiple signaling pathways such as insulin/IGF1, T17/Treg, PI3K/Akt, and NF-kB [75] are the most well-described mechanisms through which PDE5Is offer protection. The increased ERK phosphorylation boosts NOS activity and subsequent rapid NO release [30]. The repairing process following any renal injury requires energy provided by the cellular mitochondria. Mitochondria are continuously regenerated but cellular injury such as sepsis and hypoxia induce rapid biogenesis. This process is mediated by a transcriptional co-activator, peroxisome proliferator-activated receptor γ co-activator 1a (PGC-1a). PGC-1a activates the nuclear respiratory factors 1 and 2, which eventually activate mitochondrial transcription factor A that is responsible for the transcription of mitochondrial DNA [23,67]. An alternative process that PDE5Is activate to promote recovery from renal injury is the renal progenitor cell stimulation. PDE5Is, more specifically icariin, upregulates HGF, WT-1, and BMP-7, which lead to an increased number of CD133 + and CD24 + , cells that are capable of self-renew and also differentiate into podocytes or tubular cells [57,103]. In addition to the aforementioned actions, PDE5Is are likely to exert their protective effect through an alternative pathway. PDE5Is increase cGMP, which activates protein kinase G that opens mitochondrial K ATP channels that induce depolarization of the mitochondrial inner membrane and Mg 2+ release. The depolarized membrane results in reduced Ca 2+ influx; therefore, suppressed cellular death and increased Mg 2+ concentration reduces ROS and lessens p38 MAPK activation, which is responsible for apoptosis [30,88,129]. The most common reno-protective mechanisms of PDE5Is are summarized in Figure 3.
To the best of our knowledge, this is the first review that attempts in a systematic way to define the reno-protective potential of PDE5Is in the various forms of AKI. Based on our results, it appears that sildenafil is the most widely PDE5I studied in AKI among the 11 natural/synthetic agents currently available (avanafil, benzamidenafil, dasantafil, icariin, iodenafil, mirodenafil, sildenafil, tadalafil, udenafil, vardenafil, and zaprinast).
The reno-protective effects of PDE5Is have been evaluated in four human studies to date (preclinical studies using human cells: n = 2 [24,38]; clinical studies: n = 2 [17,39]) ( Table 1). In one study, human umbilical cord mesenchymal stem cells (huMSC), which have a high self-renewal/multi-directional differentiation potential, were treated with icariin and administered in an animal model of renal injury induced by adenine [38]. Blood urea nitrogen/sCr analysis showed amelioration of functional parameters. Icariin-treated huMSC increased the number of cells in injured renal tissues, reduced fibrosis, oxidative damage, inflammatory responses, and promoted expression of growth factors protecting injured renal tissue. In another study, cisplatin was added to human embryonic kidney (HEK)-293 renal cell cultures pre-treated with icariin [24]. The authors concluded that icariin prevents cisplatin-induced HEK-293 cell injury by inhibiting oxidative stress, inflammatory response, and cellular apoptosis partly via regulating nuclear factor kappa-like chain-enhancer of activated B cells (NF-κB) and PI3K/Akt signaling pathways. In a non-randomized clinical trial, 49 patients with renal tumors were submitted to open nephron-sparing surgery after renal artery clamping [17]. Twenty-two patients were pre-treated with tadalafil one day pre-and two days post-operatively and 27 patients underwent the same surgery without receiving tadalafil. Renal artery clamping induced kidney dysfunction reflected by increases in urinary NGAL and KIM-1 (two novel biomarkers for AKI) in all participants. Tadalafil reduced the urinary excretion of KIM-1, but not of NGAL. The incidence of AKI was comparable between groups but sCr elevation was significantly attenuated in the tadalafil-treated group compared to controls. It was concluded that tadalafil exerts reno-protective effects in AKI following nephron-sparing surgery. In a randomized placebo-controlled trial, 40 patients were submitted to robot-assisted partial nephrectomy after hilar clamping. The reno-protective effect of a single 100 mg oral dose of sildenafil immediately prior to clamping was evaluated [39]. GFR was similarly decreased between arms during the immediate postoperative period and at an intermediate-term follow-up of six months; the reno-protective effect of sildenafil was not evident in this study (neutral effect).  The reno-protective effects of PDE5Is have been evaluated in four human studies to date (preclinical studies using human cells: n = 2 [24,38]; clinical studies: n = 2 [17,39]) ( Table 1). In one study, human umbilical cord mesenchymal stem cells (huMSC), which have a high selfrenewal/multi-directional differentiation potential, were treated with icariin and administered in an animal model of renal injury induced by adenine [38]. Blood urea nitrogen/sCr analysis showed amelioration of functional parameters. Icariin-treated huMSC increased the number of cells in injured renal tissues, reduced fibrosis, oxidative damage, inflammatory responses, and promoted expression of growth factors protecting injured renal tissue. In another study, cisplatin was added to human embryonic kidney (HEK)-293 renal cell cultures pre-treated with icariin [24]. The authors concluded that icariin prevents cisplatin-induced HEK-293 cell injury by inhibiting oxidative stress, inflammatory response, and cellular apoptosis partly via regulating nuclear factor kappa-like chainenhancer of activated B cells (NF-κB) and PI3K/Akt signaling pathways. In a non-randomized clinical trial, 49 patients with renal tumors were submitted to open nephron-sparing surgery after renal artery clamping [17]. Twenty-two patients were pre-treated with tadalafil one day pre-and two days postoperatively and 27 patients underwent the same surgery without receiving tadalafil. Renal artery All animal studies investigating the potential reno-protective effect of sildenafil (n = 41) manifested a beneficial effect, irrespectively of the mechanism of AKI; almost all parameters evaluated (biochemical or morphological) were reported to improve (Tables 2 and A1). Similarly, all animal studies investigating the potential reno-protective effect of tadalafil (n = 19) revealed beneficial outcomes (attenuated histopathological changes/improved biochemical profile; Tables 3 and A2). Two studies provided comparative results for sildenafil and tadalafil, demonstrating the superiority of the former against tubular cell apoptosis, oxidative status, lipid peroxidation and NOS alterations [45,49]. Unique proteins, cells, and genes have been utilized to investigate the aftermath following icariin's administration as a reno-protective agent, such as connective tissue growth factor, TUNEL positive cells, nephrin, α-smooth muscle actin, E-cadherin, LY6G, F4/80, NLRP3, NF-κB, etc. All available animal studies evaluating icariin (n = 8) showed a beneficial effect (oxidative injury reversal, obliteration of renal function impairment, and improvement of renal hemodynamics/NO sensitivity; Tables 4 and A3). Similar to sildenafil/tadalafil, icariin suspends the inflammatory response initiation as well as the alteration of the cellular phase and preserves renal morphology. Finally, vardenafil, zaprinast, and udenafil have been investigated in a limited number of studies (n = 7, n = 4, and n = 2, respectively), almost all of which show antioxidant, anti-inflammatory, and reno-protective effects (Table 5, Table 6,  Table A4, and Table A5). In one study, vardenafil was compared to sildenafil and tadalafil in an animal model of partial unilateral ureteric obstruction, reporting that all agents were beneficial with sildenafil showing best results [45]. Only one study failed to demonstrate any beneficial effect from zaprinast pre-treatment in an animal model of nephrectomy and concomitant contra-lateral renal hilar occlusion [40]. Even though data are still limited, especially in humans with inconclusive or negative results of only two clinically relevant studies available at present, the results of animal studies are promising and have already fueled clinical research, which is on-going with results expected to come out in the near future [122]. Nevertheless, the potential reno-protective capacity of PDE5Is in AKI warrants further investigation in clinical trials.