Protective Role of Vitamin D in Renal Tubulopathies

Vitamin D is tightly linked with renal tubular homeostasis: the mitochondria of proximal convoluted tubule cells are the production site of 1α,25-dihydroxyvitamin D3. Patients with renal impairment or tubular injury often suffer from chronic inflammation. This alteration comes from oxidative stress, acidosis, decreased clearance of inflammatory cytokines and stimulation of inflammatory factors. The challenge is to find the right formula for each patient to correctly modulate the landscape of treatment and preserve the essential functions of the organism without perturbating its homeostasis. The complexity of the counter-regulation mechanisms and the different axis involved in the Vitamin D equilibrium pose a major issue on Vitamin D as a potential effective anti-inflammatory drug. The therapeutic use of this compound should be able to inhibit the development of inflammation without interfering with normal homeostasis. Megalin-Cubilin-Amnionless and the FGF23-Klotho axis represent two Vitamin D-linked mechanisms that could modulate and ameliorate the damage response at the renal tubular level, balancing Vitamin D therapy with an effect potent enough to contrast the inflammatory cascades, but which avoids potential severe side effects.


Introduction
Vitamin D is a fundamental agent for body homeostasis and maintaining a proper level of this hormone could make the difference in strategies against many systemic diseases; however, the best way to reach the proper level of this hormone is still debated.
The USA Endocrine Society recommends a dietary intake of 600 IU/day for adults in the general population and to double or triple the dose in patients at risk of Vitamin D deficiency; they define as adequate a level of 75-250 nmol/L (30-100 ng/mL) of Vitamin D [1]. The Institute of Medicine (IOM) identified as a proper level of Vitamin D a lower threshold of ≥50 nmol/L ( ≥ 20 ng/mL) [2]. In line with IOM findings, the National Osteoporosis Society recommends to reach a level >50 nmol/L of Vitamin D to obtain an adequate status [3].
The National Kidney Foundation in the Kidney Disease Outcomes Quality Initiative (K/DOQI) clinical practice guidelines for bone metabolism and disease in chronic kidney disease (CKD) encourages maintaining a value of Vitamin D above 30 ng/mL. The K/DOQI also suggests that for desirable serum 25(OH)D levels, supplementation with Vitamin D should be initiated using dosing regimens recommended for the general population [4].
Correct homeostasis of the renal tubular function is fundamental to guarantee the right balance of intake, production and excretion of Vitamin D. Renal tubules have a central role in the modulation of Vitamin D activity for different reasons: Vitamin D binding protein (DBP) endocytosis is linked to the interaction of Megalin, Cubilin and Amnionless complex, three key regulators of proximal renal tubules expressed in the apical membrane [5].
On the basolateral membrane of renal tubular cells, Fibroblast Growth Factor 23 (FGF23) triggers the signaling cascade that inhibits the 1α-hydroxylase activity, which is the fundamental enzyme that operates the activation of Vitamin D. FGF23 exerts its action binding to the complex of FGF receptors (FGFRs) and αKlotho hormone. Different pathologic entities that affect renal tubules could be linked to Vitamin D dysregulation. Reaching homeostasis in Vitamin D balance could be a pivotal strategy in the treatment of these diseases [6].
Vitamin D exerts numerous pleiotropic effects unrelated to plasma calcium homeostasis. Indeed, various studies demonstrated that this hormone and its active analogs perform relevant anti-proteinuric, anti-fibrotic and anti-inflammatory actions in subjects with CKD and in experimental studies on animals with end-stage renal disease [11][12][13].
The active form of Vitamin D is produced by mitochondria of the renal proximal convoluted tubules. In tubular epithelial cells, which have a preeminent function in the synthesis of active Vitamin D [14], 1α-hydroxylase converts 25-hydroxyvitamin D [25(OH)D] to [1,25 (OH)2D3] [15].
Most of the Vitamin D moves in the circulation by the Vitamin D-binding protein (DBP), in a smaller percentage it is carried by blood proteins-albumin and lipoproteins-and in a minimum part, it flows freely in its biologically active form. These complexes binding the Vitamin D are filtered through the glomeruli, after which megalin can re-uptake it in the proximal tubule; for this reason, an acute or chronic tubular injury could be associated to a loss of Vitamin D [16].
Vitamin D has many pleiotropic effects on body homeostasis, such as cardio-protective and neuro-protective functions, hypertension control and is also involved in antitumor activity as well as immunomodulation [17][18][19] through the Vitamin D receptor (VDR). VDR has a fundamental part in the kidney homeostasis, since its activation protects against renal injury in kidney diseases regulating the renin-angiotensin-aldosterone system (RAAS) activation and anti-inflammatory pathways, inhibiting renal fibrogenesis, maintaining podocyte health, restoring mitochondrial function and suppressing autoimmunity and renal cell apoptosis [20][21][22].
Several case reports underlined the link between renal tubular acidosis and deficit of Vitamin D, this condition leads to inappropriate reabsorption of bicarbonate and perturbance of electrolyte pool homeostasis [23][24][25].
A proper level of Vitamin D has a protective role on the proximal renal tubule; this status could contribute to the balance of blood pH: in fact, in this segment of nephron, bicarbonate buffer is reabsorbed through a mechanism coupled to the secretion of hydrogen into the lumen [26].

Vitamin D Protective Role in Tubular Damage in CKD
Vitamin D plays a pivotal role at a tubular level, and VDRs are present in both proximal and distal nephron [27].
A challenging question about Vitamin D action is how it can influence the tubular damage and renal fibrosis typical of CKD [32].
CKD is characterized by myofibroblast generation and activation. These mechanisms are influenced by several inflammatory mediators such as the cytokine transforming growth factor β (TGFβ), a powerful inducer of myofibroblast differentiation [33]; myofibroblast formation could also derive from epithelial and endothelial cells through the epithelial/endothelial-mesenchymal (EMT/EndMT) transition, with consequences such as renal fibrogenesis and tubular atrophy, secondary to a decreased epithelial cell mass and an increase in the matrix-producing effector cells [34].
Myofibroblast activation could be triggered by paracrine and autocrine mechanisms: lymphocyte and macrophage signals can stimulate their activation as well as autocrine factors generated even by myofibroblasts and pathogen-associated molecular patterns derived from pathogenic agents that cooperate through pattern recognition receptors on fibroblasts.
Cytokines, chemokines, angiogenic factors, acute phase proteins, caspases and components of the renin-angiotensin-aldosterone system (RAAS) have been established as significant mediators of fibrotic damage [35].
Interstitial fibrosis also impairs the ability of tubules to secrete toxins from peritubular capillaries, interferes with their passage to the capillaries and impairs the exchange of nutrients from the circulatory system [36].
In an experimental mouse model, Arfian et al. [37] observed that treatment with 1,25-dihydroxyvitamin D was significantly associated with lower fibrotic status, tubular impairment scores and myofibroblast proliferation compared with the control group.
Vitamin D deficiency intensifies tubulointerstitial injury and degeneration in interstitial fibrosis after ischemia/reperfusion insult linked to an elevation of PTH, a decrease of plasma FGF-23, VDR receptor and Klotho protein and to an increased expression of pro-inflammatory factors such as transforming growth factor β (TGF-β) [38]. Inoue et al. [39] confirmed these results in their study, showing how Vitamin D analog 22-oxacalcitriol has a protective role from tubulointerstitial fibrosis by containing the autoinduction of TGF-β1.
Moreover, Lee at al [40] outlined how paricalcitol attenuates functional deterioration and histological damage during induced AKI in mice. Vitamin D administration considerably lowed tissue neutrophil and macrophage infiltration and chemokines levels.
Proximal tubule epithelial cells (PTECs) are the most expressed cell type in the kidney and are responsible for the reabsorption of 60%-70% of water and NaCl, 80% of NaHCO3 and almost nutrients present in the glomerular ultrafiltrate, fluid-electrolyte and acid-base balance, ammoniagenesis, Vitamin D synthesis and immunomodulating functions [41]. Injured PTECs and epithelial-to-mesenchymal transformation of PTECs induce pro-inflammatory and inflammatory cytokines production [42].
Active Vitamin D is able to up-regulate the protein kinase B/mitochondrial uncoupling protein 2 (AKT/UCP2) signaling, which reduces oxidative stress of renal tubular epithelial cell line (HK2) [43]. UCP2 is a protein, mainly located in proximal convoluted tubule cells, that has been demonstrated to have a pivotal role in the preservation of mitochondria, immune regulation and modulation of oxidative stress under physiological or pathological conditions affecting the kidney [44,45]. 1,25-dihydroxyvitamin D exerts a direct protective effect, significantly decreasing IL-6 and IL-8 levels in PTECs and modulating the activity of effector T-cells [46].
Calcitriol has been demonstrated to be strongly associated with the inhibition of NF-κB and necroptotic pathway, via upregulating A20 in Ang II-induced renal injury [47]. Zinc finger protein A20 is a strong NF-κB pathway negative regulator and necroptosis inhibitor [48]; A20 induction primarily occurs in the tubular epithelial cells and its proper control leads to manage inflammation and ROS generation. In rat PTECs, 1,25-dihydroxyvitamin D tightly upregulated A20 and reduced pro-inflammatory cytokines and ROS.
Zhang et al. [49] demonstrated in vitro that 1,25-dihydroxyvitamin D and its analog Elocalcitol (BXL-628) have a relevant role in the modulation of RhoA/Rho (member of the Rho guanosine triphosphatases family), associated protein kinase pathway (ROCK) in human renal proximal tubular cells. Zhang et al. also found out in a previous study [50] involving HK-2 cells in vitro that the TGF-β1-mediated RhoA/ROCK activation contributed to the dissolution of tight junctions during EMT, which is one of the mechanisms underlying renal fibrosis [51].
Activation of VDR by paricalcitol protects against lipopolysaccharide (LPS)-induced acute kidney injury by blocking renal tubular epithelial cell apoptosis and preventing LPS-stimulated caspase 3 activation in the renal cortex of LPS-treated mice [57]. Paricalcitol administration also contributes to the modulation of renal inflammatory infiltration and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) expression by promoting VDR-mediated sequestration of NF-κB signaling [58].
AKT, a reactive oxygen species (ROS) target, has a pivotal role in controlling cell growth and proliferation, and its downregulation contributes to renal proximal tubular cell apoptosis [59]: Hong et al. [60] established the important function of paricalcitol through the anti-inflammatory and antiapoptotic effects of AKT and NF-κB signaling in LPS-induced renal proximal tubule cell injury.
In their study, Morgado-Pascual et al. [61] found out that paricalcitol could also exert an anti-inflammatory action in cultured tubular epithelial cells through the inhibition of "a disintegrin and metalloproteinase" (ADAM17)/epidermal growth factor receptor (EGFR) signaling axis. Their group demonstrated in an experimental model [62] that this ADAM17/EGFR pathway contributes to renal inflammation and the modulation of its overexpression could lead both to better regulation of fibrosis and to the inhibition of aldosterone-mediated proinflammatory factors overexpression.

Megalin, Cubilin and Vitamin D: A Synergy into Proximal Tubule Cells for Body Homeostasis
Megalin, also known as Lrp2 (low-density lipoprotein-related protein 2) or gp330, is a transmembrane receptor for serum DBP in renal cells and is essential for the uptake of the 25(OH)D3-DBP complex [63]. The 58-kD DBP is the main transporter for Vitamin D 3 metabolites in the circulation. DBP presents the highest affinity for 25-(OH) Vitamin D 3 (K d = 10 −10 to 10 −12 M). Due to this tight binding affinity and the high plasma concentration of DBP (0.3 to 0.5 mg/mL), almost all circulating 25-(OH) Vitamin D 3 molecules are present in complex with DBP [64].
Megalin also mediates the cellular uptake of many serum transport proteins, including those that act as carriers for the steroid hormone androgen and estrogen as well as lipophilic vitamins such as retinol [65,66].
Studies in rodent models showed that the DBP-Vitamin D complex is constantly filtered through the glomerulus and endocytosed via megalin into PTECs, leading to the activation of 25(OH)D(3) to the active form [67]. Chapron et al. [68], using a human cell-derived model, confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to DBP.
Loss of this receptor in congenital or acquired diseases results in multiple organ dysfunctions, including forebrain malformation (holoprosencephaly) and the Donnai-Barrow/Facio-Oculo-Acustico-Renal syndrome characterized by a multifaceted phenotype including low-molecular-weight proteinuria [69] and renal reabsorption defects (renal Fanconi syndrome) [70]. Beydoun et al. [71] also demonstrated that VDR and megalin gene variations can modify age-related cognitive decline among US adults. De et al. [72] in their study underlined how urinary full length megalin (C-megalin) excretion is increased via exocytosis from renal PTECs injured by endo-lysosomal overload caused by megalin-mediated protein metabolism.
Toi et al. [73] studied C-megalin excretion in 153 pre-dialysis CKD patients finding that its determination is clinically relevant for the assessment of renal PTECs injury. This hypothesis was based on the assessment of the correlation between C-megalin urinary excretion, excretion of β2 microglobulin/Creatinine ratio and Serum α1 microglobulin/Creatinine ratio all markers for renal PTECs dysfunction; they also enlightened that urinary loss of C-megalin might be linked to the reduction of serum 25(OH)D.
Megalin cooperates with various intracellular proteins and other membrane molecules, including the cubilin-amnionless complex [74] (Figure 1). Cubilin is a structurally unique, peripheral membrane protein [75] co-expressed with megalin in the apical endocytic compartments of the proximal tubule [76]. It is another endocytic DBP receptor and its genetic defects may induce urinary loss of DBP and a decrease in plasma Vitamin D levels [77,78].
Bonnet et al. [79] reported, in an experiment on rodents, that cubilin expression is mediated by a VDR-dependent mechanism and that cubilin has a major role in 25(OH)D3 uptake by adipocytes. In these cells, Vitamin D not only is stored in lipid droplets but could also be converted to active metabolites modulating adipocyte biology and 25(OH)D3 mobility [80].
Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal Vitamin D metabolism; in the same way, human patients with mutations causing cubilin dysfunction show abnormal urinary excretion of 25(OH)D3 [78].
Amsellem et al. [81], by employing a set of genetic models allowing inactivation of cubilin, megalin or both simultaneously, described the essential role of cubilin in albumin reabsorption by proximal tubule cells and described the mechanism of internalization of cubilin-albumin complexes. This function of cubilin has a relevant role in Vitamin D homeostasis; indeed, only 85% of Vitamin D is bound to DBP, there is a 15% bound to albumin [82].

FGF23-Klotho and Vitamin D Axis in Endocrine-Paracrine Renal Loop
FGF23, Vitamin D and Klotho have a central role in the homeostasis of tubular function, primarily regulating renal calcium and phosphate reabsorption. When the phosphate level is increased in the urine, this will contribute to tubular injury and interstitial fibrosis [83].
FGF23 is a hormone mainly produced and secreted by osteocytes and osteoblasts, even if different tissues can generate it [84,85]. FGF23 is crucial in the phosphorus homeostasis and acts through interactions with the FGF23 co-receptor α-Klotho (KL) and FGF receptors in the kidney [86]. FGF23 is a primary α-klotho-dependent endocrine regulator of mineral homeostasis, functioning to activate Vitamin D and as a phosphatonin [87].
Genetic inactivation of FGF23, klotho or both of the genes has resulted in markedly increased kidney expression of 1-alpha hydroxylase and concomitant elevated serum levels of 1,25(OH)2D3 [88].
In the early stages of CKD, serum FGF23 levels can rise 1000-fold above normal values. This condition is determined by the need to maintain acceptable phosphate levels [89]. The overexpression of FGF23 is linked to hypophosphatemic rickets/osteomalacia, in contrast, a decrease in the action of FGF23 results in hyperphosphatemic calcinosis with high 1,25(OH)2D3 levels [90]. Vitamin D, through its action on VDR, regulates the promoter region in the FGF23 gene and locally produced 1,25(OH)2D3 in bone cells regulates FGF23 generation [91,92]. 25(OH)D3 hydroxylation is inhibited by FGF23 through the action of extracellular signal-regulated kinase 1/2 (ERK 1/2) [93].
FGF23 performs a phosphaturic action through the inhibition of phosphate proximal tubular resorption by sodium phosphate cotransporters NaPi2a and NaPi2b [94]. This phosphate reabsorption inhibition is mediated by a Klotho-dependent activation of extracellular signal-regulated ERK1/2 and of serum/glucocorticoid-regulated kinase-1, leading to phosphorylation of the scaffolding protein Na + /H + exchange regulatory cofactor and internalization/degradation of sodium-phosphate cotransporters [95].
FGF23 works in close synergy with α-Klotho, a multifunctional protein that is predominantly expressed in kidney tubular epithelium and is also implicated in the metabolism of phosphate, calcium and Vitamin D. In the study of Ozeki et al. [101] on a cohort of cardiac patients with CKD, estimated glomerular filtration rate (eGFR) was correlated negatively with FGF23 and positively with α-Klotho serum levels.
There is also evidence that Klotho may regulate renal phosphate reabsorption by an FGF23 independent mechanism [102]. Hu et al. [103] reported that Klotho can act as an autocrine phosphaturic factor by altering the function of NaPi2a in renal PTECs through its glucuronidase activity.
Qian et al. [104] demonstrated that recombinant Klotho treatment protects renal tubular epithelial cells from ischemic-reperfusion injury by inhibiting the oxidative stress that can provoke necroptotic cell death in acute kidney injury.
Immunoreactivity of Klotho is closely associated with proliferation in the intercalated cells of the connecting tubules and cortical collecting ducts and may be implicated in the regulation of tubular proliferation [105].
The close link between Vitamin D and the FGF23/Klotho axis needs further investigation to fully understand the optimal strategy to prevent, retard and inhibit the comorbidity of tubular and renal dysfunction.

Vitamin D in Primitive Renal Tubular Disorders
Primitive renal tubular disorders are a group of diseases leading to fluid loss and abnormalities in the electrolyte and acid-base balance.
Many tubulopathies cause chronic dehydration, salt wasting or acidosis, while excessive phosphate loss leads to rickets in young patients and altered bone development [106].
One of the most investigated tubulopathies is Fanconi syndrome. This is characterized by a massive dysfunction of the proximal tubule that leads to glucosuria, phosphaturia, generalized aminoaciduria, and type 2 renal tubular acidosis [107]. Primary inherited Fanconi syndrome is provoked by a mutation in the NaP i -II in the proximal tubule. Recent studies have established new causes of Fanconi syndrome due to mutations in the EHHADH and the HNF4A genes [108].
Taylor et al. [109] reported the case of an adult patient showing type 2 renal tubular acidosis with Fanconi syndrome, osteomalacia, osteoporosis and secondary hyperaldosteronism. After 24 months of Cholecalciferol and calcium therapy, the lumbar spine T-score improved to −2.0, and the femoral neck T-score improved to −2.7. Bone biopsy specimens demonstrated the resolution of the osteomalacia. Ali et al. [110] also presented the case of a young female who had type 2 renal tubular acidosis and Fanconi syndrome and excellently responded to Cholecalciferol treatment. Baran et al. [111] described a case of incomplete Fanconi syndrome (without metabolic acidosis) treated with 1,25(OH)2D3 and hydrochlorothiazide with improvement in calcium and phosphorus balance.
Another inherited renal tubular disorder is Bartter syndrome, which is generated by defective salt reabsorption in the thick ascending limb of the loop of Henle, leading to salt wasting, hypokalemia and metabolic alkalosis [112]. Li et al. [113] described a case of type 2 Bartter syndrome positively treated with celecoxib and Vitamin D. Krishnamurthy et al. [114] presented a case of Bartter syndrome with Vitamin D-resistant rickets, probably secondary to the calcipenic effect of hyperprostaglandinemia or phosphate loss in urine.
In the literature, there are even two cases of Imerslund-Gräsbeck syndrome related to cubilin and amnionless (CUBAM) receptor proteins, both expressed in the small intestine as well as in the proximal tubules of the kidney. In these two cases, cubilin impairment led to loss of Vitamin D-binding protein and severe Vitamin D deficiency [115,116].
Vitamin D and phosphates are the conventional therapy for X-linked hypophosphatemia (XLH), an FGF23-mediated disorder characterized by impaired renal tubular reabsorption of phosphate. It is a rare affection caused by mutations in the PHEX gene. It encodes for an endopeptidase that is primarily expressed on the surface of osteoblasts, osteocytes, odontoblasts and cementoblasts [117]. XLH follows an X-linked mode of transmission with dominant expression and represents the first cause of hypophosphatemic rickets [118].
Liu et al. [95] treated mice suffering from XLH with daily 1,25(OH)2D3 repletion, FGF23 antibodies or biweekly high-dose of 1,25(OH)2D3 from d2 to d75 without supplemental phosphate to establish if 1,25(OH)2D3 or blocking FGF23 activity could improve the skeletal phenotype without phosphate supplementation. Their group demonstrated that daily 1,25(OH)2D3 supplementation was more efficient than other supplementations in regulating the expression of the growth plate and metaphyseal organization and had a relevant role in normalizing bone geometry and lumbar vertebral height and body weight.
The recent development and approval of an anti-FGF23 antibody, burosumab, for use in XLH provides a novel treatment option, alternative to Vitamin D therapy [119].
There is still much to be done for the treatment of tubulopathies, and atypical cases will continue to pose major challenges for physicians and researchers.

Conclusions
Chronic inflammation is a common feature in patients with renal impairment or tubular injury. This alteration originates from oxidative stress, acidosis, decreased clearance of inflammatory cytokines and stimulation of inflammatory factors. The challenge is to find the right formula for each patient to correctly modulate the landscape of treatment and preserve the essential functions of the organism without perturbing its homeostasis.
Experimental models in vitro, as well as animal and human studies, demonstrated the protective role of Vitamin D against inflammatory injuries and how the use of the different formulations of this hormone could be a tool in broader therapeutic strategies. The complexity of the counter-regulation mechanisms and the different axis involved in the Vitamin D equilibrium pose a major issue on Vitamin D as a potential effective anti-inflammatory drug. The therapeutic use of this compound should be able to inhibit the development of inflammation without interfering with normal homeostasis. Megalin-Cubilin-Amnionless and FGF23-Klotho axes represent two Vitamin D-linked mechanisms that could modulate and ameliorate the damage response at the renal tubular level, balancing Vitamin D therapy with an effect potent enough to contrast the inflammatory cascades, but which avoids potential severe side effects [120].
More research is needed to confirm these findings and to better understand the different anti-inflammatory pathways involved in renal fibrogenesis and how to preserve the tubular segments, fundamental for metabolic homeostasis. The potential benefits of Vitamin D in the prevention and treatment of immune and inflammatory conditions are encouraging; the current answers that experimentation and clinical practice provide us are still not satisfactory.
Author Contributions: Literature search G.G., R.S.; conceptualization G.G., D.S., V.C.; bias assessment; study selection G.G., D.S.; manuscript drafting G.G., V.C., A.C.; Graphical Abstract design: G.G., F.C., critical revision and approval of the manuscript, all authors. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Conflicts of Interest:
All authors have read and approved the final version of the manuscript. All authors are aware of journal's conflict-of-interest policy; to the best of our knowledge, none of the authors has any direct or indirect conflicts of interest. [1,25 (OH)