Dipeptidyl peptidase-4 inhibition and renoprotection: the role of antifibrotic effects.

PURPOSE OF REVIEW: This article analyzes the potential beneficial effects of dipeptidyl peptidase (DPP)-4 inhibitors on renal diseases. RECENT FINDINGS: The pathological significance of DPP-4, either dependent or independent on catalytic activities, on renal diseases has been reported in preclinical studies. With regard to this, we have shown that damaged endothelial cells are converted to a mesenchymal cell phenotype, which is associated with the induction of DPP-4 in endothelial cells. The endothelial mesenchymal transition may contribute to kidney fibrosis; indeed, the antifibrotic effects of DPP-4 inhibitors have been reported elsewhere. However, even though such potential benefits of DPP-4 inhibitors on renal diseases were shown in preclinical studies, clinical trials have not yet revealed significant benefits in renal hard outcomes of DPP-4 inhibitors. SUMMARY: To completely understand the beneficial effects of DPP-4 inhibitors, both the following studies are required: first, preclinical studies that analyze deeper molecular mechanisms of DPP-4 inhibition, and, second, clinical studies that investigate whether such potential beneficial effects of DPP-4 inhibitors are relevant to the patients in the clinic.

Inhibition of Dipeptidyl Peptidase-4 Activates Autophagy to Promote Survival of Breast Cancer Cells via the mTOR/HIF-1α Pathway.

Autophagy plays a complex role in breast cancer cell survival, metastasis, and chemotherapeutic resistance. Dipeptidyl peptidase (DPP)-4, a therapeutic target for type 2 diabetes mellitus, is also involved in autophagic flux. The potential influence of DPP-4 suppression on cancer biology remains unknown. Here, we report that DPP-4 deficiency promotes breast cancer cell survival via the induction of autophagy by the C-X-C motif chemokine 12 (CXCL12)/C-X-C receptor 4 (CXCR4)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor (HIF)-1α axis. DPP-4 knockdown and DPP-4 inhibitor KR62436 (KR) treatment both increased the levels of LC3II and HIF-1α in cultured human breast and mouse mammary cancer cells. The KR-induced autophagic phenotype in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 and rapamycin. HIF-1α knockdown also suppressed breast cancer autophagy induced by KR. The autophagy inhibitor 3-methyladenine significantly blocked the KR-mediated suppression of cleaved caspase-3 levels and apoptosis in breast cancer cell lines. Finally, we found that the metformin-induced apoptosis of DPP-4-deficient 4T1 mammary cancer cells was associated with the suppression of autophagy. Our findings identify a novel role for DPP-4 inhibition in the promotion of breast cancer survival by inducing CXCL12/CXCR4/mTOR/HIF-1α axis-dependent autophagy. Metformin is a potential drug that counteracts the breast cancer cell survival system.

Adenosine/adenosine type 1 receptor signaling pathway did not play dominant roles on the influence of sodium-glucose cotransporter 2 inhibitor in the kidney of bovine serum albumin-overloaded streptozotocin-induced diabetic mice.

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have been shown to display excellent renoprotective effects in diabetic kidney disease with macroalbuminuria/proteinuria. Regarding the renoprotective mechanism of SGLT2i, a sophisticated hypothesis was made by explaining the suppression of glomerular hypertension/hyperfiltration through the adenosine/adenosine type 1 receptor (A1R) signaling-mediated restoration of the tubuloglomerular feedback mechanism; however, how such A1R signaling is relevant for renoprotection by SGLT2i in diabetic kidney disease with proteinuria has not been elucidated. MATERIALS AND METHODS: Streptozotocin-induced diabetic CD-1 mice were injected with bovine serum albumin (BSA) and treated with SGLT2i in the presence/absence of A1R inhibitor administration. RESULTS: We found that the influences of SGLT2i are essentially independent of the activation of A1R signaling in the kidney of BSA-overloaded streptozotocin-induced diabetic mice. BSA-overloaded diabetic mice showed the trend of kidney damage with higher glomerular filtration rate (GFR) and the significant induction of fibrogenic genes, such as transforming growth factor-ß2 and collagen type III. SGLT2i TA-1887 suppressed diabetes-induced GFR in BSA-overloaded diabetic mice was associated with the significant suppression of transforming growth factor-ß2 and collagen type III; A1R-specific inhibitor 8-cyclopentyl-1,3-dipropylxanthine did not cancel the effects of TA-1887 on either GFR or associated gene levels. Both TA-1887 and 8-cyclopentyl-1,3-dipropylxanthine-treated BSA-overloaded diabetic mice showed suppressed glycated hemoglobin levels associated with the increased food intake. When analyzing the association among histological evaluation, GFR and potential fibrogenic gene levels, each group of mice showed distinct correlation patterns. CONCLUSIONS: A1R signaling activation was not the dominant mechanism on the influence of SGLT2i in the kidney of BSA-overloaded diabetic mice.

Novel PKD2 Missense Mutation p.Ile424Ser in an Individual with Multiple Hepatic Cysts: A Case Report.

We report a novel missense mutation, p.Ile424Ser, in the PKD2 gene of an autosomal dominant polycystic kidney disease (ADPKD) patient with multiple liver cysts. A 57-year-old woman presented to our university hospital with abdominal fullness, decreasing appetite, and dyspnea for three months. A percutaneous drainage of hepatic cysts was performed with no significant symptomatic relief. A computed tomography (CT) scan revealed a hepatic cyst in the lateral portion of the liver with appreciable compression of the stomach. Prior to this admission, the patient had undergone three drainage procedures with serial CT-based follow-up of the cysts over the past 37 years. With a presumptive diagnosis of extrarenal manifestation of ADPKD, we performed both a hepatic cystectomy and a hepatectomy. Because the patient reported a family history of hepatic cysts, we conducted a postoperative genetic analysis. A novel missense mutation, p.Ile424Ser, was detected in the PKD2 gene. Mutations in either the PKD1 or PKD2 genes account for most cases of ADPKD. To the extent of our knowledge, this point mutation has not been reported in the general population. Our in-silico analysis suggests a hereditary likely pathogenic mutation.

The PKM2 activator TEPP-46 suppresses kidney fibrosis via inhibition of the EMT program and aberrant glycolysis associated with suppression of HIF-1α accumulation.

AIMS/INTRODUCTION: Tubulointerstitial fibrosis is a hallmark of diabetic nephropathy and is associated with an epithelial-to-mesenchymal transition (EMT) program and aberrant glycolysis. Dimeric pyruvate kinase (PK) M2 (PKM2) acts as a key protein kinase in aberrant glycolysis by promoting the accumulation of hypoxia-inducible factor (HIF)-1α, while tetrameric PKM2 functions as a pyruvate kinase in oxidative phosphorylation. The aim of the research is to study the effect of PKM2 tetramer activation on preventing kidney fibrosis via suppression of aberrant glycolysis and the EMT program. MATERIALS AND METHODS: In vivo: Streptozotocin (STZ) was utilized to induce diabetes in 8-week-old CD-1 mice; 4 weeks after diabetes induction, proteinuria-induced kidney fibrosis was developed by intraperitoneal injection of bovine serum albumin (BSA: 0.3 g/30 g BW) for 14 days; The PKM2 activator TEPP-46 was also administered orally simultaneously. In vitro: HK2 cells were co-treated with high-glucose media or/and TGF-ß1 and TEPP46 for 48 h, cellular protein was extracted for evaluation. RESULTS: Diabetic mice developed kidney fibrosis associated with aberrant glycolysis and EMT; BSA injection accelerated kidney fibrosis in both the control and diabetic mice; TEPP-46 rescued the kidney fibrosis. In HK2 cells, TEPP-46 suppressed the EMT program induced by TGF-ß1 and/or high-glucose incubation. TEPP-46-induced PKM2 tetramer formation and PK activity resulted in suppression of HIF-1α and lactate accumulation. Specific siRNA-mediated knockdown of HIF-1α expression diminished high glucose-induced mesenchymal protein levels. CONCLUSION: PKM2 activation could restore the tubular phenotype via suppression of the EMT program and aberrant glycolysis, providing an alternative target to mitigate fibrosis in diabetic kidneys.

Endothelial SIRT3 regulates myofibroblast metabolic shifts in diabetic kidneys.

Defects in endothelial cells cause deterioration in kidney function and structure. Here, we found that endothelial SIRT3 regulates metabolic reprogramming and fibrogenesis in the kidneys of diabetic mice. By analyzing, gain of function of the SIRT3 gene by overexpression in a fibrotic mouse strain conferred disease resistance against diabetic kidney fibrosis, whereas its loss of function in endothelial cells exacerbated the levels of diabetic kidney fibrosis. Regulation of endothelial cell SIRT3 on fibrogenic processes was due to tight control over the defective central metabolism and linked activation of endothelial-to-mesenchymal transition (EndMT). SIRT3 deficiency in endothelial cells stimulated the TGFß/Smad3-dependent mesenchymal transformations in renal tubular epithelial cells. These data demonstrate that SIRT3 regulates defective metabolism and EndMT-mediated activation of the fibrogenic pathways in the diabetic kidneys. Together, our findings show that endothelial SIRT3 is a fundamental regulator of defective metabolism regulating health and disease processes in the kidney.

Metformin Mitigates DPP-4 Inhibitor-Induced Breast Cancer Metastasis via Suppression of mTOR Signaling.

The biological influence of antidiabetic drugs on cancer cells and diabetic cancer patients has not yet been completely elucidated. We reported that a dipeptidyl peptidase (DPP)-4 inhibitor accelerates mammary cancer metastasis by inducing epithelial-mesenchymal transition (EMT) through the CXCL12/CXCR4/mTOR axis. Metformin has been shown to inhibit the mTOR signaling pathway. In this study, we investigated whether metformin mitigates breast cancer metastasis induced by a DPP-4 inhibitor via suppression of mTOR signaling. In cultured mouse mammary and human breast cancer cells, metformin suppressed DPP-4 inhibitor KR62436 (KR)-induced EMT and cell migration via suppression of the mTOR pathway associated with AMPK activation. For the in vivo study, metformin intervention was performed in an allograft 4T1 breast cancer model mouse with or without KR. We also analyzed mice transplanted with shRNA-mediated DPP-4 knockdown 4T1 cells. Treatment with metformin inhibited the lung metastasis of DPP-4-deficient 4T1 mammary tumor cells generated by either KR administration or DPP-4 knockdown. Immunostaining of primary tumors indicated that DPP-4 suppression promoted the expression of EMT-inducing transcription factor Snail through activation of the CXCR4-mediated mTOR/p70S6K pathway in an allograft breast cancer model; metformin abolished this alteration. Metformin treatment did not alter DPP-4-deficiency-induced expression of CXCL12 in either plasma or primary tumors. Our findings suggest that metformin may serve as an antimetastatic agent by mitigating the undesirable effects of DPP-4 inhibitors in patients with certain cancers. IMPLICATIONS: Metformin could combat the detrimental effects of DPP-4 inhibitor on breast cancer metastasis via mTOR suppression, suggesting the potential clinical relevance. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/1/61/F1.large.jpg.

Sodium-glucose cotransporter 2 inhibitors in type 2 diabetes patients with renal function impairment slow the annual renal function decline, in a real clinical practice.

AIMS/INTRODUCTION: The aim of this study was to elucidate whether sodium-glucose cotransporter 2 inhibitors (SGLT2is) treatment has any renoprotective effect for type 2 diabetes mellitus patients with an estimated glomerular filtration rate (eGFR) of <60 mL/min/1.73 m2 in clinical practice. MATERIALS AND METHODS: We evaluated the annual eGFR slope in 85 type 2 diabetes mellitus patients with renal impairment, treated with SGLT2is ≥2 years. Each patient's eGFR was <60 mL/min/1.73 m2 at the start of SGLT2is therapy. The calculation of the annual change in eGFR for each patient was obtained by acquired eGFR data before and after 2 years of the initial SGLT2is administration, followed by analysis of the changes in the mean eGFR slope. RESULTS: The participants' mean age was 72.0 ± 9.4 years, and the mean eGFR was 47.1 ± 9.7 mL/min/1.73 m2 at the start of additional treatment with SGLT2is. The mean annual eGFR slope after SGLT2is administration (-0.11 ± 0.20 mL/min/1.73 m2 /year) was significantly slower than before SGLT2is administration (-2.93 ± 0.59 mL/min/1.73 m2 /year; P < 0.0001). Additionally, SGLT2is treatment slowed the annual decline of eGFR, independent of the levels of both the initial eGFR and albuminuria levels before SGLT2is therapy was started. In the patient groups who showed an annual eGFR decline of ≥3 and 1-3 mL/min/1.73 m2 , there was a significant slowing of the decline after SGLT2is therapy, compared with before the treatment (P < 0.001, respectively). CONCLUSIONS: SGLT2is administration slows the decline observed in the annual renal function in type 2 diabetes mellitus patients with eGFR of <60 mL/min/1.73 m2 in clinical practice.

Endothelial autophagy deficiency induces IL6 - dependent endothelial mesenchymal transition and organ fibrosis.

Macroautophagy/autophagy plays a vital role in the homeostasis of diverse cell types. Vascular endothelial cells contribute to vascular health and play a unique role in vascular biology. Here, we demonstrated that autophagy defects in endothelial cells induced IL6 (interleukin 6)-dependent endothelial-to-mesenchymal transition (EndMT) and organ fibrosis with metabolic defects in mice. Inhibition of autophagy, either by a specific inhibitor or small interfering RNA (siRNA) for ATG5 (autophagy related 5), in human microvascular endothelial cells (HMVECs) induced EndMT. The IL6 level was significantly higher in ATG5 siRNA-transfected HMVECs culture medium compared with the control HMVECs culture medium, and neutralization of IL6 by a specific antibody completely inhibited EndMT in ATG5 siRNA-transfected HMVECs. Similar to the in vitro data, endothelial-specific atg5 knockout mice (Atg5 Endo; Cdh5-Cre Atg5 flox/flox mice) displayed both EndMT-associated kidney and heart fibrosis when compared to littermate controls. The plasma level of IL6 was higher in Atg5 Endo compared to that of control mice, and fibrosis was accelerated in Atg5 Endo treated with a HFD; neutralization of IL6 by a specific antibody inhibited EndMT and fibrosis in HFD-fed Atg5 Endo associated with the amelioration of metabolic defects. These results revealed the essential role of autophagy in endothelial cell integrity and revealed that the disruption of endothelial autophagy could lead to significant pathological IL6-dependent EndMT and organ fibrosis. Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; EndMT: endothelial-to-mesenchymal transition; HES: hematoxylin and eosin stain; HFD: high-fat diet; HMVECs: human microvascular endothelial cells; IFNG: interferon gamma; IL6: interleukin 6; MTS: Masson's trichrome staining; NFD: normal-fat diet; siRNA: small interfering RNA; SMAD3: SMAD family member 3; TGFB: transforming growth factor ß; TNF: tumor necrosis factor; VEGFA: vascular endothelial growth factor A.

Renal protective effects of empagliflozin via inhibition of EMT and aberrant glycolysis in proximal tubules.

Sodium glucose cotransporter 2 (SGLT2) inhibitors are beneficial in halting diabetic kidney disease; however, the complete mechanisms have not yet been elucidated. The epithelial-mesenchymal transition (EMT) is associated with the suppression of sirtuin 3 (Sirt3) and aberrant glycolysis. Here, we hypothesized that the SGLT2 inhibitor empagliflozin restores normal kidney histology and function in association with the inhibition of aberrant glycolysis in diabetic kidneys. CD-1 mice with streptozotocin-induced diabetes displayed kidney fibrosis that was associated with the EMT at 4 months after diabetes induction. Empagliflozin intervention for 1 month restored all pathological changes; however, adjustment of blood glucose by insulin did not. Empagliflozin normalized the suppressed Sirt3 levels and aberrant glycolysis that was characterized by HIF-1α accumulation, hexokinase 2 induction, and pyruvate kinase isozyme M2 dimer formation in diabetic kidneys. Empagliflozin also suppressed the accumulation of glycolysis byproducts in diabetic kidneys. Another SGLT2 inhibitor, canagliflozin, demonstrated similar in vivo effects. High-glucose media induced the EMT, which was associated with Sirt3 suppression and aberrant glycolysis induction, in the HK2 proximal tubule cell line; SGLT2 knockdown suppressed the EMT, with restoration of all aberrant functions. SGLT2 suppression in tubular cells also inhibited the mesenchymal transition of neighboring endothelial cells. Taken together, SGLT2 inhibitors exhibit renoprotective potential that is partially dependent on the inhibition of glucose reabsorption and subsequent aberrant glycolysis in kidney tubules.

CD-1db/db mice: A novel type 2 diabetic mouse model with progressive kidney fibrosis.

AIMS/INTRODUCTION: To establish novel therapies to combat diabetic kidney disease, a human disease-relevant animal model is essential. However, a type 2 diabetic mouse model presenting progressive kidney fibrosis has not yet been established. Kidneys of streptozotocin-induced diabetic CD-1 mice showed severe fibrosis compared with other backgrounds of mice associated with the suppression of antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline. The BKS background (BKSdb / db ) is often utilized for diabetic kidney disease research; the kidney fibrosis in the BKSdb / db phenotype is minimal. MATERIALS AND METHODS: We generated CD-1db / db mice by backcrossing the db gene into the CD-1 background, and analyzed phenotypic differences compared with BKSdb / db and CD-1db / m mice. RESULTS: Male CD-1db / db mice appeared to have elevated blood glucose levels compared with those of BKSdb / db mice. Fasting insulin levels declined in CD-1db / db mice. Plasma cystatin C levels tended to be elevated in CD-1db / db mice from 16 to 24 weeks-of-age. Male CD-1db / db mice showed significantly progressive kidney and heart fibrosis from 16 to 24 weeks-of-age when compared with that of age-matched BKSdb / db mice. The gene expression profile showed fibrogenic program-associated genes in male CD-1db / db mice. Male CD-1db / db mice displayed significantly lower urine antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline when compared to that of BKSdb / db at 24 weeks-of-age. The gene expression of prolyl oligopeptidase, the enzyme essential for antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline production from thymosin ß4, was significantly lower in the CD-1 mice. Thymosin ß4 levels were also lower in CD-1 mice. CONCLUSIONS: These results suggest that CD-1db / db mice are a novel type 2 diabetic mouse model with progressive kidney and heart fibrosis.

Case report of superior mesenteric artery syndrome that developed in a lean type 2 diabetes patient and was associated with rapid body weight loss after sodium-glucose cotransporter 2 inhibitor administration.

A 58-year-old women who was diagnosed with type 2 diabetes 20 years earlier had been treated with antidiabetic medicines since she was aged 40 years. After sodium-glucose cotransporter 2 inhibitors administration, her bodyweight rapidly decreased from 40 to 30 kg over a period of 3 weeks. She had abdominal symptoms, including nausea, especially after a meal. On admission, physical examinations and laboratory data showed euglycemic ketoacidosis, dehydration and low insulin secretion levels. Additionally, abdominal contrast computed tomography showed the finding of superior mesenteric artery syndrome. This case urges caution, including rapid excessive bodyweight loss and euglycemic ketoacidosis, on the use of sodium-glucose cotransporter 2 for lean diabetes patients.

Dipeptidyl peptidase-4 plays a pathogenic role in BSA-induced kidney injury in diabetic mice.

Diabetic kidney disease (DKD) is appeared to be higher risk of declining kidney function compared to non-diabetic kidney disease with same magnitude of albuminuria. Epithelial-mesenchymal transition (EMT) program of tubular epithelial cells (TECs) could be important for the production of the extracellular matrix in the kidney. Caveolin-1 (CAV1), dipeptidyl peptidase-4 (DPP-4) and integrin ß1 have shown to be involved in EMT program. Here, we found diabetic kidney is prone for albuminuria-induced TECs damage and DPP-4 plays a vital role in such parenchymal damages in diabetic mice. The bovine serum albumin (BSA) injection induced severe TECs damage and altered expression levels of DPP-4, integrin ß1, CAV1, and EMT programs including relevant microRNAs in type 1 diabetic CD-1 mice when compared to non-diabetic mice; teneligliptin (TENE) ameliorated these alterations. TENE suppressed the close proximity among DPP-4, integrin ß1 and CAV1 in a culture of HK-2 cells. These findings suggest that DPP-4 inhibition can be relevant for combating proteinuric DKD by targeting the EMT program induced by the crosstalk among DPP-4, integrin ß1 and CAV1.

Inhibition of Dipeptidyl Peptidase-4 Accelerates Epithelial-Mesenchymal Transition and Breast Cancer Metastasis via the CXCL12/CXCR4/mTOR Axis.

Dipeptidyl peptidase (DPP)-4 is a multifunctional glycoprotein involved in various biological and pathologic processes. DPP-4 has been widely recognized as a therapeutic target for type 2 diabetes mellitus but is also implicated in the development of human malignancies. Here, we show that inhibition of DPP-4 accelerates breast cancer metastasis via induction of CXCL12/CXCR4, which activates mTOR to promote epithelial-mesenchymal transition (EMT). In cultured cells, DPP-4 knockdown induced EMT and cell migration. Treatment with the DPP-4 inhibitor KR62436 (KR) promoted primary tumor growth and lung metastasis in a 4T1 tumor allograft mouse model; DPP-4 knockdown in 4T1 cells displayed similar phenotypes in vivo and in vitro. KR treatment enhanced the levels of CXCL12/CXCR4 and phosphorylated mTOR, which were associated with the induction of EMT in metastatic cancer cells. KR-induced EMT in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 or the mTOR inhibitor rapamycin, and AMD3100 suppressed KR-induced metastasis in vivo. Our findings suggest that DPP-4 plays a significant role in cancer biology and that inhibition of DPP-4 promotes cancer metastasis via induction of the CXCL12/CXCR4/mTOR/EMT axis. SIGNIFICANCE: These findings reveal that inhibition of DPP-4 increases the metastatic potential of breast cancer. This is especially important given the potential use of DPP-4 inhibition as a therapeutic strategy for type 2 diabetes.

Ipragliflozin improves mitochondrial abnormalities in renal tubules induced by a high-fat diet.

AIMS/INTRODUCTION: Complete mechanisms of renoprotective effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors have not been elucidated yet. Mitochondrial biogenesis is regulated by membrane GTPases, such as optic atrophy factor 1 and mitofusion 2. Here, we investigated whether SGLT2 inhibition in mice fed with a high-fat diet (HFD) improved mitochondrial morphology and restored mitochondrial biogenesis-related molecules. MATERIALS AND METHODS: Mice were fed a control diet or HFD with or without ipragliflozin treatment. After 16 weeks, the kidneys were taken out and utilized for the analysis. RESULTS: HFD-fed mice treated with ipragliflozin showed increased caloric intake and ate more food than the control HFD-fed mice. Body and kidney weights, and blood glucose levels were not altered by ipragliflozin treatment in HFD-fed mice. Histological analysis showed that, compared with control mice, HFD-fed mice displayed tubular vacuolation, dilatation and epithelial cell detachment; ipragliflozin ameliorated these alterations. Furthermore, ultrastructural analysis showed that the tubule mitochondria of HFD-fed mice exhibited significant damage. Again, ipragliflozin reversed the damage to a normal state, and restored optic atrophy factor 1 and mitofusion 2 levels in HFD-fed mice. Increased urine 8-hydroxydeoxyguanosine levels in HFD-fed mice were suppressed by ipragliflozin as well. In vitro experiments using HK-2 cells revealed that either high glucose or high palmitate suppressed optic atrophy factor 1 and mitofusion 2 levels. Suppression of SGLT2 by a specific small interfering ribonucleic acid or ipragliflozin restored these GTPase levels to their normal values. CONCLUSIONS: SGLT2 inhibition might act directly on tubular cells and protect kidney tubular cells from mitochondrial damage by metabolic insults regardless of blood glucose levels or improvement in bodyweight reduction.

Severe electrolytes disorders with the interstitial kidney alterations in the patient with the history of total thyroidectomy and parathyroidectomy: possible role of vitamin D deficiency.

Vitamin D plays vital role for the health, and its deficiency has been implicated in the diverse pathological conditions such as hypomagnesemia and abnormal immune system. Here, we present a case of severe electrolytes disorders (hypokalemia and hypomagnesemia etc.) and kidney damages associated with vitamin D deficiency.