Nitride oxide synthase 3 and klotho gene polymorphisms in the pathogenesis of chronic kidney disease and age-related cognitive impairment: a systematic review and meta-analysis

Introduction

Aging had remained a challenging topic since the last three decades¹. Some large scale studies have been developed to clarify the precise mechanism of how aging affects the body^2,3 and also how to prevent this circumstance⁴. This is because aging is a normal condition in human life. This means that this proccess has to occur in everyone. Recently, to avoid this circumstance, studies have concerned to elucidate aging because aging is known to correlate with age-related diseases including cardiovascular disease⁵, stroke⁶, dementia⁷, and chronic kidney disease (CKD)⁸. Of these, CKD was considered the more serious disease because it was proven to associate with high risk of mortality and poor quality of life⁹. It is widely known that patients with stage V CKD should be treated with regular dialysis and or even renal transplantation¹⁰.

CKD is a fatal disease for most populations¹¹. The investigation regarding the better treatment option for this disease had not provided significant development in developing countries. In the context of aging, this might involve several mediators, including estrogen, androgen, L-arginine, nitride oxide synthase (NOS), and klotho¹². Of these, only NOS and klotho have been well reported in genetic levels and in clinical settings in the context of aging and CKD. Other mediators, during this time, were only proposed as theory or hypothesis. The absense of direct clinical investigation regarding those mediators in the context of aging and CKD led to these mediators being considered as correlated with aging and CKD. The lack of studies investigating aging in clinical practice might be due to the fact that the definition of aging is complex, and it can be difficult to determine the appropriate scope of aging. However, aging is widely to correlate with age-related cognitive impairment¹³. For this reason, in our present study, our investigation only concerned age-related cognitive impairment. Furthermore, investigating the role of NOS and klotho gene polymorphisms in the case of age-related cognitive impairment and CKD was logical and crucial for better understanding concerning the development of aging and CKD. Moreover, due to conflicting reports regarding this topic, a meta-analysis study was required to elucidate the real association.

Our current study, therefore, aimed to perform a meta-analysis concerning the role of NOS and klotho gene polymorphisms in the case of age-related cognitive impairment and CKD. Our present study might provide better understanding on which allele or genotype of NOS and klotho gene polymorphisms are associated with the risk of age-related cognitive impairment and CKD.

Methods

Results

Paper selection

Our final paper selection identified 21 papers^22–42 assessing NOS3 G894T gene polymorphisms in age-related cognitive impairment; three papers^22,43,44 assessing NOS3 T786C gene polymorphisms in age-related cognitive impairment; five papers^45–49 assessing KL G395A gene polymorphisms in age-related cognitive impairment; ten papers^50–59 evaluating NOS3 4b/4a gene polymorphisms in CKD; seven papers^{55–57,59–62} evaluating NOS3 G894T gene polymorphisms in CKD; three papers^50,55,63 assessing NOS3 T786C gene polymorphisms in CKD; six papers^64–69 assessing KL G395A gene polymorphisms in CKD; and three papers^66–68 assessing NOS3 C1818T gene polymorphisms in CKD. This number of papers were searched in PubMed, Embase, Cochrane, and Web of Science; and papers were selected in accordance with inclusion and exclusion criteria. In the initial searching, we identified 10,858 papers. Of those, 10,787 papers were excluded because of irrelevant topic. Moreover, 13 papers were also excluded because of the following reasons: review (seven), not providing required data for calculation of OR and 95%CI (four), and being of low-quality in accordance with NOS assessment (two). A flowchart describing eligibility pathway in our study is provided in Figure 1.

Figure 1. PRISMA flowchart of paper selection in our study.

Data synthesis

For age-related cognitive impairment, we identified three SNPs available for meta-analysis calculation, including NOS3 G894T, NOS3 T786C, and KL G-395A. Of those, the correlation was only found in NOS3 G894T gene variant. Conversely, we failed to clarify the correlation between the risk of age-related cognitive impairment and NOS3 T786C and KL G395A gene polymorphism. For NOS3 G894T, we found that increased risk of age-related cognitive impairment (Figure 2A) was observed in GG genotype of NOS3 G894T gene polymorphism (OR [95%CI] = 1.14 [1.01 - 1.30], p = 0.0320). On other hands, reduced risk of age-related cognitive impairment (Figure 2B) was found in GT genotype of NOS3 G894T gene variant (OR [95%CI] = 0.86 [0.75 - 0.97], p = 0.0170). The summary of the association between age-related cognitive impairment and the gene polymorphisms in NOS3 and KL is given in Table 1.

Figure 2. Forest plot of the association between NOS3 G894T gene polymorphism and the risk of age-related cognitive impairment.

(A). GG vs. GT+TT; (B). GT vs. GG+TT.

Table 1. Summary of the association between the risk of age-related cognitive impairment and both NOS3 and KL gene polymorphisms.

SNP	Allele & genotype	NS	Model	Value		OR	95%CI	pHet	pE	p-value
				Case (%)	Control (%)
NOS3 G894T	G vs. T	21	Random	75.3	75.1	1.08	0.99 - 1.18	0.0460	0.1210	0.0840
	T vs. G	21	Random	24.7	24.9	0.92	0.85 - 1.01	0.0460	0.1210	0.0840
	GG vs. GT+TT	21	Random	58.2	57.1	1.14	1.01 - 1.30	0.0100	0.1890	0.0320
	GT vs. GG+TT	21	Random	34.2	36.0	0.86	0.75 - 0.97	0.0060	0.2010	0.0170
	TT vs. GG+GT	21	Fixed	7.6	6.9	1.04	0.89 - 1.22	0.6770	<0.0001	0.6100
NOS3 T786C	T vs. C	3	Fixed	75.7	80.2	0.93	0.81 - 1.07	0.6130	<0.0001	0.3010
	C vs. T	3	Fixed	24.3	19.8	1.08	0.94 - 1.24	0.6130	<0.0001	0.3010
	TT vs. TC+CC	3	Fixed	60.0	66.1	0.94	0.79 - 1.13	0.4960	<0.0001	0.5120
	TC vs. TT+CC	3	Fixed	31.3	28.1	1.00	0.84 - 1.19	0.5520	<0.0001	0.9980
	CC vs. TT+TC	3	Fixed	8.7	5.8	1.20	0.88 - 1.64	0.6970	<0.0001	0.2500
KL G395A	G vs. A	5	Random	84.6	84.7	0.93	0.73 - 1.18	0.0160	0.2210	0.5350
	A vs. G	5	Random	15.4	15.3	1.08	0.85 - 1.37	0.0160	0.2210	0.5350
	GG vs. GA+AA	5	Random	70.3	71.1	0.92	0.72 - 1.16	0.0450	0.2070	0.4730
	GA vs. GG+AA	5	Fixed	28.6	27.2	1.08	0.93 - 1.26	0.2520	0.1020	0.3060
	AA vs. GG+GA	3	Random	1.1	1.7	1.05	0.34 - 3.27	0.0280	0.8490	0.9270

SNP, single nucleotide polymorphism; NS, number of studies; OR, odd ratio; pHet, p heterogeneity; pE, p Egger.

For NOS gene polymorphisms in CKD patients, we identified three SNPs, NOS3 4b4a, NOS3 G894T, and NOS3 T786C. For NOS3 4b4a (Figure 3A), we found that only the 4a4a genotype was associated with increased risk of CKD (OR [95%CI] = 2.09 [1.43 - 3.06], p < 0.0001). For NOS3 G894T, we found that the T allele (Figure 3B) and TT genotype (Figure 3C) were, by 1.65 and 2.08-fold, respectively, associated with increased risk of CKD. Conversely, the G allele and GG genotype were associated to decreased risk of CKD. Moreover, for NOS3 T786C, our findings failed to confirm the correlation in CKD patients.

Figure 3. Forest plot of the association between NOS3 gene polymorphism and the risk of CKD.

A). 4a4a vs. 4b4b+4b4a of NOS3 4b/4a; B). T vs. G of NOS3 G-894T; C). TT vs. GG+GT of NOS3 G-894T.

Furthermore, for klotho gene polymorphisms in CKD patients, only two SNPs were compatible for our analysis, KL G-395A and KL C1818T. For KL G395A, we included six papers consisting of 550 cases and 1131 controls. Of those, G allele and GG genotype were observed having protective effect against CKD, and A allele (Figure 4A) and GA genotype (Figure 4B) were found susceptible for CKD. Moreover, for klotho C1818T, we failed to show the correlation in CKD patients. The summary of the correlation between the risk of CKD and the gene polymorphisms in NOS3 and KL is described in Table 2.

Figure 4. Forest plot of the association between KL G395A gene polymorphism and the risk of CKD.

(A). A vs. G of KL G-395A; (B). GA vs. GG+AA of KL G395A.

Table 2. Summary of the association between the risk of CKD and both NOS3 and KL gene polymorphisms.

SNP	Allele & genotype	NS	Model	Value		OR	95%CI	pHet	pE	p-value
				Case (%)	Control (%)
NOS3 4b4a	4b vs. 4a	10	Random	83.5	86.4	0.80	0.60 - 1.07	<0.0001	0.4140	0.1300
	4a vs. 4b	10	Random	16.5	13.6	1.25	0.94 - 1.68	<0.0001	0.4140	0.1300
	4b4b vs. 4b4a+4a4a	10	Random	70.6	74.8	0.81	0.59 - 1.12	<0.0001	0.4490	0.2000
	4b4a vs. 4b4b+4a4a	10	Random	25.7	23.1	1.13	0.86 - 1.48	<0.0001	0.3610	0.3950
	4a4a vs. 4b4b+4b4a	9	Fixed	3.7	2.1	2.09	1.43 - 3.06	0.1170	0.5110	<0.0001
NOS3 G894T	G vs. T	7	Random	69.1	80.2	0.61	0.45 - 0.82	0.0030	0.3310	0.0010
	T vs. G	7	Random	30.9	19.8	1.65	1.22 - 2.23	0.0030	0.3310	0.0010
	GG vs. GT+TT	7	Random	51.0	66.0	0.59	0.42 - 0.84	0.0160	0.3570	0.0030
	GT vs. GG+TT	7	Random	36.3	28.3	1.29	0.87 - 1.93	0.0020	0.4470	0.2070
	TT vs. GG+GT	6	Fixed	12.7	5.7	2.08	1.46 - 2.97	0.3930	0.1010	<0.0001
NOS3 T786C	T vs. C	3	Random	76.4	73.5	0.80	0.45 - 1.45	0.0040	0.4720	0.4710
	C vs. T	3	Random	23.6	26.5	1.24	0.69 - 2.25	0.0040	0.4720	0.4710
	TT vs. TC+CC	3	Random	59.2	56.1	0.81	0.40 - 1.63	0.0090	0.5460	0.5570
	TC vs. TT+CC	3	Random	34.4	34.9	1.21	0.66 - 2.22	0.0290	0.4520	0.5340
	CC vs. TT+TC	3	Fixed	6.4	9.0	1.07	0.59 - 1.94	0.3240	0.2080	0.8280
KLG395A	G vs. A	6	Random	74.2	84.4	0.40	0.20 - 0.77	<0.0001	0.7720	0.0070
	A vs. G	6	Random	25.8	15.6	2.53	1.29 - 4.96	<0.0001	0.7720	0.0070
	GG vs. GA+AA	6	Random	56.0	70.0	0.36	0.17 - 0.76	<0.0001	0.8660	0.0070
	GA vs. GG+AA	6	Random	36.4	28.7	2.08	1.16 - 3.72	<0.0001	0.6230	0.0140
	AA vs. GG+GA	4	Random	7.6	1.2	2.96	0.84 - 10.42	0.0690	0.9400	0.0910
KL C1818T	C vs. T	3	Fixed	78.2	81.8	0.96	0.76 - 1.21	0.7020	<0.0001	0.7160
	T vs. C	3	Fixed	21.8	18.2	1.05	0.83 - 1.32	0.7020	<0.0001	0.7160
	CC vs. CT+TT	3	Fixed	59.3	65.4	0.96	0.72 - 1.27	0.7000	<0.0001	0.7600
	CT vs. CC+TT	3	Fixed	38.0	33.0	1.03	0.77 - 1.37	0.5620	<0.0001	0.8530
	TT vs. CC+T	2	Fixed	2.8	1.7	1.05	0.42 - 2.63	0.1800	1.0130	0.9130

SNP, single nucleotide polymorphism; NS, number of studies; OR, odd ratio; pHet, p heterogeneity; pE, p Egger.

Discussion

Our current study assessed the correlation between age-related cognitive impairment and the gene polymorphisms in NOS3 (NOS3 G894T and NOS3 T786C). Our results revealed that age-related cognitive impairment was not related to the gene polymorphism of NOS3 T786C. On other hands, we found that the GG genotype was found to correlate with susceptibility to age-related cognitive impairment, and GT genotype was found to have a protective effect against age-related cognitive impairment. Our findings were consistent with those of a previous study⁷⁰. They also found that GG genotype of NOS3 G894T was proven to associate with increased the susceptibility to age-related cognitive impairment. The evidence had confirmed that the polymorphism of NOS3 G894T had been shown to correlate with NO basal production and NOS3 enzyme activity⁷¹. Moreover, elevated NOS3 expression was also found to correlate with increased mitochondrial function in neurons⁷². Therefore, it made sense that the NOS3 G894T gene polymorphism was associated with age-related cognitive impairment as reported in our study. On other hands, we also reported the NOS3 gene polymorphism in the case of CKD. Our results identified three SNPs available for the calculation of meta-analysis. However, the association with the risk of CKD was only observed in 4b/4a and G894T NOS3 gene polymorphisms. For 4b/4a, our findings revealed that the 4a4a genotype was associated with increased risk of CKD. For the G894T gene polymorphism, we found that the T allele and TT genotype were observed to correlate with increased risk of CKD. Previous meta-analysis regarding this topic had been conducted^73,74. However, our current study provided the update, and our current results were consistent with previous studies. Additionally, supporting our results, a previous study confirmed that the TT genotype and T allele of the G894T polymorphism, but not 4b/4a, were associated with the lower level of NO in circulation⁷⁵ and enzyme activity⁷⁶. Furthermore, lower NO levels compared to control was found in patients with CKD⁷⁷, although increased levels of NO were observed in CKD patients after dialysis⁷⁸. The similarity of the dominant role of the NOS3 G894T gene polymorphism, both in age-related cognitive impairment and CKD, might explain the bridging mechanism between aging and CKD with NO involvement, in the context of gene-disease and gene-gene interactions.

The precise mechanism of NO in age-related cognitive impairment and CKD is undefined. However, some speculation may be proposed. Briefly, NO plays a significant role in cell growth and renal vasculature. It is widely known that NO plays as a vascular vasodilator. Additionally, NO may also inhibit the growth of mesangial cell and matrix production. The decreased level of NO in aging may cause to renal vasoconstriction, sodium retention, and increased matrix production and mesangial fibrosis⁷⁹. Moreover, NO isoforms are observed at higher levels in the medullary region than other regions. On other hands, in the renal cortex, the levels of NO isoforms are reduced. Therefore, they may contribute to the reduced perfusion of renal cortex in the elderly⁸⁰. The precise pathway of decreased level of NO in elderly remains confusing. However, several mechanisms have been proposed. First, oxidative stress is known to increase with age. It may stimulate to decrease the key factors for normal NO production, for example tetrahydrobiopterrin⁸¹. Second, L-arginine is known to be key for the production of NO. The availability of this substrate may decline with advance age. While L-arginine is not an essential amino acid, a study had reported that the level of L-arginine was observed decreased in older rats. This indicates that L-arginine may play a crucial role as an essential amino acid in advance age, and therefore sufficient dietary intake may be required to maintain the NO production⁷⁹. Moreover, L-arginine level in circulation was also found to be significantly lower in patients with CKD than controls, and it was consistent with the level of NO⁷⁷. This suggested the pivotal role of L-arginine and NO in aging and CKD. Third, it is known that NOS is degraded by asymmetric dimethyl arginine (ADMA). Previous study in a rat model revealed that ADMA levels were observed higher in advance age. This suggests that elevated ADMA level may increase the degradation of NOS and cause lower NO production⁸². Supporting this explanation, a study found that ADMA levels in circulation were higher in CKD patients than control, was contrary to the levels of NO and L-arginine⁷⁷. This explanation might bridge the mechanism between NO, aging, and CKD as reported in our present study.

While klotho was considered as one of the important mediators in aging, our findings failed to confirm the association between the KL G395A gene polymorphism and risk of age-related cognitive impairment. However, due to limited sample size, further investigation to assess this correlation was required. On other hands, correlating to CKD, our searching strategy identified KL G395A and C1818T as available for meta-analysis calculation. Our analysis confirmed that the association with CKD was only found in klotho G-395A gene polymorphism. We revealed that the A allele and GA genotype were correlated with increased risk of CKD. Until now, we have failed to obtain a systematic review or meta-analysis in the topic of either klotho in aging or in CKD. Therefore, a direct comprehensive comparison was unable to perform. However, it had been reported that α-klotho protein was related to the G395A polymorphism⁸³, and α-klotho protein in circulation was also proven by a large scale meta-analysis study to have positive correlation with renal function⁸⁴. It means that the lower level of klotho protein, the lower the renal function. Therefore, it might explain the results of our study confirming that the G395A gene polymorphism was correlated with the risk of CKD.

The theory explaining the exact mechanism between klotho, aging, and CKD is complicated and may involve genes, proteins, and target organ damage. At the genetic level, KL is expressed in limited tissues and cell types, and the highest expression is observed in distal convoluted tubules in the kidney and choroid plexus in the brain⁸⁵. Therefore, the klotho protein exists in two forms. One is the trans-membrane form expressed primarily in renal tubular cells, and the other is the secreted form circulating in the blood⁸⁶. Klotho protein level has been shown to correlate with human longevity⁸⁷. However, in advance age, the level of klotho protein is decreased⁸⁸, and this decreased level is associated with increased oxidative stress, proinflammatory cytokine production, and activation of endothelin signal transduction⁸⁹. Furthermore, the interaction between klotho and CKD is complex. It may involve specific signaling axis, defined as the klotho-fibroblast growth factor-23 (FGF23) signaling axis. Briefly, when the body has excessive amounts of phosphate, FGF23 is secreted. Subsequently, in the kidney, FGF23 may promote phosphate excretion into urine and suppress vitamin D synthesis. Consequently, it may induce negative phosphate balance. However, in this circumstance, FGF23 requires klotho to bind and activate FGF receptors. After FGF receptor activation by klotho, FGF23 binds to its receptor⁹⁰. There are four type of receptors for FGF23, such as FGFR1, FGFR2, FGFR3, and FGFR4. However, FGFR1 is the dominant receptor playing in this signaling pathway⁹¹. When the binding between FGF23 and FGFR1 occurs, it may activate extracellular signals - regulated kinase (ERK) and serum/glucocorticoid-regulated kinase (SGK) signals. Furthermore, the phosphorylation of the Na⁺/H⁺ exchange regulatory cofactor (NHERF)-1 by SGK-1 was established to down-regulate membrane expression of sodium phosphate co-transporter NaPi-2a. Consequently, it may cause increasing urinary phosphate excretion⁹². On other hands, the binding between FGF23 and FGFR1 may also suppress the expression of 1α-hydroxylase, the enzyme responsible for the production of 1.25(OH)₂D. Therefore, it may participate to systemic mineral homeostasis and regulate the excretion of phosphate⁹³. In this context, if the level of klotho is decreased, it may lead to lower level of FGF23 and stimulate to hyperphosphatemia, one of the pathological states widely observed in CKD⁹⁴. Moreover, it was also reported that the level of klotho protein was found to decline and it was also accompanied by renal insufficiency in patients with CKD⁹⁵. Additionally, the gene-interaction studies also revealed that the decline of klotho level in subjects with CKD involved specific phenotypes, suggesting that klotho was independently involved in the pathogenesis of CKD^85,95. This explanation might be a benchmark for the results of our study that klotho is an important mediator involved in the development of aging and CKD.

Our results have identified SNPs potentially involved in the pathogenesis of age-related cognitive impairment and CKD. Therefore, our current findings might help to elucidate the precise mechanism of aging and CKD in the perspective of clinical evidence and gene-disease interaction. Despite the limitations of our study, our findings might be as the initial step to develop further investigation for the management of aging and CKD. However, more studies on this topic are required to establish further due to some limitations, especially the wide context of aging that may make it difficult to conduct analysis and also may lead to high potency for bias.

In our current study, several limitations were noted. First, some factors that might influence NOS3 and klotho level including multiple sclerosis⁹⁶, asthma⁹⁷, chronic obstructive pulmonary disease⁹⁸, and cardiovascular disease⁵ were not controlled for. Second, several potential confounding factors, mediators, and compensatory factors that might implicate the final findings of our study were not analyzed. Third, due to relatively small sample size, our findings should be interpreted with caution, considering the potency for bias. Fourth, most of study design in our included studies were cross-sectional. Thus, further studies with involving better study design might be required.

Conclusion

Our present study has identified that NOS3 G894T plays an important role in the pathogenesis of both age-related cognitive impairment and CKD. On other hand, while we have found an association between KL G395A gene polymorphism and the risk of CKD, its correlation with age-related cognitive impairment has not been clarified. Our current study may contribute to better understanding regarding the role of NOS3 and KL in the pathogenesis of age-related cognitive impairment and CKD.

Data availability