Drosophila buzzatii exposed to a hallucinogenic columnar cactus, also consumed by humans, to identify ortholog genes exhibiting adaptive footprints of alkaloid tolerance. Genomic analyses in human populations revealed a suite of ortholog genes evolving under recent positive selection in indigenous populations of the Central Andes. Our results provide evidence of selection in genetic variants related to alkaloids toxicity, xenobiotic metabolism, and neuronal plasticity in Aymara and Quechua populations, suggesting a possible process of gene-culture coevolution driven by religious practices.Cultural transformations of lifestyles and dietary practices have been key drivers of human evolution. However, while most of the evidence of genomic adaptations is related to the hunter-gatherer transition to agricultural societies, little is known on the influence of other major cultural manifestations. Shamanism is considered the oldest religion that predominated throughout most of human prehistory and still prevails in many indigenous populations. Several lines of evidence from ethno-archeological studies have demonstrated the continuity and importance of psychoactive plants in South American cultures. However, despite the well-known importance of secondary metabolites in human health, little is known about its role in the evolution of ethnic differences. Herein, we identified candidate genes of adaptation to hallucinogenic cactus in Native Andean populations with a long history of shamanic practices. We used genome-wide expression data from the cactophilic fly  Populations responded both culturally and genetically to the specific environmental conditions, resulting in further dramatic changes with the advent of agricultural and horticultural societies ~ 8,500 years ago2. One of the best pieces of evidence is the ability to digest novel foods such as lactose of dairy cattle3 and carbohydrates from crops4, or to tolerate potentially toxic substances like alcohol5, salt6 and arsenic7. Despite these examples illustrating how lifestyles and dietary factors can shape global patterns of genetic variation in human populations, the role of ancient traditions has received little attention.The dispersal of human populations out of Africa almost 100,000 years ago has been accompanied by the colonization of almost every terrestrial habitat, resulting in conspicuous ethnic differences across regions8. Shamanic practices employing hallucinogenic plants are found in almost every indigenous group around the world; from the Siberian Koryaks using fungi of the genus Amanita, the Australian aborigines using Pituri herbs (Duboisia hopwoodii), to the popular use of Cannabis by Mongols, Indians and Chinese, the ingestion of Iboga roots (Tabernanthe iboga) in west Africa, or the consumption of Mandrake (Mandragora spp.) in Europe9. In the New World, archaeological investigations have shown that psychotropic cacti have been used in shamanic rituals for almost 10,000 years12. The use of cacti was both common and widespread across the Americas, ranging from southern United States to the northern regions of Chile and Argentina. These plants played a vital role in religion, medicine, ritual life and folklore of major ancient civilizations such as Aztecs, Mayans, and Toltecs, which remain until now e.g.,9. In South America, the consumption of giant columnar cacti of the genus Trichocereus, rich in phenylethylamine alkaloids, is common throughout the cultural continuum of the Andean region that can be traced back almost 6,000 years15. In the regions of Ecuador and Perú, T. pachanoi and T. peruvianus are among the most consumed species, whereas T. bridgesii is also consumed in central Bolivia16, and T. terscheckii is the most commonly ingested cactus at higher latitudes between southern Bolivia and northern Argentina18. All these four species have been equally named “San Pedro” in Spanish, or "Achuma" and other similar variants derived from the Quechua term “Kachum” (Cactus)20 and likely used indistinctly due to their high mescaline content21.Shamanism is an extremely ancient system of religious practices that predominated throughout most of human prehistory, starting at least 30,000 years ago23. The use of these cacti has been especially important to consolidate the politico-religious organization of early Central Andean civilizations. Dominant cultures often implemented cosmological beliefs and shamanic rituals aided by psychotropic plants in sophisticated religious complexes as expansionist strategies24. However, the use of these plants was not limited to religious activities, since its consumption was widely spread and practiced by all members of the society, with a special role of the female gender20. The mode of consumption included brews, dried buttons, raw tissues, cigars, enemas or even through the ingestion of cactophilic snails of the genus Scutalus, which concentrates the hallucinogenic alkaloids14.Common representations of San Pedro and its use are consistently found in stone sculptures, reliefs, ceramics and textiles throughout major cultures of the Central Andes: Caral (3000 BCE), Chavin (1500 BCE), Cupisnique, (900 BCE), Wari (600 BCE), Moche (400 BCE), Nazca (200 BCE), Salinar (200 BCE), Lambayeque (800 CE), Chimu  and Inca 25, including the teratogenic effects of mescaline in hamsters27, it is uncertain if the ancient use of psychotropic cactus has left detectable genetic footprints in Central Andean populations. Currently, alkaloids are a health problem of global importance, with authorities interested in avoiding the exposure of consumers to these toxins in food products28, and as expected, the religious use of psychotropic plants by children and pregnant women in Native communities have raised important concerns30. Notably, early cytogenetic studies in the Huichol Native Mexican population suggested the possibility that their long cultural tradition of consuming hallucinogenic cacti may have selected against inherited cytogenetic abnormalities31 and recent analyses have revealed an important genetic predisposition for drug responses and addictions33. However, the identification of genetic variants related to drug susceptibility has been challenging in human studies.Despite several studies have addressed the cytotoxicity of alkaloids not only in humans, but in several vertebratesDrosophila , and the possibility to perform complex physiological experiments in the latter, provides a valuable tool to identify candidate genes, that would otherwise be difficult to pinpoint. For instance, several studies have utilized D. melanogaster to detect genes implicated in human diseases, to understand the genetic architecture of quantitative traits, or to explore the genetic basis of alcoholism and drug addiction35. However, an important drawback of D. melanogaster is its poorly understood ecology36, hindering the extrapolation of ecological traits to other species.One powerful approach to address physiological adaptation in human genetics is to gain experimental insights from genetic model species. The highly evolutionary conservation among genetic pathways of humans and Drosophila, on the other hand, provide a well-known model of evolutionary ecology, especially useful to elucidate the role of chemical adaptation39. The evolution of cactophily in Drosophila was a major ecological transition resulting in the ability of many species to exploit cacti with high levels of secondary metabolites. In North America, for example, D. mettleri exhibit genomic signatures of positive selection in P-450 gene family related to the detoxification of isoquinoline alkaloids present in Senita (Lophocereus schottii) and Saguaro (Carnegiea gigantea) cacti, while adaptation to Agria (Stenocereus gummosus) and Organ Pipe cacti (Stenocereus thurberi) in D. mojavensis also included GST and UGT gene families38. In South America, the cactophilic D. buzzatii stands as an emerging model species in adaptation genomics41. Originally from the arid zones of central South America, this species utilizes prickly pear cacti of the genus Opuntia as main hosts and columnar cacti of the genus Trichocereus as secondary hosts, representing an interesting transition between tolerance and adaptation to the use of columnar cacti, especially useful to elucidate signatures of natural selection associated to generalized detoxification systems43. Moreover, the main host of D. buzzatii  is a widely consumed food by ancient and contemporary human populations across the Americas, allowing to assess the genetic consequences of switching from a nutritional to a hallucinogenic cactus with relevance in humans. Recently, we have shown that the transcriptional response of D. buzzatti to host shifts is mostly modulated by the phenylethylamine alkaloids of T. terscheckii41. We found that D. buzzatti deploys a wide array of genetic products to mitigate the harmful consequences of ingesting alkaloids, of which many were shown to be under positive selection39.The combined genomic and transcriptomic data of D. buzzatii related to the use of hallucinogenic cacti, also consumed by humans, provides an excellent opportunity to explore selection signatures in Native populations of South America. Herein, we characterized the alkaloid profile of T. terscheckii and used whole-genome expression data related to alkaloid tolerance in D. buzzatii to provide insights into the potential evolutionary role of the ancient shamanic lifestyle of indigenous human populations of the Central Andes  analysis revealed consistent retention times in all samples, with detectable levels of nine identified phenylethylamine alkaloids  spectra confirmed the presence of mescaline and trichocereine as the major components of the chloroform and ether fractions, respectively , and in higher doses of alkaloids. i.e., T. terscheckii vs O. sulphurea (DEG I); and T. terscheckii 2 × alkaloids vs T. terscheckii (DEG II). We found a total of 127 protein-coding genes differentially expressed across both comparisons (Table D. melanogaster (showing informative annotations) for downstream analyses. Specifically, 23 genes were over-expressed and 58 genes were under-expressed in comparison DEG I, while 33 genes were over-expressed and only 3 genes were under-expressed in the comparison DEG II  were enriched in Gene Ontology (GO) terms mostly related to the detoxification metabolism , neuronal processes such as neurotransmitter clearance and serotonergic mechanisms (Aldh), or related to general metabolism   and ATP synthesis (ATPsynC)  Fig.  and S5. nC) Fig. .Figure 3Homo sapiens , nervous system development , oxidative stress , exogenous drug catabolic processes, , alkaloid detoxification (CYP3 and CYP4 family genes), general metabolism  and response to narcotics  , nervous system development , muscle contraction  and response to toxic substances . Overall, our analysis of enrichment of biological pathways in H. sapiens showed similar results to those obtained for D. melanogaster , we searched orthologs sequences in the Human genome. Our analysis revealed a total of 70 genes exhibiting moderate to high orthology confidence with ens Fig. D. The GO) Tables  and S7. er Table  and S8.To investigate potential signatures of recent selection for alkaloids tolerance in human populations, we analyzed the extent of genetic differentiation and haplotype homozygosity in Native communities of the Central Andes exhibiting a long history of consumption of hallucinogenic cacti. For this, we used two genomic datasets including our target groups (within the area of influence of Andean shamanic practices) and reference populations . Data-set 1 included the Aymara and Quechua as target populations and Yukpa and Bari as reference populations, while our Data-set 2 consisted of Aymara and Quechua as target groups, and Wichi and Yanesha as references Fig. -S9. NotiP-values into a Fisher’s combination score (ZF). We identified ten candidate genes showing evidence of evolution under the influence of positive selection in at least one comparison; i.e., genes with identified signal either one , two , three (ALDH2), or four comparisons . Our results were highly consistent when considering either the median or mean values to summarize at the gene level the |iHS| and LRT scores estimated at the variant level, especially when comparing against Bari, Yukpa and Wichi reference populations  for Extended Haplotype Homozigosity, and Likelihood Ratio Test (LRT) score for genetic differentiation . Then, we combined the indexes into gene-level summary statistics (mean and median) to finally merge the resulting ons Fig. . Finallyons Fig. .Figure 4Fst test revealed low genetic differentiation respect to Central Andean groups , while our admixture analysis confirmed a certain degree of shared ancestry with the Andean population44  responsible of alkaloids detoxification and clearance of psychotropic medication in humans54. These findings are in line with our detection of several psychotropic and toxic alkaloids in T. terscheckii. Mescaline, for example, the major hallucinogenic alkaloid of the genus Trichocereus, and 2-phenylethylamine, a precursor of mescaline with mild psychotropic properties55, have both been associated with lethal and teratogenic effects in rodent embryos56. Although little is known on the action of the mescaline derivative alkaloids , early studies suggested no or mild psychoactive effects57, whereas tyramine, N-methyltyramine, hordenine and dopamine are likely precursors of these alkaloids, displaying neuromodulatory properties with possible adverse effects58. In particular, tyramine was reported to exert oxidative changes in the brain of rats similar to that of narcotics59, while excessive intake in humans results in a toxicological response known as the “cheese reaction”, leading to hypertension, migraines, neurological problems and respiratory disorders that can induce heart failure and brain hemorrhage60. Further, hordenine and 3,4-dimethoxyphenethylamine exhibit inhibitory effects on monoamine oxidases responsible for alkaloids degradation, potentiating the physiological effects of cactus alkaloids 62. Moreover, the preparation of San Pedro is usually performed by boiling the plant for several hours to potentiate its effects9, implying that even minority alkaloids could accumulate in significant quantities, influencing both toxic and psychotropic properties50. Indeed, although the hallucinogenic experience is the most relevant attribute of San Pedro cacti, other common physiological effects include respiratory failure, diffuse anxiety, motor dysfunction, partial or total cardiac arrest, or even death50. Further, regular use of hallucinogenic plants by children, pregnant, and breastfeeding women has been widely reported in Native American populations, raising important health concerns30. For instance, medical investigations reported significant associations between drug abuse and maternal and fetal morbidity63, while recent family studies suggest an important inherited predisposition driving the variability to drug response and addiction33. Taken together, our results suggest that the detection of subtle but significant shifts in allele frequencies of genes implicated in alkaloids metabolism could likely be related to their fitness value in the Central Andean region where humans have been consuming San Pedro cacti for millennia.Our in vivo study of transcriptomic responses in the genetic model of cactophilic 66. In fact, dietary adaptation has been a major contributor to human evolution, especially important during the transition from generalized plant diets of early hominids to the increased meat consumption and mastery of fire in Homo, leading to reduced ingestion of plant toxins68. Later cultural transitions to intensive agriculture, horticulture and animal domestication starting ~ 10,000 years ago resulted in further changes of the selective pressures on the human genome. Some common examples of modern instances representing gene-culture coevolution include lactase persistence during adulthood3, tolerance to alcoholic fermentation5, and protection against plant secondary metabolites69. Thus, the standing genetic variation derived from our early hominid diet may have provided the basis for rapid selective responses in shamanic societies with a deep history of consumption of plant secondary metabolites. Our findings are consistent with previous population genomic analyses combining ecological information, which found subtle shifts in allele frequencies of genes related to diets rich in roots and tubers in boreal human populations, suggesting that several SNPs may be involved in the detoxification of plant secondary metabolites65. Indeed, an increasing number of studies are discovering ethnic distributions of selected detoxification genes70, likely associated to novel toxins derived from modern lifestyles71, and recent advances in the field of pharmacogenomics have revealed important variation across populations in SNPs associated with opioid dose variability32. Our finding of signatures of selective sweeps associated with CYP3A43 and especially in CYP3A4 is in agreement with several studies where signals of positive selection in CYP3A genes were found in African, Asian and European populations, suggesting that this locus is sensitive to natural selection66. Moreover, CYP3A43 has been associated with the clearance of neuroleptic drugs (antipsychotics), while CYP3A4 is considered the most important drug metabolizing CYP enzyme in humans , and a major responsible for the metabolism of opioids and other alkaloids with important clinical implications in drug addiction73. Although little is known on the phenotypic effect of most polymorphisms, previous studies have provided evidence of higher activity of the wild-type CYP3A4*1 than that of most isoforms when exposed to the quinine alkaloid72. Thus, the higher metabolic rate associated to the wild-type found in Quechua and Aymara populations could have provided a selective advantage to detoxify cactus alkaloids, contributing to the allele fixation in the Central Andes.It has been argued that genetic differentiation between ethnic groups is largely related to the ingestion of substances and their detoxification24. More than twenty years of archaeological research in northern Peru revealed that the civilization of Caral, the oldest in the Americas , exhibited one of the largest early urban complexes in the world probably based on communal spiritual exaltation74. Detailed studies on the archaeological site have found evidence of cactus trade and numerous shells of the cactophilic snail Scutalus proteus used for concentrating the alkaloids of San Pedro14, suggesting the deep and widespread history of cactus use in the region . Shamanic traditions extended far in time, to the recent Inca Empire, and even survived the suppression of European colonization, which failed to eradicate the use of San Pedro that still persists today50.Elucidating the antiquity and extent of the use of psychotropic cacti in the Andean culture is a difficult task, if not impossible. The lack of writing systems in early Andean societies only allows us to infer the relevance of shamanic traditions through the indirect evidence left in archaeological settlements or in the inertia of the cultural legacy. Notwithstanding, most studies agree that the use of San Pedro seems to have been a critical element of the cultural evolution throughout most of the prehistory of Andean civilizations, even prior to the first expansion associated with the religious power and theocratic rule of Chavın and Cupisnique cultures almost 3,500 years ago76. Notwithstanding, our study has several methodological limitations that require discussion. First, we cannot rule out that the signals of selection detected in our analysis could have been driven by adaptive evolution in response to a complex combination of selective pressures that could also be potentially important for population health. For instance, missense of CTSF has been related to the Kufs neuronal disease47 while variants of COX5A result in lactic acidemia, pulmonary arterial hypertension and failure to thrive77. Some mutations in ATP2A1 can result in a rare autosomal recessive myopathy (Brody disease), characterized by exercise-induced muscle stiffness78, whereas variations in FADS1 have been associated with ethnic differences and medical phenotypes such as metabolic syndrome, abnormal lipid metabolism, and attention deficit disorder/hyperactivity79. In particular, CYP3A is not only a critical genetic contributor to drug clearance, but also has important implications in the endogenous metabolism of several hormones such as estrogens, cortisol and corticosterone associated with cancer risk and sodium retention related to hypertension and pregnancy complications80. The diversity of exogenous and endogenous substrates acting on CYP3A suggests that multiple and even antagonist selective pressures have likely shaped pleiotropic functions and potential trade-offs, resulting in complex genotype–phenotype relationships. Thus, further studies on the endogenous-exogenous metabolism interaction are needed to better understand their relative importance for phenotypic associations and ethnic considerations. Secondly, despite recent studies in Drosophila are significantly increasing our overall understanding of the genetic basis to the susceptibility to psychotropic drugs, such as cocaine, morphine, amphetamine and methamphetamine82, it is worth to note that several genes are likely to be overlooked, partly due to the divergent functionalities and physiological differences between flies and humans. Third, we acknowledge that many human orthologs of our candidate genes might not be shared across our studied datasets, resulting in the underestimation of the number of genes. Indeed, recent comparative studies of flies and humans recognize that the overall low number of reported genes associated to drug response, is likely due to the current low representation of human studies 33. This is especially critical in our case, as the reduced genotyping effort of South American Native communities implied a limited sampling for both our target and reference populations84. Thus, a larger collection of whole-genome sequences is necessary to supply larger sample sizes and SNP density to capture a larger number of genes and discriminate whether positive selection preferentially acted on protein-coding or regulatory regions.Although it is important to note that the effects of selection and demography are difficult to disentangle, the examination of multiple loci should be informative of these processes because population fluctuations result in similar random effects across loci, while positive selection tends to be locus-specific. Furthermore, our outlier approach and the combination of our two tests considering both genetic differentiation and linkage disequilibrium should be robust to the confounding effects of demographyAlthough more efforts are certainly needed to test our hypotheses, this study provides a first step in addressing the complex interplay between cultural and genetic co-evolution in the Central Andes, that would otherwise be extremely difficult to investigate. Our identification of candidate genes for alkaloid tolerance in Andean populations not only contributes to a better understanding of how ancient practices may have contributed to recent human evolution, but also provides insight on the genetic basis of ethnic differences in many disease risks. Several genetic variants related to alkaloid responses are also implicated in addictions, neurological disorders, and the metabolism of many important drugs. Therefore, the identification of ancient adaptive footprints to natural drugs across different ecosystems is fundamental for human health and especially important for the development of personalized genomic medicine. A larger representation of South American genomes combined with functional validation studies will be key to demonstrate the putative role of shamanism in the recent evolution of the human genome.40. Briefly, we collected individuals of D. buzzattii from a wild population of Northwestern Argentina to generate nearly isogenic lines of three conspicuous genotypes . We also collected fresh pieces of their hosts present in the area: the giant columnar cactus Trichocereus terscheckii and the prickly pear cactus Opuntia sulphurea. To assess the effect of the columnar cactus and the hallucinogenic alkaloids on the genomic expression of D. buzzattii, we exposed 1st instar larvae to each cactus, including the addition of the alkaloid fraction of T. terscheckii (2 × the native concentration). Thus, we employed three rearing media  to perform two pairwise comparisons  for differential gene expression analyses (see below). We extracted total RNA from 10 batches of 3rd instar larvae for each genotype , using a TRIzol/RNeasy protocol specific to Drosophila. We used the Illumina paired-end library (insert size: 150–450 bp) and sequenced in a HiSeq 2000 platform with 101-cycle reads, obtaining a mean of 16 Gbp of raw reads per genotype and treatment (NCBI Accession: PRJNA314520).The experimental set up has been previously described in detailT. terscheckii, we collected fresh samples from five individuals at different times of the year to account for temporal variation in alkaloid concentration. The plant material was collected in the northwest of Argentina, in the Valle Fertil Natural Park (public land of the province of San Juan) and the identification of the cactus was performed by Alejandro Saint-Esteven et al85. We isolated alkaloids from plant tissues through the alkaline-CH2Cl2 extraction method40. The chemical profile of the extracted fraction was accomplished by GC–MS (Thermo Scientific EM/DSQ II—Trace GC Ultra AI3000). Alkaloids identification was performed by comparing the retention times and mass spectra with reference standards and database spectra (UMYMFOR and NIST). The relative variation and abundance of each alkaloid was quantified based on the integration of the peaks area across the gas chromatograms. We analyzed five GC quality standards  of the most representative alkaloids present in the genus Trichocereus21: Tyramine; Hordenine; 3,5-dimethoxy-4-hydroxyphenethylamine; 3,4-dimethoxyphenethylamine; 3-methoxytyramine. Given the occurrence of dopamine as a major biosynthetic precursor of mescaline and other substituted phenethylamines in giant columnar cacti21, we performed an acid extraction to pH 3 with 0.1 M HCl solution. Dopamine identification was performed by comparing the retention time and mass spectra of the acidic extract and a dopamine reference standard  through HPLC–tandem Mass Spectrometry (Waters Quattro Premier XE spectrometer). To identify mescaline and trichocereine  alkaloids, we used their solubility differences, extracting with chloroform and ether, respectively86 and purified the solutions by column chromatography (silica gel G 60 Merck) with a mixture of CH2Cl2:MeOH. Major compounds were determined by thin-layer chromatography and identified via 1H-NMR in a Bruker AM-200 MHz spectrometer (details in Supplementary Information). All methods were carried out in accordance with relevant guidelines and regulations. The holotype of the cactus collected in our sampling region is housed at the Herbarium of the Institute of Botany Darwinion in Buenos Aires (voucher No. 8958). Collection permits were issued by the Environmental Ministry of the government of San Juan Province (Argentina), under permit No. 1300–0236.To thoroughly characterize the alkaloid fraction of D. buzzatii, we analyzed differential gene expression between flies reared in: T. terscheckii vs O. sulphurea (DEG I); and T. terscheckii 2 × alkaloids vs T. terscheckii (DEG II). Given that Native communities do not isolate the alkaloids, but rather concentrate all the characteristics of the plant, our first comparison (DEG I) allowed us to account for the differential expression caused by the columnar cactus , while our second comparison (DEG II) allowed us to elucidate the specific effects of high doses of hallucinogenic alkaloids . For the search human orthologs, we combined the significantly differentially expressed genes of both comparisons to contemplate the distinctive effect of the columnar cactus and especially its alkaloid fraction. Raw reads of our nine transcriptomes (three genotypes per treatment) were quality controlled using FASTQC v.0.10.187 and filtered for quality scores ≥ 25 and minimum lengths ≥ 25 bp, resulting in a mean of 12 Gbp per genotype in each treatment. We used the program RSEM v1.2.3088 to estimate gene expression levels in fragments per kilobase of transcript per million mapped reads of protein-coding genes using the reference genome of D. buzzatii39. To analyze the differential gene expression, we used the NOISeqBIO method of the R package NOISeq v2.18.089 with a false discovery rate (FDR) < 0.01, and applied a posteriori filter using a custom script to further reduce possible spurious “noise”. The statistical strategy of NOISeq considers the differences in both the mean expression level and in the order of magnitude to measure changes in gene expression between conditions, and thus identify significantly differentially expressed genes. We searched human ortholog genes from our filtered gene list through the DRSC Integrative Ortholog Prediction Tool (DIOPT) v6.0.190 which facilitates the identification of orthologs by calculating a score for the support of a given orthologous gene-pair relationship, as well as by a weighted score based on the functional assessment of molecular function annotation of all fly-human orthologous pairs predicted by each tool and algorithm. We retained the genes with moderate (best score in the forward or reverse search and DIOPT score >  = 2 or DIOPT score >  = 4) and high confidence rank (best score in both forward/reverse search and DIOPT score >  = 2). Because this tool uses D. melanogaster genes as input, we first performed a cross-species analysis to identify homologous genes with D. buzzatii and carried out a detailed exploration of the functional annotation using g:Profiler91. To interpret the biological information in the context of Homo sapiens , we analyzed our ortholog genes in the Reactome Knowledgebase92.To evaluate the effects of the columnar cactus and its alkaloid fraction on 93) and two reference populations from Northern Andes/Caribbean (Yukpa and Bari from Venezuela94). Data-set 2 consists of three focal ethnic groups of the Central Andes , and two reference populations from the Gran Chaco in Argentina (Wichi), and western Amazonia (Yanesha) in Peru44. While Aymarans, Quechuans and Uros represented our target groups, the reference populations represented our “control” groups  as they constitute putatively culturally divergent and relatively isolated populations95 outside the distribution range of Trichocereus species , resulting in a best model of K = 8 for both data sets  data Table . Data-seies Fig. . We filts Figure . We remo97, after phasing the data using the 1000 Genomes haplotypes as reference96. This analysis revealed the genetic clustering of all Central Andean individuals (both data sets), Bari, Yukpa (Data-set 1), Yanesha and Wichi (Data-set 2) individuals , we performed an additional local fine-scale analysis of genetic structure using FineStructure v4s Figure . We furt Figures  and S8 t Figures . Altoget98 to contrast whether allele frequencies in any of the evaluated populations are significantly differentiated from the putative ancestral genetic background. We performed the TreeSelect test in the Central Andean population, using the reference population of Han Chinese96 in all cases, and one reference population of South America  as summary statistics at the protein-coding gene level. In order to exclude possible stochastic signals of non-selective events, such as genetic drift or demographic fluctuations, we implemented a genome scan approach100 by taking into account the gene-level background for protein coding regions. We computed the median and the mean summary statistics for both |iHS| and LRT scores and estimated gene-level empirical distributions for each genomic background as previously implemented76. Empirical P-values were calculated using the gene-level score distributions generated from the genes in the background genome set. Finally, LRT and |iHS| based scores were combined using the Fisher combination test101:Pi stands for the empirical P-value obtained from the test i. Gene-level formal P-values were finally derived from the X 2 distribution with 4 degrees of freedom101. We thus obtained a total of eight ZFscores per gene for both the mean or the median as summary statistics: two for Data Set 1 (with either Yukpa or Bari as the 2nd reference population for the TreeSelect test), and two for Data Set 2 .For the analyses of positive selection, we employed two alternative approaches: the degree of genetic differentiation and the extended haplotype homozygosity. For the genetic differentiation test, we used the method implemented in the TreeSelect softwareSupplementary Information 1.Supplementary Information 2.


The results showed that the vegetation indices fluctuated smoothly in the time series within the validation sites, and the vegetation indices of multisource satellite images were good measures of long-term vegetation cover in the region; the vegetation indices of the same satellite images showed significant correlations (both R2 and ρ exceeded 0.8), and the vegetation indices of different satellite images  showed more significant correlations (both R2 and ρ exceeded 0.7); the smaller the difference between the original resolutions of satellite images, the more significant the correlation between the extracted NDVI and RVI.Extracting vegetation cover information by combining multisource satellite images can improve the time scale of vegetation cover monitoring, realize encrypted observation in short period, and shorten the regional vegetation remote sensing monitoring cycle. The NDVI and RVI datasets from 2007–2019 were extracted using 9 phases of multisource satellite images  covering Xiaxi, Sichuan. Three typical validation sites representing higher vegetation cover in mountains and no vegetation cover in water bodies in the region, respectively, were selected to extract NDVI and RVI at the corresponding locations. Linear regression and Spearman correlation coefficient ( Vegetation cover is a comprehensive quantitative index indicating the ground cover status of vegetation communities. Meanwhile, vegetation cover is also an important geoenvironmental factor to control soil erosion, soil invasion, and soil desertification. The traditional vegetation cover survey is generally based on manual sampling and observation. Although the measurement accuracy of this method is high, it is difficult to achieve quasi-real-time dynamic monitoring for large-scale vegetation cover survey. Secondly, manual sampling observation cannot obtain historical vegetation cover data. With the development of remote sensing observation technology and the open access to multisource satellite images , the dynamic measurement of vegetation cover over large areas in quasi-real time provides strong support. Several studies have shown that the vegetation indices extracted from satellite images can be a good measure of surface vegetation cover and its spatial and temporal evolution characteristics. For example, the extraction of vegetation indices using Landsat TM/OLI images , SPOT saAt present, studies generally focus on single satellite images to extract vegetation indices, while there are fewer relevant studies on joint multisource satellite images to extract vegetation indices. The differences between satellite payload indicators  make it difficult to combine multisource satellite images for long time-series vegetation cover observation. For example, there are large differences in spatial resolution, spectral range, and revisit period  among Landsat-8, Sentinel-2, and HMS-1/2 satellite images, which make it difficult to unify the joint multisource satellite images on the scale. However, the significance of joint multisource satellite images to extract vegetation cover is (1) to realize the complementary advantages of multisource satellite images, so as to obtain high-resolution and higher-accuracy vegetation cover data; (2) to improve the time scale of satellite images for vegetation cover monitoring and realize long time series regional vegetation cover monitoring; (3) to provide dense satellite images in a short period and shorten the regional vegetation cover monitoring cycle. Therefore, this paper aims to explore the extraction of long time-series vegetation indices based on multisource satellite images . Taking Xiaxi Township in Sichuan Province as an example, the feasibility of joint multisource satellite images to obtain long time series vegetation indices is discussed.2, and the GF-1 fusion image (2m) and digital elevation model show that the middle water system of the area is developed with rivers and sufficient water supply, and the surrounding area is mainly mountainous with good vegetation cover . The administrative area of the test area is 70.98 kmon cover . In addihttps://scihub.copernicus.eu), with 2 image phases imaged in September 2017 and June 2018. GF-1 PMS images are from the China Resources Satellite Center's Land Observation Satellite Data Service Platform, and the imaging time is June 2019. All satellite images (9 issues) covered the test area spatially and the plant growth period temporally, and the total cloudiness in the area was controlled below 5%.Landsat TM/OLI images were obtained from the Earth Resources Observation and Science Center (EROS) of the United States Geological Survey (USGS), with 6 image periods imaged in September 2007, June 2009, June 2011, May 2013, and October 2015 Sentinel-2 MSI images from ESA , FLASSH atmospheric correction , geometric correction , orthorectification correction (tilt and projection aberration correction), image fusion , and image alignment . Finally, a multisource satellite image dataset covering the test area for the long time series from 2007 to 2019 was obtained. The image preprocessing process is shown in (1)NDVI  is calculated by (2)RVI (ratio vegetation index) is calculated by where and denote the near-infrared band and infrared band feature reflectance, respectively.Vegetation Index (VI) refers to various values with certain intellectual significance to vegetation, which are formed by linear or nonlinear combination of multispectral information acquired by satellite sensors. The vegetation index is a characteristic index calculated based on the characteristics of vegetation reflection band to reflect the growth status, coverage, and vegetation volume of the ground. In this paper, two vegetation indices, NDVI and RVI , which aIn this paper, the normalized vegetation index NDVI;  and ratiIn addition, although the 9-period NDVI and RVI extracted from four types of satellite images  cover 12 years (2007–2019) in time scale. However, the NDVI and RVI values within the typical area fluctuated smoothly in the time series, which reasonably reflects the pattern of ground vegetation cover distribution during the growing season in a relatively stable geographic environment. This also indicates that the vegetation index by combining multisource satellite images is still a good measure of the long-term vegetation cover status. Therefore, based on the typical regional statistical results and the spatial distribution of NDVI and RVI of the four types of satellite images in the test area, there is a strong correlation between the long-term serial vegetation cover and the vegetation indices extracted from the multisource satellite images in the test area .R of the linear regression relationship, the higher the correlation between the two indices. Meanwhile, Spearman's correlation coefficient was introduced as a measure of the correlation between the two vegetation indices for the following reasons: (1) Spearman's correlation coefficient is a rank correlation coefficient, which estimates the position of the variables within the overall data for correlation calculation. Because of the positive correlation that the larger the vegetation index value (NDVI > 0) is, the larger the ground vegetation cover is, the introduction of Spearman's correlation coefficient can better measure the correlation between vegetation indices; (2) Spearman's correlation coefficient has no limitation on whether the data conform to normal distribution and the sample size. The formula for calculating Spearman's correlation coefficient (ρ) is as follows:In order to verify the correlation and stability between the vegetation indices extracted using multisource satellite images, a one-dimensional linear regression analysis was performed between the vegetation indices calculated from the same satellite and different satellite images. Theoretically, the higher the slope K and the higher the correlation coefficient p > 0.05) and were statistically significant, and the coefficient of determination R2 and Spearman correlation coefficient ρ were both higher than 0.6. This indicates that all four types of image NDVI showed significant correlation between NDVI at the confidence level of 0.05. By longitudinal comparison, it can be seen that the determination coefficients R2 between Landsat TM, Landsat OLI, and Sentinel MSI images with different imaging times reach 0.8043, 0.8513, and 0.8383, respectively (R2 and Spearman's correlation coefficients are over 0.7), which represent the strong correlation between NDVI of different satellite images in different time phases. This also indicates that different satellite images of different phases can be used jointly for vegetation cover monitoring in the same area, thus extending the temporal coverage of regional vegetation cover status and achieving encrypted observation in short time period to a certain extent and obtaining quasi-real-time vegetation cover observation results with longer time series or shorter revisit period than that of a single satellite. As shown in R2 and correlation coefficient ρ between Landsat TM and Landsat OLI image NDVI, Landsat OLI and Sentinel MSI image NDVI, and GF-1 PMS and Landsat TM image NDVI are basically lower than 0.7, indicating that the correlation between these three images NDVI is low. This low correlation will make the uncertainty of NDVI results increase throughout the monitoring cycle and introduce bias to further constrain the vegetation cover.The correlation analysis of NDVI of the four types of images produced eight linear regression equations and corresponding Spearman correlation coefficients to represent the correlation between NDVI of the same satellite and different satellite satellite images, respectively. Specifically, between Landsat TM images, between Landsat OLI images, between Landsat TM and Landsat OLI images, between Sentinel MSI images, between GF-1 PSM and Sentinel MSI images, between GF-1 PSM and Landsat OLI images, between Landsat OLI and Sentinel MSI images, and the correlation between GF-1 PSM and Landsat TM images. The linear regression equations all passed the significance test  analysis were used to verify the correlation between NDVI and RVI from multisource satellite images. The results show that the NDVI and RVI extracted from the validation sites can well distinguish vegetation cover from nonvegetation cover, and the two vegetation indices fluctuate smoothly along the time series, and the vegetation indices of multisource satellite images can well measure the long-term vegetation cover in the region; the vegetation indices of the same satellite images show significant correlation (R2 and ρ exceed 0.8), and the vegetation indices between different satellite images  showed significant correlation (R2 and ρ exceeded 0.7), while the correlation between vegetation indices of other different images was low; through horizontal and vertical comparison of correlation coefficients.The NDVI and RVI datasets for 2007–2019 were extracted using 9-phase multisource satellite images  covering the experimental area. Three typical validation sites representing higher vegetation cover in mountains (R1 and R3) and no vegetation cover in water bodies (R2) in the region were selected to extract NDVI and RVI at the corresponding locations. Linear regression and Spearman correlation coefficient (


Astrocytes and microglia are non-neuronal cells that maintain homeostasis within the central nervous system via their capacity to regulate neuronal transmission and prune synapses. Both astrocytes and microglia can undergo morphological and transcriptomic changes in response to infection with human immunodeficiency virus (HIV). While both astrocytes and microglia can be infected with HIV, HIV viral proteins in the local environment can interact with and activate these cells. Given that both astrocytes and microglia play critical roles in maintaining neuronal function, it will be critical to have an understanding of their heterogeneity and to identify genes and mechanisms that modulate their responses to HIV. Heterogeneity may include a depletion or increase in one or more astrocyte or microglial subtypes in different regions of the brain or spine as well as the gain or loss of a specific function. Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool that can be used to characterise these changes within a given population. The use of this method facilitates the identification of subtypes and changes in cellular transcriptomes that develop in response to activation and various disease processes. In this review, we will examine recent studies that have used scRNA-seq to explore astrocyte and microglial heterogeneity in neurodegenerative diseases including Alzheimer’s disease and amyotrophic lateral sclerosis as well as in response to HIV infection. A careful review of these studies will expand our current understanding of cellular heterogeneity at homeostasis and in response to specific disease states. Until recently, researchers in neuroscience fields focused their efforts primarily on neurons and performed experiments designed to elucidate their structure, function, and responses to injury and infection. By contrast, the roles of non-neuronal cells and tissues have been largely overlooked. Although Rudolf Virchow discovered and provided the first description of glial cells as early as the mid-1800s, these cells were believed to function solely as support for essential neuronal functions. Thus, many of the unique properties and critical functions of glial cells remain to be explored. Various types of glial cells identified within the central nervous system (CNS)  exhibit different functions, localisation, and morphology. For example, microglia have been identified as immune cells that promote phagocytosis of foreign material and cellular debris and facilitate synaptic pruning. By contrast, astrocytes perform surveillance and regulate neurotransmission, whilst oligodendrocytes myelinate axons and radial glia serve as progenitor cells ,2. GivenAstrocytes and microglia undergo functional and transcriptomic changes in association with neurodegenerative disorders that include Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and HIV-associated chronic pain ,4,5,6. WThe goal of this review is to discuss microglial and astrocyte heterogeneity at homeostasis and in response to disease conditions with a particular focus on results from recent studies that feature the use of single-cell (sc) or single-nucleus (sn) RNA sequencing (scRNA-seq).Microglia are primary components of the immune regulatory system of the CNS. However, in contrast to macrophages and other immune cells, microglia do not originate in the bone marrow. Studies carried out in mouse models revealed that microglia originate from myeloid progenitor cells during early embryonic development . AlthougMicroglial morphology and function are closely related phenomena. Microglia are highly ramified in the absence of external stimulation. Whilst the microglial body is comparatively small, several large protruding processes increase its surface area and facilitate sampling to detect changes in the environment. Microglia are highly motile when in this ramified state. In the absence of disease or injury, microglial processes undergo rapid and frequent retraction and growth .Microglia perform multiple roles within the CNS. While they are best known for their capacity to migrate through the CNS to facilitate the detection of foreign material and cellular debris, they are also capable of antigen presentation, phagocytosing myelin, and providing support for neural stem cell proliferation ,16,17,18Cx3cr1, P2ry12, and Tmem119 have been used to identify microglia in experimental studies featuring single-cell RNA sequencing (scRNA-seq) [The task of identifying specific microglial subtypes is somewhat difficult and complex largely because of their overlapping patterns of gene expression. This problem is further complicated by the fact that these profiles also overlap with those of macrophages infiltrating from the periphery. Microglia have been identified in immunofluorescence and fluorescence-activated cell sorting studies via their expression of specific proteins, including ionised calcium-binding adapter molecule 1 (IBA1), transmembrane protein 119 (TMEM119), and purinergic receptor P2Y12 (P2RY12) as well as cluster of differentiation (CD) markers CD16, CD68, and CD11b ,22,23,24The results of a recent scRNA-seq study published by Zheng et al.  revealedIn addition to their region specific subtype heterogeneity, microglia can be activated via numerous proinflammatory pathways. These pathways can be initiated via microglial exposure to pathogen-associated molecular patterns (PAMPs) such as LPS and/or via damage-associated molecular patterns (DAMPs) that include free DNA fragments and extracellular ATP ,28,29. AThe use of scRNA-seq approaches has expanded our understanding of microglia both at homeostasis and in various disease conditions. Microglia have been frequently classified using the well-established binary system in which microglia expressing inflammatory genes are considered M1, whilst M2 microglia are associated with a neuroregenerative phenotype. M1 microglia are defined as cells that express proinflammatory cytokines including TNFα, IL-1β, and IL-6, whilst M2 microglia are identified by the expression of TGFβ. Some investigators have divided these two classifications into additional subgroups based on gene expression patterns . HoweverStudies featuring the use of sc/snRNA-seq have characterised transcriptomic changes in microglia over time and in response to various diseases including AD, ALS, and HIV infection .Several publications describe the results of scRNA-seq studies designed to explore microglial heterogeneity in AD. Collectively, these studies have identified multiple transcriptionally-distinct microglial subtypes that are unique amongst patients with AD. Mathys et al.  used a sen-Shaul  presenteALS is a disease in which muscle atrophy ultimately leads to respiratory failure typically within several years after the initial diagnosis . SimilarHIV infects immune cells and ultimately leads to the destruction of the host’s immune system unless identified promptly and treated continuously. HIV virions initially target CD4-positive immune cells; these infected cells produce and release progeny virions, thus permitting the infection to spread. Infected cells can enter the CNS via transit through the blood-brain barrier, where they release nascent HIV particles that proceed to infect immune cells in the brain. Microglia express CD4 and other HIV co-receptors, including CCR5 and CXCR4, and thus can be infected with HIV . AdditioLpl, Cst7, and Igf1 and displayed overall increased expression of genes associated with both demyelination and remyelination. This result suggests that this microglial subtype may promote altered myelination within the CNS and thus contribute to the development of pain. Another microglial subtype identified in the gp120 transgenic mouse model was classified as interferon-related and was characterised by increased expression of Ifit2, and Ifit3. These genes are associated with the activation of cellular interferon response and viral clearance [Two new microglial subtypes were identified in the brain and spinal cord of the gp120 transgenic mouse model of HIV-related pain by scRNA-seq . A subtylearance ,47. Genelearance , and thulearance . Howeverlearance ,49,50,51Microglial activation serves an important immunological function in the CNS. However, prolonged activation can lead to neurodegeneration and disease progression. Once they have detected an injury or infection, microglia move to the affected area and proceed to phagocytose cellular debris and infectious particles. However, if the microglia are unable to resolve the injury, they may remain in a prolonged state of activation and continue to release proinflammatory cytokines. These responses can lead to a prolonged inflammatory state and may exacerbate existing symptoms. Therefore, whilst microglial activation is necessary for neuronal maintenance and defense against infection, prolonged activation may cause or exacerbate neurodegeneration.Astrocytes are non-neuronal cells that are derived from radial glial cells in the embryonic ectoderm ,53. The Astrocytes perform numerous functions within the CNS. Historically, astrocytes were believed to contribute structural support and served as indicators of neurological disease. However, recent studies have shown that, in the absence of disease or injury, astrocytes may extend processes to cover specific regions of the brain and perform a variety of functions that regulate neuronal homeostasis. In studies in which electron microscopy was used to visualise their fine processes, protoplasmic astrocytes within the CA1 region of the hippocampus were found in non-overlapping domains. This finding suggests that astrocytes are capable of neuronal monitoring in distinct regions of the brain . HoweverAstrocytes can be identified by their expression of specific marker proteins including glial fibrillary acidic protein (GFAP), S100 calcium-binding protein-β (S100β), aldehyde dehydrogenase family 1 member L1 (Aldh1l1), and excitatory amino acid transporter 2  ,70,71,72Gfap and angiotensinogen (Agt). The increase in Agt expression suggests that this astrocyte subtype may contribute directly to synaptic plasticity and may interact closely with neurons [Unc13c and was found primarily within the cortical layers. Although UNC13C is involved in the process of membrane fusion, additional studies will be needed to understand the specific role of this pathway in astrocytes. The third new subtype of mature astrocytes exhibited higher levels of Agt and reduced levels of Gfap and Unc13c expression and was found throughout the cortex and hippocampus. In conclusion, single-cell sequencing and clustering analysis can provide detailed insight into region-specific expression patterns that can be used to identify subtypes and, more importantly, presents a powerful tool for the identification and evaluation of specific changes that develop in response to disease.Single-cell and single-nuclei studies of astrocytes , LINGO1, RASGEF1B were observed as were patterns associated with pathways involved in protein localisation to the endoplasmic reticulum, protein stabilisation, translational initiation, cellular response to mechanical stimulus, and neutrophil degranulation. Although further investigation will be needed to determine the significance of these findings, the amplification of pathways related to protein translation, localisation, and stabilisation suggest that this new astrocyte subtype may be more biologically active. The authors also reported a general decrease in the expression of genes associated with myelination; these findings suggest that astrocytes may be partially responsible for the demyelination observed during the progression of AD. Another snRNA-seq study targeting AD patients found that whilst the overall number of astrocytes remained unchanged, there was a significant decrease in the population of a neuroprotective astrocyte subtype in tissues from AD patients [HES5, NTRK2, SLC1A2, and SPARCL1 in association with diminished synaptic signaling and glutamate secretion [Results of a recent snRNA-seq study of human post-mortem tissue identified a cluster of astrocytes that were enriched in patients with AD characterised by increased expression of oping AD . Increaspatients . These recretion . These lMt1, Mt2, and Fam107a and decreased expression of Malat1 in astrocytes from this mouse model. While further studies will be needed to characterise the impact of these genes and their role in the pathogenesis of ALS, these results reveal the potential of scRNA-seq as a means to evaluate various disease states at the cellular and molecular levels.Changes in astrocyte expression have also been reported in association with ALS. A scRNA-seq study targeting the brainstem in a mouse model for ALS revealed enrichment of previously identified pathways in astrocytes that are related to neurogenesis and CNS development. This study also identified novel pathways including one involved in the regulation and organisation of cell projections . ResultsAlthough astrocytes do not express the cellular receptors known to mediate direct infection with HIV, the results of numerous studies suggest that HIV can infect and ultimately activate these cells. Postmortem tissues from HIV-infected individuals revealed that 1–7% of astrocytes contained HIV DNA or mRNA ,82. ThesOne earlier study used microarrays to examine transcriptomic changes associated with immune and neuronal function in cultured human astrocytes that were HIV-infected or exposed to HIV protein gp120. Both conditions resulted in similar patterns of differential gene regulation, although HIV infection alone resulted in significantly increased expression of chemokine and cytokine-related genes . AmongstSc/snRNA-seq is a powerful approach for studying transcriptomic alterations in response to disease at the single cell resolution. This approach has the potential to identify hundreds of genes within a specific type of cell that are differentially regulated in response to specific conditions, including diseases . Compare


Diabetic kidney disease (DKD) is one of the most common causes of end-stage renal disease worldwide. The treatment of DKD is strongly associated with clinical outcomes in patients with diabetes mellitus. Traditional therapeutic strategies focus on the control of major risk factors, such as blood glucose, blood lipids, and blood pressure. Renin–angiotensin–aldosterone system inhibitors have been the main therapeutic measures in the past, but the emergence of sodium–glucose cotransporter 2 inhibitors, incretin mimetics, and endothelin-1 receptor antagonists has provided more options for the management of DKD. Simultaneously, with advances in research on the pathogenesis of DKD, some new therapies targeting renal inflammation, fibrosis, and oxidative stress have gradually entered clinical application. In addition, some recently discovered therapeutic targets and signaling pathways, mainly in preclinical and early clinical trial stages, are expected to provide benefits for patients with DKD in the future. This review summarizes the traditional treatments and emerging management options for DKD, demonstrating recent advances in the therapeutic strategies for DKD. Diabetic kidney disease (DKD) is one of the most common and serious complications of diabetes mellitus (DM) and remains the leading cause of end-stage renal disease (ESRD) in Western countries . The gloAlthough there were no specific drugs with the exception of renin–angiotensin–aldosterone system (RAAS) inhibitors for a long time, the therapeutic strategies for DKD have advanced significantly over the last decade . With clLifestyle intervention is an essential component of the strategy for managing DM and its complications. For all patients with DM, lifestyle modifications, including weight loss, smoking cessation, adequate physical exercise, and restriction on sodium and protein intake, are crucial to preventing the onset of DKD and other adverse events .Obesity is a powerful risk factor for albuminuria, and DKD is more prevalent in patients with DM who are obese . ObesitySmoking is an independent risk factor for the occurrence and development of DKD . A meta-Clinical data of patients with T1DM or T2DM have demonstrated that regular physical activity improves insulin sensitivity and cardiovascular outcomes . One aniSodium restriction has been demonstrated to afford a potent benefit with respect to albuminuria in patients with DM . StandarIn addition, recent studies have illustrated that the influence of lifestyle on the occurrence and development of certain metabolic diseases may begin as early as embryonic stage, and the fetal programming hypothesis has been derived ,19. FetaPoor glycemic control is an independent predictor of albuminuria and ESRD progression in patients with DM . SeveralThe importance of intensive glycemic control in reducing albuminuria and improving the reduction in eGFR were initially confirmed in the Diabetes Control and Complications Trial (DCCT) and the Epidemiology of Diabetes Interventions and Complications (EDIC) study ,30. In tHowever, a retrospective analysis demonstrated that the renoprotective effect of intensive glycemic control was predominantly observed in patients with early DM and subjects without kidney diseases . For patHyperlipidemia is another risk factor for DKD progression, especially increases in triglycerides, low-density lipoprotein cholesterol (LDL-C), and apolipoprotein-B-100, which may increase the incidence of cardiovascular events in patients with DM with persistent albuminuria . Thus, tThe renoprotective effects of statin-based treatments remain to be demonstrated. The Anglo-Danish-Dutch Study of Intensive Treatment in People with Screen Detected Diabetes in Primary Care (ADDITION-Europe) claimed no obvious renoprotective effect of statins in patients with DM , but theHypertension is an important factor associated with DKD progression . One aniIn addition to increasing the risk of cardiovascular events, hypertension can activate the production of multiple cytokines, chemokines, and growth factors through hemodynamic mechanisms, leading to renal inflammation and fibrosis . GlomeruHyperglycemia can lead to glomerular hypertension through multiple pathways, such as dilating afferent arterioles by releasing vasoactive substances and tubuloglomerular feedback, and constricting efferent arterioles by increasing local angiotensin II (Ang II) levels . The RAARAAS inhibitors, which mainly include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), have been used to treat DKD for several decades. RAAS inhibitors have been widely demonstrated to be effective in protecting kidney function in several landmark studies ,49,50. TThe renoprotective effect of RAAS inhibitors was initially considered to be achieved by controlling blood pressure and was not recommended for use in patients with DKD without hypertension . HoweverSince the combination of ACEI and ARB did not provide more beneficial effects than monotherapy and instead increased the risk of hyperkalemia and acute kidney injury (AKI), the enthusiasm for enhanced RAAS blockade shifted to combining mineralocorticoid receptor antagonists (MRAs) with RAAS inhibitors . AldosteSpironolactone and eplerenone are the first studied MRAs. Animal studies have demonstrated that spironolactone could improve glomerulosclerosis in rats with DM, and eplerenone could decrease intraglomerular pressure and albuminuria in mice with DM ,59. EpleThe underlying mechanism of the renoprotective function of MRAs is still not fully understood, and some preclinical studies have shown that it may be related to the reduction of renal neutrophil gelatinase-associated lipocalin (NGAL) and the inhibition of Rac1 ,71. HoweAs mentioned above, the antialbuminuric effects of aldosterone antagonists have been established in clinical studies, and while the efficacy of other diuretics for DKD has been less well-studied, evidence suggests that they may have similar antialbuminuric properties.Several studies have shown that thiazide diuretics, such as hydrochlorothiazide and losalidone, have obvious albuminuria-reducing effects in patients with DKD, possibly by reducing intraglomerular pressure, but there are also results supporting that their renoprotective effects may be independent of their antihypertensive effects ,73,74. AOther diuretics have also been reported to have renoprotective effects. One study showed that the addition of furosemide on the basis of half-dose RAAS inhibitor had a better antialbuminuric efficacy than full-dose RAAS inhibitor therapy . ThiazidAlthough the antialbuminuric effects of most diuretics and their mechanisms require more research to elucidate and the fluid and electrolyte disturbances caused by them are nonnegligible side effects, diuretics should be considered an essential component of combination therapy.RAAS inhibitors were the only approved pharmacotherapy for patients with DKD until the advent of SGLT2 inhibitors. SGLT2 inhibitors are oral hypoglycemic drugs that exert hypoglycemic effects by inhibiting SGLT2, which is a cotransporter located in proximal tubule that transports sodium and glucose in a 1:1 ratio via the sodium-potassium ATPase-mediated sodium concentration gradient ,84. NearThe first major study validating the effects of SGLT2 inhibitors was the Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes (EMPA-REG) Outcome Trial . This trIt is worth mentioning that the effects of SGLT2 inhibitors on the risk of AKI in patients with DKD are heterogeneous. The cardiovascular safety data from large clinical studies of SGLT2 inhibitors showed significant differences in the effects of different SGLT2 inhibitors on the risk of AKI ,86,87. FThe physiological mechanisms underlying the cardiorenal benefits of SGLT2 inhibitors have not been thoroughly clarified. It is widely accepted that SGLT2 inhibitors correct afferent arteriole dilatation and glomerular hyperfiltration by increasing the sodium concentration in the macula densa. SGLT2 is upregulated in hyperglycemia and results in increased the reabsorption of sodium in the proximal tubule via the sodium–glucose co-transporter, which in turn leads to the low sodium concentration in the macula densa and activates tubuloglomerular feedback, causing the vasodilation of afferent arterioles and increased intraglomerular pressure and filtration; SGLT2 inhibitors reverse these changes, improve sodium and fluid retention, and reduce intraglomerular pressure . AnotherAdmittedly, SGLT2 inhibitors are limited by potential side effects such as ketoacidosis and urinary tract infections . This maIncretins are peptides that are secreted by intestinal cells and are involved in glycemic regulation. Incretins exert hypoglycemic effects by stimulating pancreatic β-cell proliferation to increase insulin and inhibit glucagon release from pancreatic α-cells . GLP-1 iApart from their hypoglycemic effects, emerging evidence illustrated that GLP-1R agonists may have complex renoprotective effects. GLP-1R agonists can induce natriuresis and diuresis by suppressing the sodium-hydrogen exchanger 3 (NHE3) in the brush border of proximal tubular cells, and they also reduce Ang II levels and thereby inhibit renal RAAS activation ,101. TheAt least six GLP-1R agonists have been tested in clinical research  106,107,110,111.A comparison of data on emplagiflozin and liraglutide revealed that SGLT2 inhibitors had a more pronounced benefit on renal endpoints (doubling SCr levels and ESRD progression) than GLP-1R agonists . This diTo date, several DPP-4 inhibitors have been tested in large clinical studies: sitagliptin, saxagliptin, linagliptin, and alogliptin  115,116,118. TheIt was previously thought that the effects of DPP-4 inhibitors were mainly dependent on GLP-1 and its receptor, but the evidence that DPP-4 inhibitors also benefit the kidneys of mice lacking the GLP-1R suggests that the incretin-independent effects of DPP-4 inhibitors need to be explored . EmerginThe increased risk of hypoglycemia and reduced insulin clearance make the application of insulin in patients with advanced DKD still controversial . IncretiThe ET family was identified in the late 1980s, and three isoforms of ET have been described: ET-1, ET-2, and ET-3 . ET-1 isPreclinical and clinical studies have described that ET-1 activity is increased in patients with DM and that ET-1 receptor antagonists (ERAs) reduce albuminuria and protect renal function ,134,135.Although available clinical trials have shown that ERAs may be novel drugs for DKD, no consistent conclusions regarding their effectiveness and safety in patients with DKD have been reached. The Avosentan for Overt Diabetic Nephropathy (ASCEND) study showed that patients treated with avosentan demonstrated a reduction in doubled SCr and albuminuria compared with the placebo group, demonstrating that avosentan has renal benefit . UnfortuMetabolic surgery occupies an important position in the interventions of patients with severe obesity with DM; however, emerging evidence implies that metabolic surgery improves the albuminuria and reduces the progression of ESRD in patients with obesity, whereas this renoprotective effect occurs independent of body weight ,141.Roux-en-Y gastric bypass (RYGB) is the metabolic surgery with the most prominent renal protective effects. Preclinical studies have demonstrated improved glomerular morphology, reduced mechanical stretch of podocytes, and reduced macrophage infiltration and fibrosis after metabolic surgery . One cliBecause the clinical specimens of kidneys from patients with DM receiving metabolic surgery are not easily available, the molecular mechanism of its renoprotective effects has not been fully clarified. Additionally, no studies have examined changes in dyslipidemia, blood pressure, and eGFR before and after metabolic surgery, and these deficiencies should be the focus of future research in this field.Protein kinase C (PKC) is a key intracellular signaling molecule involved in the pathogenesis of DKD. DM can activate PKC through glucose itself, Ang II, and advanced glycation end products (AGEs), and elevated PKC can in turn participate in the pathophysiology of DKD . The effThe PKC-β inhibitor ruboxistaurin is renoprotective according to preclinical and clinical studies. In animal experiment, ruboxistaurin ameliorated the increase of albuminuria and SCr in rats with DKD . In a clAs an adipokine, adiponectin improves insulin sensitivity and reduces serum glucose levels in obesity-related metabolic disorders, including DM, by promoting glucose transporter-4-mediated glucose uptake in muscle and adipose tissue and inhibiting hepatic gluconeogenesis . In an aDKD is generally considered a noninflammatory disease. However, a genome-wide transcriptome profiling study identified strong inflammatory signaling pathways in the pathogenesis of DKD . This fiThe proinflammatory factor IL-20 is recognized as a causative factor in kidney injury and may be an important therapeutic target for slowing the progression of DKD . The useChemokines and chemokine receptors also play important roles in the progression of DKD. C-C motif chemokine ligand 2 (CCL2), a monocyte chemoattractant protein, has been identified to be overexpressed in kidneys of animals and patients with DM, and inhibition of CCL2 is associated with renoprotective effects ,167. EmaThe Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is a family of intracellular signaling molecules activated by binding extracellular cytokine, chemokine, growth factor, and hormone ligands to glomerular cell surface receptors, and their persistent activation is closely related to many inflammatory diseases . The anaBaricitinib is a selective inhibitor of JAK-1 and JAK-2. One clinical study designed to investigate the effects of baricitinib in patients with T2DM showed that treatment with baricitinib in combination with RAAS inhibitors improved albuminuria and reduced inflammatory biomarkers, such as serum TNF receptor 1, TNF receptor 2, and urinary CCL2 . HoweverApoptotic signal-regulated kinase 1 (ASK1) is a stress-responsive mitogen-activated protein kinase (MAPK) that signals through a series of downstream kinases to regulate the expression of target genes, including inflammatory cytokine genes . Kidney Selonsertib is a selective ASK1 inhibitor and its renoprotective effect in patients with T2DM was investigated in a clinical study with change of eGFR as the primary endpoint; although selonsertib showed no significant effect on eGFR and UACR compared with placebo, the post hoc analysis suggested that selonsertib had potential renal benefits .Oxidative stress caused by the formation of reactive oxygen species (ROS) due to cellular respiratory dysfunction under diabetic conditions is thought to exert a crucial effect in the development of DKD . HyperglNuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that was confirmed to prevent damage from oxidative stress . Under sThe most promising Nrf2 agonist at present is bardoxolone methyl, a synthetic derivative of oleanolic acid. One early clinical trial with patients with T2DM designed to investigate the efficiency and safety of bardoxolone methyl demonstrated robust increases in eGFR in subjects, but the trial was terminated prematurely due to the risk of cardiorenal events . In the NADPH oxidase (NOX) is an enzyme family proven to be an important source of ROS that is upregulated in patients with DM . NOX4, aStudies have shown that some bioactive compounds with antioxidant properties, either from the diet or from plants, protect against DM and its complications, and these natural products are generally considered to have better safety profiles than other drugs.Several vitamins are effective antioxidants and are closely linked to the onset of DM. Vitamin C can directly scavenge ROS, and its effect on T2DM has been evaluated in numerous studies. Both animal models and clinical studies have shown that vitamin C significantly inhibits oxidative stress and improves albuminuria and glomerulosclerosis ,191. StuCurcumin, which is found in turmeric, has antioxidant and anti-inflammatory properties . One aniResveratrol is a natural polyphenolic compound that is commonly found in peanuts and berries . ResveraRenal fibrosis is the final common pathway of pathophysiological processes such as abnormal renal hemodynamics, a high-glucose environment, oxidative stress, RAAS hyperactivation, ischemia, and inflammation, and it is the main reason for the progression of DKD and the occurrence of ESRD. Some of the therapeutic strategies mentioned above, such as RAAS inhibition, reduce renal fibrosis but with limited efficacy . To achiPirfenidone, which is an antifibrotic drug used to treat idiopathic pulmonary fibrosis, was recently also evaluated for its antifibrotic effects in DKD. An animal model suggested that pirfenidone reduced mesangial matrix expansion associated with renal fibrosis but had no effect on albuminuria . One ranTGF-β is thought to be a key driver of renal fibrosis in many progressive kidney diseases because of its ability to lead to myofibroblast activation and the overproduction of ECM . TherefoBecause TGF-β has important immunomodulatory effects and it is difficult to directly target TGF-β, many studies have been devoted to finding new antifibrotic targets and drugs in recent years.Deficiencies in nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) have been shown to directly promote the progression of renal fibrosis . SolubleHypoxia-inducible factor (HIF) is a nuclear transcription factor activated under hypoxic conditions, consisting of HIF-α and HIF-1β in the form of heterodimers . HIF-α hApelin, which is an adipokine found in adipose tissue, has been shown to alleviate clinical symptoms in patients with DM by regulating blood glucose . In the Pyrroloquinoline quinone (PQQ), which is a natural bioactive compound, was previously shown to protect human renal tubular epithelial cells under high-glucose conditions, but the underlying mechanism is unclear . In a reSIRT3, which is a major mitochondrial deacetylase, blocks organ fibrosis by modulating TGF-β/Smad signaling . One stuAlthough most of these treatments have not been clinically evaluated, their potential to inhibit diabetes-related renal fibrosis has brought renewed enthusiasm to the field of DKD therapeutics.Autophagy is a highly conserved lysosomal degradation pathway that clears damaged proteins and organelles to maintain cellular homeostasis . AutophaThe mammalian target of rapamycin (mTOR) signaling pathway is an important element of autophagy, and an animal model showed that rapamycin has beneficial effects on the histopathological changes in rats with DKD by inhibiting the mTOR signaling pathway, but it has some side effects, including immunosuppression and renal toxicity ,244. TheIdentifying other autophagy-related signaling pathways is also a potential renoprotective approach for future studies. One recent study demonstrated that hyperglycemia increased the expression of microRNA-214 (miR-214) in the kidney, which in turn led to the downregulation of unc-51-like autophagy-activating kinase 1 (ULK1), which may be responsible for the autophagy damage in DM. Removing the miR-214 of renal proximal tubules reduced the downregulation of ULK1 in kidneys and prevented autophagy damage, whereas p53 inhibition decreased the induction of miR-214 and ameliorated renal injury . This exDKD was previously thought to have a uniform natural history characterized by the development of persistent albuminuria with a decline in eGFR and eventually leading to renal failure . HoweverThe heterogeneity of nonalbuminuric DKD makes its diagnosis crucial and challenging. UACR is generally considered to be an important criterion for early diagnosis of DKD, but for patients without albuminuria, eGFR is clearly a more appropriate diagnostic indicator . A varieOne meta-analysis demonstrated that renal function declined more rapidly in men than in women amo patients with kidney diseases caused by other causes, but it was opposite in populations with nonalbuminuric DKD . One cliPharmacological therapies for nonalbuminuric DKD should focus on improving eGFR rather than traditional antialbuminuric therapy. Many patients with nonalbuminuric DKD have a usage history of RAAS inhibitors, and the discontinuation of RAAS inhibitors often leads to the appearance of proteinuria . The cliIn recent years, the adverse effects of DKD in patients with DM have received increasing attention, and the importance of developing new treatments for DKD has been widely recognized. The demonstration of the renoprotective effects of SGLT2 inhibitors, GLP-1R agonists, and DPP-4 inhibitors has introduced a new era in the field of DKD treatment. Novel drugs targeting inflammation, fibrosis, oxidative stress, and other new targets also show promise for patients with DKD, although a large number of clinical studies are needed to assess their safety and efficacy. In addition, a single class of drug therapy may not produce significant therapeutic effects, but with the continued emergence of agents that work through different mechanisms, combination therapy with multiple drugs is expected to achieve a breakthrough in the treatment of DKD in the future.